IL12B Recombinant Monoclonal Antibody

What is IL12B and what role does it play in immune function?

IL12B (p40) is the 40 kDa subunit of interleukin-12 that performs multiple crucial immunological functions. IL12B can form a heterodimer with either the p35 subunit (creating IL-12) or with the p19 subunit (creating IL-23) . As part of IL-12, it induces production of IFN-gamma and TNF by resting and activated T and NK cells, enhances cytotoxic activity of NK and T cells, and acts as a co-mitogen to stimulate T cell proliferation . The p40 subunit also exists as a monomer and homodimer, with the latter functioning as a potent IL-12 antagonist .

IL-12 represents a pivotal mediator of cell-mediated immunity through its actions on TH1 cell development, proliferation, and activity. It is primarily produced by macrophages, dendritic cells, activated phagocytes, and to a lesser extent, B cells in response to infectious agents .

How are IL12B recombinant monoclonal antibodies generated?

IL12B recombinant monoclonal antibodies are produced through sophisticated in vitro cloning techniques. The general methodology involves:

• Gene cloning and vector construction: The genes encoding both heavy and light chains of the IL12B antibody are isolated and integrated into expression vectors .

• Host cell transfection: The vectors are introduced into appropriate host cells (commonly mammalian cell lines or insect cells like Spodoptera frugiperda) .

• Cell culture expression: The transfected cells express the recombinant antibody in a controlled cell culture environment .

• Purification: Following expression, the antibody undergoes affinity chromatography purification to isolate the target protein .

• Quality control: The purified antibody is characterized through techniques such as SDS-PAGE and Size Exclusion Chromatography to verify purity and structural integrity .

This process allows for consistent production of high-quality monoclonal antibodies with specific binding characteristics and functional properties.

What are the structural components of IL12B antibodies and how do they affect function?

IL12B antibodies typically consist of standard immunoglobulin structures with two heavy chains and two light chains, forming antigen-binding Fab regions and an Fc region that can recruit immune effector functions . The specific binding regions are engineered to recognize distinct epitopes on the IL12B protein.

For example, the monoclonal antibody described in catalog number ABIN948031 targets amino acids 229-328 of human IL12B , while other antibodies may target different regions such as Val30~Ser320 . The choice of binding epitope significantly influences the antibody's functional properties, including:

• Neutralization capability: Antibodies binding to crucial functional domains may block interactions with receptors or with the p35 subunit

• Cross-reactivity: Some antibodies like the one described in search result cross-react with murine IL-12

• Application suitability: Different epitope binding affects performance in specific applications like ELISA, flow cytometry, or Western blotting

The clonality, isotype (commonly IgG1, IgG2a), and species origin (mouse, rabbit, etc.) of the antibody further define its functional properties and experimental utility .

What are the optimized applications for IL12B recombinant monoclonal antibodies?

IL12B recombinant monoclonal antibodies have been validated for multiple research applications with specific optimization parameters:

For optimal detection of IL-12 p70 heterodimer by ELISA, researchers should consider combining complementary antibodies, such as using purified B-T21 antibody (targeting p35) for capture and biotinylated C8.6 antibody for detection .

How should experimental conditions be optimized when using IL12B antibodies in flow cytometry?

For optimal flow cytometry results with IL12B antibodies, comprehensive optimization of experimental conditions is essential:

• Cell stimulation protocols:

  • For optimal IL-12 detection in PBMC monocytes, employ sequential stimulation with IFN-gamma (10 ng/mL for 2 hours), followed by LPS (100 ng/mL for 12 hours), and lastly monensin (3 μM for 3 hours)

• Fixation and permeabilization:

  • Use appropriate fixation buffers (e.g., Flow Cytometry Fixation Buffer)

  • Follow with suitable permeabilization solutions (e.g., Flow Cytometry Permeabilization/Wash Buffer I)

  • These steps are critical since IL12B is predominantly an intracellular target

• Antibody selection and staining procedure:

  • Primary antibody: Anti-IL12B at optimized concentration (typically 1:50-1:200 dilution)

  • Secondary antibody: Fluorophore-conjugated detection antibody (e.g., Allophycocyanin-conjugated Anti-species IgG)

  • Consider co-staining with cell surface markers (e.g., CD14 for monocytes) using PE-conjugated antibodies

• Controls:

  • Include unstimulated cells as negative controls

  • Use isotype controls to confirm specificity

  • Consider fluorescence minus one (FMO) controls for multicolor panels

A properly optimized flow cytometry protocol allows for sensitive detection of IL12B in various cell populations while minimizing background and non-specific signals.

What are the critical parameters for validating IL12B antibody specificity in research applications?

Validating IL12B antibody specificity requires a multi-faceted approach:

• Subunit specificity determination:

  • Test reactivity against recombinant human IL-12 (p70 heterodimer)

  • Confirm binding to p40 subunit using isolated p40 protein

  • Test against chimeric murine/human IL-12 to map binding regions

  • Verify absence of cross-reactivity with p35 subunit unless explicitly designed for p35

• Cross-species reactivity assessment:

  • Systematically test against IL12B from multiple species (human, mouse, rat, etc.)

  • Only 3 of 22 monoclonal antibodies in one study cross-reacted with murine IL-12

• Functional validation:

  • Neutralization assays: Evaluate the antibody's ability to block IL-12 biological activity in cell-based assays

  • Of 22 monoclonal antibodies tested in one study, only 7 demonstrated neutralizing activity against IL-12

  • Assess IFN-γ production inhibition in appropriate bioassays

• Application-specific controls:

  • Use knockout/knockdown systems as negative controls

  • Compare multiple antibodies targeting different epitopes

  • Include recombinant IL12B as positive control

These validation steps ensure experimental reliability and help researchers select the appropriate antibody for their specific research questions.

How have IL12B antibodies contributed to understanding tumor microenvironment interactions?

IL12B antibodies have been instrumental in developing novel immunotherapeutic approaches that leverage IL-12's potent antitumor properties while addressing toxicity concerns:

• Antibody-cytokine fusion proteins:
  Researchers have developed innovative IL12-antibody fusion constructs that selectively target the tumor microenvironment:

  • The IL12-F8-F8 fusion protein combines IL-12 with antibodies specific to the alternatively spliced EDA domain of fibronectin (a tumor neovasculature marker)

  • This construct demonstrated potent tumor growth inhibition in three different immunocompetent syngeneic models of cancer

  • Quantitative biodistribution analysis confirmed selective localization to tumor neovasculature in vivo

  • The approach allows production of both murine IL-12 (mIL12) and human IL-12 (hIL12) versions

• FAP-targeted IL-12 delivery:

  • The mIL12-7NP2 fusion protein combines IL-12 with antibodies targeting human fibroblast activation protein (FAP)

  • Selective accumulation was observed in FAP-positive tumors but not in FAP-negative tumors or healthy organs

  • Immunofluorescence-based biodistribution analysis confirmed preferential accumulation in FAP-positive neoplastic lesions 24 hours after intravenous administration

These approaches demonstrate how IL12B antibodies can be engineered to overcome systemic toxicity limitations while preserving antitumor efficacy, providing crucial insights into tumor-immune interactions.

What challenges exist in IL-12 pharmacokinetics and how do antibody-based approaches address them?

IL-12 pharmacokinetic desensitization represents a significant challenge in therapeutic applications, characterized by reduced IL-12 concentrations and biological effects following repeated exposure. Recent research has identified two potential mechanisms:

• Increased clearance model:

  • Previously proposed to occur due to upregulation of IL-12 receptor on T-cells causing increased receptor-mediated clearance

  • Modeling studies demonstrated this mechanism failed to capture trends in clinical trial data

  • The model predicts changes in maximal time and half-life that weren't observed clinically

• Reduced bioavailability model:

  • More accurately predicts IL-12 pharmacokinetics across clinical trials

  • The model suggests issues with IL-12 bioavailability and transport into blood rather than accelerated clearance

  • Mathematical modeling demonstrated this mechanism could fit clinical datasets accurately across a wide range of parameter values

Antibody-based approaches address these challenges through:

• Targeted delivery: Fusion proteins like IL12-F8-F8 and mIL12-7NP2 reduce systemic exposure while maintaining local efficacy

• Altered pharmacokinetics: Antibody components can extend half-life and modify distribution

• Controlled release: Antibody-mediated binding to tissue components can create local reservoirs for sustained activity

These strategies represent promising avenues to overcome the pharmacokinetic limitations that have historically hindered IL-12's therapeutic potential.

How do IL12B antibodies enhance combination immunotherapy approaches?

IL12B antibodies significantly augment combination immunotherapy strategies through several mechanisms:

• Rituximab combination therapy:
  A phase I clinical study combining IL-12 with rituximab (anti-CD20 antibody) in B-cell lymphoma demonstrated:

  • Objective responses in 29 of 43 patients (69%)

  • 8 of 11 complete responses at IL-12 doses ≥300 ng/kg

  • Optimal immunologic dose: 300 ng/kg subcutaneously twice weekly

  • 20-fold increase in serum IFN-γ levels and 2.5-5 fold increase in IP-10 levels at IL-12 doses ≥100 ng/kg

  The mechanism involves IL-12 enhancing rituximab efficacy by:

  • Increasing cytotoxicity of T and NK cells

  • Promoting antibody-dependent cellular cytotoxicity (ADCC)

  • Recruiting immune cells to tumor sites through chemokine induction

• Paclitaxel combination:

  • The murine IL12-F8-F8 fusion protein demonstrated enhanced efficacy when combined with paclitaxel

  • This combination showed significant activity across multiple tumor models

• Immune checkpoint inhibitor synergy:

  • IL-12's ability to stimulate Th1 responses can complement checkpoint inhibitor activity

  • Local IL-12 delivery may overcome resistance to checkpoint blockade

The success of these approaches highlights the potential of IL12B antibodies to enhance existing immunotherapies by promoting more robust and directed immune responses against malignancies.

What strategies optimize detection sensitivity for low abundance IL12B in complex biological samples?

Detecting low-abundance IL12B in complex samples requires specialized methodological approaches:

• Enhanced stimulation protocols for cellular IL12B expression:

  • Optimized sequential stimulation with IFN-gamma (10 ng/mL for 2 hours), LPS (100 ng/mL for 12 hours), and monensin (3 μM for 3 hours) maximizes IL-12 production in monocytes

  • This protocol significantly increases detection sensitivity in flow cytometry and immunoassays

• Advanced immunoassay techniques:

  • Sandwich ELISA using complementary antibodies targeting different epitopes:

    • Use B-T21 antibody (targeting p35) for capture at 1-4 μg/mL

    • Combine with biotinylated C8.6 antibody for detection

    • Include recombinant human IL-12 p70 standards ranging from 15-2000 pg/mL

• Signal amplification methods:

  • Tyramide signal amplification for immunohistochemistry

  • Polymer-based detection systems

  • Biotin-streptavidin amplification

• Sample preparation optimization:

  • For cell lysates: Use appropriate detergents (RIPA buffer with protease inhibitors)

  • For tissue samples: Consider antigen retrieval methods

  • For serum/plasma: Pre-clearing with protein A/G can reduce background

• Technical considerations for Western blotting:

  • Extended transfer times for optimal protein migration

  • Blocking with 5% BSA rather than milk to reduce background

  • Overnight primary antibody incubation at 4°C

These approaches significantly enhance detection sensitivity and reliability when analyzing IL12B in biological specimens where the target may be present at low concentrations.

How can researchers effectively differentiate between free IL12B and heterodimeric forms (IL-12/IL-23) in experimental systems?

Differentiating between free IL12B and its heterodimeric cytokine forms requires specific analytical approaches:

• Antibody selection strategy:

  • Use antibodies with known epitope specificity:

    • Antibodies targeting p35 (like B-T21) exclusively detect IL-12 heterodimer

    • Antibodies targeting p19 exclusively detect IL-23 heterodimer

    • Antibodies targeting p40 (IL12B) detect all forms (free IL12B, IL-12, and IL-23)

• Sequential immunoprecipitation approach:

  • First immunoprecipitation with anti-p35 antibodies captures IL-12 heterodimer

  • Second immunoprecipitation with anti-p19 antibodies captures IL-23 heterodimer

  • Final immunoprecipitation with anti-p40 antibodies captures remaining free IL12B

• Size-based differential analysis:

  • Native PAGE or size exclusion chromatography to separate proteins based on size

  • Western blotting with anti-IL12B antibodies will detect different molecular weight bands:

    • ~40 kDa: Free IL12B monomer

    • ~70-80 kDa: IL-12 or IL-23 heterodimers

    • ~80 kDa: IL12B homodimer

• Functional bioassays:

  • IL-12-specific bioassays using cells expressing only IL-12 receptors (IL-12Rβ1/IL-12Rβ2)

  • IL-23-specific bioassays using cells expressing only IL-23 receptors (IL-12Rβ1/IL-23R)

  • Comparing results with these selective bioassays can distinguish which heterodimer is present

By employing these techniques, researchers can accurately quantify the distribution of IL12B between its free and heterodimeric forms, providing crucial insights into the biological significance of each form in various experimental systems.

What are the methodological considerations for analyzing IL12B-mediated signaling pathways in different cell types?

Analyzing IL12B-mediated signaling requires tailored approaches for different cell populations:

• Cell type-specific receptor expression profiling:

  • IL-12 signals through IL-12Rβ1/IL-12Rβ2 heterodimeric receptors

  • IL-23 signals through IL-12Rβ1/IL-23R heterodimeric receptors

  • Flow cytometric characterization of receptor expression helps predict responsiveness

• Phospho-protein analysis optimization:

  • For JAK-STAT pathway activation:

    • Optimal stimulation timepoints: pJAK2 (5-15 min), pSTAT4 (15-30 min)

    • Quick sample processing is essential (immediate fixation)

    • Use phosphatase inhibitors in all buffers

  • Recommended antibody panels:

    • For T cells: anti-pSTAT4 + anti-CD3/CD4/CD8

    • For NK cells: anti-pSTAT4 + anti-CD56/CD16

    • For monocytes: anti-pSTAT4 + anti-CD14

• Downstream effect measurement:

  • IFN-γ production assays:

    • ELISA measurement at 24-48 hours post-stimulation

    • Intracellular cytokine staining at 6-12 hours (with protein transport inhibitors)

  • Cytotoxicity assays:

    • 51Cr release assays for NK and T cell cytotoxicity

    • Flow cytometry-based killing assays

• Pathway inhibition strategies:

  • JAK inhibitors (e.g., tofacitinib) to block STAT activation

  • Anti-IL12B neutralizing antibodies to block initial signaling

  • siRNA knockdown of receptor components

• Cell-specific considerations:

  • T cells: Pre-activation with anti-CD3/CD28 enhances IL-12 responsiveness

  • NK cells: IL-15 priming increases receptor expression

  • Monocytes: LPS pre-treatment induces IL-12 production and alters signaling dynamics

These methodological considerations enable detailed characterization of IL12B-mediated signaling in different immune cell populations, providing insights into cytokine-specific effects on immune function.

How can researchers address IL12B antibody cross-reactivity issues in multi-species studies?

Cross-reactivity issues present significant challenges in comparative immunology research. Researchers can implement several strategies to address these limitations:

• Comprehensive cross-reactivity testing:

  • Systematically validate antibodies against recombinant IL12B from multiple species

  • From historical data, only 3 of 22 monoclonal antibodies tested cross-reacted with murine IL-12

  • Document exact epitope binding regions that determine cross-reactivity

• Species-specific antibody development approaches:

  • Target conserved epitopes when cross-reactivity is desired

  • Select species-unique regions when specificity is required

  • Consider developing species-specific detection panels

• Alternative detection strategies when cross-reactivity is limited:

  • Species-specific PCR for mRNA detection

  • Mass spectrometry-based protein identification

  • Use of species-matched detection systems

• Data interpretation with cross-reactivity limitations:

  • Adjust sensitivity expectations based on known affinity differences

  • Include appropriate controls for each species

  • Consider parallel validation with multiple antibody clones

• Technical optimization for cross-reactive antibodies:

  • Adjust antibody concentrations based on species-specific affinity testing

  • Modify incubation conditions (time, temperature, buffer composition)

  • Evaluate different detection systems for optimal signal-to-noise ratios

When selecting antibodies for multi-species studies, researchers should prioritize clones with demonstrated cross-reactivity or develop species-specific detection strategies when necessary.

What are the key considerations for long-term storage and handling of IL12B antibodies to maintain functionality?

Proper storage and handling are critical for maintaining IL12B antibody functionality:

Recommendations from manufacturers indicate that properly stored antibodies remain stable for one year after shipment . Researchers should monitor antibody performance over time, particularly for critical applications like neutralization assays or therapeutic studies.

How can inconsistent results with IL12B antibodies across different experimental systems be reconciled?

Inconsistent results with IL12B antibodies across experimental systems can be systematically addressed:

• Sample preparation variability:

  • Standardize lysis buffers and protein extraction protocols

  • Control for post-translational modifications that might affect epitope recognition

  • Consider native vs. denatured conditions affecting epitope accessibility

• Antibody validation across applications:

  • Recognize that antibodies optimized for one application may not perform equally in others

  • Validate each application independently (WB, ELISA, IHC, flow cytometry)

  • Example: The antibody described in ABIN948031 is validated for Western Blotting and ELISA but may not perform optimally in other applications

• Technical optimization strategies:

  • Dilution optimization: "Optimal dilutions should be determined by each laboratory for each application"

  • Blocking buffer composition affects background and signal-to-noise ratio

  • Incubation conditions (time, temperature) require application-specific adjustment

• Control integration:

  • Include positive controls (recombinant IL12B protein)

  • Utilize negative controls (IL12B knockout/knockdown samples)

  • Implement isotype controls for immunostaining applications

• Data interpretation frameworks:

  • Triangulate results using multiple antibody clones targeting different epitopes

  • Compare results across complementary detection methods

  • Consider biological variability in IL12B expression across different experimental systems

By implementing these systematic approaches, researchers can identify the source of inconsistencies and develop standardized protocols that yield reproducible results across experimental systems.

What novel modifications of IL12B antibodies are being developed to enhance their research and therapeutic utility?

Cutting-edge research is expanding the capabilities of IL12B antibodies through innovative engineering approaches:

• Advanced tumor-targeting immunocytokine formats:

  • The IL12-F8-F8 format fuses IL-12 with two F8 antibodies in single-chain Fv (scFv) format targeting the EDA domain of fibronectin

  • The mIL12-7NP2 format targets fibroblast activation protein (FAP) in the tumor microenvironment

  • These designs demonstrate superior tumor selectivity compared to conventional IL-12 administration

• Novel antibody engineering strategies:

  • Tandem diabody formats enhance tumor accumulation and retention

  • Sequential fusion of IL-12 as a single polypeptide with targeting antibodies

  • Integration of cleavable linkers for conditional activation in tumor microenvironments

• Cell therapy integration approaches:

  • Cell membrane-anchored and tumor-targeted IL-12-T (attIL12-T) cell products avoid systemic toxicity

  • These approaches seek to prevent cytokine release syndrome in peripheral tissues while maintaining antitumor efficacy

• Combination therapy optimization:

  • IL12B antibody fusions synergize with established therapies:

    • Combination with rituximab enhances efficacy in B-cell lymphoma

    • Paclitaxel combinations demonstrate enhanced activity in multiple tumor models

These innovations represent promising avenues for enhancing the research and therapeutic utility of IL12B antibodies, especially in challenging areas like cancer immunotherapy.

How are researchers addressing challenges in studying IL12B heterodimeric interactions with novel antibody approaches?

Researchers are developing innovative antibody-based approaches to study the complex biology of IL12B heterodimeric interactions:

• Bispecific antibody technologies:

  • Dual-targeting antibodies simultaneously binding p40 and p35 (for IL-12) or p40 and p19 (for IL-23)

  • These constructs allow specific isolation and functional analysis of intact heterodimers

  • Enable comparative studies between heterodimers sharing the common p40 subunit

• Conformation-specific antibody development:

  • Antibodies recognizing unique epitopes formed only in the assembled heterodimer

  • These reagents distinguish between monomeric p40 and heterodimeric forms

  • Enable precise quantification of assembled cytokines vs. free subunits

• Proximity-based detection systems:

  • Pairs of antibodies targeting different subunits coupled with proximity ligation assay (PLA) technology

  • FRET-based antibody pairs for real-time monitoring of heterodimer assembly

  • These approaches provide spatial and temporal information about heterodimer formation

• Structural biology integration:

  • Antibodies that stabilize specific conformations for crystallography studies

  • Fragment antibodies facilitating cryo-EM analysis of heterodimeric complexes

  • These tools advance our understanding of the structural basis for IL12B interactions

These approaches are transforming our ability to study the complex biology of IL12B-containing heterodimers and their distinct roles in immune regulation and disease pathogenesis.

What are the prospects for developing antibodies targeting post-translational modifications of IL12B?

Emerging research is exploring the role of post-translational modifications (PTMs) in IL12B biology and the development of modification-specific antibodies:

• Key IL12B post-translational modifications under investigation:

  • Glycosylation patterns affecting heterodimer assembly and stability

  • Phosphorylation events potentially regulating secretion and bioactivity

  • Proteolytic processing modifying biological functions

• Development challenges for PTM-specific antibodies:

  • Generating antibodies with exquisite specificity for modified epitopes

  • Validating specificity across different experimental systems

  • Ensuring recognition of physiologically relevant modifications

• Methodological innovations enabling PTM-specific detection:

  • Synthetic peptide antigens incorporating defined modifications

  • Recombinant expression systems with controlled modification patterns

  • Mass spectrometry validation of modification-specific antibody binding

• Research applications:

  • Studying the kinetics of IL12B modification in different activation states

  • Analyzing tissue-specific modification patterns

  • Investigating the role of modifications in disease contexts

• Therapeutic implications:

  • Targeting specifically modified forms of IL12B for enhanced selectivity

  • Developing antibodies that modulate modification-dependent protein-protein interactions

  • Engineering therapeutic antibodies that recognize disease-associated modification patterns

These emerging approaches promise to reveal new layers of IL12B regulation through post-translational modifications and provide novel tools for both basic research and therapeutic development.