Recombinant Human Interleukin-12 subunit alpha protein (IL12A) (Active)

What is the structural composition of IL-12α protein?

IL-12α (also known as p35) is a 35 kDa protein subunit composed of a bundle of four alpha helices . It forms part of heterodimeric cytokines, most notably IL-12 (when combined with the 40 kDa IL-12β/p40 subunit) and IL-35 (when paired with Ebi3) . Structurally, IL-12α shows significant sequence similarity to IL-6, G-CSF, and chicken MGF, suggesting evolutionary relationships with these cytokines .

The IL-12α subunit contains several critical regions that affect its function:

• The protein contains disulfide linkages important for maintaining its tertiary structure

• Post-translational modifications may occur, including potential cleavage sites (e.g., between Lys260 and Arg261)

• N-glycosylation sites contribute to heterogeneity in molecular weight

How does IL-12α function within the broader IL-12 family of cytokines?

IL-12α exists as a component of multiple cytokines with contrasting functions:

The IL-12 family is unique in comprising the only heterodimeric cytokines, which includes IL-12, IL-23, IL-27, and IL-35 . Despite sharing structural features and molecular partners, these cytokines mediate diverse and sometimes opposing immunological effects. While IL-12α is broadly expressed in many cell types, the IL-12β subunit expression is primarily limited to hematopoietic cells , creating a regulatory mechanism for IL-12 production.

What are the established detection methods for IL-12α in experimental settings?

The most widely used method for IL-12α detection is Enzyme-Linked Immunosorbent Assay (ELISA). A typical sandwich ELISA for IL-12α involves:

• A microplate pre-coated with an antibody specific to IL-12α

• Addition of standards or samples to appropriate wells

• Addition of HRP-conjugated antibody specific for IL-12α

• Development with TMB substrate solution, producing a colorimetric reaction

• Measurement of optical density at 450 nm

Commercial ELISA kits offer sensitivity around 46.2 pg/ml with detection ranges typically between 66-1200 pg/ml .

For protein characterization, researchers commonly employ:

• SDS-PAGE under reducing and non-reducing conditions

• Size-exclusion chromatography (SEC-HPLC)

• UPLC/MS for precise molecular weight determination

• N-terminal sequencing for fragment analysis and confirmation of protein identity

What are critical considerations when designing experiments with recombinant human IL-12α?

When working with recombinant human IL-12α, researchers should consider:

Protein quality assessment:

• Verify protein integrity using SDS-PAGE under both reducing and non-reducing conditions

• Check for potential cleavage products, as fragmentation can occur between specific amino acids (e.g., Lys260 and Arg261)

• Confirm molecular weight using mass spectrometry (expected ~35 kDa for IL-12α)

Bioactivity evaluation:

• Assess functional activity through induction of IFN-gamma secretion in NK-92 human natural killer cells (when combined with IL-12β)

• Consider that fragmented IL-12 shows approximately half the specific activity of intact IL-12

Experimental design factors:

• Dosing range: Clinical studies have utilized doses from 50-250 ng/kg to establish dose-response relationships

• Expression system selection: Mammalian expression systems (e.g., HEK293 cells) in serum-free media yield higher quality protein

• Storage conditions: Proper aliquoting and storage at -80°C to prevent freeze-thaw cycles

What purification approaches yield the highest quality recombinant IL-12α protein?

The most effective purification strategy for recombinant IL-12α leverages its intrinsic binding properties:

Heparin affinity chromatography:

• IL-12 demonstrates strong intrinsic heparin binding properties

• A one-step heparin affinity purification method can yield large amounts of highly pure IL-12

• This approach eliminates issues associated with affinity tags such as:

  • Extra amino acids that could create undesirable antigenic epitopes

  • Contamination with proteases used for tag removal

  • Low protein expression yields

Expression system optimization:

• High-density culture of IL-12-producing HEK293 cells in serum-free media using Hollow Fiber bioreactors

• Animal component-free systems reduce contamination risks and increase reproducibility

Quality control checkpoints:

• SEC-HPLC to assess aggregation and purity

• SDS-PAGE under non-reducing conditions to verify heterodimer formation

• Mass spectrometry to confirm exact molecular weight and detect modifications

How can recombinant IL-12α be modified to enhance therapeutic efficacy while reducing toxicity?

Several protein engineering approaches have been developed to optimize IL-12α for therapeutic applications:

Signal peptide modification:

• Deletion of the N-terminal signal peptide prevents IL-12 secretion from cells

• This modification maintains anti-tumor efficacy while eliminating toxic systemic effects

Targeted delivery systems:

• Tumor-targeted oncolytic adenovirus (Ad-TD) delivery of non-secreting IL-12

• This approach significantly enhanced survival in pancreatic cancer models without toxic side effects observed with unmodified IL-12

Site-directed mutagenesis:

• IL-12α C96S mutation creates a form with distinct anti-inflammatory properties

• This variant reduces transcription of proinflammatory cytokines IL1B, IL6, CXCL8, and TNFA

Prevention of proteolytic cleavage:

• Identifying and modifying susceptible regions (e.g., between Lys260 and Arg261)

• This approach maintains full bioactivity, as fragmentation reduces activity by approximately 50%

How does IL-12α deficiency affect different disease models and what mechanisms explain these contradictory effects?

IL-12α deficiency demonstrates dramatically opposing effects depending on disease context:

Protective effect in heart failure models:

• IL-12α knockout significantly attenuates pressure overload-induced cardiac inflammation

• Reduces cardiac fibrosis, hypertrophy, and dysfunction

• Decreases pulmonary accumulation of macrophages and dendritic cells

• Prevents transition from left ventricular failure to lung remodeling and right ventricular hypertrophy

Detrimental effect in sepsis models:

• IL-12α deletion aggravates lipopolysaccharide (LPS) and cecal ligation and puncture (CLP)-induced cardiac dysfunction

• Increases serum and cardiac levels of lactate dehydrogenase (LDH) and cardiac creatine kinase-myocardial band (CK-MB)

• Enhances LPS-induced macrophage accumulation and shifts toward pro-inflammatory M1 phenotype

• Downregulates AMPK activity while increasing phosphorylation of p65 and IκBα

This dichotomy suggests that IL-12α's immunomodulatory effects are highly context-dependent, with researchers proposing that "the regulatory roles of IL-12α in the inflammatory response are related to different inflammatory microenvironments" .

What is the significance of IL12A genetic polymorphisms in disease susceptibility and progression?

The rs568408 polymorphism in IL12A has been associated with multiple disease conditions:

In COVID-19 patients, different IL12A rs568408 genotypes were associated with specific clinical parameters:

• GG genotype: Elevated D-dimer (1458 ng/ml)

• AA genotype: High potassium levels (5.2 mmol/L) and very low SpO₂

• AG genotype: Elevated total protein (62 g/L)

These associations highlight IL12A as a potential genetic marker for disease risk assessment and prognostication across multiple conditions.

What are the functional differences between the IL-12 heterodimer and isolated IL-12α subunit?

The biological activities of the IL-12 heterodimer differ significantly from those of the isolated IL-12α subunit:

IL-12 heterodimer (p70):

• Potent pro-inflammatory cytokine

• Mediates antitumor activity in preclinical models and clinical studies

• Essential for T cell-independent IFN-gamma production

• Critical for development of Th1 and Th2 cells

• Activates the JAK-STAT pathway, particularly STAT4, through binding to IL-12Rβ1/IL-12Rβ2 receptor complex

Isolated IL-12α subunit:

• Demonstrates anti-inflammatory properties

• Reduces transcription of proinflammatory cytokines IL1B, IL6, CXCL8, and TNFA

• May act independently from its heterodimeric partners

• Has distinct receptor binding patterns separate from complete IL-12

These contrasting activities suggest that IL-12α has evolved functionally independent roles beyond its participation in heterodimeric cytokines, opening new avenues for therapeutic development targeting the isolated subunit rather than complete IL-12.

What challenges exist in translating IL-12α research from preclinical models to clinical applications?

Despite promising results in experimental models, several challenges hinder clinical translation:

Toxicity concerns:

• Systemic administration of IL-12 can lead to potentially lethal inflammatory syndrome

• Rapid development of toxic effects has precluded broader clinical application

• Potential for triggering cytokine storm through excessive immune activation

Therapeutic window optimization:

• Finding effective dosages that balance efficacy with safety

• Clinical studies have explored ranging doses from 50-250 ng/kg without clear consensus on optimal levels

Autoimmunity risk:

• Administration of IL-12 to patients with autoimmune diseases worsens autoimmune phenomena

• IL-12 gene knockout or treatment with IL-12-specific antibodies ameliorates autoimmune conditions in mouse models

• Systemic administration may increase the risk of developing new autoimmune conditions

Delivery and targeting limitations:

• Need for tumor-specific or tissue-specific delivery systems

• Engineering requirements for modified versions that maintain efficacy while reducing toxicity

• Challenges in achieving stable expression in target tissues

How effective is recombinant human IL-12α in treating radiotherapy-induced complications?

Recombinant human IL-12 (rhIL-12) has shown promising results in addressing radiotherapy complications:

A randomized controlled trial with 100 patients who received high-dose, short-course precision radiotherapy (Cyber knife or IGRT) evaluated rhIL-12's efficacy in treating:

• Severe myelosuppression/pancytopenia

• Decline or imbalance of immune function

The treatment group (n=50) received rhIL-12 at varying doses (50, 100, 150, 200, or 250 ng/kg), while the control group (n=50) received only symptomatic and supportive therapy .

Results demonstrated that rhIL-12:

• Prevented radiation damage

• Improved hematopoietic function

• Regulated immunity

• Reduced radiotherapy side effects

• Improved patient quality of life

This study highlighted rhIL-12's unique advantage as "the only agent which could not only restore hematopoietic function but also improve immune function" .

What is the evidence for IL-12α's role in cardiovascular conditions?

IL-12α has emerged as a significant modulator of cardiovascular pathologies:

Heart failure:

• IL-12α knockout significantly attenuates pressure overload-induced cardiac inflammation

• Reduces cardiac fibrosis, hypertrophy, and dysfunction

• Prevents transition to right ventricular hypertrophy

• Has no detectable detrimental effects on normal cardiac development, structure, or function

Sepsis-induced cardiac dysfunction:

• IL-12α deletion aggravates sepsis-induced cardiac dysfunction through:

  • Enhancing macrophage accumulation

  • Driving macrophages toward pro-inflammatory M1 phenotype

  • Downregulating AMPK activity

  • Increasing phosphorylation of NF-κB pathway components

Myocardial infarction:

• Inhibition of IL-12α by genetic deletion significantly attenuates:

  • Macrophage polarization

  • T cell-derived interferon-γ production

  • Cardiac injury repair after myocardial infarction

These contradictory effects suggest IL-12α functions are highly context-dependent, with studies noting that "IL-12α exerts different immune-regulatory functions determined by its heterodimeric cytokines under the specific disease conditions" .

How does isolated IL-12α compare to the IL-12 heterodimer in terms of anti-inflammatory properties?

Recent research has revealed unexpected anti-inflammatory properties of isolated IL-12α:

When tested on primary human peripheral blood mononuclear cells (PBMCs) stimulated with lipopolysaccharide (LPS), IL-12α C96S demonstrated:

• Reduced transcription of proinflammatory cytokines IL1B, IL6, CXCL8, and TNFA

• Similar effects to EBI3 (its partner in IL-35) when added individually

• No additional effect when added simultaneously with EBI3

These effects align with IL-35's known immunosuppressive role but suggest IL-12α can function independently to suppress inflammation.

In contrast, the IL-12 heterodimer (IL-12α/IL-12β) is predominantly pro-inflammatory, suggesting that IL-12α's functional properties are significantly influenced by its heterodimeric partner .