CAT5 Antibody

What is the relationship between CAT5/COQ7 and antibody research?

CAT5 (also known as COQ7) is a gene involved in ubiquinone biosynthesis, particularly studied in yeast models. While not directly an antibody target itself, research suggests that CLD1 can reverse the ubiquinone insufficiency caused by hypomorphic mutations in the cat5/coq7 gene. Suppressors of coq7 mutants have been identified through high-copy genomic DNA suppressor screens, using selection media comprised of non-fermentable ethanol supplemented with small amounts of dextrose (YEPE 3% + 0.1% DEX) to facilitate isolation of potential suppressors .

What are the main types of C5-targeting antibodies used in research?

Based on current research, three major types of C5-targeting antibodies have been developed and characterized:

BB5.1 was the original anti-C5 function-blocking antibody, which catalyzed enthusiasm for anti-complement drug development and eventually led to eculizumab, the most successful anti-complement drug to date .

How do researchers measure antibody responses against C5-related proteins?

Researchers commonly employ enzyme-linked immunosorbent assays (ELISAs) to measure antibodies against C5-related proteins. For example, ELISAs have been developed to measure both IgG and IgA antibodies against streptococcal C5a peptidase (SCP) in human sera and saliva. These assays allow for comparing antibody levels between different populations (e.g., children vs. adults) and between patient samples (e.g., acute vs. convalescent sera) .

What are the optimal methods for purifying C5 for research applications?

Traditional C5 purification methods involving precipitation or pH shifts often result in functional loss and low yield. A superior approach uses the novel anti-C5 monoclonal antibody RO7112689 (SKY59), which has been pH-switch engineered to induce antibody-antigen dissociation in acidic conditions (pH ~5.5):

• Create an affinity column with bound RO7112689 antibody

• Apply serum to the column (results in complete depletion of C5)

• Elute at pH 5.0

• This method produces fully active C5 at 98% yield

This antibody-based purification approach works efficiently for both human and mouse C5, making it a versatile tool for comparative studies .

How can researchers engineer antibodies for improved pH-dependent binding to C5?

Engineering antibodies for pH-dependent binding to C5 involves several sophisticated approaches:

• Comprehensive substitution for multidimensional optimization (COSMO): This approach systematically substitutes all residues in complementarity-determining regions (CDRs) and key residues in framework regions with different natural amino acids (except cysteine). For example, SKY59 was developed by expressing over 1,000 antibody variants to identify mutations that improved C5-binding properties .

• Non-histidine mutations: While histidine residues are commonly used for pH-dependent binding due to their pKa values, researchers have identified non-histidine mutations that further improve pH dependency of C5-antibody interactions .

• Surface charge engineering: Modifying the surface charge or isoelectric point (pI) of antibodies can optimize the balance between antibody pharmacokinetics and C5 clearance. This approach successfully suppressed C5 accumulation by accelerating the cycle of C5 sweeping - a process where immune complexes are taken up into cells, C5 dissociates from the antibody in the endosome, and C5-free antibody is salvaged back to plasma .

What methods can be used to evaluate C5 inhibition by experimental antibodies?

Researchers can employ several complementary approaches to evaluate C5 inhibition:

• Hemolytic assays: Using antibody-sensitized sheep erythrocytes (ShEA) or rabbit erythrocytes (RbE) to assess complement-mediated lysis in the presence of inhibitory antibodies

• C5a generation assays: Measuring the production of C5a anaphylatoxin after complement activation in the presence of inhibitory antibodies

• Western blotting: Detecting C5 cleavage products to confirm inhibition of C5 processing

• Surface plasmon resonance: Determining binding kinetics (on-rate, off-rate) and affinity of antibodies to C5 at different pH values

• In vivo disease models: Evaluating the efficacy of C5-inhibitory antibodies in relevant animal models of complement-mediated diseases

How do pH-dependent anti-C5 antibodies achieve longer duration of action?

pH-dependent anti-C5 antibodies like SKY59 achieve longer-lasting neutralization of C5 through a unique mechanism:

• Conventional anti-C5 antibodies can cause C5
  accumulation in plasma by reducing C5 clearance when C5 forms immune complexes (ICs) with the antibody

• These immune complexes are salvaged from endosomal vesicles by neonatal Fc receptor (FcRn)-mediated recycling

• pH-dependent antibodies release C5 in the acidic endosome (pH ~5.5)

• Released C5 is trafficked to lysosomes for degradation, while the C5-free antibody returns to plasma

• This "C5 sweeping" cycle (uptake of ICs into cells → C5 dissociation in endosomes → return of C5-free antibody to plasma) prevents C5 accumulation and extends the duration of action

This engineering approach has demonstrated long-lasting neutralization of C5 in cynomolgus monkeys, showing potential for subcutaneous delivery or less frequent administration compared to conventional anti-C5 antibodies .

What are the mechanisms by which anti-C5 antibodies inhibit complement activation?

Anti-C5 antibodies can inhibit complement activation through multiple mechanisms:

• Prevention of C5 cleavage: Most anti-C5 antibodies, including BB5.1 and eculizumab, prevent the cleavage of C5 by C5 convertase, thereby inhibiting generation of both C5a (anaphylatoxin) and C5b (initiator of membrane attack complex formation)

• Binding to C5b in the C5b6 complex: Some antibodies, such as RO7112689, can also bind C5b in the C5b6 complex, preventing C5b6 binding to target membranes. This represents an additional mechanism of membrane attack complex inhibition

• Chain specificity: Anti-C5 antibodies may target either the α-chain (as with BB5.1) or other regions of C5. The specific binding site determines the mechanism and efficacy of inhibition

How can researchers address species specificity when developing anti-C5 antibodies?

Species specificity is a critical consideration in anti-C5 antibody development:

• Cross-species reactivity evaluation: Systematically test antibody binding and functional inhibition across species (human, non-human primates, rodents) to determine specificity profiles. For example, BB5.1 efficiently inhibits C5 in mouse serum but not in human or other rodent sera

• Structural analysis of C5 binding interfaces: Use X-ray crystallography or cryo-electron microscopy to determine the epitopes recognized by anti-C5 antibodies and compare these regions across species

• Humanization strategies: For rabbit-derived antibodies like SKY59, humanization through framework shuffling can be employed, where each rabbit framework (FR1, FR2, FR3, and FR4) is individually replaced with a human germline framework that maintains structural compatibility with binding activity

• Cysteine residue management: When cysteine residues are found in CDRs (as in the case of SKY59's precursor), they can be substituted with alanine or serine to promote developability while minimizing impact on binding affinity

How can researchers address C5 accumulation problems when using anti-C5 antibodies?

C5 accumulation is a common challenge when using anti-C5 antibodies. Researchers can address this issue through:

• Surface charge engineering: Modify the surface charge or isoelectric point (pI) of the antibody to achieve optimal balance between antibody pharmacokinetics and C5 clearance. This approach was successful with SKY59, suppressing C5 accumulation without compromising the antibody's plasma half-life

• Optimizing IC uptake rate: If accumulation occurs, the immune complex uptake rate may be too slow. Engineering antibodies to accelerate uptake can enhance C5 clearance

• Monitoring C5 levels: Regularly assess C5 concentrations during treatment using sensitive assays such as those utilizing RO7112689 as a capture antibody, which can provide specific quantification of human and mouse C5

What controls should be included when evaluating anti-C5 antibody efficacy in experimental systems?

When evaluating anti-C5 antibody efficacy, researchers should include:

• Positive controls: Include known C5 inhibitors (e.g., eculizumab for human samples, BB5.1 for mouse samples) to validate assay performance

• Isotype controls: Include matched isotype control antibodies to ensure effects are specific to C5 targeting

• C5-deficient samples: When available, include C5-deficient serum or plasma as a reference for complete C5 inhibition

• Dose-response evaluation: Test a range of antibody concentrations to establish dose-response relationships and determine IC50 values

• Functional readouts: Include both biochemical (C5 cleavage) and functional (hemolysis, C5a generation) endpoints to comprehensively assess inhibition

How do researchers interpret conflicting data between in vitro and in vivo anti-C5 antibody performance?

When faced with discrepancies between in vitro and in vivo results:

• Consider pharmacokinetic differences: In vivo, antibody half-life and tissue distribution significantly impact efficacy. pH-dependent antibodies may perform differently in vivo due to their unique "sweeping" mechanisms

• Evaluate species specificity: Many anti-C5 antibodies show species-specific activity. For example, BB5.1 efficiently inhibits mouse C5 but not human C5

• Assess target accessibility: In complex biological environments, C5 may be less accessible than in purified systems

• Consider complement regulation differences: The complement system is regulated differently across species and tissues, which may affect antibody performance

• Evaluate immune complex formation: Some antibodies may cause C5 accumulation in vivo, requiring higher doses for sustained inhibition than predicted by in vitro studies

What are promising approaches for next-generation anti-C5 antibody development?

Several innovative approaches are being explored:

• Bispecific antibodies: Targeting C5 along with another complement component or cell surface receptor to enhance efficacy or tissue specificity

• Antibody fragments: Developing smaller anti-C5 constructs with improved tissue penetration while maintaining inhibitory function

• Advanced engineering for pH dependency: Further refinement of pH-dependent binding to optimize the balance between target engagement at physiological pH and release in endosomes

• Combination with CAT5/COQ7 pathways: Exploring potential synergies between C5 inhibition and modulation of ubiquinone biosynthesis pathways, particularly in conditions where both complement activation and mitochondrial dysfunction contribute to pathology

How can researchers leverage the pH-switch properties of antibodies like RO7112689 beyond C5 purification?

The pH-switch properties of antibodies like RO7112689 offer several research applications:

• Purification of other complement components: Adapting the methodology to purify additional complement proteins or their complexes (such as C5b6)

• Development of sensitive and specific assays: Creating quantitative assays for both human and mouse C5, potentially adaptable to other complement components

• Studying C5 conformational changes: Using pH-dependent binding to probe structural transitions of C5 under different conditions

• Developing similar antibodies for other targets: Applying the pH-switch engineering principles to create antibodies against other abundant serum proteins for purification and analytical applications

What methodological approaches can improve the study of CAT5/COQ7 in relation to complement biology?

Integrating CAT5/COQ7 research with complement biology could be advanced through:

• Genetic suppressor screens: Using approaches similar to the high-copy suppressor screen for coq7 hypomorphic mutants to identify novel regulators that might connect ubiquinone biosynthesis with complement function

• Metabolic profiling: Examining whether complement activation affects ubiquinone metabolism and whether CAT5/COQ7 function influences complement activity

• Conditional expression systems: Developing systems where CAT5/COQ7 function can be modulated during different phases of the immune response to study temporal relationships

• Cross-species comparative studies: Examining the relationship between ubiquinone biosynthesis and complement function across different model organisms to identify conserved regulatory mechanisms