Apoc3 Antibody

Basic Research Questions

• What is apolipoprotein C-III (APOC3) and why is it an important target for antibody development?

  Apolipoprotein C-III (APOC3) is a critical inhibitor of triglyceride (TG) lipolysis and remnant triglyceride-rich lipoprotein (TRL) clearance. It is a 99 amino acid protein with a molecular weight of approximately 10.9 kDa that is primarily synthesized in the liver . APOC3 functions as a key regulator of plasma triglycerides by inhibiting very-low-density-lipoprotein uptake by the liver and inhibiting the activity of lipoprotein lipase .

  APOC3 is an important target for antibody development because genetic studies have established a causal link between APOC3 and cardiovascular disease (CVD). Loss-of-function mutations in the APOC3 gene are associated with lower plasma TG levels and a reduced risk of coronary heart disease (CHD) . For instance, subjects carrying rare mutations in APOC3 had 39% lower plasma TG levels and 40% lower risk of CHD compared with non-carriers among 110,970 study participants from 14 studies . This suggests that targeting APOC3 with antibodies could potentially provide therapeutic benefits for dyslipidemia and cardiovascular disease.

• What are the different proteoforms of APOC3 and how do they relate to cardiovascular risk?

  APOC3 exists in four major proteoforms, which differ in their glycosylation patterns:

  • Native peptide (C-III 0a)

  • Non-glycosylated proteoform (C-III 0b)

  • Mono-sialylated proteoform (C-III 1, most abundant)

  • Di-sialylated proteoform (C-III 2)

  Studies have shown that these proteoforms have different relationships with cardiovascular disease (CVD) risk. In the Multiethnic Study of Atherosclerosis, researchers found that C-III 0b/III 1 ratio was inversely associated with CVD risk, with a hazard ratio of 0.86 (95% CI: 0.79–0.93), even after full adjustment including plasma lipids . This suggests that not only the total concentration of APOC3 but also the relative distribution of its proteoforms may play a role in determining cardiovascular risk.

  The proteoform composition shows variations associated with demographic and clinical characteristics. For example, C-III 2 was higher in older participants, C-III 0b and C-III 1 were higher in women, and compared to Whites, C-III 2 was higher in Blacks, C-III 0a and C-III 1 were higher in Hispanics, and C-III 0a and C-III 2 were higher in Chinese . These differences may have implications for the development of antibodies targeting specific proteoforms.

• What methodologies are available for measuring APOC3 in research samples?

  Several methodologies are available for measuring APOC3 in research samples:

  Mass Spectrometry Immunoassay:
  This technique combines antibody-based purification with mass spectrometric detection, allowing for the quantification of specific APOC3 proteoforms. In the Multiethnic Study of Atherosclerosis, APOC3 proteoforms were measured using this approach, where APOC3 protein was captured using immunoaffinity columns derivatized with anti-APOC3 antibody .

  Immunoblotting (Western Blot):
  Western blot analysis is commonly used to measure APOC3 protein levels in tissue samples, such as liver, or plasma. This method allows for the semi-quantitative assessment of APOC3 protein expression .

  Metabolic Labeling:
  For measuring APOC3 secretion rates, researchers have used [35S]methionine labeling to track newly synthesized, metabolically labeled APOC3 released into circulation .

  Internal Standard Peptide-Based Quantification:
  This approach involves adding isotopically labeled peptides to samples to facilitate quantification of wild-type and mutant APOC3 protein over a range of concentrations, based on comparison of product ion intensities for light versus heavy peptides .

  Each methodology has specific advantages and limitations, and the choice depends on the research question and available resources.

• How do different types of anti-APOC3 antibodies vary in their applications and specificity?

  Anti-APOC3 antibodies vary significantly in their applications, specificity, and functional properties:

  Monoclonal vs. Polyclonal Antibodies:
  Both monoclonal and polyclonal antibodies against APOC3 are available for research applications. Monoclonal antibodies provide high specificity for a single epitope, while polyclonal antibodies recognize multiple epitopes, potentially offering higher sensitivity but sometimes lower specificity .

  Application-Specific Antibodies:
  Anti-APOC3 antibodies are designed for various applications, including:

  • Western blot analysis (250+ antibodies available from 26 suppliers)

  • Enzyme-linked immunosorbent assay (ELISA)

  • Flow cytometry

  • Immunohistochemistry (IHC)

  • Immunofluorescence

  Species Reactivity:
  Some antibodies are specific to human APOC3, while others may cross-react with APOC3 from other species such as mouse, rat, bovine, chimpanzee, and chicken. For example, the STT505 antibody showed potent abrogation of human APOC3 function with no cross-reactivity to mouse APOC3 .

  Functional Antibodies:
  Some antibodies, like STT505, are designed to have functional effects, such as abrogating the inhibitory effect of human APOC3 on DiI-labeled VLDL uptake by HepG2 hepatocytes . These antibodies can promote APOC3 clearance and lower triglyceride-rich lipoproteins in animal models.

  Epitope-Specific Antibodies:
  Antibodies can be designed to target specific regions of APOC3, such as lipoprotein-bound forms versus free APOC3, which can have different functional implications .

  When selecting an antibody for research, considerations should include the specific application, target species, required specificity, and whether functional effects are desired.

Intermediate Research Questions

• What is the mechanism by which monoclonal antibodies targeting APOC3 reduce triglyceride levels?

  Monoclonal antibodies targeting APOC3 reduce triglyceride levels through several mechanisms:

  Promotion of APOC3 Clearance:
  Studies have shown that monoclonal antibodies to lipoprotein-bound human APOC3 can promote the catabolism of APOC3 from lipoproteins. This leads to accelerated clearance of APOC3 in the circulation, particularly through increased splenic uptake .

  Enhanced Triglyceride-Rich Lipoprotein (TRL) Catabolism:
  By reducing the APOC3 content on TRLs, these antibodies enhance the catabolism of TRLs. In a study published in Nature Medicine, a novel monoclonal antibody targeting lipoprotein-bound human APOC3 was shown to promote the clearance of circulating APOC3 in mice expressing human APOC3, thereby increasing the catabolism of TRLs and reducing circulating triglyceride levels .

  Abrogation of APOC3 Inhibitory Function:
  APOC3 normally inhibits lipoprotein lipase activity and hepatic uptake of TRLs. Antibodies like STT505 have been shown to abrogate this inhibitory function. In an in vitro assay, STT505 potently abrogated human APOC3's inhibitory effect on DiI-labeled VLDL uptake by HepG2 hepatocytes .

  Mimicking the A43T Variant Protective Mechanism:
  The development of these antibodies was inspired by the protective mechanism of the A43T missense variant of APOC3. Carriers of this variant display decreased fasting TG levels and reduced plasma APOC3 levels (approximately 50% of those measured in non-carriers) . The reduced APOC3 in these carriers is due to impaired binding of A43T APOC3 to lipoproteins and accelerated renal catabolism of free APOC3, which the antibodies aim to mimic .

  The therapeutic potential of this approach is supported by both genetic evidence and preclinical studies, suggesting that antibodies targeting APOC3 could be effective in treating dyslipidemia and reducing cardiovascular risk.

• How do loss-of-function mutations in the APOC3 gene affect cardiovascular risk, and what insights do they provide for antibody development?

  Loss-of-function mutations in the APOC3 gene have significant effects on cardiovascular risk and provide valuable insights for antibody development:

  Reduction in Triglyceride Levels:
  Carriers of APOC3 loss-of-function mutations exhibit substantially lower plasma triglyceride levels. Among 110,970 study participants from 14 studies, subjects with rare mutations in APOC3 had 39% lower plasma TG levels compared to non-carriers . Similarly, data from 72,725 participants in 2 general populations showed that heterozygosity for loss-of-function mutations in the APOC3 gene was associated with a 44% mean reduction in plasma TG levels .

  Decreased Cardiovascular Disease Risk:
  These mutations are associated with a significant reduction in cardiovascular disease risk. Carriers had a 40% lower risk of coronary heart disease (CHD) compared to non-carriers . Heterozygosity for APOC3 loss-of-function mutations was associated with 41% and 36% reductions in the risk of ischemic vascular disease and ischemic heart disease, respectively .

  Mechanism of Risk Reduction:
  A meta-analysis of approximately 137,000 subjects from 8 study cohorts showed that the lower risk of ischemic vascular disease observed in carriers of APOC3 loss-of-function mutations is mainly mediated by the associated low remnant (VLDL) cholesterol rather than by low LDL-C . This suggests that targeting APOC3 may provide additional cardiovascular benefits beyond traditional LDL-C-lowering approaches.

  Types of Protective Mutations:
  Four protective APOC3 variants have been identified:

  • R19* (nonsense variant)

  • IVS2+1G>A (splice-site variant)

  • IVS3+1G>T (splice-site variant)

  • A43T (missense variant)

  Insights for Antibody Development:
  The A43T missense variant has been particularly instructive for antibody development. Unlike the other three classic loss-of-function variants, A43T affects the protein's function rather than its expression. Detailed investigation revealed that A43T carriers had reduced plasma APOC3 due to impaired binding of the variant APOC3 to lipoproteins and accelerated renal catabolism of free APOC3 . This led to the development of monoclonal antibodies that target lipoprotein-bound APOC3 to promote its dissociation and clearance, mimicking the natural protective mechanism of the A43T variant .

  These genetic insights have provided a strong rationale for targeting APOC3 with antibodies as a therapeutic approach for reducing triglyceride levels and cardiovascular risk.

• What experimental models are commonly used to evaluate anti-APOC3 antibody efficacy?

  Several experimental models are used to evaluate anti-APOC3 antibody efficacy across different research stages:

  In Vitro Models:

  • HepG2 Hepatocyte Uptake Assay: This assay measures the ability of antibodies to abrogate APOC3's inhibitory effect on DiI-labeled VLDL uptake by HepG2 hepatocytes. The STT505 antibody was screened using this assay and showed potent abrogation of human APOC3 function .

  • Biochemical Binding Assays: These assess the binding affinity and specificity of antibodies to different forms of APOC3 (lipoprotein-bound versus free).

  In Vivo Mouse Models:

  • AAV-Mediated APOC3 Expression: Adeno-associated virus (AAV) vectors encoding either wild-type or variant (e.g., A43T) human APOC3 are used to express human APOC3 in mice. This approach allows comparison of the effects of wild-type versus mutant APOC3 expression on triglyceride metabolism .

  • APOC3 Knockout Mice: Targeted disruption of APOC3 results in low levels of TGs and a reduced post-prandial response in mice, providing a negative control for antibody studies .

  • APOC3 Transgenic Mice: Transgenic mice expressing human APOC3 exhibit hypertriglyceridemia, with severity proportional to the number of gene copies. Mice with 1-2 copies show mild hypertriglyceridemia, while those with ~100 copies display severe hypertriglyceridemia .

  • Diabetic Mouse Models: Black and tan, brachyury (BT) wild-type and leptin-deficient (OB; diabetic) mice treated with antisense oligonucleotides to APOC3 have been used to study the role of APOC3 in diabetic kidney disease and associated atherosclerosis .

  Metabolic Studies in Mouse Models:

  • Triglyceride Metabolism: Measurement of fasting and postprandial triglyceride levels in mice treated with anti-APOC3 antibodies.

  • APOC3 Clearance: Assessment of APOC3 clearance rates in circulation using metabolic labeling techniques, such as [35S]methionine administration .

  • Lipoprotein Profiling: Analysis of the distribution of cholesterol and triglycerides among different lipoprotein fractions.

  Disease-Specific Models:

  • Atherosclerosis Models: Mice prone to atherosclerosis (e.g., ApoE-deficient mice) treated with anti-APOC3 antibodies to assess effects on plaque development.

  • Diabetic Kidney Disease Models: Diabetic mice with human-like dyslipidemia to study the effects of APOC3 inhibition on kidney disease progression .

  These diverse experimental models provide a comprehensive framework for evaluating the efficacy and safety of anti-APOC3 antibodies at different stages of development, from initial screening to preclinical proof-of-concept studies.

• How do different apoC-III proteoforms correlate with clinical outcomes and demographic characteristics?

  The apoC-III proteoforms show distinct correlations with clinical outcomes and demographic characteristics, highlighting the complexity of apoC-III biology:

  Proteoform Distribution and Relative Abundance:

  In the Multiethnic Study of Atherosclerosis (MESA), C-III 1 was the most abundant proteoform (median 60% of total peak area), followed by C-III 2 (21%), C-III 0b (12%), and C-III 0a (7%) . These percentages correlated with total apoC-III concentration to varying degrees: inversely for C-III 2 (r= −0.30) and positively for other proteoforms (r= 0.07 for C-III 0a; r= 0.14 for C-III 0b; and r= 0.25 for C-III 1) .

  Demographic Associations:

  Age: C-III 2 was higher in older participants .

  Gender: C-III 0b and C-III 1 were higher in women .

  Race/Ethnicity: Compared with Whites, C-III 2 was higher in Blacks, C-III 0a and C-III 1 were higher in Hispanics, and C-III 0a and C-III 2 were higher in Chinese .

  Menopausal Status: Among women aged 45-54 years, those who had gone through menopause had significantly lower C-III 2 and higher C-III 1 .

  Variance Explanation:

  The Pillai's trace values indicated that 9% of the variance in apoC-III proteoform composition was explained by age, 6% by gender, and 18% by race/ethnicity .

  Cardiovascular Disease (CVD) Risk:

  The C-III 0b/III 1 ratio was inversely associated with CVD risk with a hazard ratio of 0.86 (95% CI: 0.79–0.93), even after full adjustment including plasma lipids . This suggests that specific proteoform ratios may be more informative for risk assessment than total apoC-III levels alone.

  Diabetic Kidney Disease Progression:

  Elevated baseline apoC-III levels predicted greater loss of renal function in people with type 2 diabetes from the Veterans Affairs Diabetes Trial. After adjustment for nonlipid clinical and demographic covariates, the hazard ratio for renal function loss was 1.23 (95% CI: 1.05–1.44, P = 0.0098) .

  These correlations between apoC-III proteoforms and clinical/demographic characteristics have important implications for both risk assessment and therapeutic development. They suggest that targeting specific proteoforms or modulating proteoform ratios might be more effective than reducing total apoC-III levels in certain populations or disease states.