PCSK9 Monoclonal Antibodies Have Come a Long Way.

PURPOSE OF THE REVIEW: This review examines the pivotal role of monoclonal antibodies against PCSK9 in lipid-lowering therapy, emphasizing their biological and clinical impact. RECENT FINDINGS: Randomized controlled trials have validated that PCSK9 monoclonal antibodies (Mabs) effectively reduce LDL-c levels by approximately 50%, even when added to maximal statin therapy. They moreover produce a notable 15-20% relative decrease in major cardiovascular events, with a greater reduction among high-risk patients and no evidence for serious adverse effects, assuaging previous concerns. This review highlights the benefits of PCSK9 Mabs in high cardiovascular risk patients. Despite their efficacy and safety, these therapies are hindered by limited access, and require broader integration into clinical practice to optimize therapeutic outcomes.

Transforming hypercholesterolemia management: Spotlight on PCSK9 peptide vaccines.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a therapeutic target for dyslipidemia and atherosclerotic cardiovascular disease (ASCVD). Two recent studies published by Fang et al.1 and Zhang et al.2 in Cell Reports Medicine and Cell Reports, respectively, show the efficacy of peptide vaccines in eliciting an antibody response against PCSK9 and reducing plasma cholesterol levels.

Optimization of Hypercholesterolemia Treatment after Heart Transplant: The Role of PCSK9 Inhibitors.

BACKGROUND: Previous studies have reported the benefit of statins after heart transplant (HT). However, the use of high-dose statins might be limited in some HT patients due to intolerance and interactions with immunosuppression or might not be enough to achieve low-density lipoprotein (LDL) cholesterol goals. Hyperlipidemia has been associated with coronary allograft vasculopathy. Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors might be a safe and effective option in HT patients with suboptimal lipid control. METHODS: In a retrospective study, we identified HT patients in our center with LDL cholesterol >100 mg/dL, after diet modifications and up-titration of statins to maximum tolerated dose, treated with PCSK9i. The primary endpoint was LDL reduction one month after, and secondary endpoints were the development of donorspecific HLA antibodies (DSA) and the presence of coronary allograft vasculopathy or rejection. RESULTS: From January, 2018, to January, 2024, we identified five HT patients treated with PCSK9 inhibitors. In all cases, evolocumab was used. A significant reduction in LDL cholesterol was observed (151.6 ± 13.5 mg/dl to 72.4 ± 14.6 mg/dl; p = 0.04, mean reduction 75.7 ± 14.1 mg/dl), as well as in total cholesterol (231 ± 34.6 mg/dl to 152.2 ± 38.9 mg/dl; p < 0.01, mean reduction 78.8 ± 22.2 mg/dl). A significant increase in HDL cholesterol was not observed (45.4 ± 10.9 mg/dl to 46.2 ± 11.1 mg/dl; p = 0.60). One patient developed DSA five years after treatment onset. Rejection and coronary allograft vasculopathy were not observed. CONCLUSION: PCSK9 inhibitors are safe and effective in reducing LDL in HT patients. However, larger studies are needed to clarify if they can reduce the development of coronary allograft vasculopathy.

Biparatopic anti-PCSK9 antibody enhances the LDL-uptake in HepG2 cells.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a promising therapeutic target to reduce lipids. In 2020, we reported a chimeric camelid-human heavy chain antibody VHH-B11-Fc targeting PCSK9. Recently, it was verified that VHH-B11 binds one linear epitope in the PCSK9 hinge region. To enhance its druggability, we have developed a novel biparatopic B11-H2-Fc Ab herein. Thereinto, surface plasmon resonance (SPR) confirmed the epitope differences in binding-PCSK9 among VHH-B11, VHH-H2 and the approved Repatha. Additionally, SPR revealed the B11-H2-Fc exhibits an avidity of approximately 0.036 nM for PCSK9, representing a considerable increase compared to VHH-B11-Fc (~ 0.69 nM). Moreover, we found the Repatha and B11-H2-Fc exhibited > 95% PCSK9 inhibition efficiency compared to approximately 48% for the VHH-Fc at 7.4 nM (P < 0.0005). Further, we verified its biological activity using the human hepatoma cells G2 model, where the B11-H2-Fc exhibited almost 100% efficiency in PCSK9 inhibition at only 0.75 µM. The immunoblotting results of low-density lipoprotein cholesterol (LDL-c) uptake assay also demonstrated the excellent performance of B11-H2-Fc on recovering the LDL-c receptor (LDLR), as strong as the Repatha (P > 0.05). These findings provide the first evidence of the efficacy of a novel Ab targeting PCSK9 in the field of lipid-lowering drugs.

The multifaceted role of PCSK9 in cancer pathogenesis, tumor immunity, and immunotherapy.

Proprotein convertase subtilisin/kexin type 9 (PCSK9), a well-known regulator of cholesterol metabolism and cardiovascular diseases, has recently garnered attention for its emerging involvement in cancer biology. The multifunctional nature of PCSK9 extends beyond lipid regulation and encompasses a wide range of cellular processes that can influence cancer progression. Studies have revealed that PCSK9 can modulate signaling pathways, such as PI3K/Akt, MAPK, and Wnt/ß-catenin, thereby influencing cellular proliferation, survival, and angiogenesis. Additionally, the interplay between PCSK9 and cholesterol homeostasis may impact membrane dynamics and cellular migration, further influencing tumor aggressiveness. The central role of the immune system in monitoring and controlling cancer is increasingly recognized. Recent research has demonstrated the ability of PCSK9 to modulate immune responses through interactions with immune cells and components of the tumor microenvironment. This includes effects on dendritic cell maturation, T cell activation, and cytokine production, suggesting a role in shaping antitumor immune responses. Moreover, the potential influence of PCSK9 on immune checkpoints such as PD1/PD-L1 lends an additional layer of complexity to its immunomodulatory functions. The growing interest in cancer immunotherapy has prompted exploration into the potential of targeting PCSK9 for therapeutic benefits. Preclinical studies have demonstrated synergistic effects between PCSK9 inhibitors and established immunotherapies, offering a novel avenue for combination treatments. The strategic manipulation of PCSK9 to enhance tumor immunity and improve therapeutic outcomes presents an exciting area for further investigations. Understanding the mechanisms by which PCSK9 influences cancer biology and immunity holds promise for the development of novel immunotherapeutic approaches. This review aims to provide a comprehensive analysis of the intricate connections between PCSK9, cancer pathogenesis, tumor immunity, and the potential implications for immunotherapeutic interventions.

Development of a PCSK9-targeted nanoparticle vaccine to effectively decrease the hypercholesterolemia.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low-density lipoprotein receptor (LDLR) and mediates its internalization and degradation, resulting in an increase in LDL cholesterol levels. Recently, PCSK9 emerged as a therapeutic target for hypercholesterolemia and atherosclerosis. In this study, we develop a PCSK9 nanoparticle (NP) vaccine by covalently conjugating the catalytic domain (aa 153-aa 454, D374Y) of PCSK9 to self-assembled 24-mer ferritin NPs. We demonstrate that the PCSK9 NP vaccine effectively induces interfering antibodies against PCSK9 and reduces serum lipids levels in both a high-fat diet-induced hypercholesterolemia model and an adeno-associated virus-hPCSK9D374Y-induced hypercholesterolemia model. Additionally, the vaccine significantly reduces plaque lesion areas in the aorta and macrophages infiltration in an atherosclerosis mouse model. Furthermore, we discover that the vaccine's efficacy relied on T follicular help cells and LDLR. Overall, these findings suggest that the PCSK9 NP vaccine holds promise as an effective treatment for hypercholesterolemia and atherosclerosis.

Efficacy and Safety of Ongericimab in Chinese Patients With Primary Hypercholesterolemia and Mixed Dyslipidemia.

BACKGROUND: A phase 3 trial was conducted to evaluate the efficacy and safety of ongericimab, a monoclonal antibody that inhibits proprotein convertase subtilisin/kexin type 9, as an add-on treatment to optimized lipid-lowering therapy in Chinese patients with primary hypercholesterolemia and mixed dyslipidemia. METHODS AND RESULTS: A total of 806 patients who were receiving stable and optimized lipid-lowering therapy but did not achieve their low-density lipoprotein cholesterol (LDL-C) targets were enrolled and randomly assigned in a 2:1:2:1 ratio to receive either ongericimab 150 mg or matching placebo every 2 weeks, or ongericimab 300 mg or matching placebo every 4 weeks for 52 weeks. Efficacy and safety were evaluated in 802 patients who received at least 1 dose of ongericimab or placebo. The primary end point was the percentage change in LDL-C from baseline to week 24. Our findings demonstrated that the least-squares mean difference of percentage change in LDL-C from baseline to week 24 was -67.7% (95% CI, -72.5% to -63.0%; P<0.0001) in the ongericimab 150 mg every 2 weeks group compared with the placebo every 2 weeks group, and -61.2% (95% CI, -67.1% to -55.2%; P<0.0001) in the ongericimab 300 mg every 4 weeks group compared with the placebo every 4 weeks group. These reductions were sustained up to week 52. Furthermore, treatment with ongericimab favorably altered other lipid parameters. A similar incidence of adverse events was observed in the ongericimab and placebo groups. CONCLUSIONS: Ongericimab, as an add-on treatment to optimized lipid-lowering therapy, significantly reduced LDL-C and was well-tolerated in Chinese patients with primary hyperlipidemia and mixed dyslipidemia who did not achieve their LDL-C targets. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT04781114.

How to verify the analytical and clinical performance of ELISA immunoanalysis in the real laboratory practice. PCSK9 as an example.

BACKGROUND: Manufacturers and diagnostic companies often recommend on-site verification of analytical performance in the clinical laboratory. The validation process used by manufacturers is rarely described in detail, and certain information on analytical performance is missing from the product sheet, especially for immunoanalytical methods. We describe an approach to the detailed validation of an ELISA method for the measurement of proprotein convertase subtilisin/kexin type 9 (PCSK9) plasma concentrations. We compared manufacturers' claims of analytical performance with data obtained in the field laboratory using several approaches. METHODS: We used the Human Proprotein Convertase 9/PCSK9 Quantikine ELISA diagnostic kit (R&D systems, Bio-Techne Ltd., Abingdon Science Park, Abingdon, UK) and three levels of quality control solution Quantikine Immunoassay Control Group 235 (R&D systems, Bio-Techne Ltd., Abingdon Science Park, Abingdon, UK) to verify precision. We measured the concentration of PCSK9 using the DS2 ELISA Reader (Dynex Technologies GmbH, Denkendorf, Germany). We used analysis of variance (ANOVA) and the R statistical package (R core team, version 1.4.5). Statistical analysis and terminology were performed according to protocol CLSI EP15-A3, and the reference interval was checked according to CLSI/IFCC C28-A3c. RESULTS: We found a significant difference between the manufacturer's claims of analytical performance and real data measured in the routine clinical laboratory. The calculated CV (%) for repeatability (calculated by simple estimation of the mean and SD, as used by the manufacturer) was between 5.5% and 7.4%, but the manufacturer's claim was between 4.1% and 6.5%. Using ANOVA, the true repeatability was between 5.0% and 6.9%. Similarly, ANOVA revealed values of CV (%) for within-laboratory imprecision between 6.5% and 9.1%, while the manufacturer's claims were between 4.1% and 5.9%. The recovery ranged from 105.5% to 121.8%. The manufacturer's recommended reference interval was checked and we didn't find any significant difference between men and women. CONCLUSIONS: We describe a comprehensive approach to verify the analytical performance of an ELISA method using the measurement of PCSK9 plasma concentration as an example. We found differences between the results of this approach based on the CLSI EP15-A3 protocol and data provided by the manufacturer. We recommend the verification of analytical performance by more complex statistical tools in laboratory practice.

Cholesterol reduction by immunization with a PCSK9 mimic.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a plasma protein that controls cholesterol homeostasis. Here, we design a human PCSK9 mimic, named HIT01, with no consecutive 9-residue stretch in common with any human protein as a potential heart attack vaccine. Murine immunizations with HIT01 reduce low-density lipoprotein (LDL) and cholesterol levels by 40% and 30%, respectively. Immunization of cynomolgus macaques with HIT01-K21Q-R218E, a cleavage-resistant variant, elicits high-titer PCSK9-directed antibody responses and significantly reduces serum levels of cholesterol 2 weeks after each immunization. However, HIT01-K21Q-R218E immunizations also increase serum PCSK9 levels by up to 5-fold, likely due to PCSK9-binding antibodies altering the half-life of PCSK9. While vaccination with a PCSK9 mimic can induce antibodies that block interactions of PCSK9 with the LDL receptor, PCSK9-binding antibodies appear to alter homeostatic levels of PCSK9, thereby confounding its vaccine impact. Our results nevertheless suggest a mechanism for increasing the half-life of soluble regulatory factors by vaccination.

Safety of the PCSK9 inhibitor alirocumab: insights from 47 296 patient-years of observation.

The ODYSSEY OUTCOMES trial, comprising over 47 000 patient-years of placebo-controlled observation, demonstrated important reductions in the risk of recurrent ischaemic cardiovascular events with the monoclonal antibody to proprotein convertase subtilisin/kexin type 9 alirocumab, as well as lower all-cause death. These benefits were observed in the context of substantial and persistent lowering of low-density lipoprotein cholesterol with alirocumab compared with that achieved with placebo. The safety profile of alirocumab was indistinguishable from matching placebo except for a ∼1.7% absolute increase in local injection site reactions. Further, the safety of alirocumab compared with placebo was evident in vulnerable groups identified before randomization, such as the elderly and those with diabetes mellitus, previous ischaemic stroke, or chronic kidney disease. The frequency of adverse events and laboratory-based abnormalities was generally similar to that in placebo-treated patients. Thus, alirocumab appears to be a safe and effective lipid-modifying treatment over a duration of at least 5 years.

Comparing PCSK9 Monoclonal Antibody Treatment Strategies Following Myocardial Infarction Using Negative Control Outcomes: A Target Trial Emulation Study.

BACKGROUND: Initiation of proprotein convertase subtilisin/kexin type 9 monoclonal antibody (PCSK9 mAb) for lipid-lowering following myocardial infarction (MI) is likely affected by patients' prognostic factors, potentially leading to bias when comparing real-world treatment effects. METHODS: Using target-trial emulation, we assessed potential confounding when comparing two treatment strategies post-MI: initiation of PCSK9 mAb within 1 year and no initiation of PCSK9 mAb. We identified MI hospitalizations during July 2015-June 2020 for patients aged ≥18 years in Optum's de-identified Clinformatics Data Mart (CDM) and MarketScan, and those aged ≥66 in the US Medicare claims database. We estimated a 3-year counterfactual cumulative risk and risk difference (RD) for 10 negative control outcomes using the clone-censor-weight approach to address time-varying confounding and immortal person-time. RESULTS: PCSK9 mAb initiation within 1-year post-MI was low (0.7% in MarketScan and 0.4% in both CDM and Medicare databases). In CDM, there was a lower risk for cancer (RD = -3.6% [95% CI: -4.3%, -2.9%]), decubitus ulcer (RD = -7.7% [95% CI: -11.8%, -3.7%]), fracture (RD = -8.1% [95% CI: -9.6%, -6.6%]), influenza vaccine (RD = -9.3% [95% CI: -17.5%, -1.1%]), and visual test (RD = -0.6% [95% CI: -0.7%, -0.6%]) under the PCSK9 mAb initiation versus no initiation strategy. Similar differences persisted in the MarketScan and Medicare databases. In each database, ezetimibe and low-density lipoprotein testing were unbalanced between treatment strategies. CONCLUSION: A comparative effectiveness study of these treatments using the current approach would likely bias results due to the low number of PCSK9 mAb initiators.

Safety of Monoclonal Antibodies Inhibiting PCSK9 in Pregnancy: Disproportionality Analysis in VigiBase®.

Safety data on the use of monoclonal antibodies inhibiting proprotein convertase subtilisin/kexin type 9 in pregnancy are scarce. This study queried VigiBase®, the World Health Organization global pharmacovigilance database, to search for signals of disproportionate reporting for pregnancy outcomes with alirocumab and evolocumab. As of November 22, 2023, there were 45 safety reports of exposure to evolocumab (N = 31) and alirocumab (N = 14) in pregnancy. Most of them originated from Europe (N = 25, 55.6%) and were more frequently reported by healthcare professionals (N = 35, 77.8%). Median patient age was 37 years (25th-75th percentiles; 32-41 years). Drug exposure occurred during pregnancy in 36 (80.0%) safety reports, via paternal exposure during pregnancy in four (8.9%), during lactation in three (6.7%), and in two safety reports the time of drug exposure remained unknown. Twenty safety reports (57.8%) merely reported drug exposure, while 19 (42.2%) also reported pregnancy outcomes, however, without specific maternal toxicities or patterns of birth defects. Spontaneous abortion was reported in eight safety reports without representing a signal of disproportionate reporting compared with either the full database (reporting odds ratio, ROR, 0.06 95% confidence interval, CI 0.03-0.12) or statins (ROR 0.16, 95% CI 0.08-0.32). In conclusion, this study showed that, currently, there are no signals of increased reporting of spontaneous abortion with alirocumab and evolocumab compared with the full database and statins in VigiBase®. Notwithstanding, lack of disproportionality is not synonymous with safety and, as disproportionality analyses depend on the number of safety reports that progressively accumulate in VigiBase®, they should be repeated at regular intervals to confirm the results of the present study.

PCSK9 inhibition protects mice from food allergy.

The Proprotein Convertase Subtilisin Kexin of type 9 (PCSK9) has been identified in 2003 as the third gene involved in familial hypercholesterolemia. PCSK9 binds to the membrane low-density lipoprotein receptor (LDLR) and promotes its cellular internalization and lysosomal degradation. Beyond this canonical role, PCSK9 was recently described to be involved in several immune responses. However, to date, the contribution of PCSK9 in food allergy remains unknown. Here, we showed that Pcsk9 deficiency or pharmacological inhibition of circulating PCSK9 with a specific monoclonal antibody (m-Ab) protected mice against symptoms of gliadin-induced-food allergy, such as increased intestinal transit time and ear oedema. Furthermore, specific PCSK9 inhibition during the elicitation steps of allergic process was sufficient to ensure anti-allergic effects in mice. Interestingly, the protective effect of PCSK9 inhibition against food allergy symptoms was independent of the LDLR as PCSK9 inhibitors remained effective in Ldlr deficient mice. In vitro, we showed that recombinant gain of function PCSK9 (PCSK9 D374Y) increased the percentage of mature bone marrow derived dendritic cells (BMDCs), promoted naïve T cell proliferation and potentiated the gliadin induced basophils degranulation. Altogether, our data demonstrate that PCSK9 inhibition is protective against gliadin induced food allergy in a LDLR-independent manner.

Proprotein convertase subtilisin/kexin type 9-inhibition across different patient populations.

PURPOSE OF REVIEW: Monoclonal antibodies (mAb) targeting proprotein convertase subtilisin/kexin type 9 (PCSK9) have been established in cardiovascular risk prevention. The purpose of this review is to summarize the effects of PCSK9 inhibitors across different patient populations. RECENT FINDINGS: Long-term data on the use of evolocumab and alirocumab shows persisting low- density lipoprotein cholesterol (LDL-C) lowering and good tolerability. PCSK9 inhibitors are effective and safe in both sexes, in pediatric patients as well as in the elderly. Initiation of PCSK9 mAb during acute myocardial infarction is safe and leads to beneficial morphological plaque changes. The PCSK9 inhibitors evolocumab, alirocumab and inclisiran lower LDL-C in patients with heterozygous familial hypercholesterolemia (FH), while the response of patients with homozygous FH is heterogeneous. New areas of application beyond lipid lowering are currently investigated. SUMMARY: PCSK9 inhibitors are safe, well tolerated, and effective in primary and secondary prevention in a wide range of patient populations.

Optimal follow-up schedule for patients taking PCSK9 monoclonal antibodies in a health system: Analysis of specialty pharmacy clinical interventions.

PURPOSE: The objective of this study was to determine if and when it is clinically appropriate to consider a reduction in the frequency of health-system specialty pharmacy (HSSP) clinical pharmacist assessments for patients taking a proprotein convertase subtilisin/kexin type 9 (PCSK9) monoclonal antibody (mAb) after they are deemed clinically stable on therapy. METHODS: A single-center, retrospective, observational study of adult patients on PCSK9 mAb therapy enrolled in the University of Rochester Specialty Pharmacy Cardiology Patient Management Program was performed between October 24, 2016, and April 30, 2022. The primary outcome was the number of clinical pharmacist interventions per interval within the baseline 12 months compared to 12-month intervals for up to 72 months after initiation of PCSK9 mAb therapy. RESULTS: A total of 368 patients on PCSK9 mAb therapy were included in the study. A significantly lower percentage of patients had more than 2 interventions during the 12- to 24-month interval (24.3%) as compared to the baseline 12-month interval (80.2%) (P < 0.001); this represented a 70% reduction in the chance of a patient requiring more than 2 interventions (relative risk, 0.30; 95% CI, 0.24-0.38). A similar trend was demonstrated in the 24- to 36-month and 36- to 48-month intervals when compared to the first year of therapy. The most commonly documented clinical pharmacist interventions were in the categories of safety (29.2%), effectiveness (28.4%), and adherence (19.9%). CONCLUSION: Patients beyond 1 year of PCSK9 mAb therapy required less clinical pharmacist interventions. Therefore, stable patients receiving a PCSK9 mAb may be considered for less frequent clinical assessments to allow for HSSP growth to nontraditional clinical areas.

PCSK9-directed therapies: an update.

PURPOSE OF REVIEW: Two large cardiovascular outcomes trials of monoclonal antibodies against proprotein convertase subtilisin/kexin type 9 (PCSK9) demonstrated that therapeutic inhibition of extracellular PCSK9 markedly reduces LDL cholesterol concentration and cardiovascular risk. Several novel strategies to inhibit PCSK9 function are in development. Different mechanisms of action may determine specific properties with potential relevance for patient care. RECENT FINDINGS: For the monoclonal antibodies evolocumab und alirocumab as first-generation PCSK9 inhibitors, follow-up data of up to 8 years of exposure complement the information on efficacy and safety available from outcome trials. For the small-interfering RNA inclisiran as second-generation PCSK9 inhibitor, several phase III trials have been published and a cardiovascular outcome trial has completed recruitment and is ongoing. Third-generation PCSK9 inhibitors encompass, among others, orally available drugs such as MK-0616 and the fusion protein lerodalcibep. Additional strategies to inhibit PCSK9 include vaccination and gene editing. SUMMARY: Long-term inhibition of PCSK9 with monoclonal antibodies is safe and conveys sustained cardiovascular benefit. Novel strategies to inhibit PCSK9 function such as orally available drugs, RNA targeting, and one-time treatment with gene editing may further enhance the therapeutic armamentarium and enable novel preventive strategies.

Beneficial effects of PCSK9 inhibition with alirocumab in familial hypercholesterolemia involve modulation of new immune players.

Familial hypercholesterolemia (FH) is associated with low-grade systemic inflammation, a key driver of premature atherosclerosis. We investigated the effects of inhibiting proprotein convertase subtilisin/kexin type 9 (PCSK9) function on inflammatory state, endothelial dysfunction and cardiovascular outcomes in patients with FH. Fourteen patients with FH were evaluated before and 8 weeks after administration of a PCSK9 blocking monoclonal antibody (alirocumab, 150 mg/subcutaneous/14 days). In vivo and ex vivo analysis revealed that alirocumab blunted the attachment of leukocytes to TNFα-stimulated human umbilical arterial endothelial cells (HUAEC) and suppressed the activation of platelets and most leukocyte subsets, which was accompanied by the diminished expression of CX3CR1, CXCR6 and CCR2 on several leukocyte subpopulations. By contrast, T-regulatory cell activation was enhanced by alirocumab treatment, which also elevated anti-inflammatory IL-10 plasma levels and lowered circulating pro-inflammatory cytokines. Plasma levels of IFNγ positively correlated with levels of total and LDL-cholesterol, whereas circulating IL-10 levels negatively correlated with these key lipid parameters. In vitro analysis revealed that TNFα stimulation of HUAEC increased the expression of PCSK9, whereas endothelial PCSK9 silencing reduced TNFα-induced mononuclear cell adhesion mediated by Nox5 up-regulation and p38-MAPK/NFκB activation, concomitant with reduced SREBP2 expression. PCSK9 silencing also decreased endothelial CX3CL1 and CXCL16 expression and chemokine generation. In conclusion, PCSK9 inhibition impairs systemic inflammation and endothelial dysfunction by constraining leukocyte-endothelium interactions. PCSK9 blockade may constitute a new therapeutic approach to control the inflammatory state associated with FH, preventing further cardiovascular events in this cardiometabolic disorder.

Impact of PCSK9 Immunization on Glycemic Indices in Diabetic Rats.

METHODS: To prepare the anti-PCSK9 vaccine, a peptide construct called Immunogenic Fused PCSK9-Tetanus (IFPT) was linked to the surface of nanoliposome carriers. Healthy rats received four subcutaneous injections of the vaccine at biweekly intervals. Two weeks after the last vaccination, anti-PCSK9 antibody titers, PCSK9 targeting, and inhibition of PCSK9-low-density lipoprotein receptor (LDLR) interaction were evaluated. After verification of antibody generation, the immunized rats were intraperitoneally treated with a single dose (45 mg/kg) of streptozotocin (STZ) to induce diabetes mellitus. The levels of fasting blood glucose (FBG) were measured, and the oral glucose tolerance test (OGTT) as well as the insulin tolerance test (ITT) were carried out to assess glycemic status. At the end of the study, the total cholesterol, low-density lipoprotein cholesterol (LDL-C), triglyceride, and high-density lipoprotein cholesterol concentrations were assayed. Histopathology examination of the liver and pancreas was also performed using the hematoxylin-eosin staining method. RESULTS: The prepared nanoliposomal vaccine could strongly induce anti-PCSK9 antibodies in the vaccinated rats. Within one week following the STZ injection, the FBG level was lower in the vaccinated group vs. diabetic control group (49% (-171.7 ± 35 mg/dL, p < 0.001)). In the OGTT, the injected rats showed improved glucose tolerance as reflected by the reduction of blood glucose levels over 180 min, compared with the diabetic controls. Moreover, the ITT demonstrated that, after the insulin injection, blood glucose concentration declined by 49.3% in the vaccinated group vs. diabetic control group. Expectedly, the vaccinated rats exhibited lower (-26.65%, p = 0.03) plasma LDL-C levels compared with the diabetic controls. Histopathology examination of pancreas tissue demonstrated that the pancreatic islets of the vaccinated rats had a slight decline in the population of ß-cells and few α-cells. Normal liver histology was also observed in the vaccinated rats. CONCLUSION: PCSK9 inhibition through the liposomal IFPT vaccine can improve the glucose and insulin tolerance impairments as well as the lipid profile in diabetes.

Development of a Bispecific Antibody-Based Platform for Retargeting of Capsid Modified AAV Vectors.

A major limiting factor for systemically delivered gene therapies is the lack of novel tissue specific AAV (Adeno-associated virus) derived vectors. Bispecific antibodies can be used to redirect AAVs to specific target receptors. Here, we demonstrate that the insertion of a short linear epitope "2E3" derived from human proprotein-convertase subtilisin/kexin type 9 (PCSK9) into different surface loops of the VP capsid proteins can be used for AAV de-targeting from its natural receptor(s), combined with a bispecific antibody-mediated retargeting. We chose to target a set of distinct disease relevant membrane proteins-fibroblast activation protein (FAP), which is upregulated on activated fibroblasts within the tumor stroma and in fibrotic tissues, as well as programmed death-ligand 1 (PD-L1), which is strongly upregulated in many cancers. Upon incubation with a bispecific antibody recognizing the 2E3 epitope and FAP or PD-L1, the bispecific antibody/rAAV complex was able to selectively transduce receptor positive cells. In summary, we developed a novel, rationally designed vector retargeting platform that can target AAVs to a new set of cellular receptors in a modular fashion. This versatile platform may serve as a valuable tool to investigate the role of disease relevant cell types and basis for novel gene therapy approaches.

Utilization of the human gamma-satellite insulator for the enhancement of anti-PCSK9 monoclonal antibody expression in Chinese hamster ovary cells.

Monoclonal antibodies (mAbs) are widely employed as invaluable therapeutics for a vast number of human disorders. Several approaches have been introduced for the improvement of mAb production in Chinese hamster ovary (CHO) cells due to the increasing demand for these products. In this regard, various chromatin-modifying elements such as insulators have been incorporated in the expression vectors to augment mAb expression. In this study, human gamma-satellite insulator containing vectors were utilized for the expression of an anti-proprotein convertase subtilisin/kexin type 9 (PCSK9) mAb in CHO-K1 cells. To this aim, dual expression vectors encoding the antibody light chain (LC) and heavy chain (HC) with or without the insulator element were constructed, and mAb expression was evaluated in transient and stable expression. Based on the results, mAb expression significantly increased in the stable cell pool, and clonal cells developed using the human gamma-satellite insulator. In contrast, transient antibody expression was not affected by the insulator element. Finally, the enhancement of LC and HC mRNA levels was found in the insulator containing stable cell pools using the quantitative real-time-polymerase chain reaction (qRT-PCR). Our findings showed the positive effect of the human gamma-satellite insulator on the stable expression of an anti-PCSK9 immunoglobulin G1 (IgG1) mAb in CHO-K1 cells using dual expression vectors.