Evaluation of the Structural, Physicochemical, and Biological Characteristics of SB11, as Lucentis® (Ranibizumab) Biosimilar.

INTRODUCTION: SB11 was recently approved as a ranibizumab biosimilar by the US Food and Drug Administration (FDA) and the European Commission (EC) as a therapy for retinal vascular disorders under the brand name Byooviz™. This study was performed to assess the analytical similarity between SB11 and reference products from the European Union (EU-ranibizumab) and United States (US-ranibizumab). METHODS: A comprehensive structural, physicochemical, and biological characterization was performed utilizing state-of-the-art analytical methods. Comparisons included the following: primary structure related to amino acid sequence and post-translational modifications; higher order structure; product-related substances and purity/impurity including size and charge variants. In addition, biological characterization included a series of mechanism of action (MoA)-related bioassays such as vascular endothelial growth factor (VEGF)-A binding assay (VEGF-A 165 and its isoforms), cell-based VEGF-A 165 neutralization assay, and anti-proliferation assay using human umbilical vein endothelial cells (HUVEC). RESULTS: The amino acid sequence of SB11 was identical to that of reference products, and post-translational modification profiles and higher order structures of SB11 were shown to be indistinguishable from the reference products. Product-related size and charge variants and aggregates were also similar. Using a broad range of VEGF-related functional assays, we demonstrated that SB11 has similar biological properties to reference products in VEGF-A binding activities (VEGF-A 165 and isoforms (VEGF-A 110, VEGF-A 121, and VEGF-A 189)), VEGF-A 165 neutralization, and HUVEC anti-proliferation. Overall, SB11 exhibits high similarity compared to EU/US-ranibizumab. CONCLUSION: Based on the comprehensive analytical similarity assessment, SB11 is highly similar to the EU/US-ranibizumab with respect to structural, physicochemical, and biological properties.

Assessment of the Molecular Mechanism of Action of SB3, a Trastuzumab Biosimilar.

BACKGROUND: SB3, a biosimilar of Herceptin® (trastuzumab, hereinafter referred to as reference product) is currently approved in the EU, Korea, Australia, the USA, and Brazil for the treatment of human epidermal growth factor receptor (HER) 2-positive early and metastatic breast cancer and HER2-positive metastatic gastric cancer. Previously, the biological similarity of SB3 to EU- or US-sourced reference product was assessed using various cell-based and binding assays. OBJECTIVE: In this paper, as a part of its similarity assessment, SB3 was evaluated for additional characteristics related to its molecular mechanism of action (MoA). METHODS: For extracellular effects of SB3, HER2-overexpressing cancer cell lines were used to assess expression of surface HER2, shedding of the extracellular domain of HER2, and antibody-dependent cell-mediated phagocytosis (ADCP) activity. For intracellular effects, Akt phosphorylation and vascular endothelial growth factor (VEGF) release were assessed. Additionally, in vitro docetaxel or pertuzumab combination experiments were performed for further characterization; anti-proliferation, HER2/HER3 dimerization inhibition, apoptosis, and antibody-dependent cell-mediated cytotoxicity (ADCC) assays were used. RESULTS: It was confirmed that SB3 is highly similar to the reference products on quality attributes related to extracellular/intracellular efficacy. This similarity was also confirmed during combination studies with docetaxel and pertuzumab. CONCLUSION: Overall, the equivalence of SB3 with reference product in MoA-related qualities in in vitro mono- and combination therapy experiments may support clinical bioequivalence of the two substances.

Biological Characterization of SB3, a Trastuzumab Biosimilar, and the Influence of Changes in Reference Product Characteristics on the Similarity Assessment.

BACKGROUND: SB3 has been developed as a trastuzumab biosimilar, a therapeutic monoclonal antibody targeted to human epidermal growth factor receptor 2 (HER2), and approved by the European Commission and United States (US) Food and Drug Administration (FDA). During the developmental period of a biosimilar, setting an appropriate quality target is critical for assessing the similarity of the biosimilar product to the reference product. A stepwise approach should be taken to assessing similarity, beginning with extensive characterization of the reference product to establish the quality target. OBJECTIVE: In this study, we evaluated the similarity of SB3 to the reference product and the impact of changes in the biological profile of the reference product on similarity assessment. METHODS: Analytical similarity was assessed with defined test procedures in terms of critical quality attributes (CQAs) that could affect efficacy, potency, and safety, as well as for the non-CQAs that are related to process consistency. The quality target was established using up to 154 lots of European Union (EU)- and US-sourced Herceptin® (reference product), analyzed during the developmental period of SB3. RESULTS: Trends of the EU- and US-sourced reference product showed that the biological profile exhibited two marked changes for Fc-related attributes, and then recovered to pre-change quality level. Since the similarity range set by pre-change lots was considered most relevant, the changed lots were excluded from establishing the similarity range, which resulted in tightened acceptance criteria. As shown in the results of similarity assessment using the stringent quality target ranges, SB3 exhibits highly similar functional activities compared to the reference product in terms of both CQAs and non-CQAs. CONCLUSION: SB3 has been developed as a trastuzumab biosimilar approved in the EU and USA, and its manufacturing process is deemed to be robust and well-controlled within stringent quality target ranges.

Body Mass Index and Risk of Intestinal Metaplasia: A Cohort Study.

BACKGROUND: We examined the association between body mass index (BMI) and development of endoscopic intestinal metaplasia. METHODS: This retrospective cohort study included 142,832 Korean adults free of endoscopic intestinal metaplasia and atrophic gastritis who underwent upper endoscopy at baseline and subsequent visits and were followed for up to 5 years. A parametric proportional hazards model was used to estimate the adjusted HR with 95% confidence interval (CI) for incident intestinal metaplasia. RESULTS: In more than 444,719.1 person-years of follow-up, 2,281 participants developed endoscopic intestinal metaplasia (incidence rate, 5.1 per 1,000 person-years). Increased BMI categories were associated with increased risk of new-onset intestinal metaplasia in a dose-response manner. After adjustment for age, sex, center, year of screening exam, smoking status, alcohol intake, exercise, total calorie intake, history of diabetes and hypertension, and history of Helicobacter pylori infection, the multivariable adjusted HRs (95% CIs) for incident intestinal metaplasia comparing BMIs of <18.5, 23-24.9, 25.0-29.9, and >30 kg/m2 with a BMI of 18.5-22.9 kg/m2 were 0.84 (0.64-1.09), 1.03 (0.93-1.16), 1.07 (0.96-1.20), and 1.48 (1.20-1.83), respectively. These associations did not differ by clinically relevant subgroups. Risk of endoscopic atrophic gastritis also increased as the baseline BMI category increased. CONCLUSIONS: In a large cohort of Korean men and women, obesity was independently associated with increased incidence of endoscopic atrophic gastritis and intestinal metaplasia. IMPACT: Excessive adiposity appears to play a role in development of stomach precursor lesions of stomach cancer, requiring further studies to determine whether strategies to reduce obesity will also help reduce precancerous lesions and, in turn, gastric cancer.

Smoking and Urinary Cotinine Levels Are Predictors of Increased Risk for Gastric Intestinal Metaplasia.

Studies on a longitudinal relationship between smoking status and intestinal metaplasia (IM), a premalignant lesion of stomach cancer, are limited. Here we examined the association of smoking status and urinary cotinine levels, an objective measure of smoking, with the development of endoscopic IM. This cohort study included 199,235 Korean adults free of endoscopic IM who underwent upper endoscopy at baseline and subsequent visits and who were followed for up to 6.8 years (median, 3.7 years). Former and current smoking status and pack-years based on self-reports were associated with an increased risk of new-onset IM in men but not in women. However, urinary cotinine levels were positively associated with incident IM in a dose-response manner in both men and women. For men, the multivariable-adjusted HR [95% confidence interval (CI)] for incident IM comparing the urinary cotinine levels of 50 to 99 ng/mL, 100 to 499 ng/mL, and ≥500 ng/mL with <50 ng/mL were 1.20 (0.94-1.55), 1.26 (1.14-1.40), and 1.54 (1.44-1.64), respectively, whereas for women, corresponding HR (95% CI) were 0.75 (0.19-2.99), 1.86 (1.20-2.88), and 1.57 (1.07-2.30), respectively. These associations were observed when changes in smoking status and other confounders were updated during follow-up as time-varying covariates. In this large cohort of young and middle-aged men and women, urinary cotinine levels were independently associated with an increased incidence of endoscopic IM in a dose-response manner. Collectively, these data confirm smoking as an independent risk factor for the development of gastric IM, a precursor lesion of stomach cancer. SIGNIFICANCE: A large-scale cohort study of nearly 200,000 adults associates smoking with increased risk for gastric intestinal metaplasia, a precursor lesion of stomach cancer.

Drifts in ADCC-related quality attributes of Herceptin®: Impact on development of a trastuzumab biosimilar.

A biosimilar product needs to demonstrate biosimilarity to the originator reference product, and the quality profile of the latter should be monitored throughout the period of the biosimilar's development to match the quality attributes of the 2 products that relate to efficacy and safety. For the development of a biosimilar version of trastuzumab, the reference product, Herceptin®, was extensively characterized for the main physicochemical and biologic properties by standard or state-of-the-art analytical methods, using multiple lots expiring between March 2015 and December 2019. For lots with expiry dates up to July 2018, a high degree of consistency was observed for all the tested properties. However, among the lots expiring in August 2018 or later, a downward drift was observed in %afucose (G0+G1+G2). Furthermore, the upward drift of %high mannose (M5+M6) was observed in the lots with expiry dates from June 2019 to December 2019. As a result, the combination of %afucose and %high mannose showed 2 marked drifts in the lots with expiry dates from August 2018 to December 2019, which was supported by the similar trend of biologic data, such as FcγRIIIa binding and antibody-dependent cell-mediated cytotoxicity (ADCC) activity. Considering that ADCC is one of the clinically relevant mechanisms of action for trastuzumab, the levels of %afucose and %high mannose should be tightly monitored as critical quality attributes for biosimilar development of trastuzumab.

Amelioration of sepsis by TIE2 activation-induced vascular protection.

Protection of endothelial integrity has been recognized as a frontline approach to alleviating sepsis progression, yet no effective agent for preserving endothelial integrity is available. Using an unusual anti-angiopoietin 2 (ANG2) antibody, ABTAA (ANG2-binding and TIE2-activating antibody), we show that activation of the endothelial receptor TIE2 protects the vasculature from septic damage and provides survival benefit in three sepsis mouse models. Upon binding to ANG2, ABTAA triggers clustering of ANG2, assembling an ABTAA/ANG2 complex that can subsequently bind and activate TIE2. Compared with a conventional ANG2-blocking antibody, ABTAA was highly effective in augmenting survival from sepsis by strengthening the endothelial glycocalyx, reducing cytokine storms, vascular leakage, and rarefaction, and mitigating organ damage. Together, our data advance the role of TIE2 activation in ameliorating sepsis progression and open a potential therapeutic avenue for sepsis to address the lack of sepsis-specific treatment.

Gln-362 of angiopoietin-2 mediates migration of tumor and endothelial cells through association with α5ß1 integrin.

Angiopoietin-2 (Ang-2) not only regulates angiogenesis by binding to its well known receptor Tie2 on endothelial cells but also controls sprouting of Tie2-negative angiogenic endothelial cells and invasion of Tie2-negative non-endothelial cells by binding to integrins. However, the molecular mechanism of the Ang-2/integrin association has been unclear. In this study, we found that the Gln-362 residue of Ang-2 was essential for binding to α5ß1 integrin. A Q362E Ang-2 mutant, which still bound to Tie2, failed to associate with α5ß1 integrin and was unable to activate the integrin downstream signaling of focal adhesion kinase. In addition, unlike wild-type Ang-2, the Q362E Ang-2 mutant was defective in mediating invasion of Tie2-negative glioma or Tie2-positive endothelial cells. Furthermore, the tailpiece domain of the α5 subunit in α5ß1 integrin was critical for binding to Ang-2. Taken together, these results provide a novel insight into the mechanism of integrin regulation by Ang-2, which contributes to tumor invasion and endothelial cell migration in a Tie2-independent manner.

Identification of Heat Shock Protein 60 as a Regulator of Neutral Sphingomyelinase 2 and Its Role in Dopamine Uptake.

Activation of sphingomyelinase (SMase) by extracellular stimuli is the major pathway for cellular production of ceramide, a bioactive lipid mediator acting through sphingomyelin (SM) hydrolysis. Previously, we reported the existence of six forms of neutral pH-optimum and Mg(2+)-dependent SMase (N-SMase) in the membrane fractions of bovine brain. Here, we focus on N-SMase ε from salt-extracted membranes. After extensive purification by 12,780-fold with a yield of 1.3%, this enzyme was eventually characterized as N-SMase2. The major single band of 60-kDa molecular mass in the active fractions of the final purification step was identified as heat shock protein 60 (Hsp60) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Proximity ligation assay and immunoprecipitation study showed that Hsp60 interacted with N-SMase2, prompting us to examine the effect of Hsp60 on N-SMase2 and ceramide production. Interestingly, Hsp60 siRNA treatment significantly increased the protein level of N-SMase2 in N-SMase2-overexpressed HEK293 cells. Furthermore, transfection of Hsp60 siRNA into PC12 cells effectively increased both N-SMase activity and ceramide production and increased dopamine re-uptake with paralleled increase. Taken together, these results show that Hsp60 may serve as a negative regulator in N-SMase2-induced dopamine re-uptake by decreasing the protein level of N-SMase2.