Physicochemical and biological similarity assessment of LBAL, a biosimilar to adalimumab reference product (Humira®).

LBAL was developed as an adalimumab (Humira®) biosimilar using Chinese hamster ovary cell lines. Comparable quality, safety, and efficacy between a biosimilar and its reference product should be ensured for regulatory approval. Here, we present the results of a comprehensive physicochemical and biological characterization between LBAL and Humira®. As physicochemical attributes, primary and higher-order structure, N-glycan profile, and disulfide linkage were investigated. Biological attributes were evaluated by target/receptor binding analysis and in vitro/ex vivo cell-based assays, which are linked to mechanisms of action. As a result, LBAL had the identical amino acid sequence, similar post-translational modifications and N-/C-terminal variants, and comparable primary, secondary, and tertiary structures and disulfide linkage profile. However, some differences in N-glycan profiles were observed. Biological activities, including tumor necrosis factor (TNF) binding, TNF-neutralization, apoptosis, Fc receptor binding, and complement-dependent cytotoxicity, were largely consistent. Despite a slightly lower antibody-dependent cellular cytotoxicity activity in LBAL, this difference was not significant under physiological conditions. As indicated, this extensive analytical characterization and functional comparison assessment showed that LBAL was similar to Humira®, with minor differences of no clinical relevance. Taken together, our comparative assessment of physicochemical and biological attributes demonstrated that LBAL is structurally and functionally very similar to Humira®, supporting the biosimilarity of clinical efficacy and safety.

[Quality characteristics and nonclinical/clinical profiles of Etanercept BS SC [MA]].

Etanercept is a dimeric genetic recombinant glycoprotein consisting of Fc domain of human Immunoglobulin G1 and the extracellular domain of human tumor necrosis factor (TNF) receptor type II. Etanercept exerts therapeutic effects on inflammatory diseases such as rheumatoid arthritis and juvenile idiopathic arthritis by neutralizing biological activities of TNFα/Lymphotoxin (LT) α. Mochida Pharmaceutical and LG Chem have developed syringe, pen, and vial products of Etanercept BS (biosimilar) as the first biosimilar of Enbrel in Japan. The active ingredient of those products "Etanercept biosimilar 1" has the identical primary structure to that of Enbrel. The development of the Etanercept BS, including evaluations of quality attributes, nonclinical and clinical studies was performed in accordance with "Policies on Assurance of Quality, Safety and Efficacy of Biosimilars". The quality attributes of Etanercept BS were similar to those of Enbrel, and the binding affinities to TNFα/LTα, TNFα neutralizing activity, nonclinical pharmacokinetics and toxicological profiles of Etanercept BS were comparable to Enbrel. Additionally, the pharmacokinetic profile and efficacy of Etanercept BS were equivalent to those of Enbrel and there was no clinically significant difference in safety profiles between them in Phase I and Phase III clinical studies. The marketing approval application of the Etanercept BS with the same indications as Enbrel filed by Mochida Pharmaceutical was approved in January 2018 and the products will be launched by Ayumi Pharmaceutical in the near future. The Etanercept BS, which is as highly effective as Enbrel is expected to make beneficial therapies more easily accessible to patients.

Cell type-specific activation of intracellular transglutaminase 2 by oxidative stress or ultraviolet irradiation: implications of transglutaminase 2 in age-related cataractogenesis.

Transglutaminase (TGase) 2 is a ubiquitously expressed enzyme that modifies proteins by cross-linking or polyamination. An aberrant activity of TGase 2 has implicated its possible roles in a variety of diseases including age-related cataracts. However, the molecular mechanism by which TGase 2 is activated has not been elucidated. In this report, we showed that oxidative stress or UV irradiation elevates in situ TGase 2 activity. Neither the expression level nor the in vitro activity of TGase 2 appeared to correlate with the observed elevation of in situ TGase 2 activity. Screening a number of cell lines revealed that the level of TGase 2 activation depends on the cell type and also the environmental stress, suggesting that unrecognized cellular factor(s) may specifically regulate in situ TGase 2 activity. Concomitantly, we observed that human lens epithelial cells (HLE-B3) exhibited about 3-fold increase in in situ TGase 2 activity in response to the stresses. The activated TGase 2 catalyzed the formation of water-insoluble dimers or polymers of alphaB-crystallin, betaB(2)-crystallin, and vimentin in HLE-B3 cells, providing evidence that TGase 2 may play a role in cataractogenesis. Thus, our findings indicate that in situ TGase 2 activity must be evaluated instead of in vitro activity to study the regulation mechanism and function of TGase 2 in biological and pathological processes.

Transglutaminase 2 inhibits Rb binding of human papillomavirus E7 by incorporating polyamine.

Transglutaminase 2 (TGase 2) is one of a family of enzymes that catalyze protein modification through the incorporation of polyamines into substrates or the formation of protein crosslinks. However, the physiological roles of TGase 2 are largely unknown. To elucidate the functions of TGase 2, we have searched for its interacting proteins. Here we show that TGase 2 interacts with E7 oncoprotein of human papillomavirus type 18 (HPV18) in vitro and in vivo. TGase 2 incorporates polyamines into a conserved glutamine residue in the zinc-binding domain of HPV18 E7 protein. This modification mediates the inhibition of E7's Rb binding ability. In contrast, TGase 2 does not affect HPV16 E7, due to absence of a glutamine residue at this polyamination site. Using E7 mutants, we demonstrate that TGase 2-dependent inhibition of HPV E7 function correlates with the presence of the polyamination site. Our results indicate that TGase 2 is an important cellular interfering factor and define a novel host-virus interaction, suggesting that the inability of TGase 2 to inactivate HPV16 E7 could explain the high prevalence of HPV16 in cervical cancer.

Improved immunodetection of human papillomavirus E7.

Human papillomavirus E7 (HPV E7) is a viral oncoprotein that plays an important role in cervical carcinogenesis through binding with retinoblastoma protein (Rb). Inactivation of Rb by E7 is necessary but not sufficient for cellular transformation, suggesting other protein-protein interactions are required for E7-mediated cellular transformation aside from the interaction with Rb. However, studies on the oncogenic function of HPV E7 have been limited by its poor immunoreactivity. In this report, we show that the fixation of purified recombinant HPV E7 on blotted nitrocellulose membrane with glutaldehyde markedly enhanced the immunoreactivity of HPV E7 protein. Using HeLa and Caski cell lines which are infected with HPV 18 and HPV 16, respectively, we demonstrated that native HPV E7 proteins also could be detected by this method. These results therefore can provide the experimental conditions for detection of HPV E7 proteins with greater sensitivity and may help to analyze E7 functions.