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T cell immunoglobulin and ITIM domain (TIGIT) is a novel immune checkpoint that has been considered as a target in cancer immunotherapy. Current available bioassays for measuring the biological activity of therapeutic antibodies targeting TIGIT are restricted to mechanistic investigations because donor primary T cells are highly variable. Here, we designed a reporter gene assay comprising two cell lines, namely, CHO-CD112-CD3 scFv, which stably expresses CD112 (PVRL2, nectin-2) and a membrane-bound anti-CD3 single-chain fragment variable (scFv) as the target cell, and Jurkat-NFAT-TIGIT, which stably expresses TIGIT as well as the nuclear factor of activated T-cells (NFAT) response element-controlled luciferase gene, as the effector cell. The anti-CD3 scFv situated on the target cells activates Jurkat-NFAT-TIGIT cells through binding and crosslinking CD3 molecules of the effector cell, whereas interactions between CD112 and TIGIT prevent activation. The presence of anti-TIGIT mAbs disrupts their interaction, which in turn reverses the inactivation and luciferase expression. Optimization and validation studies have demonstrated that this assay is superior in terms of specificity, accuracy, linearity, and precision. In summary, this reliable and effective reporter gene assay may potentially be utilized in lot release control, stability assays, screening, and development of novel TIGIT-targeted therapeutic antibodies., Graphical abstract Here we developed one reporter gene assay comprising two cell lines, CHO-CD112-CD3 scFv and Jurkat-NFAT-TIGIT, which can be adopted in the bioactivity determination of anti-TIGIT antibody with high accuracy and specificity.Image 1

Since the outbreak of COVID-19, a variety of vaccine platforms have been developed. Amongst these, inactivated vaccines have been authorized for emergency use or conditional marketing in many countries. To further enhance the protective immune responses in populations that have completed vaccination regimen, we investigated the immunogenic characteristics of different vaccine platforms and tried homologous or heterologous boost strategy post two doses of inactivated vaccines in a mouse model. Our results showed that the humoral and cellular immune responses induced by different vaccines when administered individually differ significantly. In particular, inactivated vaccines showed relatively lower level of neutralizing antibody and T cell responses, but a higher IgG2a/IgG1 ratio compared with other vaccines. Boosting with either recombinant subunit, adenovirus vectored or mRNA vaccine after two-doses of inactivated vaccine further improved both neutralizing antibody and Spike-specific Th1-type T cell responses compared to boosting with a third dose of inactivated vaccine. Our results provide new ideas for prophylactic inoculation strategy of SARS-CoV-2 vaccines.

The outbreak of COVID-19 has posed a huge threat to global health and economy. Countermeasures have revolutionized norms for working, socializing, learning, and travel. Importantly, vaccines have been considered as most effective tools to combat with COVID-19. As of the beginning of 2021, >200 COVID-19 vaccine candidates, covering nearly all existing technologies and platforms, are being research and development (R&D) by multiple manufacturers worldwide. This has posed a huge obstacle to the quality control and evaluation of those candidate vaccines, especially in China, where five vaccine platforms are deployed in parallel. To accelerate the R&D progress of COVID-19 vaccines, the guidances on R&D of COVID-19 vaccine have been issued by National Regulatory Authorities or organizations worldwide. The Center for Drug Evaluation and national quality control laboratory in China have played a leading role in launching the research on quality control and evaluation in collaboration with relevant laboratories involved in the vaccine R&D, which greatly supported the progression of vaccines R&D, and accelerated the approval for emergency use and conditional marketing of currently vaccine candidates. In this paper, the progress and experience gained in quality control and evaluation of COVID-19 vaccines developed in China are summarized, which might provide references for the R&D of current and next generation of COVID-19 vaccines worldwide.

Although enormous success has been achieved with anti-PD-1/PD-L1 and anti-CTLA-4 monoclonal antibodies (mAbs), their unsatisfactory response rate in cancer patients has been driving the research and development of novel immune checkpoint inhibitors (ICIs). Anti-LAG-3 mAbs, as one of the most promising candidates, are now being tested for various human cancers at different stages of clinical trials. Here, we describe the development and validation of a reporter gene assay (RGA) to measure the bioactivity of anti-LAG-3 mAbs. We established the bioassay based on parental Raji cells and a Jurkat cell line stably transfected with human LAG-3 gene and luciferase reporter elements controlled by nuclear factor of activated T cell (NFAT) from the IL-2 promoter. After optimization of key parameters, the established RGA showed excellent precision, specificity, accuracy, and stability. The mechanism of action (MOA) relatedness and the excellent assay performance make the RGA suitable for the characterization, lot release, and stability test of anti-LAG-3 mAbs.

Our previous studies have demonstrated that trametenolic acid B (TAB) extracted from the Laetiporus sulphureus (Fr.) Murrill owned effective anti-proliferation of HepG2/2.215 cells and induced autophagy activity. The present aim was to further investigate its mechanisms involved by proteomic analysis. The iTRAQ of TAB on HepG2/2.215 was carried out and the western blot was used to verify the results of the proteomics analysis. According to the peptide segment quantitative standard (FDR ≤ 1%), a total of 5324 proteins were identified in HepG2/2.215 by proteomic analysis. The results identified that the major up-regulated proteins were HSP90AA4P, MYB, SERPINE1, and down-regulated proteins were Rho C, SERPINA1, and PIK3R4, which were related to PI3K/Akt signaling pathway, cell metastasis, and autophagy. HSP90AA4P and Rho C’s proteomics analysis were further confirmed by the western blot. The proteomic results demonstrated that the anti-hematoma effect of TAB was closely related to the increase of HSP90AA4P protein expressions and autophagy, which may be a critical target of TAB, which was expected to be a candidate drug for the treatment liver cancer.

Severe fever with thrombocytopenia syndrome (SFTS) is an acute infectious disease caused by severe fever with thrombocytopenia syndrome virus (SFTSV). SFTS is mainly characterized by severe fever with thrombocytopenia and has a high mortality rate. The virus has been found in China, South Korea, and Japan. Effective antiviral drugs or vaccines still have been unavailable. Now, two vaccine manufacturers in China are actively engaged in the development of the vaccine. To promote the development of SFTS vaccines and ensure their effective quality control, we developed national antigen and antibody references. We collaborative calibrated the standards; evaluated the homogeneity and stability of the national SFTS standards. The national SFTS vaccine antigen and antibody references met the Chinese national standards and can be used to standardize quality control for the manufacture of SFTS vaccines. And also can be used into the study the dose-response relationship of SFTS vaccines, determine clinical doses, and evaluate vaccine immunogenicity.

Background Patient delay in seeking help has been reported to be a major factor that related to delay in care of acute myocardial infarction (AMI). Lack the knowledge of symptoms may translate to behavioral deficiencies which lead up to failing to seek medical assistant at early stage of AMI. Purpose The aim of present study is to investigate the awareness of AMI symptoms and the emergency responses among community residents over 35 years old. Methods The sample was proportionally distributed by age according to the national census data. The sex ratio was 1:1. The sample size is calculated according to the significance level of 0.05, the allowable error of 0.1, and the estimated Excellent awareness level of 10%. The final sample size is adjusted to 4200, considering non-response rate of 20%. Multi-stage stratified random sampling was used. On the first stage, two districts each in urban and rural regions were randomly selected. On the second stage, 3–10 community health service centers were randomly selected in each district. On the third stage, residents over 35 managed by the community health service center were proportionally sampled to be interviewed. A structured questionnaire was used and the survey was conducted in a face-to-face interview by clinical physicians. Logistic regression was applied to analyze factors related to Good knowledge. Results The top three symptom recognized by public is “pain or discomfort in the chest” (71.3%), followed by “difficulty breathing” (65.1%) and “pain or discomfort in the jaw, neck, or back” (60.9%). 85.0% chose to call an ambulance as first response when witness others having an AMI. Those who lives alone (OR=1.408; 95% CI, 1.005–1.972) and whose immediate family had been diagnosed with AMI (OR=1.510; 95% CI, 1.040–2.192) has better knowledge. A significant positive correlation was observed for those with hypertension (OR=1.199; 95% CI, 1.007–1.429), while a negative correlation was observed for those with diabetes (OR=0.788; 95% CI, 0.626–0.992). Public education could improve Good knowledge (OR=1.662; 95% CI, 1.388–1.990), while doctoral advise has been shown negative impact (OR=0.824; 95% CI, 0.691–0.984). Conclusions Our data provide first population-based estimates of public awareness in our country. Further promotional strategies to increase overall awareness in general public are seriously needed. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Major Science and Technology Projects of Tianjin Science and Technology Commission Multivariable logistic regression

The effects of phloridzin (PHL), main component of Malus hupehensis (MH) tea leaves, on blood glucose (BG) and glucose-6-phosphatase (G-6-Pase) were investigated to provide a basis for finding a scheme of stabilizing BG. Glucose uptake of insulin resistant HepG2 cells was measured by glucose oxidase method. Glucose tolerance, fasting BG (FBG) and postprandial BG (PBG) were determined by BG test strips. The expression of G-6-Pase was detected by Western blot. The results showed that glucose uptake was enhanced and the expression of G-6-Pase was inhibited by PHL in insulin resistant HepG2 cells. Glucose tolerance was enhanced, FBG level was increased and PBG level was decreased by PHL in mice. The expression of G-6-Pase in the liver was enhanced under fasting state, and was inhibited by the low and medium dose under postprandial state. It indicated that PHL has a positive effect on stabilizing BG in mice, which is related to bidirectional regulation of G-6-Pase activity. PRACTICAL APPLICATIONS: Malus hupehensis, edible and medicinal plant, which has been proved by long-term application and experiments that it has a good effect on stabilizing blood glucose, preventing diabetes and adjuvant treatment. Its effect is closely related to its main component PHL. Thus, MH can be used as a dietary regulating drink for daily life to maintain blood glucose. Its main ingredient is PHL, which can be developed as a candidate drug for diabetes treatment.

Malus hupehensis (Pamp.) Rehd (MH) is a dual-purpose plant used as medicine and food, which possesses various biological activities including hepatoprotective effect. In this study, the hepatoprotective activity of extracts of MH (MHE) against RFP + INH-induced liver injury was investigated. Animals in model group were given saline and INH + RFP. Animals in MHE groups were given MHE and INH + RFP. Animals in normal control group were given equal volume of saline. Subsequently, blood and liver samples were collected and assessed. MHE significantly alleviated the liver injury, as evidenced by decreased activities of the ALT, AST, TBIL, MDA and GSH-ST, and increased levels of SOD and GSH. MHE also effectively reduced the pathological tissue damage. Moreover, the CYP450 levels, and expression of CYP3A4, CYP2C9 and NADPH4 protein were inhibited by MHE. MHE exerts a protective effect against RFP + INH-induced liver injury in mice, and could be developed as a liver protectant for treatment of DILI.

CTLA-4 is an important immune checkpoint for the regulation of T cell activation, and anti-CTLA-4 monoclonal antibodies (mAbs) are being developed as mono- or combination therapy for various tumors with reliable clinical efficacy. Ipilimumab is the first approved inhibitor of immune checkpoint, and many other anti-CTLA-4 mAbs, including ipilimumab biosimilars, are in different stages of clinical trials. However, due to the immunomodulating nature of the mAbs targeting CTLA-4, mode of action (MoA) and cell-based bioassay to determine their bioactivities as the lot release or stability test has been a great challenge to quality control laboratories. In this study, we have developed and validated a reporter gene assay (RGA), in which two kinds of cell lines were engineered to measure the bioactivity of anti-CTLA-4 mAbs. Raji cells were stably transfected with the membrane-anchored anti-CD3 single chain antibody fragment (scFv) as antigen-presenting cells (APCs, Raji-CD3scFv cells), while Jurkat cells were stably transfected with CTLA-4 with Y201V mutation and NFAT controlled luciferase as the effector cells (Jurkat-CTLA-4-NFAT-luc cells). The ligation of CD80/CD86 on the APCs with CTLA-4 could reduce the luciferase expression accompanied with the activation of effector cells, while the anti-CTLA-4 mAb could reverse the reduction, which resulted in good dose response curve to determine its bioactivity. After optimizing various assay conditions, we performed full validation according to ICH-Q2 (R1), which demonstrated the excellent specificity, accuracy, precision, linearity, and the cell passage stability. The satisfied performance characteristics render the RGA a good bioassay in the bioactivity determination of anti-CTLA-4 mAbs, as applied in characterization, batch release control, stability study, and biosimilar assessment.

The world has entered the second wave of the COVID-19 pandemic, and its intensity is significantly higher than that of the first wave of early 2020. Many countries or regions have been forced to start the second round of lockdowns. To respond rapidly to this global pandemic, dozens of COVID-19 vaccine candidates have been developed and many are undergoing clinical testing. Evaluating and defining effective vaccine candidates for human use is crucial for prioritizing vaccination programs against COVID-19. In this review, we have summarized and analyzed the efficacy, immunogenicity and safety data from clinical reports on different COVID-19 vaccines. We discuss the various guidelines laid out for the development of vaccines and the importance of biological standards for comparing the performance of vaccines. Lastly, we highlight the key remaining challenges, possible strategies for addressing them and the expected improvements in the next generation of COVID-19 vaccines.

The high global burden of tuberculosis (TB) underscores the urgent need for an effective TB vaccine since the only licensed Bacillus Calmette-Guérin (BCG) vaccine is ineffective in preventing adult pulmonary TB and affords no protection against latent TB infection (LTBI). Herein we investigated the potential of Mycobacterium tuberculosis (Mtb) antigen proteins AEC comprised of Ag85b and ESAT6-CFP10 proteins in conjunction with aluminum (Al) and polyriboinosinic-polyribocytidylic acid (poly-IC) as a novel subunit vaccine against TB. The immunogenicity and protection induced by the adjuvanted vaccine were evaluated in two animal models. Mice vaccinated with AEC/Al/poly-IC exhibited significant antigen-specific humoral immune responses and cell-mediated immunity as determined by immunoassay and multicolor flow cytometric assay, and the protective effect of the vaccine was demonstrated in a guinea pig model of latent Mtb infection. Compared to the control group, the mean pathological scores and bacterial loads in lungs and spleens of AEC/Al/poly-IC-immunized guinea pigs were significantly reduced. These data indicate that the AEC/Al/poly-IC is highly immunogenic in mice and can effectively protect guinea pigs against latent Mtb infection; it may represent a promising candidate vaccine for the control of latent TB.

Influenza vaccine potency is determined by the quantification of immunologically active hemagglutinin capable of eliciting neutralizing antibodies upon immunization. Currently, the single radial immunodiffusion (SRID) method is the standard in vitro potency assay used for lot release of seasonal inactivated influenza vaccines. Despite the proven usage of SRID, significant limitations such as the time-consuming preparation of reagents and limited dynamic range warrant the need for the development of alternative potency assays. Such alternative approaches need to discriminate and quantify relevant hemagglutinin material, provide strain identity, and be independent of strain-specific and seasonal reagents. Herein, we present a proof of concept method that combines the capture of conformationally well-folded hemagglutinin via a sialic acid binding step with the resolving power of reversed-phase high-performance liquid chromatography for strain identity and determination. Details of the protocol for the selective capture of receptor-binding hemagglutinin, its release from the receptor, and its relative determination are presented. This approach was found to provide flexibility for the reagents to be used and was adaptable to varying strain compositions of influenza vaccines. This proof of concept approach was developed as an antibody-independent methodology.

Background The high cost and insufficient supply of human papillomavirus (HPV) vaccines have slowed the pace of controlling cervical cancer. A phase III clinical trial was conducted to evaluate the efficacy, safety, and immunogenicity of a novel Escherichia coli-produced bivalent HPV-16/18 vaccine. Methods A multicenter, randomized, double-blind trial started on November 22, 2012 in China. In total, 7372 eligible women aged 18–45 years were age-stratified and randomly assigned to receive three doses of the test or control (hepatitis E) vaccine at months 0, 1, and 6. Co-primary endpoints included high-grade genital lesions and persistent infection (over 6 months) associated with HPV-16/18. The primary analysis was performed on a per-protocol susceptible population of individuals who were negative for relevant HPV type-specific neutralizing antibodies (at day 0) and DNA (at day 0 through month 7) and who received three doses of the vaccine. This report presents data from a prespecified interim analysis used for regulatory submission. Results In the per-protocol cohort, the efficacies against high-grade genital lesions and persistent infection were 100.0% (95% confidence interval = 55.6% to 100.0%, 0 of 3306 in the vaccine group vs 10 of 3296 in the control group) and 97.8% (95% confidence interval = 87.1% to 99.9%, 1 of 3240 vs 45 of 3246), respectively. The side effects were mild. No vaccine-related serious adverse events were noted. Robust antibody responses for both types were induced and persisted for at least 42 months. Conclusions The E coli-produced HPV-16/18 vaccine is well tolerated and highly efficacious against HPV-16/18–associated high-grade genital lesions and persistent infection in women.

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen that causes substantial morbidity and mortality in livestock and humans. To date, there are no licensed human vaccines or therapeutics available. Here, we report the isolation of monoclonal antibodies from a convalescent patient, targeting the RVFV envelope proteins Gn and Gc. The Gn-specific monoclonal antibodies exhibited much higher neutralizing activities in vitro and protection efficacies in mice against RVFV infection, compared to the Gc-specific monoclonal antibodies. The Gn monoclonal antibodies were found to interfere with soluble Gn binding to cells and prevent infection by blocking the attachment of virions to host cells. Structural analysis of Gn complexed with four Gn-specific monoclonal antibodies resulted in the definition of three antigenic patches (A, B and C) on Gn domain I. Both patches A and B are major neutralizing epitopes. Our results highlight the potential of antibody-based therapeutics and provide a structure-based rationale for designing vaccines against RVFV. The isolation and characterization of glycoprotein-specific monoclonal antibodies from a patient with Rift Valley fever (RVF) provide insights into the humoral immune responses elicited by the RVF virus as well as the therapeutic potential of antibody-based strategies.

Adoptive cell therapy using chimeric antigen receptor T (CAR-T) cells, which is a promising cancer immunotherapy strategy, has been developing very rapidly in recent years. CAR-T cells are genetically modified T cells that can specifically recognize tumor specific antigens on the surface of tumor cells, and then effectively kill tumor cells. At present, exciting results are being achieved in clinical applications of CAR-T cells for patients with hematological malignancies. The research and development of CAR-T cells for various targets and for the treatment of solid tumors have become a hot topic worldwide, so an increasing number of investigational new drug applications (INDAs) and new drug applications (NDAs) of CAR-T cell products are expected to be submitted in future. The quality control and nonclinical research of these products are of great significance in ensuring the safety and effectiveness of these products; however, they also present great challenges and difficulties. This article discusses the general principles of and key issues regarding the quality control and nonclinical research of CAR-T cell products based on their product characteristics and on relevant guidelines for gene and cell therapy products. Keywords: Chimeric antigen receptor T cells, Quality control, Nonclinical research, Safety, Efficacy, Clinical trials, Cancer immunotherapy

RANKL (receptor activator of nuclear factor κB ligand) plays a key role in the differentiation, activation and survival of osteoclasts. Denosumab, which targets RANKL, is approved for osteoporosis or bone loss that has a high risk for fracture and bone metastases from solid tumors. Bioactivity determination is essential for the safety and efficacy of therapeutic antibodies. At present, the mechanism of action (MOA) based bioassay for anti-RANKL monoclonal antibodies (mAbs) is the measurement of tartrate resistant acid phosphatase (TRAP) activity, which takes about five days and has complex operation and relatively high variation. In this study, we developed a reporter gene assay (RGA) based on a RAW264.7 cell line stably expressing luciferase reporter under the control of nuclear factor-κB (NF-κB) response elements. After optimizing the key parameters, the validation results based on ICH-Q2 not only show superior specificity, precision, linearity, accuracy and passage stability, but also a short duration and simple operation. These results demonstrate the RGA based on the RANKL-RANK-NF-κB pathway can be an excellent alternative for measuring the bioactivity of anti-RANKL mAbs.

We studied Phellinus lonicerinus to determine the cytotoxic effect and the dual estrogenic activities of methyl-hispolon and their relation to estrogen signals in vivo and in vitro. The Glide scores of methyl-hispolon-estrogen receptor α (ERα) and methyl-hispolon-ERβ docked complexes were -7.29 kcal/mol and -6.68 kcal/mol in docking simulations. Methyl-hispolon had a significant antiproliferative effect for estrogen-sensitive ER(+) MCF-7 cells in the absence of estrogen, and it exhibited dual estrogen activities. Methyl-hispolon increased the serum E2 in rats with premature ovarian failure and fulfilled the estrogenic function in the uterus and ovary. Methyl-hispolon significantly inhibited the expression of Ras, API, ERα, C-myc, and cyclinDl, as well as their gene transcription in RL95-2 cells. The phosphorylation of ERK1/2 was inhibited by methyl-hispolon. Thus, methyl-hispolon has potential use in treating estrogen deficiency-related diseases, with good antitumor effects and estrogenic activity.

The aim of this study was to elucidate the gastroprotective activity and possible mechanism of involvement of araloside A (ARA) against ethanol- and aspirin-induced gastric ulcer in mice. The experimental mice were randomly divided into control, model, omeprazole (20 mg/kg, orally) and ARA (10, 20 and 40 mg/kg, orally). Gastric ulcer in mice was induced by intragastric administration of 80% ethanol (10 mL/kg) containing 15 mg/mL aspirin 4 h after drug administration on day 7. The results indicated that ARA could significantly raise gastric juice volume and acidity; ameliorate gastric mucosal blood flow, gastric binding mucus volume, ulcer index and ulcer inhibition rate; suppress H+/K+-ATPase activity, which was confirmed by computer-aided docking simulations; inhibit the release of mitochondrial cytochrome c into the cytoplasm; inhibit caspase-9 and caspase-3 activities and down-regulate mRNA expression levels; down-regulate the mRNA and protein expressions of apoptosis protease-activating factor-1 and protein expression of cleaved poly(ADP ribose) polymerase-1; and up-regulate Bcl-2 mRNA and protein expressions and down-regulate Bax mRNA and protein expressions, thus elevating the Bcl-2/Bax ratio in a dose-dependent manner. Histopathological observations further provided supportive evidence for the aforementioned results. The results demonstrated that ARA exerted beneficial gastroprotective effects on alcohol- and aspirin-induced gastric ulcer in mice, which was related to suppressing H+/K+-ATPase activity as well as pro-apoptotic protein expression, and promoting anti-apoptotic protein expression, thus alleviating gastric mucosal injury and cell death.

Accurate determination of biological activity is essential in quality control of recombinant human brain natriuretic peptide (rhBNP). In previous study, we successfully developed a genetically modified cell line 293GCAC3-based ELISA assay for rhBNP. But ELISA procedure is still tedious, so this study was aimed to develop a rapid and simple bioassay for rhBNP using GloSensor technology, which provides a platform of flexible luciferase-based biosensors for real-time detection of signaling events in live cells, including cGMP production. A reporter cell line 293GCAGlo-G1 was constructed by transfecting pGloSensor™ 40 F plasmid into 293GCAC3. The reporter assay based on 293GCAGlo-G1 showed high precision with intra-assay CV being 8.3% and inter-assay CV being 14.1%; high accuracy with 80%, 100% and 120% recovery rate being 99.2%, 102.4% and 99.0% respectively; and great linearity with R2 of linear fitting equation being 0.99. Besides, no significant difference was found in test results of reporter assay and 293GCAC3-based ELISA assay (paired t test, p = 0.630). All these results suggested that the reporter assay was a viable assay for biological determination of rhBNP. Keywords: RhBNP, cGMP, GloSensor technology, Reporter assay

Calcitonin gene-related peptide (CGRP) is critical for the pathophysiology of migraine, and four therapeutic antibodies targeting CGRP and its corresponding receptors have been approved by the Food and Drug Administration (FDA), while many others are in the different stages of clinical trials. Bioactivity determination is essential for the quality control and clinical application of therapeutic monoclonal antibodies (mAbs). However, no bioassay has been reported to date. In this study, we developed a reporter gene assay (RGA) based on SK-N-MC cells stably expressing firefly luciferase driven by cAMP response element (CRE). The key assay parameters were optimized according to signal-to-noise (SNR), the response value, and the fitted dose-response curve. Validation of the RGA in accordance with ICH-Q2 guidelines showed that the method had good specificity, accuracy, linearity, and precision. The established RGA can be utilized as a reference method for release testing and stability studies of relevant antibodies.

The tumor necrosis factor alpha (TNF-α)/nuclear factor-kappa B (NF-κB) signaling pathway plays a crucial role in the pathogenesis of inflammatory diseases. Several therapeutic monoclonal antibodies (mAbs) and biosimilars against TNF-α have been developed to treat patients who suffer from inflammatory diseases caused by disordered expression of TNF-α. Hence, quality control of biopharmaceuticals is crucial during research and development. The high-order structure of these complex molecules cannot be entirely identified by physiochemical attributes; however, they can be inferred by observing biological activities. Thus, we developed a U937-based bioassay to determine the biological activities of mAbs and biosimilars against TNF-α using a low-basal NF-κB-inducible lentiviral reporter gene. The reporter gene assay (RGA) can be induced with a high signal-to-noise ratio (SNR) in a short time by TNF-α. Validation of the RGA showed accuracy (% relative standard deviation [RSD] = 4.64%), linearity (r2 = 0.9856), and precision (Interday RSD = 4.6%, between analysts RSD = 3.51%) as well as reasonable specificity and robustness. The measured potency values of a biosimilar to adalimumab were between 90% and 110%. Results showed our RGA is suitable for mAb quality control and lot release, and for evaluation of the biological activity similarity of the biosimilar.

OX40 plays a prominent role in the onset and development of solid tumors, and OX40-targeted monoclonal antibodies (mAbs) have entered clinical trials for various tumors. Bioactivity determination of therapeutic mAbs is of great significance in product quality, however, mechanism of action-based bioassays to determine the bioactivity of anti-OX40 mAbs is still lacking. Here, we established a reporter gene assay system based on two cell lines, namely Jurkat-OX40-NFκB-Luc which stably expresses NFκB-controlled luciferase, and Raji cells which inherently express FcγRs. In the model, FcγRs on Raji cells could crosslink the Fc of anti-OX40 mAbs, which leads to the further crosslinking between Fab of anti-OX40 mAbs and OX40 on Jurkat-OX40-NFκB-Luc cells. OX40 crosslinking could activate Jurkat-OX40-NFκB-Luc cells, and induce the expression of NFκB-controlled luciferase, the extent of which could reflect the bioactivity of anti-OX40 mAbs in a dose-dependent manner. After the optimization of various assay conditions, the validation of the cell-based bioassay showed good assay performance characteristics, including specificity, accuracy, precision, linearity, and stability. This innovative assay that is based on the OX40-NFκB pathway can be a powerful pool to measure the bioactivity of OX40-targeted mAbs.

Type 2 inflammatory cytokines, including IL-4, IL-5 and IL-13, contribute considerably to the pathogenesis of asthma. Anti-IL-4R monoclonal antibody (mAb) has been approved for the therapeutic treatment of asthma, and many mAbs with the same target are in the different stages of R&D and clinical trials. Bioactivity determination is required to ensure the quality control of mAbs. However, current ELISA and SPR assays or cell-based anti-proliferation assays for IL-4R mAbs are either not mechanism-of-action (MOA) representative or tedious and time consuming. Therefore, we developed a reporter gene assay (RGA) based on the HEK-293 cell line that stably expressed signal transducer and activator of transcription 6 (STAT6) and the luciferase reporter controlled by STAT6 binding elements. Anti-4R mAb could bind to IL-4R, and block the interaction between IL-4 and IL-4R, resulting in the reduction of IL-4 induced STAT6 controlled luciferase expression. After careful optimization of the experiment parameters, the RGA method demonstrated optimal dose-response curve between anti-IL-4R mAb concentration and luciferase expression level. Validation according ICH-Q2 proved the excellent assay performance characteristics of the established RGA, including specificity, accuracy, precision, linearity and range. The established transgenic cell line was stable for the bioactivity determination of anti-IL-4R mAb up to 46 generations, and the RGA was also suitable for the bioactivity determination of anti-IL-4 mAbs, and potentially of anti-IL-13 mAbs. The established RGA could be adopted to determine the bioactivity during the development, characterization, lot release, stability, and comparability studies of anti-IL-4R mAbs.

Environmental disturbances may result in dysregulation of interleukin-23 (IL-23), which is a crucial modulator of immunity. Several therapeutic monoclonal antibodies (mAbs) have been developed for treating IL-23-related autoimmune inflammation, such as ustekinumab, guselkumab, tildrakizumab, and risankizumab. Accurate bioactivity determination of therapeutic mAbs is essential for their quality control and clinical application. However, the current methods are tedious and complicated. In the present study, we employed low-background lentivirus carrying sis-inducible element (SIE)-driven firefly luciferase to generate a stable DB-SIE-Luc cell line that expresses endogenous IL-23 receptors and developed a sensitive and straightforward reporter gene assay (RGA) based on DB-SIE-Luc cells. After the optimization of various assay parameters, we set up a bioassay with the best fit of a four-parameter model and an appropriate signal-to-noise ratio (SNR) for bioactivity determination of guselkumab. We further verified the excellent assay performance characteristics of our RGA, including specificity, linearity, accuracy, precision, and stability, according to ICH-Q2. Taken together, we established a reliable and robust cell-based RGA, which potentially serves as a valubale alternative bioactivity determination assay for the release control and stability study of anti-IL-23 mAbs.

In this study, the estrogenic effect of GL-1, a component of the Ganoderma lucidum, was studied, and the possible mechanism was discussed preliminarily. The binding ability of GL-1 to estrogen receptor was calculated by computer aided simulation. The effects of GL-1 on the proliferation of estrogen sensitive estrogen receptor (ER) (+) MCF-7 cells and estrogen insensitive ER (−) MDA-MB-231 cells were detected by MTT method. The effects of GL-1 on the proliferation of estrogen-induced MCF-7 cells, and the effects of the estrogen receptor inhibitor ICI182780 on the proliferation of GL-1-induced MCF-7 cells were detected by MTT assay. The expression of ERα and ERβ monoclonal antibody were detected by Western blot. The results showed that GL-1 has a good binding ability to estrogen receptor β, and has estrogen-like effect, which might be related to secretion of estrogen and expression of ERβ by binding to ERs.

The management of hand-foot-and-mouth disease(HFMD) epidemic is difficult due to the frequent emergence of non-EV71 and non-CVA16 enteroviruses and some cases testing negative for HFMD-associated causative agents. To clarify the virus spectrum of mild and severe HFMD, a comprehensive virome analysis of 238 samples was performed using next-generation sequencing (NGS). The data revealed total thirteen mammalian- and plant- virus families and diverse viral populations including enteroviruses, common respiratory viruses, diarrhea-related viruses, plant viruses and anelloviruses. A total of 18 viruses from 7 virus families were identified in severe cases, versus 37 viruses from 12 virus families in mild cases. Moreover, complicated mixed-infections of enteroviruses with common respiratory viruses were mainly found in severe cases(P = 0.013), while diarrhea-related viruses were mainly found in mild cases(P 0.001). This study provides the preliminary understanding of viromes both in mild and severe cases, which may benefit the detection of etiologic agents and prevention of HFMD.