Discovery and characterization of hydroxylysine O-glycosylation in an engineered IL-2 fusion protein.

In the present study, an engineered interleukin-2 (IL-2) fusion protein consisting of an anti-human serum albumin nanobody linked by ASTKG and a (G4S)2 linker to IL-2 was constructed. Liquid chromatography-mass spectrometry (LC-MS) characterization was performed on the intact molecule and at the peptide level. The LC-MS molecular mass analysis for the engineered fusion protein showed the appearance of unreported +340 Da peaks, apart from the expected O-glycosylation-related peaks in the IL-2 domain. Through a combination analysis of a K120R mutated molecule (The lysine at the position of 120 was mutated to arginine while the rest amino acid sequence remain unchanged), the possibility of a non-cleaved valine-histidine-serine signal peptide was ruled out and the presence of hydroxylysine (HyK) O-glycosylation in the ASTKG linker was confirmed. HyK O-glycosylation have been reported in other proteins such as collagen, which occurs in the conserved Gly-Xaa-HyK motif and is catalyzed by lysyl hydroxylase-3 complex. The present study showed high similar conserved motif of HyK-O-glycosylation in collagen, implying the HyK O-glycosylation in the engineered IL-2 possibly was catalyzed by the Chinese hamster ovary homolog of enzymes promoting HyK O-glycosylation in collagen. Bioactivity testing results revealed that HyK-O-glycosylation had no obvious effect on the in vitro activity of engineered IL-2. Our study is the first to report HyK-O-glycosylation modifications in therapeutic proteins through LC-MS characterization and in vitro activity analysis, which expands the scope of post-translational modification knowledge of therapeutic proteins.

Metabolomic analysis reveals the metabolic disturbance in aortic dissection: Subtype difference and accurate diagnosis.

BACKGROUND AND AIMS: Aortic dissection (AD), a severe clinical emergency with high mortality, is easily misdiagnosed as are other cardiovascular diseases. This study aimed at discovering plasma metabolic markers with the potential to diagnose AD and clarifying the metabolic differences between two subtypes of AD. METHODS AND RESULTS: To facilitate the diagnosis of AD, we investigated the plasma metabolic profile by metabolomic approach. A total 482 human subjects were enrolled in the study: 80 patients with AD (50 with Stanford type A and 30 with Stanford type B), 198 coronary artery disease (CAD) patients, and 204 healthy individuals. Plasma samples were submitted to targeted metabolomic analysis. The partial least-squares discriminant analysis models were constructed to illustrate clear discrimination of AD patients with CAD patients and healthy control. Subsequently, the metabolites that were clinically relevant to the disturbances in AD were identified. Twenty metabolites induced the separation of AD patients and healthy control, 9 of which caused the separation of CAD patients and healthy control. There are 11 metabolites specifically down-regulated in AD group. Subgroup analysis showed that the levels of glycerol and uridine were dramatically lower in the plasma of patients with Stanford type A AD than those in the healthy control or Stanford type B AD groups. CONCLUSION: This study characterized metabolomic profiles specifically associated with the pathogenesis and development of AD. The findings of this research may potentially lead to earlier diagnosis and treatment of AD.

[Clinical Application of "Three-Low" Technique Combined with Artificial Intelligence Iterative Reconstruction Algorithm in Aortic CT Angiography].

Objective: To explore the application value of the "three-low" technique (low radiation dose, low contrast agent dosage and low contrast agent flow rate) combined with artificial intelligence iterative reconstruction (AIIR) in aortic CT angiography (CTA). Methods: A total of 33 patients who underwent aortic CTA were prospectively enrolled. Based on the time of their follow-up examinations, the imaging data were divided into Group A and Group B, with Group A being the control group (100 kV, 0.8 mL/kg, 5 mL/s) and Group B being the "three-low" technique group (70 kV, 0.5 mL/kg, 3 mL/s). In group A, the images were reconstructed by Karl iterative algorithm. Group B was divided into B1 and B2 subgroups, with their images being reconstructed by Karl iterative algorithm and AIIR, respectively. The CT and SD values of the ascending aorta, descending aorta, abdominal aorta, left common iliac artery and right common iliac artery were measured, and the signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) were calculated. The subjective scoring of image quality was performed. The radiation dose parameters were documented. Results: Differences in the CT value, SD value, SNR and CNR of the three groups were statistically significant ( P<0.001). The CT value, SNR and CNR of group B2 were significantly higher than those of group B1, while the SD value of group B2 was significantly lower than that of group B1 ( P<0.017). There was no significant difference between the CT values of group A and those of group B2 ( P>0.017). The SD values, SNR and CNR in group B2 were better than those in group A ( P>0.017). There was significant difference in the subjective evaluation of image quality among the three groups ( P<0.05), but there was no significant difference between group A and group B2 ( P>0.017). The radiation dose and contrast medium dosage in group B decreased 84.14% and 37.08%, respectively, compared with those of group A. Conclusion: With the "three-low" technique combined with AIIR algorithm, the image quality of aortic CTA obtained is comparable to that of conventional dose scanning, while the radiation dose, contrast agent dosage and contrast agent flow rate of patients are significantly reduced.

Antiinfective therapy with a small molecule inhibitor of Staphylococcus aureus sortase.

Methicillin-resistant Staphylococcus aureus (MRSA) is the most frequent cause of hospital-acquired infection, which manifests as surgical site infections, bacteremia, and sepsis. Due to drug-resistance, prophylaxis of MRSA infection with antibiotics frequently fails or incites nosocomial diseases such as Clostridium difficile infection. Sortase A is a transpeptidase that anchors surface proteins in the envelope of S. aureus, and sortase mutants are unable to cause bacteremia or sepsis in mice. Here we used virtual screening and optimization of inhibitor structure to identify 3-(4-pyridinyl)-6-(2-sodiumsulfonatephenyl)[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole and related compounds, which block sortase activity in vitro and in vivo. Sortase inhibitors do not affect in vitro staphylococcal growth yet protect mice against lethal S. aureus bacteremia. Thus, sortase inhibitors may be useful as antiinfective therapy to prevent hospital-acquired S. aureus infection in high-risk patients without the side effects of antibiotics.

The conserved Lys-95 charged residue cluster is critical for the homodimerization and enzyme activity of human ribonucleotide reductase small subunit M2.

Ribonucleotide reductase (RR) catalyzes the reduction of ribonucleotides to deoxyribonucleotides for DNA synthesis. Human RR small subunit M2 exists in a homodimer form. However, the importance of the dimer form to the enzyme and the related mechanism remain unclear. In this study, we tried to identify the interfacial residues that may mediate the assembly of M2 homodimer by computational alanine scanning based on the x-ray crystal structure. Co-immunoprecipitation, size exclusion chromatography, and RR activity assays showed that the K95E mutation in M2 resulted in dimer disassembly and enzyme activity inhibition. In comparison, the charge-exchanging double mutation of K95E and E98K recovered the dimerization and activity. Structural comparisons suggested that a conserved cluster of charged residues, including Lys-95, Glu-98, Glu-105, and Glu-174, at the interface may function as an ionic lock for M2 homodimer. Although the measurements of the radical and iron contents showed that the monomer (the K95E mutant) was capable of generating the diiron and tyrosyl radical cofactor, co-immunoprecipitation and competitive enzyme inhibition assays indicated that the disassembly of M2 dimer reduced its interaction with the large subunit M1. In addition, the immunofluorescent and fusion protein-fluorescent imaging analyses showed that the dissociation of M2 dimer altered its subcellular localization. Finally, the transfection of the wild-type M2 but not the K95E mutant rescued the G1/S phase cell cycle arrest and cell growth inhibition caused by the siRNA knockdown of M2. Thus, the conserved Lys-95 charged residue cluster is critical for human RR M2 homodimerization, which is indispensable to constitute an active holoenzyme and function in cells.

Construction, expression, purification, and characterization of a dual-targeting PD-1/VEGF-A fusion protein (P-V).

Targeting programmed death-1 (PD-1) is regarded as a novel and promising means for the treatment of many types of solid tumor. In the tumor microenvironment (TME), VEGF expression is dramatically up-regulated, and compounds that neutralize VEGF or block the interaction of VEGF with its receptors exhibit potent antitumor activity, and blocking PD-1 might promote T cell infiltration into TME and significantly enhance local immune activation. Thus, we fused domain II and domain III of kinase-insert domain receptor (KDR), the receptor of VEGF-A, to the Fc side of an anti-PD-1 monoclonal antibody with a (Gly4Ser)3 linker to generate a dual targeting fusion protein. The recombinant plasmid was successfully constructed and the fusion protein was expressed in 293E cells. Protein purification was performed in a single step by using protein A affinity chromatography. The molecular weight of the fusion protein was approximately 220kDa, and the yield was approximately 2.97g/L. Specific binding of recombinant protein to PD-1 and VEGF was detected by enzyme-linked immunosorbent assay (ELISA) analysis. Half maximal effective concentration (EC50) values were 0.561nM for PD-1 and 0.682nM for VEGF-A; accordingly, half maximal inhibitory concentration (IC50) values were 0.914nM and 0.583nM, respectively. Proliferation inhibition assays indicated that the fusion protein could inhibit the growth of human umbilical vein endothelial cells effectively. Taken together, the results indicate that this novel fusion protein can simultaneously target PD-1 and VEGF and may be beneficial for combining anti-angiogenesis with immunotherapeutic approaches for the treatment of patients with cancer.

Helix unfolding/refolding characterizes the functional dynamics of Staphylococcus aureus Clp protease.

The ATP-dependent Clp protease (ClpP) plays an essential role not only in the control of protein quality but also in the regulation of bacterial pathogen virulence, making it an attractive target for antibacterial treatment. We have previously determined the crystal structures of Staphylococcus aureus ClpP (SaClpP) in two different states, extended and compressed. To investigate the dynamic switching of ClpP between these states, we performed a series of molecular dynamics simulations. During the structural transition, the long and straight helix E in the extended SaClpP monomer underwent an unfolding/refolding process, resulting in a kinked helix very similar to that in the compressed monomer. As a stable intermediate in the molecular dynamics simulation, the compact state was suggested and subsequently identified in x-ray crystallographic experiment. Our combined studies also determined that Ala(140) acted as a "hinge" during the transition between the extended and compressed states, and Glu(137) was essential for stabilizing the compressed state. Overall, this study provides molecular insights into the dynamics and mechanism of the functional conformation changes of SaClpP. Given the highly conserved sequences of ClpP proteins among different species, these findings potentially reflect a switching mechanism for the dynamic process shared in the whole ClpP family in general and thus aid in better understand the principles of Clp protease assembly and function.

Machine learning reveals serum myristic acid, palmitic acid and heptanoylcarnitine as biomarkers of coronary artery disease risk in patients with type 2 diabetes mellitus.

BACKGROUND: Coronary heart disease (CHD) is the most important complication of type 2 diabetes mellitus (T2DM) and the leading cause of death. Identifying the risk of CHD in T2DM patients is important for early clinical intervention. METHODS: A total of 213 participants, including 81 healthy controls (HCs), 69 T2DM patients and 63 T2DM patients complicated with CHD were recruited in this study. Serum metabolomics were conducted by using ultra-high performance liquid chromatography coupled with tandem mass spectrometry (UHPLC-MS/MS). Demographic information and clinical laboratory test results were also collected. RESULTS: Metabolic phenotypes were significantly altered among HC, T2DM and T2DM-CHD. Acylcarnitines were the most disturbed metabolites between T2DM patients and HCs. Lower levels of bile acids and higher levels of fatty acids in serum were closely associated with CHD risk in T2DM patients. Artificial neural network model was constructed for the discrimination of T2DM and T2DM complicated with CHD based on myristic acid, palmitic acid and heptanoylcarnitine, with accuracy larger than 0.95 in both training set and testing set. CONCLUSION: Altogether, these findings suggest that myristic acid, palmitic acid and heptanoylcarnitine have a good prospect for the warning of CHD complications in T2DM patients, and are superior to traditional lipid, blood glucose and blood pressure indicators.

The Effect of Dihydromyricetin on the Pharmacokinetics of Fluconazole in Sprague-Dawley Rat Plasma, Based on High-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Background: The synergistic effect of dihydromyricetin (DHM) and fluconazole (FLC) can improve the killing effect of FLC-resistant Candida albicans in vitro and in vivo. However, it is not clear whether DHM affects the pharmacokinetic characteristics of FLC. Methods: In this study, 12 Sprague-Dawley (SD) rats were randomly divided into two groups as follows: (1) an FLC group in which rats were administered FLC only (42 mg/kg orally); (2) an FLC with the combined administration of DHM group, in which rats received an equivalent FLC dose immediately following the administration of DHM (100 mg/kg). Blood samples were collected from the ocular choroid vein of rats and converted into plasma. The concentrations of FLC in the rat plasma were then determined by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), and the related pharmacokinetic parameters were analysed. The initial mobile phase included 0.1% acetonitrile and water with gradient elution. Multiple reaction monitoring modes of m/z 307.2→220.1 for FLC, and m/z 237.1→194.2 for carbamazepine, were utilised to conduct quantitative analysis. Results: The calibration curve of FLC in rat plasma demonstrated good linearity in the range of 0.1-30 µg/mL (r > 0.99), and the lower limit of quantification was 0.1 µg/mL. Moreover, the intra- and inter-day precision relative standard deviation of FLC was less than 9.09% and 6.51%, respectively. There were no significant differences in the pharmacokinetic parameters between the two groups. Conclusion: The results showed that DHM administration did not significantly alter FLC pharmacokinetics in SD rat plasma.

Simultaneous determination of four antibiotics in human plasma by high-performance LC-MS/MS: application to therapeutic drug monitoring.

Background: Aimed to simultaneously measure linezolid, voriconazole, cefoperazone and fluconazole in human plasma suitable for therapeutic drug monitoring applications, a robust, rapid and easy-to-use HPLC-MS/MS approach was developed and validated. Materials & methods: Protein precipitation was used to prepare analytes from 100 µl plasma. HPLC was employed for analyte separation, and quantification was conducted via multiple-reaction monitoring in positive ion mode. The methodology was fully validated. Results & conclusion: All four antibiotics were found to be stable under the tested conditions, and accuracy values ranged from 90.96 to 113.25% and CV values were <14.0%. This HPLC-MS/MS method can be used for routine clinical therapeutic drug monitoring of linezolid, voriconazole, cefoperazone and fluconazole simultaneously.

Enhanced chemoimmunotherapy of breast cancer in mice by apolipoprotein A1-modified doxorubicin liposomes combined with interleukin-21.

Backgroud: Breast cancer is a prevalent malignancy among women, with triple-negative breast cancer (TNBC) comprising approximately 15-20% of all cases, possessing high invasiveness, drug resistance and poor prognosis. Chemotherapy, the main treatment for TNBC, is limited by toxicity and drug resistance. Apolipoprotein A1 modified doxorubicin liposome (ApoA1-lip/Dox) was constructed in our previous study, with promising anti-tumour effect and improved safety been proved. However, during long-term administration, the problem of cumulative toxicity and insufficient tumour inhibition is still inevitable. Interleukin-21 is a small molecule protein secreted by T cells with various immune regulatory functions. IL-21 has significantly curative effects in numerous solid tumours, but it has the disadvantages of low response rate and short half-life. The combination of chemotherapy and immunotherapy has received increasing attention.Purpose: In this study, ApoA1 drug loading system and long-acting IL-21 are innovatively combined for tumour treatment.Methods: We combined ApoA1-lip/Dox and IL-21 for treatment and evaluated their impact on tumor-infiltrating lymphocytes and CD8+ T and NK cell cytotoxicity.Results: Combined administration significantly improved the tumour-infiltrating lymphocytes and enhanced the cytotoxicity of CD8+ T and NK cells. The combination of ApoA1-lip/Dox and IL-21 exhibits significantly enhanced anti-tumour efficacy with lower toxicity of ApoA1-lip/Dox, providing a new strategy for TNBC treatment with enhanced anti-tumour response and reduced toxicity.

Characterization of the high-affinity anti-CTLA-4 monoclonal antibody JS007 for immune checkpoint therapy of cancer.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a critical inhibitory checkpoint molecule, and monoclonal antibodies (mAbs) targeting CTLA-4 that restore anti-tumor T cell immunity have achieved clinical success. Here, we report a humanized IgG1 mAb, namely JS007, with high binding affinity to CTLA-4. JS007 shows superior binding affinity and T-cell activating efficiency over ipilimumab. Moreover, it demonstrates substantial in vivo tumor suppression efficacy at low doses. The crystal structure of JS007/CTLA-4 complex (PDB: 8HIT) shows JS007 adopts a heavy-chain-dominant binding mode, and mainly contacts the BC loop, DE loop and FG loop of CTLA-4. Notably, two Tyr residues (VH-Y100 and VL-Y32) from the complementarity-determining region loops insert into the two cavities formed by the residues from the loops of CTLA-4, which may contribute to the stabilization of the binding. Comparative analysis with other anti-CTLA-4 mAbs indicates that the double "wedge-into-hole" binding mode is unique for JS007 and may be responsible for the high-affinity binding to CTLA-4. These findings have provided an important molecular understanding of the high-affinity CTLA-4 blockade mAbs and shed light on future development of agents targeting CTLA-4.

Inhibition of PCSK9 enhances the antitumor effect of PD-1 inhibitor in colorectal cancer by promoting the infiltration of CD8+ T cells and the exclusion of Treg cells.

Immunotherapy especially immune checkpoint inhibitors (ICIs) has brought favorable clinical results for numerous cancer patients. However, the efficacy of ICIs in colorectal cancer (CRC) is still unsatisfactory due to the poor median progression-free survival and overall survival. Here, based on the CRC models, we tried to elucidate novel relapse mechanisms during anti-PD-1 therapy. We found that PD-1 blockade elicited a mild antitumor effect in these tumor models with both increased CD8+ T cells and Treg cells. Gene mapping analysis indicated that proprotein convertase subtilisin/kexin type 9 (PCSK9), low-density lipoprotein receptor, transforming growth factor-ß (TGF-ß), and CD36 were unexpectedly upregulated during PD-1 blockade. To investigate the critical role of these proteins especially PCSK9 in tumor growth, anti-PCSK9 antibody in combination with anti-PD-1 antibody was employed to block PCSK9 and PD-1 simultaneously in CRC. Data showed that neutralizing PCSK9 during anti-PD-1 therapy elicited a synergetic antitumor effect with increased CD8+ T-cell infiltration and inflammatory cytokine releases. Moreover, the proportion of Treg cells was significantly reduced by co-inhibiting PCSK9 and PD-1. Overall, inhibiting PCSK9 can further enhance the antitumor effect of anti-PD-1 therapy in CRC, indicating that targeting PCSK9 could be a promising approach to potentiate ICI efficacy.

Simultaneous Quantitation of Clevidipine and Its Active Metabolite H152/81 in Human Whole Blood by LC-MS/MS: Application to Bioequivalence Study.

Clevidipine is an ultrashort-acting dihydropyridine calcium antagonist, which can control blood pressure accurately. It is necessary to develop a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantitate clevidipine and its active metabolite H152/81 for clinical pharmacokinetic study and therapeutic drug monitoring. Liquid-liquid extraction was used for sample preparation, and clevidipine-d7 and H152/81-13C-d3 were chosen as the isotope internal standard. The chromatographic separation was performed on an ACE Excel 2 Phenyl column (50 × 2.1 mm). Mass quantification was carried out on the multiple reaction monitoring of the transitions of m/z 473.1→338.1, 480.1→338.1, 356.0→324.0, and 362.2→326.2 for clevidipine, clevidipine-d7, H152/81, and H152/81-13C-d3. The validated method gave an excellent linearity over a concentration range of 0.1-30 ng/ml for clevidipine and 2-600 ng/ml for H152/81. Other fully validated content such as accuracy, precision, extraction recovery, matrix effect, and stability were also investigated and showed satisfactory results. It was strongly recommended that whole blood is the first choice for clinical bioanalysis. Using whole blood for sample analysis can reduce the whole blood collection volume (1 ml vs. 4 ml) and shorten the time from sample collection to storage to 5 min, and there is no centrifugation process and precooling in the ice water bath, which can further reduce the instability caused by exposure. The method was successfully applied to a bioequivalence study of clevidipine butyrate-injectable emulsion.

Etesevimab in combination with JS026 neutralizing SARS-CoV-2 and its variants.

The neutralizing antibody is a potential therapeutic for the ongoing COVID-19 pandemic. As an antiviral agent, numerous mAbs recognize the epitopes that overlap with ACE2-binding sites in the SARS-CoV-2-RBD. Some studies have shown that residual changes on the spike protein can significantly decrease the efficiency of neutralizing antibodies. To address this issue, a therapeutic cocktail could be an effective countermeasure. In the present study, we isolated a fully human neutralizing antibody, JS026, from a convalescent patient. The comparative analysis revealed that JS026 binding to SARS-CoV-2-RBD mainly located between epitopes for class 2 and class 3 mAbs as opposed to that of class 1 (etesevimab) antibodies. A cocktail of etesevimab and JS026 increased neutralizing efficacy against both wild-type SARS-CoV-2 and the recent emergence of Alpha, Beta, Gamma, and Delta variants. JS026 and the cocktail reduced virus titers in the infected lungs of hACE2 transgenic mice and relieved pathological changes. These findings would benefit antibody-based therapeutic countermeasures in the treatment of COVID-19.

Quality comparability assessment of a SARS-CoV-2-neutralizing antibody across transient, mini-pool-derived and single-clone CHO cells.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has triggered a serious public health crisis worldwide, and considering the novelty of the disease, preventative and therapeutic measures alike are urgently needed. To accelerate such efforts, the development of JS016, a neutralizing monoclonal antibody directed against the SARS-CoV-2 spike protein, was expedited from a typical 12- to 18-month period to a 4-month period. During this process, transient Chinese hamster ovary cell lines are used to support preclinical, investigational new drug-enabling toxicology research, and early Chemistry, Manufacturing and Controls development; mini-pool materials to supply Phase 1 clinical trials; and a single-clone working cell bank for late-stage and pivotal clinical trials were successively adopted. Moreover, key process performance and product quality investigations using a series of orthogonal and state-of-the-art techniques were conducted to demonstrate the comparability of products manufactured using these three processes, and the results indicated that, despite observed variations in process performance, the primary and high-order structures, purity and impurity profiles, biological and immunological functions, and degradation behaviors under stress conditions were largely comparable. The study suggests that, in particular situations, this strategy can be adopted to accelerate the development of therapeutic biopharmaceuticals and their access to patients.

Study on the Relationship between Rivaroxaban and Factor Xa Activity in Blood Based on HPLC-MS/MS.

OBJECTIVE: The aim of the study was to investigate a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the determination of rivaroxaban and evaluate the correlation between plasma concentration and anti-Xa activity in patients using oral rivaroxaban. METHODS: In this study, the plasma concentration of rivaroxaban and anti-Xa factor activities was determined in 125 patients, and the relationship between the two variables was analysed by SPSS 21.0 software. RESULTS: The results showed that the plasma concentrations of oral rivaroxaban patients were significantly correlated with the activity of the anti-Xa factor (Spearman's r = 0.990, P < 0.05). CONCLUSION: The plasma concentrations of rivaroxaban are a potentially useful monitoring indicator to assess the patient's bleeding risk if testing for plasma anti-Xa activity is not available.

A reporter gene assay for determining the biological activity of therapeutic antibodies targeting TIGIT.

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.

Pharmacokinetics, Bioavailability, Excretion and Metabolism Studies of Akebia Saponin D in Rats: Causes of the Ultra-Low Oral Bioavailability and Metabolic Pathway.

Background: Akebia saponin D (ASD) has a variety of biological activities and great medicinal potential, but its oral bioavailability is so low as to limit its development. Its pharmacokinetic profiles and excretion and metabolism in vivo have not been fully elucidated. This study was an attempt in this area. Methods: A simple LC-MS/MS method to simultaneously quantify ASD and its metabolites M1∼M5 in rat plasma, feces, urine and bile was established with a negative ESI model using dexketoprofen as the internal standard. Meanwhile, the UPLC-HR/MS system was used to screen all possible metabolites in the urine, feces and bile of rats, as compared with blank samples collected before administration. Absolute quantitative analysis was for M0, M3, M4, and M5, while semi-quantitative analysis was for M1, M2, and Orbitrap data. Results: The AUC0-t values after intravenous administration of 10 mg/kg and intragastrical administration of 100 mg/kg ASD were 19.05 ± 8.64 and 0.047 ± 0.030 h*µg/ml respectively. The oral bioavailability was determined to be extremely low (0.025%) in rats. The exposure of M4 and M5 in the oral group was higher than that of M0 in the terminal phase of the plasma concentration time profile, and ASD was stable in the liver microsome incubation system of rats, but metabolism was relatively rapid during anaerobic incubation of intestinal contents of rats, suggesting that the low bioavailability of ASD might have been attributed to the poor gastrointestinal permeability and extensive pre-absorption degradation rather than to the potent first pass metabolism. This assertion was further verified by a series of intervention studies, where improvement of lipid solubility and intestinal permeability as well as inhibition of intestinal flora increased the relative bioavailability to different extents without being changed by P-gp inhibition. After intravenous administration, the cumulative excretion rates of ASD in the urine and bile were 14.79 ± 1.87%, and 21.76 ± 17.61% respectively, but only 0.011% in feces, suggesting that the urine and bile were the main excretion pathways and that there was a large amount of biotransformation in the gastrointestinal tract. Fifteen possible metabolites were observed in the urine, feces and bile. The main metabolites were ASD deglycosylation, demethylation, dehydroxylation, decarbonylation, decarboxylation, hydroxylation, hydroxymethylation, hydroxyethylation and hydrolysis. Conclusion: The pharmacokinetics, bioavailability, metabolism and excretion of ASD in rats were systematically evaluated for the first time in this study. It has been confirmed that the ultra-low oral bioavailability is due to poor gastrointestinal permeability, extensive pre-absorption degradation and biotransformation. ASD after iv administration is not only excreted by the urine and bile, but possibly undergoes complex metabolic elimination.

An engineered IL-21 with half-life extension enhances anti-tumor immunity as a monotherapy or in combination with PD-1 or TIGIT blockade.

Interleukin-21 (IL-21) has exhibited anti-tumor activity in preclinical and clinical studies; however, its modest efficacy and short half-time has limited its therapeutic utility as a monotherapy. Therefore, we engineered a fusion protein (IL-21-αHSA) in which a nanobody targeting human serum albumin (HSA) was fused to the C-terminus of rhIL-21. The αHSA nanobody displayed broad species cross-reactivity and bound to a HSA epitope that does not overlap with the FcRn binding site, thus providing a strategic design for half-life extension. The IL-21-αHSA fusion protein showed increased stability compared to rhIL-21, while retaining its bioactivity in a liquid solution for at least 6 months. Moreover, IL-21-αHSA showed a dramatically extended half-life and prolonged exposure in cynomolgus monkeys, with the t1/2 and AUC nearly 10 and 50 times greater than that of rhIL-21, respectively. Furthermore, IL-21-αHSA displayed enhanced anti-tumor efficacy in two syngeneic mouse models. Notably, IL-21-αHSA increased the anti-tumor effect of programmed cell death protein 1 (PD-1) and T cell immunoglobulin and ITIM domain (TIGIT) blockades when used in combination, with a protection against tumor rechallenge, suggesting the formation of long-term anti-tumor memory response. KEGG analysis identified significantly enriched pathways associated with anti-tumor immune response, with increased expression of genes associated with CD8+ T and NK cell cytotoxicity. Overall, these data support further clinical evaluation of IL-21-αHSA as a monotherapy or in combination with immune checkpoint blockades.