Extension of human GCSF serum half-life by the fusion of albumin binding domain

Granulocyte colony stimulating factor (GCSF) can decrease mortality of patients undergo chemotherapy through increasing neutrophil counts. Many strategies have been developed to improve its blood circulating time. Albumin binding domain (ABD) was genetically fused to N-terminal end of GCSF encoding sequence and expressed as cytoplasmic inclusion bodies within Escherichia coli. Biological activity of ABD-GCSF protein was assessed by proliferation assay on NFS-60 cells. Physicochemical properties were analyzed through size exclusion chromatography, circular dichroism, intrinsic fluorescence spectroscopy and dynamic light scattering. Pharmacodynamics and pharmacokinetic properties were also investigated in a neutropenic rat model. CD and IFS spectra revealed that ABD fusion to GCSF did not significantly affect the secondary and tertiary structures of the molecule. DLS and SEC results indicated the absence of aggregation formation. EC50 value of the ABD-GCSF in proliferation of NFS-60 cells was 75.76 pg/ml after 72 h in comparison with control GCSF molecules (Filgrastim: 73.1 pg/ml and PEG-Filgrastim: 44.6 pg/ml). Animal studies of ABD-GCSF represented improved serum half-life (9.3 ± 0.7 h) and consequently reduced renal clearance (16.1 ± 1.4 ml/h.kg) in comparison with Filgrastim (1.7 ± 0.1 h). Enhanced neutrophils count following administration of ABD-GCSF was comparable with Filgrastim and weaker than PEG-Filgrastim treated rats. In vitro and in vivo results suggested the ABD fusion as a potential approach for improving GCSF properties.

Search for a way to improve the serum albumin binding function and the functional state of the liver when hypertension combined with non-alcoholic fatty liver disease

Recently, there has been a significant increase in interest in research on hypertension (HT), primarily due to its high prevalence. The interest in studying this problem is also exacerbated by the often insufficient effectiveness of existing treatments. The effect of concomitant pathologies on HT, in particular non-alcoholic fatty liver disease (NAFLD), remains poorly understood. The aim of the study – to evaluate the changes in the serum albumin binding function (SABF) and its relationship with the biochemical parameters of the blood when HT and HT combined with NAFLD and to suggest ways of medical correction of the detected disorders. Material and methods. 76 individuals with stage 2 HT with degree 2–3 arterial hypertension were examined. They were divided into two groups. Group 1 included 28 patients with HT without concomitant diseases who received basic hypertension therapy, and group 2 included patients with concomitant NAFLD. The latter in turn was divided into two subgroups: 2a – 27 patients who in addition to basic HT therapy received additional Antral hepatoprotector 200 mg three times a day for 2 months, and 2b – 21 patients who received only basic HT therapy. All of them underwent a standard clinical examination, as well as SABF, protein fractions, and liver function indicators. The comparison group consisted of 25 healthy individuals, comparable in age and sex. Results and Discussion. Patients in group 1 showed moderate changes in the functional state of the liver, but they did not exceed the norm, patients in group 2 – a significant decrease in SABF, as well as protein metabolism (decrease in total protein, albumin, albumin-globulin ratio and increase globulins) and liver function (increased activity of aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltranspeptidase, thymol levels, alkaline phosphatase and total bilirubin). After treatment, the majority of patients in subgroup 2 had a statistically significant increase in SABF and a quantitative improvement in protein fractions and functional state of the liver. In subgroup 2-b, where hepatoprotective treatment was not performed, significant changes in most indicators did not occur. The results may be related to the positive effect of the drug on the liver, which leads to improved functional status of hepatocytes and their protein-synthesizing ability. In subgroup 2 b, where hepatoprotective treatment was not performed, significant changes in most indicators did not occur. The results may be related to the positive effect of the drug on the liver leading to improved functional status of hepatocytes and their protein-synthesizing ability. Conclusions. Changes of the functional state of the liver are observed when HT without concomitant pathology. HT with NAFLD is accompanied by a significant decrease in SABF, changes in protein metabolism and the functional state of the liver. Prescribing Antral to such patients helps to increase SABF, normalize protein metabolism and improve the functional state of the liver.

Cathepsin B-Overexpressed Tumor Cell Activatable Albumin-Binding Doxorubicin Prodrug for Cancer-Targeted Therapy

Prodrugs are bioreversible medications that should undergo an enzymatic or chemical transformation in the tumor microenvironment to release active drugs, which improve cancer selectivity to reduce toxicities of anticancer drugs. However, such approaches have been challenged by poor therapeutic efficacy attributed to a short half-life and low tumor targeting. Herein, we propose cathepsin B-overexpressed tumor cell activatable albumin-binding doxorubicin prodrug, Al-ProD, that consists of a albumin-binding maleimide group, cathepsin B-cleavable peptide (FRRG), and doxorubicin. The Al-ProD binds to in situ albumin, and albumin-bound Al-ProD indicates high tumor accumulation with prolonged half-life, and selctively releases doxorubicin in cathepsin B-overexpressed tumor cells, inducing a potent antitumor efficacy. Concurrently, toxicity of Al-ProD toward normal tissues with innately low cathepsin B expression is significantly reduced by maintaining an inactive state, thereby increasing the safety of chemotherapy. This study offers a promising approach for effective and safe chemotherapy, which may open new avenues for drug design and translational medicine.

The Influence of Domain Permutations of an Albumin-Binding Domain-Fused HER2-Targeting Affibody-Based Drug Conjugate on Tumor Cell Proliferation and Therapy Efficacy

Human epidermal growth factor receptor 2 (HER2) is a clinically validated target for breast cancer therapy. Previously, a drug-fused HER2-targeting affinity protein construct successfully extended the survival of mice bearing HER2-expressing xenografts. The aim of this study was to evaluate the influence of the number and positioning of the protein domains in the drug conjugate. Seven HER2-targeting affibody-based constructs, including one or two affibody molecules (Z) with or without an albumin-binding domain (ABD), namely Z, Z-ABD, ABD-Z, Z-Z, Z-Z-ABD, Z-ABD-Z, and ABD-Z-Z, were evaluated on their effects on cell growth, in vivo targeting, and biodistribution. The biodistribution study demonstrated that the monomeric constructs had longer blood retention and lower hepatic uptake than the dimeric ones. A dimeric construct, specifically ABD-Z-Z, could stimulate the proliferation of HER2 expressing SKOV-3 cells in vitro and the growth of tumors in vivo, whereas the monomeric construct Z-ABD could not. These two constructs demonstrated a therapeutic effect when coupled to mcDM1; however, the effect was more pronounced for the non-stimulating Z-ABD. The median survival of the mice treated with Z-ABD-mcDM1 was 63 days compared to the 37 days for those treated with ABD-Z-Z-mcDM1 or for the control animals. Domain permutation of an ABD-fused HER2-targeting affibody-based drug conjugate significantly influences tumor cell proliferation and therapy efficacy. The monomeric conjugate Z-ABD is the most promising format for targeted delivery of the cytotoxic drug DM1.

Targeting HER2 Expressing Tumors with a Potent Drug Conjugate Based on an Albumin Binding Domain-Derived Affinity Protein

Albumin binding domain derived affinity proteins (ADAPTs) are a class of small and folded engineered scaffold proteins that holds great promise for targeting cancer tumors. Here, we have extended the in vivo half-life of an ADAPT, targeting the human epidermal growth factor receptor 2 (HER2) by fusion with an albumin binding domain (ABD), and armed it with the highly cytotoxic payload mertansine (DM1) for an investigation of its properties in vitro and in vivo. The resulting drug conjugate, ADAPT6-ABD-mcDM1, retained binding to its intended targets, namely HER2 and serum albumins. Further, it was able to specifically bind to cells with high HER2 expression, get internalized, and showed potent toxicity, with IC50 values ranging from 5 to 80 nM. Conversely, no toxic effect was found for cells with low HER2 expression. In vivo, ADAPT6-ABD-mcDM1, radiolabeled with 99mTc, was characterized by low uptake in most normal organs, and the main excretion route was shown to be through the kidneys. The tumor uptake was 5.5% ID/g after 24 h, which was higher than the uptake in all normal organs at this time point except for the kidneys. The uptake in the tumors was blockable by pre-injection of an excess of the monoclonal antibody trastuzumab (having an overlapping epitope on the HER2 receptor). In conclusion, half-life extended drug conjugates based on the ADAPT platform of affinity proteins holds promise for further development towards targeted cancer therapy.