Cytokine/Antibody Fusion Protein Design and Evaluation.

Cytokines constitute a class of secreted proteins that activate transmembrane receptors to coordinate a vast array of physiological processes, particularly those related to immune activity. Due to their vital role in immune regulation, cytokines have garnered great interest as potential therapeutic agents. Unfortunately, the clinical success of cytokine drugs has been limited by their multifunctional activities, which hinder therapeutic performance and lead to harmful toxicities. In addition, the strikingly short circulation half-life of cytokines further hampers their efficacy as drugs. To overcome the translational challenges associated with natural cytokines, significant efforts have focused on engineering cytokines to target their activities and improve their pharmacological properties. One such strategy is the design of fusion proteins that tether a cytokine to an anti-cytokine antibody that selectively biases its functions and extends its serum half-life. These cytokine/antibody fusion proteins (termed immunocytokines) assemble intramolecularly to bias cytokine signaling behavior through multi-layered structural and molecular effects. Here, we present a detailed workflow for the design, production, and functional validation of intramolecularly assembled immunocytokines. In-depth procedures are presented for gene manipulation, mammalian cell-based expression and purification, binding analysis via bio-layer interferometry, and interrogation of cytokine signaling activity on human primary cells. In contrast with immunocytokines in which the tethered cytokine and antibody do not bind one another, intramolecularly assembled immunocytokines require special considerations with respect to their production to avoid oligomerization and/or aggregation. The protocol herein was developed based on experience with immunocytokines that incorporate interleukin-2 (IL-2); however, this modular approach can be extended to any cytokine of interest for a broad range of biomedical applications. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Design and generation of immunocytokine genes Basic Protocol 2: Immunocytokine expression and purification Basic Protocol 3: Validation of immunocytokine assembly and binding by bio-layer interferometry Basic Protocol 4: Analysis of immunocytokine signaling on human primary cells.

Reprofiling synthetic glucocorticoid-induced leucine zipper fusion peptide as a novel and effective hair growth promoter.

Hair is a biofilament with unique multi-dimensional values. In human, in addition to physiologic impacts, hair loss and hair related disorders can affect characteristic features, emotions, and social behaviors. Despite significant advancement, there is a dire need to explore alternative novel therapies with higher efficacy, less side effects and lower cost to promote hair growth to treat hair deficiency. Glucocorticoid-induced leucine zipper (GILZ) is a protein rapidly induced by glucocorticoids. Studies from our group and many others have suggested that a synthetic form of GILZ, TAT-GILZ, a fusion peptide of trans-activator of transcription and GILZ, can function as a potent regulator of inflammatory responses, re-establishing and maintaining the homeostasis. In this study, we investigate whether TAT-GILZ could promote and contribute to hair growth. For our pre-clinical model, we used 9-12 week-old male BALB/c and nude (athymic, nu/J) mice. We applied TAT-GILZ and/or TAT (vehicle) intradermally to depilated/hairless mice. Direct observation, histological examination, and Immunofluorescence imaging were used to assess the effects and compare different treatments. In addition, we tested two current treatment for hair loss/growth, finasteride and minoxidil, for optimal evaluation of TAT-GILZ in a comparative fashion. Our results showed, for the first time, that synthetic TAT-GILZ peptide accelerated hair growth on depilated dorsal skin of BALB/c and induced hair on the skin of athymic mice where hair growth was not expected. In addition, TAT-GILZ was able to enhance hair follicle stem cells and re-established the homeostasis by increasing counter inflammatory signals including higher regulatory T cells and glucocorticoid receptors. In conclusion, our novel findings suggest that reprofiling synthetic TAT-GILZ peptide could promote hair growth by increasing hair follicle stem cells and re-establishing homeostasis.

Phase 1 first-in-human dose-escalation study of ANV419 in patients with relapsed/refractory advanced solid tumors.

Patients with advanced cancer, previously treated with immune checkpoint blockade therapy, may retain residual treatment when undergoing the initial infusion of experimental monotherapy in phase 1 clinical trials. ANV419, an antibody-cytokine fusion protein, combines interleukin-2 (IL-2) with an anti-IL-2 monoclonal antibody, aiming to stimulate the expansion of CD8 T and natural killer lymphocytes while restricting regulatory T lymphocytes. In the recent publication of the phase 1 dose escalation study of ANV419, a notable gap exists in detailed information regarding patients' prior antitumoral treatments, specifically programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) targeted monoclonal antibodies. Some patients likely retained residual anti-PD-1/PD-L1 monoclonal antibodies, potentially influencing the outcomes of ANV419. In a separate clinical cohort, we retrospectively measured the residual concentration of nivolumab and pembrolizumab, revealing persistent serum concentrations of anti-PD-1/PD-L1 antibodies even months after treatment cessation. This underscores the importance of comprehensively documenting prior immunotherapy details in clinical trials. Such information is crucial for understanding potential interactions that may impact both immunological and clinical effects.

Efficient production of human interleukin-3 from Escherichia coli using protein disulfide isomerase b'a' domain.

Human interleukin-3 (IL3) is a multifunctional cytokine essential for both clinical and biomedical research endeavors. However, its production in Escherichia coli has historically been challenging due to its aggregation into inclusion bodies, requiring intricate solubilization and refolding procedures. This study introduces an innovative approach employing two chaperone proteins, maltose binding protein (MBP) and protein disulfide isomerase b'a' domain (PDIb'a'), as N-terminal fusion tags. Histidine tag (H) was added at the beginning of each chaperone protein gene for easy purification. This fusion of chaperone proteins significantly improved IL3 solubility across various E. coli strains and temperature conditions, eliminating the need for laborious refolding procedures. Following expression optimization, H-PDIb'a'-IL3 was purified using two chromatographic methods, and the subsequent removal of the H-PDIb'a' tag yielded high-purity IL3. The identity of the purified protein was confirmed through liquid chromatography coupled with tandem mass spectrometry analysis. Biological activity assays using human erythroleukemia TF-1 cells revealed a unique two-step stimulation pattern for both purified IL3 and the H-PDIb'a'-IL3 fusion protein, underscoring the protein's functional integrity and revealing novel insights into its cellular interactions. This study advances the understanding of IL3 expression and activity while introducing novel considerations for protein fusion strategies.

CYP116B5-SOX: An artificial peroxygenase for drug metabolites production and bioremediation.

CYP116B5 is a class VII P450 in which the heme domain is linked to a FMN and 2Fe2S-binding reductase. Our laboratory has proved that the CYP116B5 heme domain (CYP116B5-hd) is capable of catalyzing the oxidation of substrates using H2O2. Recently, the Molecular Lego approach was applied to join the heme domain of CYP116B5 to sarcosine oxidase (SOX), which provides H2O2 in-situ by the sarcosine oxidation. In this work, the chimeric self-sufficient fusion enzyme CYP116B5-SOX was heterologously expressed, purified, and characterized for its functionality by absorbance and fluorescence spectroscopy. Differential scanning calorimetry (DSC) experiments revealed a TM of 48.4 ± 0.04 and 58.3 ± 0.02°C and a enthalpy value of 175,500 ± 1850 and 120,500 ± 1350 cal mol-1 for the CYP116B5 and SOX domains respectively. The fusion enzyme showed an outstanding chemical stability in presence of up to 200 mM sarcosine or 5 mM H2O2 (4.4 ± 0.8 and 11.0 ± 2.6% heme leakage respectively). Thanks to the in-situ H2O2 generation, an improved kcat/KM for the p-nitrophenol conversion was observed (kcat of 20.1 ± 0.6 min-1 and KM of 0.23 ± 0.03 mM), corresponding to 4 times the kcat/KM of the CYP116B5-hd. The aim of this work is the development of an engineered biocatalyst to be exploited in bioremediation. In order to tackle this challenge, an E. coli strain expressing CYP116B5-SOX was employed to exploit this biocatalyst for the oxidation of the wastewater contaminating-drug tamoxifen. Data show a 12-fold increase in tamoxifen N-oxide production-herein detected for the first time as CYP116B5 metabolite-compared to the direct H2O2 supply, equal to the 25% of the total drug conversion.

Affinity Purification of a 6X-His-Tagged Protein using a Fast Protein Liquid Chromatography System.

Functional characterization of proteins requires them to be expressed and purified in substantial amounts with high purity to perform biochemical assays. The Fast Protein Liquid Chromatography (FPLC) system allows high-resolution separation of complex protein mixtures. By adjusting various parameters in FPLC, such as selecting the appropriate purification matrix, regulating the protein sample's temperature, and managing the sample's flow rate onto the matrix and the elution rate, it is possible to ensure the protein's stability and functionality. In this protocol, we will demonstrate the versatility of the FPLC system to purify 6X-His-tagged flap endonuclease 1 (FEN1) protein, produced in bacterial cultures. To improve protein purification efficiency, we will focus on multiple considerations, including proper column packing and preparation, sample injection using a sample loop, flow rate of sample application to the column, and sample elution parameters. Finally, the chromatogram will be analyzed to identify fractions containing high yields of protein and considerations for proper recombinant protein long-term storage. Optimizing protein purification methods is crucial for improving the precision and reliability of protein analysis.

[Application of optical coherence tomography in the assessment of the posterior lens capsule during anti-angiogenic therapy]. / Primenenie opticheskoi kogerentnoi tomografii v otsenke zadnei kapsuly khrustalika na fone provedeniya antiangiogennoi terapii.

Intravitreal injection (IVI) of anti-angiogenic drugs is one of the most common therapeutic procedures in ophthalmology. In recent years, a new non-contact study method has been developed - anterior segment optical coherence tomography (AS-OCT), which allows the formation of three-dimensional images of the lens and provides more detailed information about its structure and morphology. PURPOSE: This study uses optical coherence tomography method to analyze the risks of developing changes in the posterior lens capsule in patients after IVI of an anti-angiogenic drug. MATERIAL AND METHODS: The study involved 100 people (14 men and 86 women) with a natural lens and neovascular age-related macular degeneration (nAMD). The average age was 70.57±7.98 years. During the study (12 months), all patients underwent IVI of an anti-angiogenic drug aflibercept in the treat-and-extend (T&E) mode. All subjects were divided into 2 groups: with a total number of IVI less than 10 - group 1 (50 patients), and more than 10 IVI - group 2 (50 patients, of which 49 were included in the study). All patients underwent OCT using the Optopol REVO NX device (Poland) with the Anterior B-scan Wide protocol before inclusion in the study, as well as after 3, 6 and 12 months. RESULTS: It was found that the risk of developing a posterior lens capsule rupture, visualized using OCT, depends on the total number of IVI (correlation coefficient 0.473 p=0.001): the more IVI, the higher the probability that damage to the posterior capsule will occur after the next IVI, and after the 15th injection the risk of developing damage to the posterior capsule increases sharply. CONCLUSION: The astudy analyzed the risk factors for the development of posterior lens capsule damage that can be detected using OCT, and presented three risk groups for the development of rupture (or damage) of the posterior lens capsule depending on the number of intravitreal injections performed.

Effects of Antithymocyte Globulin, Basiliximab, and Induction-Free Treatment in Living Donor Kidney Transplant Recipients on Tacrolimus-Based Immunosuppression.

OBJECTIVES: Induction treatment in renal transplant is associated with better graft survival. However, intensified immunosuppression is known to cause unwanted side effects such as infection and malignancy. Furthermore, the effects of the routine use of immunosuppressants in low-risk kidney transplant recipients are still not clear. In this study, we assessed the first-year safety and efficacy of induction treatment. MATERIALS AND METHODS: We examined first living donor kidney transplant patients who were on tacrolimus based immunosuppression therapy. We formed 3 groups according to the induction status: antithymocyte globulin induction, basiliximab induction, and no induction. We collected outcome data on delayed graft function, graft loss, creatinine levels, estimated glomerular filtration rates, acute rejection episodes, hospitalization episodes, and infection episodes, including cytomegalovirus infection and bacterial infections. RESULTS: We examined a total of 126 patients (age 35 ± 12 years; 65% male). Of them, 25 received antithymocyte globulin, 52 received basiliximab, and 49 did notreceive any induction treatment. We did not observe any statistically significant difference among the 3 groups in terms of acute rejection episodes, delayed graft function, and first-year graft loss. The estimated glomerular filtration rates were similar among the groups. Overall bacterial infectious complications and cytomegalovirus infection showed similar prevalence among all groups. Hospitalization was less common in the induction-free group. CONCLUSIONS: In low-risk patients, induction-free regimens could be associated with a better safety profile without compromising graft survival. Therefore, induction treatment may be disregarded in first living donor transplant patients who receive tacrolimusbased triple immunosuppression treatment.

Tebentafusp in the treatment of metastatic uveal melanoma - the first patient treated in the Czech Republic.

BACKGROUND: Uveal melanoma is a rare cancer, in which metastases occur in approximately one half of cases. In metastatic disease, the prognosis is unfavorable and the median of survival does not exceed 6 months. Effective treatment options were very limited up to date. Tebentafusp is a bispecific fusion protein, which as the first drug proved efficacy in uveal melanoma. CASE: The patient was referred for suspected uveal melanoma of the left eye. She was treated for Hodgkin's disease in the past. Primarily, the tumor was treated by radiosurgery with radiotherapy of a small lesion of the vertebral body. However, later the patient had to undergo bulbus enucleation with confirmation of a large tumor category pT4b. PET/CT revealed metastases of the bones and the liver; simultaneously, haplotype A*02: 01 was confirmed. The patient underwent radiotherapy of the sternum and later, after confirmation of payment from the health insurance company, she started treatment with tebentafusp. The first three doses were administered during admission to the hospital, with a need to treat cytokine release syndrome by corticosteroids. Later, the administration was performed in an out-patient regimen, without complications, except for a transient elevation of transaminases. The first CT restaging confirmed stable disease; however, the second restaging confirmed a new osteolytic lesion in the processus of Th11. Because of progression, the treatment with tebentafusp was withdrawn after 6 months. Unfortunately, the lesion could not be treated by radiotherapy. Two months later, the patient was urgently admitted to the hospital because of right-sided hemiplegia; MRI revealed bleeding metastatic lesion in the brain stem. CONCLUSION: In this case report, we present the case of the first patient treated with this drug in the Czech Republic.

An Elastin-like Polypeptide-fusion peptide targeting capsid-tegument interface as an antiviral against cytomegalovirus infection.

The tegument protein pp150 of Human Cytomegalovirus (HCMV) is known to be essential for the final stages of virus maturation and mediates its functions by interacting with capsid proteins. Our laboratory has previously identified the critical regions in pp150 important for pp150-capsid interactions and designed peptides similar in sequence to these regions, with a goal to competitively inhibit capsid maturation. Treatment with a specific peptide (PepCR2 or P10) targeted to pp150 conserved region 2 led to a significant reduction in murine CMV (MCMV) growth in cell culture, paving the way for in vivo testing in a mouse model of CMV infection. However, the general pharmacokinetic parameters of peptides, including rapid degradation and limited tissue and cell membrane permeability, pose a challenge to their successful use in vivo. Therefore, we designed a biopolymer-stabilized elastin-like polypeptide (ELP) fusion construct (ELP-P10) to enhance the bioavailability of P10. Antiviral efficacy and cytotoxic effects of ELP-P10 were studied in cell culture, and pharmacokinetics, biodistribution, and antiviral efficacy were studied in a mouse model of CMV infection. ELP-P10 maintained significant antiviral activity in cell culture, and this conjugation significantly enhanced P10 bioavailability in mouse tissues. The fluorescently labeled ELP-P10 accumulated to higher levels in mouse liver and kidneys as compared to the unconjugated P10. Moreover, viral titers from vital organs of MCMV-infected mice indicated a significant reduction of virus load upon ELP-P10 treatment. Therefore, ELP-P10 has the potential to be developed into an effective antiviral against CMV infection.

Fusion of histone variants to Cas9 suppresses non-homologous end joining.

As a versatile genome editing tool, the CRISPR-Cas9 system induces DNA double-strand breaks at targeted sites to activate mainly two DNA repair pathways: HDR which allows precise editing via recombination with a homologous template DNA, and NHEJ which connects two ends of the broken DNA, which is often accompanied by random insertions and deletions. Therefore, how to enhance HDR while suppressing NHEJ is a key to successful applications that require precise genome editing. Histones are small proteins with a lot of basic amino acids that generate electrostatic affinity to DNA. Since H2A.X is involved in DNA repair processes, we fused H2A.X to Cas9 and found that this fusion protein could improve the HDR/NHEJ ratio by suppressing NHEJ. As various post-translational modifications of H2A.X play roles in the regulation of DNA repair, we also fused H2A.X mimicry variants to replicate these post-translational modifications including phosphorylation, methylation, and acetylation. However, none of them were effective to improve the HDR/NHEJ ratio. We further fused other histone variants to Cas9 and found that H2A.1 suppressed NHEJ better than H2A.X. Thus, the fusion of histone variants to Cas9 is a promising option to enhance precise genome editing.

Dulaglutide restores endothelial progenitor cell levels in diabetic mice and mitigates high glucose-induced endothelial injury through SIRT1-mediated mitochondrial fission.

Type 2 diabetes mellitus (T2DM) significantly impairs the functionality and number of endothelial progenitor cells (EPCs) and resident endothelial cells, critical for vascular repair and regeneration, exacerbating the risk of vascular complications. GLP-1 receptor agonists, like dulaglutide, have emerged as promising therapeutic agents due to their multifaceted effects, including the enhancement of EPC activity and protection of endothelial cells. This study investigates dulaglutide's effects on peripheral blood levels of CD34+ and CD133+ cells in a mouse model of lower limb ischemia and its protective mechanisms against high-glucose-induced damage in endothelial cells. Results demonstrated that dulaglutide significantly improves blood flow, reduces tissue damage and inflammation in ischemic limbs, and enhances glycemic control. Furthermore, dulaglutide alleviated high-glucose-induced endothelial cell damage, evident from improved tube formation, reduced reactive oxygen species accumulation, and restored endothelial junction integrity. Mechanistically, dulaglutide mitigated mitochondrial fission in endothelial cells under high-glucose conditions, partly through maintaining SIRT1 expression, which is crucial for mitochondrial dynamics. This study reveals the potential of dulaglutide as a therapeutic option for vascular complications in T2DM patients, highlighting its role in improving endothelial function and mitochondrial integrity.

Functionality of chimeric TssA proteins in the type VI secretion system reveals sheath docking specificity within their N-terminal domains.

The genome of Pseudomonas aeruginosa encodes three type VI secretion systems, each comprising a dozen distinct proteins, which deliver toxins upon T6SS sheath contraction. The least conserved T6SS component, TssA, has variations in size which influence domain organisation and structure. Here we show that the TssA Nt1 domain interacts directly with the sheath in a specific manner, while the C-terminus is essential for oligomerisation. We built chimeric TssA proteins by swapping C-termini and showed that these can be functional even when made of domains from different TssA sub-groups. Functional specificity requires the Nt1 domain, while the origin of the C-terminal domain is more permissive for T6SS function. We identify two regions in short TssA proteins, loop and hairpin, that contribute to sheath binding. We propose a docking mechanism of TssA proteins with the sheath, and a model for how sheath assembly is coordinated by TssA proteins from this position.

Efficacy and safety of onasemnogene abeparvovec for the treatment of patients with spinal muscular atrophy type 1: A systematic review with meta-analysis.

BACKGROUND: Onasemnogene abeparvovec has been approved for the treatment of spinal muscular atrophy 5q type 1 in several countries, which calls for an independent assessment of the evidence regarding efficacy and safety. OBJECTIVE: Conduct a meta-analysis to assess the efficacy and safety of onasemnogene abeparvovec in patients diagnosed with SMA type 1, based on the available evidence. METHODS: This article results from searches conducted on databases up to November 2022. Outcomes of interest were global survival and event-free survival, improvement in motor function and treatment-related adverse events. Risk of bias assessment and certainty of evidence were performed for each outcome. Proportional meta-analysis models were performed when applicable. RESULTS: Four reports of three open-label, non-comparative clinical trials covering 67 patients were included. Meta-analyses of data available in a 12-month follow-up estimate a global survival of 97.56% (95%CI: 92.55 to 99.86, I2 = 0%, n = 67), an event-free survival of 96.5% (95%CI: 90.76 to 99.54, I2 = 32%, n = 66) and a CHOP-INTEND score ≥ 40 points proportion of 87.28% (95%CI: 69.81 to 97.83, I2 = 69%, n = 67). Proportion of 52.64% (95%CI: 27.11 to 77.45, I2 = 78%, n = 67) of treatment-related adverse events was estimated. CONCLUSION: The results indicate a potential change in the natural history of type 1 SMA, but the methodological limitations of the studies make the real extent of the technology's long-term benefits uncertain.

Case report: Recombinant human type II tumour necrosis factor receptor-antibody fusion protein induced occult hepatitis B virus reactivation leading to liver failure.

Recombinant human type II tumour necrosis factor receptor-antibody fusion protein (rh TNFR:Fc) is an immunosuppressant approved for treating rheumatoid arthritis (RA). This case report describes a case of hepatitis B reactivation in a patient with drug-induced acute-on-chronic liver failure. A 58-year-old woman with a history of RA was treated with rh TNFR:Fc; and then subsequently received 25 mg rh TNFR:Fc, twice a week, as maintenance therapy. No anti-hepatitis B virus (HBV) preventive treatment was administered. Six months later, she was hospitalized with acute jaundice. HBV reactivation was observed, leading to acute-on-chronic liver failure. After active treatment, the patient's condition improved and she recovered well. Following careful diagnosis and treatment protocols are essential when treating RA with rh TNFR:Fc, especially in anti-hepatitis B core antigen antibody-positive patients, even when the HBV surface antigen and the HBV DNA are negative. In the case of HBV reactivation, liver function parameters, HBV surface antigen and HBV DNA should be closely monitored during treatment, and antiviral drugs should be used prophylactically when necessary, as fatal hepatitis B reactivation may occur in rare cases. A comprehensive evaluation and medication should be administered in a timely manner after evaluating the patient's physical condition and closely monitoring the patient.

Significant elevation of serum CA19-9 and CA242 levels induced by dulaglutide.

The use of dulaglutide, a common medication for managing type 2 diabetes, rarely causes elevated pancreatic tumour markers. Here, we report the case of a woman in her mid-60s with diabetes for over 10 years. The patient presented with markedly elevated serum CA19-9 and CA242 levels revealed during a routine health examination despite being asymptomatic. She had been receiving dulaglutide injections for 16 months. Imaging and interventional assessments did not reveal any hepatobiliary, gastrointestinal or pancreatic neoplasm. After excluding alternate diagnoses, the patient was determined to exhibit an adverse reaction to dulaglutide use. Management involved the discontinuation of dulaglutide, which resulted in normalisation of serum CA19-9 and CA242 levels within 6 weeks. This case underscores the importance of discontinuing dulaglutide and monitoring changes in the biomarker levels in asymptomatic patients receiving dulaglutide, rather than immediately resorting to imaging and endoscopic examinations.

Safety, tolerability, and pharmacokinetics of single- and multiple-ascending doses of olamkicept: Results from randomized, placebo-controlled, first-in-human phase I trials.

Olamkicept selectively inhibits the cytokine interleukin-6 (IL-6) trans-signaling pathway without blocking the classic pathway and is a promising immunoregulatory therapy for inflammatory bowel disease (IBD). These first-in-human, randomized, placebo-controlled, single- (SAD) and multiple-ascending dose (MAD) trials evaluated olamkicept safety, tolerability, pharmacokinetic, and pharmacodynamic characteristics. Doses tested in the SAD trial included seven single intravenous doses (0.75, 7.5, 75, 150, 300, 600, and 750 mg) and one subcutaneous (SC) dose (60 mg) given to healthy subjects (N = 64), and three intravenous doses (75 mg, 300 mg, and 750 mg) given to patients with Crohn's disease (CD; N = 24). Doses tested in the MAD trial included multiple intravenous doses (75, 300, and 600 mg once weekly for 4 weeks) given to healthy subjects (N = 24). No severe or serious treatment-emergent adverse events (TEAEs) were recorded. The most common TEAEs were headache, nasopharyngitis, and myalgia in the SAD trial, and diarrhea, headache, and cough in the MAD trial. Infusion-related reactions occurred in one and two subjects in the SAD and MAD trial, respectively, leading to treatment discontinuation in the MAD trial. Olamkicept showed dose-independent pharmacokinetics after single and multiple administrations, and there was no major difference in systemic exposure between healthy subjects and patients with CD. Complete target engagement (inhibition of phosphorylation of signal transducer and activator of transcription-3) was achieved in blood around or above olamkicept serum concentrations of 1-5 µg/mL. Overall, these results suggest that olamkicept is safe and well-tolerated in healthy subjects and patients with CD after single intravenous/SC and multiple intravenous administrations.

Application of SUMO fusion technology for the enhancement of stability and activity of lysophospholipase from Pyrococcus abyssi.

Heterologous production of proteins in Escherichia coli has raised several challenges including soluble production of target proteins, high levels of expression and purification. Fusion tags can serve as the important tools to overcome these challenges. SUMO (small ubiquitin-related modifier) is one of these tags whose fusion to native protein sequence can enhance its solubility and stability. In current research, a simple, efficient and cost-effective method is being discussed for the construction of pET28a-SUMO vector. In order to improve the stability and activity of lysophospholipase from Pyrococcus abyssi (Pa-LPL), a 6xHis-SUMO tag was fused to N-terminal of Pa-LPL by using pET28a-SUMO vector. Recombinant SUMO-fused enzyme (6 H-S-PaLPL) works optimally at 35 °C and pH 6.5 with remarkable thermostability at 35-95 °C. Thermo-inactivation kinetics of 6 H-S-PaLPL were also studied at 35-95 °C with first order rate constant (kIN) of 5.58 × 10- 2 h-1 and half-life of 12 ± 0 h at 95 °C. Km and Vmax for the hydrolysis of 4-nitrophenyl butyrate were calculated to be 2 ± 0.015 mM and 3882 ± 22.368 U/mg, respectively. 2.4-fold increase in Vmax of Pa-LPL was observed after fusion of 6xHis-SUMO tag to its N-terminal. It is the first report on the utilization of SUMO fusion tag to enhance the overall stability and activity of Pa-LPL. Fusion of 6xHis-SUMO tag not only aided in the purification process but also played a crucial role in increasing the thermostability and activity of the enzyme. SUMO-fused enzyme, thus generated, can serve as an important candidate for degumming of vegetable oils at industrial scale.

[Pharmacoeconomic analysis of anti-angiogenic drugs for diabetic macular edema]. / Farmakoekonomicheskii analiz primeneniya antiangiogennykh preparatov pri diabeticheskom makulyarnom oteke.

Diabetic macular edema (DME) is a degenerative disease of the macular area in diabetes mellitus and can lead to vision loss, disability, and significantly reduced quality of life. Faricimab is the only bispecific antibody for DME therapy that targets two pathogenic pathways (Ang-2 and VEGF-A). PURPOSE: This study comparatively evaluates the clinical and economic feasibility of faricimab and other angiogenesis inhibitors in patients with DME. MATERIAL AND METHODS: This article analyzed literature on the efficacy and safety of intravitreal injections (IVI) of ranibizumab 0.5 mg, aflibercept 2 mg, and faricimab 6 mg. A model of medical care was developed for patients with DME receiving anti-angiogenic therapy. Pharmacoeconomic analysis was performed using cost minimization and budget impact analysis (BIA) methods. Modeling time horizon was 2 years. The research was performed from the perspective of the healthcare system of the Russian Federation. RESULTS: The efficacy and safety of faricimab in a personalized regimen (up to one IVI in 16 weeks) are comparable to those of aflibercept and ranibizumab, administered in various regimens. The use of faricimab is associated with the lowest number of IVIs. Over 2 years, the maximum costs of drug therapy were associated with the use of ranibizumab (about 914 thousand rubles), while the minimum costs were associated with the use of faricimab (614 thousand rubles). The reduction in inpatient care costs with faricimab therapy was 36% compared to aflibercept (216 and 201 thousand rubles in inpatient and day hospitals, respectively) and 82% compared to ranibizumab (486 and 451 thousand rubles in inpatient and day hospitals, respectively). BIA demonstrated that the use of faricimab will reduce the economic burden on the healthcare system by 11.3 billion rubles (9.8%) over 2 years. CONCLUSION: The use of faricimab is a cost-effective approach to treatment of adult patients with DME in Russia.