A Novel Anti-CD22 scFv.Bim Fusion Protein Effectively Induces Apoptosis in Malignant B cells and Promotes Cytotoxicity.

CD22 is a B-cell surface antigen which is highly expressed in cancerous B-cell lineages. Anti-CD22 antibodies are currently under focus as promising biologics against hematologic B-cell malignancies. Herein, we introduce a novel active recombinant anti-CD22 scFv.Bim fusion protein for targeting this cancerous antigen. An expression cassette encoding anti-CD22 scFv.Bim fusion protein was expressed in Pichia pastoris. The binding ability, cytotoxicity, and apoptotic activity of the purified recombinant protein against CD22+ Raji cell line were assessed by flow cytometry, microscopy, and MTT assay. Using bioinformatics, the 3D structure of the fusion protein and its interaction with CD22 were assessed. The in vitro binding analysis by immunofluorescence microscopy and flow cytometry demonstrated the specific binding of scFv.Bim to CD22+ Raji cells but not to CD22- Jurkat cells. MTT data and Annexin V/PI flow cytometry analysis confirmed the apoptotic activity of anti-CD22 scFv.Bim against Raji cells but not Jurkat cells. In silico analysis also revealed the satisfactory stereochemical quality of the 3D model and molecular interactions toward CD22. This novel recombinant anti-CD22 scFv.Bim fusion protein could successfully deliver the pro-apoptotic peptide, BIM, to the target cells and thus nominates it as a promising molecule in treating B-cell malignancies.

Targeted Deletion of Los1 Homologue Affects the Production of a Recombinant Model Protein in Pichia pastoris.

Background: The methylotrophic yeast Pichia pastoris is an appealing production host for a variety of recombinant proteins, including biologics. In this sense, various genetic- and non-genetic-based techniques have been implemented to improve the production efficiency of this expression platform. Loss of supression (Los1) encodes a non-essential nuclear tRNA exporter in Saccharomyces cerevisiae, which its deletion extends replicative lifespan. Herein, a los1-deficient strain of P. pastoris was generated and characterized. Methods: A gene disruption cassette was prepared and transformed into an anti-CD22-expressing strain of P. pastoris. A δ los1 mutant was isolated and confirmed. The drug sensitivity of the mutant was also assessed. The growth pattern and the level of anti-CD22 single-chain variable fragment (scFv) expression were compared between the parent and mutant strains. Resuults: The los1 homologue was found to be a non-essential gene in P. pastoris. Furthermore, the susceptibility of los1 deletion strain to protein synthesis inhibitors was altered. This strain showed an approximately 1.85-fold increase in the extracellular level of anti-CD22 scFv (p < 0.05). The maximum concentrations of total proteins secreted by δ los1 and parent strains were 125 mg/L and 68 mg/L, respectively. Conclusion: The presented data suggest that the targeted disruption of los1 homologue in P. pastoris can result in a higher expression level of our target protein. Findings of this study may improve the current strategies used in optimizing the productivity of recombinant P. pastoris strains.

Oral Administration of Recombinant Saccharomyces boulardii Expressing Ovalbumin-CPE Fusion Protein Induces Antibody Response in Mice.

Saccharomyces boulardii, a subspecies of Saccharomyces cerevisiae, is a well-known eukaryotic probiotic with many benefits for human health. In the present study, a recombinant strain of S. boulardii was prepared to use as a potential oral vaccine delivery vehicle. In this sense, a ura3 auxotroph strain of S. boulardii CNCM I-745 (known as S. cerevisiae HANSEN CBS 5926, Yomogi®) was generated using CRISPR/Cas9 methodology. Then a gene construct encoding a highly immunogenic protein, ovalbumin (OVA), was prepared and transformed into the ura3- S. boulardii. To facilitate the transport of the recombinant immunogen across the intestinal barrier, a claudin-targeting sequence from Clostridium perfringens enterotoxin (CPE) was added to the C-terminus of the expression cassette. The recombinant S. boulardii strain expressing the OVA-CPE fusion protein was then administered orally to a group of mice, and serum IgG and fecal IgA levels were evaluated by ELISA. Our results demonstrated that anti-OVA IgG in serum significantly increased in test group (P < 0.001) compared to control groups (receiving wild type S. boulardii or PBS), and the fecal IgA titer was significantly higher in test group (P < 0.05) than control groups. In parallel, a recombinant S. boulardii strain expressing the similar construct lacking C-terminal CPE was also administered orally. The result showed an increased level of serum IgG in group receiving yeasts expressing the CPE negative construct compared to control groups; however, the fecal IgA levels did not increase significantly. In conclusion, our findings indicated that the yeast S. boulardii, as a delivery vehicle with possible immunomodulatory effects, and c-CPE, as a targeting tag, synergistically assist to stimulate systemic and local immunity. This proposed recombinant S. boulardii system might be useful in the expression of other antigenic peptides, making it as a promising tool for oral delivery of vaccines or therapeutic proteins.

Expression Optimization of Anti-CD22 scFv-Apoptin Fusion Protein Using Experimental Design Methodology

Background: Design of experiments is a rapid and cost-effective approach for optimization of recombinant protein production process. In our previous study, we generated a potent dual-acting fusion protein, anti-CD22 scFv-apoptin, to target B-cell malignant cell lines. In the present investigation, we report the effect of different variables on the expression levels of this fusion protein. Methods: Four variables (cell optical density at induction, IPTG concentration, induction temperature, and induction time) were tested using experimental design. Results: Our findings demonstrated that among the examined variables, only the induction time had a significant positive effect on the protein expression yield. Conclusion: Experimental design was successfully applied in this study. The optimized condition obtained in the current study can be applied in future commercial production of this novel fusion protein.

A novel anti-CD22 scFv-apoptin fusion protein induces apoptosis in malignant B-cells.

CD22 marker is a highly internalizing antigen which is located on the surface of B-cells and is being used as a promising target for treatment of B cell malignancies. Monoclonal antibodies targeting CD22 have been introduced and some are currently under investigation in clinical trials. Building on the success of antibody drug conjugates, we developed a fusion protein consisting of a novel anti-CD22 scFv and apoptin and tested binding and therapeutic effects in lymphoma cells. The recombinant protein was expressed in E. coli and successfully purified and refolded. In vitro binding analysis by immunofluorescence and flow cytometry demonstrated that the recombinant protein specifically binds to CD22 positive Raji cells but not to CD22 negative Jurkat cells. The cytotoxic properties of scFv-apoptin were assessed by an MTT assay and Annexin V/PI flow cytometry analysis and showed that the recombinant protein induced apoptosis preferentially in Raji cells with no detectable effects in Jurkat cells. Our findings indicated that the recombinant anti-CD22 scFv-apoptin fusion protein could successfully cross the cell membrane and induce apoptosis with high specificity, make it as a promising molecule for immunotherapy of B-cell malignancies.

Human-Yeast Hybrids: New Visions to Genetic Disorders and Drug Discovery

Yeast has been a very helpful organism for centuries, especially with respect to fermentation of sugars and production of bread. However, for an even longer time, yeast has been a distant relative of humans having diverged from a common ancestor, about one billion years ago. More than one third of the yeast genes have human counterparts, despite this evolutionary distance. Yeast and human orthologs perform the same or similar functions. Investigations have demonstrated that 9-92% of the amino acid sequences in similar human and yeast proteins overlap. However, even if two genes perform similar functions in two different organisms, it may not be possible to replace some of yeast genes with their human counterpart...

Electrocardiographic changes in patients with tramadol-induced idiosyncratic seizures.

OBJECTIVES: To assess ECG changes in patients with tramadol-induced seizure(s) and compare these changes in lower and higher than 500 mg tramadol doses as a main goal. MATERIAL AND METHODS: In an analytical-cross sectional manner over 1 year, 170 patients with idiosyncratic seizure(s) after using tramadol, were studied. Full data were recorded for each patient. ECGs were taken from all the patients on admission and 1 h later and were assessed for findings. RESULTS: 70 of 170 patients (41.2%) had used lower than 500 mg doses of tramadol while 90 patients (52.9%) were included in the high dose group. Rate of female patients in the high dose group was significantly higher. The average age of patients in the high dose group was significantly lower (22.04 vs 25.76). The high dose group had significantly higher heart rates. There was no history of cardiovascular diseases; two patients had previous history of seizure. No significant difference was shown between low dose and high dose groups from the point of ECG changes. DISCUSSION AND CONCLUSION: Using doses higher than 500 mg is more frequently seen in women, young people and those who have not experienced previous use of tramadol. Terminal S wave, sinus tachycardia, and terminal R wave in the lead aVR are among the most common ECG changes in tramadol users.

Design of a novel chimeric tissue plasminogen activator with favorable Vampire bat plasminogen activator properties.

Fibrinolytic agents are widely used in treatment of the thromboembolic disorders. The new generations like recombinant tissue plasminogen activator (t-PA, alteplase) are not showing promising results in clinical practice in spite of displaying specific binding to fibrin in vitro. Vampire bat plasminogen activator (b-PA) is a plasminogen activator with higher fibrin affinity and specificity in comparison to t-PA resulting in reduced probability of hemorrhage. b-PA is also resistant to plasminogen activator inhibitor-1 (PAI-1) showing higher half-life compared to other variants of t-PA. However, its non-human origin was a driving force to design a human t-PA with favorable properties of b-PA. In the present study, we designed a chimeric t-PA with desirable b-PA properties and this new molecule was called as CT-b. The construct was prepared through kringle 2 domain removal and replacement of t-PA finger domain with b-PA one. In addition, the KHRR sequence at the initial part of protease domain was replaced by four alanine residues. The novel construct was integrated in Pichia pastoris genome by electroporation. Catalytic activity was investigated in the presence and absence of fibrin. The purified protein was analyzed by western blot. Fibrin binding and PAI resistance assays were also conducted. The activity of the recombinant protein in the presence of fibrin was 1560 times more than its activity in the absence of fibrin, showing its higher specificity to fibrin. The fibrin binding of CT-b was 1.2 fold more than t-PA. In addition, it was inhibited by PAI enzyme 44% less than t-PA. Although the presented data demonstrate a promising in vitro activity, more in vivo studies are needed to confirm the therapeutic advantage of this novel plasminogen activator.

High efficient expression of a functional humanized single-chain variable fragment (scFv) antibody against CD22 in Pichia pastoris.

Single-chain variable fragments (scFvs) have recently emerged as attractive candidates in targeted immunotherapy of various malignancies. The anti-CD22 scFv is able to target CD22, on B cell surface and is being considered as a promising molecule in targeted immunotherapy of B cell malignancies. The recombinant anti-CD22 scFv has been successfully expressed in Escherichia coli; however, the insufficient production yield has been a major bottleneck for its therapeutic application. The methylotrophic yeast Pichia pastoris has become a highly popular expression host for the production of a wide variety of recombinant proteins such as antibody fragments. In this study, we used the Pichia expression system to express a humanized scFv antibody against CD22. The full-length humanized scFv gene was codon optimized, cloned into the pPICZαA and expressed in GS115 strain. The maximum production level of the scFv (25 mg/L) were achieved at methanol concentration, 1 %; pH 6.0; inoculum density, OD600 = 3 and the induction time of 72 h. The correlation between scFv gene dosage and expression level was also investigated by real-time PCR, and the results confirmed the presence of such correlation up to five gene copies. Immunofluorescence and flow cytometry studies and Biacore analysis demonstrated binding to CD22 on the surface of human lymphoid cell line Raji and recombinant soluble CD22, respectively. Taken together, the presented data suggest that the Pichia pastoris can be considered as an efficient host for the large-scale production of anti-CD22 scFv as a promising carrier for targeted drug delivery in treatment of CD22(+) B cell malignancies.

Cloning and expression of Aspergillus flavus urate oxidase in Pichia pastoris.

Urate oxidase is an important enzyme with therapeutic and diagnostic applications. Rasburicase is a recombinant urate oxidase enzyme approved by FDA to use in the treatment of hyperuricemia conditions. Various hosts such as Saccharomyces cerevisiae, Hansenula polymorpha and Escherichia coli have been used to express the enzyme. Today, Pichia pastoris is considered as an important host for heterologous protein expression since it has beneficial characteristics such as strong promoters, simple scale up, post translational modifications, high cell density cultivation and simple genetic manipulation. In this study, Aspergillus flavus urate oxidase gene was cloned in pPICZαA expression vector and expressed in P. pastoris. The recombinant urate oxidase was expressed in secretory form and was confirmed through RT-PCR, SDS-PAGE analysis and western blotting. The enzyme activity was determined using a colorimetric assay. A production yield of 0.43 U/ml of culture supernatant was obtained.

Expression of a novel chimeric-truncated tPA in Pichia pastoris with improved biochemical properties.

Thrombolytic therapy by plasminogen activators (PAs) has been a main goal in the treatment of acute myocardial infarction. Despite improved outcomes of currently available thrombolytic therapies, all these agents have different drawbacks that may result in less than optimal outcomes. In order to make tissue plasminogen activator (tPA) more potent, while being more resistant to plasminogen activator inhibitor-1 (PAI-1) and having a higher affinity to fibrin, a new chimeric-truncated form of tPA (CT tPA) was designed and expressed in Pichia pastoris. This novel variant consists of a finger domain of Desmoteplase, an epidermal growth factor (EGF) domain, a kringle 1 (K1) domain, a kringle 2 (K2) domain, in which the lysine binding site (LBS) was deleted, and a protease domain, where the four amino acids lysine 296, arginine 298, arginine 299, and arginine 304 were substituted by aspartic acid. The chimera CT tPA showed 14-fold increase in its activity in the presence of fibrin compared to the absence of fibrin. Furthermore, CT tPA showed about 10-fold more potency than commercially available full-length tPA (Actylase(®)) and provided 1.2-fold greater affinity to fibrin. A residual activity of only 68 % was observed after incubation of Actylase(®) with PAI-1, however, 91 % activity remained for CT tPA. These promising findings suggest that the novel CT tPA variant might be an acceptable PA with superior characteristics and properties.

A simple and sensitive HPLC-UV method for quantitation of lovastatin in human plasma: application to a bioequivalence study.

An available, simple, sensitive, and rapid method has been developed for determination of the 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase inhibitor, lovastatin in human plasma. The analytical procedure involves a one-step liquid-liquid extraction method using atorvastatin as internal standard. Chromatographic separation was carried out on a reversed phase C(18) column using a mixture of 0.05 M phosphate buffer (pH 7) and acetonitrile (44.5 : 55.5, v/v) as mobile phase with UV detection set at 238 nm. The total run time of analysis was 6 min with the retention time of lovastatin being 4.3 min. A complete set of analytical method validation tests were carried out on the method. Accordingly, the method was linear in the wide range of 1-100 ng/ml. The limit of detection (LOD) and limit of quantification (LOQ) for lovastatin were 0.5 and 1 ng/ml, respectively. The method was shown to be precise with average within-run and between-run variations of 10.45+/-6.88 and 8.68+/-5.13%, respectively. The average within-run and between-run accuracy of the method throughout its linear range was 113.33+/-3.98 and 105.72+/-5.07%, respectively. The mean relative recovery of lovastatin from human plasma by the developed method was 88.61+/-7.00%. The applicability of the method in real pharmacokinetic situations was evaluated successfully during a bioequivalence study in 14 fasting healthy male volunteers.

A reversed-phase high-performance liquid chromatography method for bovine serum albumin assay in pharmaceutical dosage forms and protein/antigen delivery systems.

Bovine serum albumin (BSA) is among the most widely used proteins in protein formulations as well as in the development of novel delivery systems as a typical model for therapeutic/diagnostic proteins and the new versions of vaccines. The development of reliable and easily available assay methods for quantitation of this protein would therefore play a crucial role in these types of studies. A simple gradient reversed-phase high-performance liquid chromatography with ultra-violet detection (HPLC-UV) method has been developed for quantitation of BSA in dosage forms and protein delivery systems. The method produced linear responses throughout the wide BSA concentration range of 1 to 100 micro g/mL. The average within-run and between-run variations of the method within the linear concentration range of BSA were 2.46% and 2.20%, respectively, with accuracies of 104.49% and 104.58% for within-run and between-run samples, respectively. The limits of detection (LOD) and quantitation (LOQ) of the method were 0.5 and 1 microg/mL, respectively. The method showed acceptable system suitability indices, which enabled us to use it successfully during our particulate vaccine delivery research project.

Preparation and validation of carrier human erythrocytes loaded by bovine serum albumin as a model antigen/protein.

Erythrocytes as the most readily available and abundant cells within the body have been studied extensively for their potential application as drug delivery carries. In this study, human erythrocytes were loaded by bovine serum albumin (BSA) as a model antigen/protein using hypotonic preswelling method for targeted delivery of this antigen-to antigen-presenting cells. The average loaded amount, efficiency of entrapment, and cell recovery upon loading procedure were 1979.25 +/- 9.4 microg, 30.06 +/- 0.20%, and 87.53 +/- 0.66%, respectively. The total BSA recovery upon loading procedure was 97.20 +/- 4.90%. The apparent mechanism of entrapment was simple concentration-based gradient in/out the cells with some minor limiting factors against protein entry into the cells. We have shown that the intra- and intersubject variations of the method were interestingly low (i.e., less than 5% in all cases).