single chain fragment variable
در نشریات گروه پزشکی-
Introduction
Monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs) have changed the face of cancer treatment methods drastically. Herein, I attempted to conjugate a podoplanin (PDPN)-single-chain fragment variable (scFv) to granzyme B (GrB) to design an antibody conjugate (named LpMab-2-(G4S)3-GrB) using in-silico techniques.
Materials and MethodsThe 3D models of the PDPN -specific scFv (LpMab-2), LpMab-2-(G4S)3-GrB, and PDPN were predicted by GalaxyWEB, assessed through Ramachandran plot analysis, and refined by 3Drefine. The physicochemical properties, solubility value, and Tm of LpMab-2-(G4S)3-GrB were estimated/predicted and compared with LpMab-2 and GrB. Finally, the binding capacity of LpMab-2-(G4S)3-GrB to PDPN was compared with that of LpMab-2 through docking and affinity prediction alongside identifying the residues that participate in the binding through 2D interaction plots using the LigPlot+ software.
ResultsThe 3D models were predicted to be of high quality and refined successfully. The solubility and Tm of LpMab-2 were predicted to decrease and increase following its conjugation with GrB, respectively, whereas its estimated half-life was not affected. The docking results indicated that LpMab-2-(G4S)3-GrB targets PDPN in the same orientation as that of LpMab-2 and with the exact same residues as indicated by their 2D plots. Moreover, the affinity of LpMab-2-(G4S)3-GrB to PDPN was predicted to be similar to that of the single scFv.
ConclusionThe conjugation of LpMab-2 to GrB does not affect its binding capacity or characteristics in a major negative fashion. In-silico techniques could be utilized for antibody engineering, and future studies could focus on the assessment of LpMab-2-(G4S)3-GrB in vitro and in vivo.
Keywords: Antibody Drug Conjugate, Cancer Immunotherapy, Granzyme B, Podoplanin, Single Chain Fragment Variable -
Objective (s)
Exosomes are nanovesicular vehicles capable of transporting different cargoes. Based on their characteristics, exosomes are proposed as a class of vehicles for targeted delivery of therapeutics. We aimed to establish a HEK293T stable cell line capable of secreting GRP78-specific scFv-targeted exosomes.
MethodsThe pLEX-LAMP2b-GRP78 construct was developed by enzymatic replacement of DARPin in pLEX-LAMP2b-DARPin with a GRP78-specific scFv. pLEX-LAMP2b-GRP78 (or TurboGFP as control), psPAX2, and pMD2.G plasmids were co-transfected into HEK293T cells, and produced lentiviruses were harvested. Different multiplicities of infection (MOI; 10, 20, 30, 60, 120, and 240) were used for the transduction of HEK293T to select the most appropriate one as assessed by flow cytometry. Transduced HEK293T cells were subject to puromycin selection and the presence of the scFv was assessed in the established cell line at the DNA, transcript, and protein levels by PCR, RT-PCR, and Western blotting, respectively.
ResultspLEX-LAMP2b-GRP78 was successfully developed. Co-transfection resulted in the expression of GFP by HEK293T in the control group 48 hours following transfection. The MOI of 60 was selected as 10% of cells were GFP+ 72 hours following transduction. Following puromycin selection, the presence of the integrated scFv DNA and transcript was confirmed. Moreover, Western blotting results confirmed the presence of the His-tagged scFv in the established cell line.
ConclusionsHEK293T cells can be engineered for the production of targeted exosomes which could be applied for therapeutic purposes. Moreover, scFvs are potent targeting domains that could be leveraged for the development of targeted exosomes.
Keywords: nanovesicles, Grp78, Single-Chain Fragment Variable, Targeted therapy, Exosome -
مجله علمی دانشگاه علوم پزشکی کردستان، سال بیست و هشتم شماره 4 (پیاپی 127، مهر و آبان 1402)، صص 12 -23زمینه و هدف
درمان های مرسوم موفقیت کمی در درمان سرطان نشان داده اند. استفاده از قطعات آنتی بادی کنژوگه شده با پروتیین های خاص و هدفمند کردن آن یک روش نوین در تولید داروهای ضد سرطان است. علیرغم مزایای میزبان های پروکاریوتی، بیان پروتیین به شکل اجسام انکلوزیون بادی (سیتوپلاسمی) چالش برانگیز بوده و منجر به سختی های بعدی می شود. تولید پروتیین به صورت محلول تا حد زیادی به حل این مشکل کمک می کند. هدف اصلی این مطالعه شبیه سازی و بیان لیگاند القاکننده آپوپتوز مرتبط با فاکتور نکروز دهنده تومور (TRAIL) کونژوگه با قطعه متغیر تک زنجیر آنتی بادی (scFV) ضد CD20 آنتی بادی ضد CD20 به عنوان یک پروتیین محلول با استفاده از تعدیل کننده کوچک وابسته به یوبیکویتین بود. سیستم پروتیین فیوژن اصلاح کننده (SUMO) در E. Coli به عنوان روشی جدید برای تولید بهینه داروهای ضد سرطان است.
مواد و روش هاپرایمرهای مناسب برای یک توالی DNA از قبل طراحی شده که پروتیین فیوژن را کد می کند برای تکثیر قطعه استفاده شد و در پلاسمید pSUMO در ناحیه پایین دست توالی برچسب ژنیSUMO ساب کلون گردید. پس از تایید با روش های استاندارد، پلاسمید نوترکیب به سویه E. Coli strain BL21 (DE3) منتقل شد. بیان توسط ایزوپروپیل بتا-دی-1-تیوگالاکتوپیرانوزید القا شد. پروتیین های بیان شده توسط SDS-PAGE ارزیابی و سپس با کروماتوگرافی میل ترکیبی جداسازی شدند. برای تایید بیان پروتیین از وسترن بلات استفاده شد.
یافته هانتایج نشان داد که DNA پروتیین نوترکیب فیوژن به درستی ساب کلون شده و آنالیزهای مربوطه نشان داد که فرآیند بیان موفقیت آمیز بوده است. پروتیین فیوژن نوترکیب با تجزیه و تحلیل مناسب تایید شد.
نتیجه گیریتولید پروتیین های نوترکیب به صورت محلول در میزبان پروکاریوتی E. coli می تواند هزینه های مصروف در فرآیندهای پایین دستی را کاهش دهد و با توجه به نتایج مناسب این تحقیق در تولید پروتیین فیوژن AntiCD20 scFV-TRAIL به صورت محلول می توان از روش استفاده شده در این تحقیق به منظور تولید محلول و عملکردی این گونه پروتیین های نوترکیب بهره برد.
کلید واژگان: پروتئین هم جوش، بیان، تخلیص، نوترکیب، قطعه متغیر تک زنجیر آنتی بادی، لیگاند القاکننده آپوپتوز وابسته به فاکتور نکروز دهنده تومورBackground and AimConventional treatments have shown little success in cancer treatment. Using antibody fragments conjugated with specific proteins and targeting specific receptors is a new strategy for producing anticancer drugs. Despite the advantages of prokaryotic hosts, protein expression in the form of inclusion bodies (cytoplasmic) has been challenging and leads to subsequent difficulties. Production of proteins as soluble forms will significantly help to solve this problem. The primary purpose of this study was to clone and express Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) conjugated with anti CD20 single chain fragment variable (scFV) region of anti-CD20 antibody as a soluble protein using a small ubiquitin-related modifier (SUMO) fusion protein system in E. coli as a new method for optimal production of anticancer drugs.
Materials and MethodsAppropriate primers for a previously designed DNA sequence encoding the fusion protein were used to amplify the fragment. The amplified fragment was sub-cloned downstream of the SUMO tag in pSUMO vector. Once confirmed by standard methods, the recombinant plasmid was transformed into E. coli strain BL21 (DE3). Expression was induced by isopropyl β- d-1-thiogalactopyranoside. The expressed proteins were assessed by SDS-PAGE and then isolated by affinity chromatography. Western blotting was used to confirm protein expression.
ResultsThe results showed the DNA of the recombinant fusion protein was sub-cloned correctly, and the relevant analyses indicated that the expression process was successful. The recombinant fusion protein was confirmed by appropriate analysis.
ConclusionThe production of recombinant proteins in a soluble state in the prokaryotic host E. coli can reduce the costs of the downstream process. Production of soluble antiCD20 scFV-TRAIL fusion protein can be considered a proper method to produce recombinant protein in a soluble state in the E. coli system and can decrease the cost of downstream processes.
Keywords: Fusion protein, Expression, Purification, Recombinant, Single chain fragment variable, Tumor necrosis factor-related apoptosis-inducing ligand
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