single chain antibody
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eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Keith F DeLuca ◽  
Jeanne E Mick ◽  
Amy Hodges Ide ◽  
Wanessa C Lima ◽  
Lori Sherman ◽  
...  

Antibodies are indispensable tools used for a large number of applications in both foundational and translational bioscience research; however, there are drawbacks to using traditional antibodies generated in animals. These include a lack of standardization leading to problems with reproducibility, high costs of antibodies purchased from commercial sources, and ethical concerns regarding the large number of animals used to generate antibodies. To address these issues, we have developed practical methodologies and tools for generating low-cost, high-yield preparations of recombinant monoclonal antibodies and antibody fragments directed to protein epitopes from primary sequences. We describe these methods here, as well as approaches to diversify monoclonal antibodies, including customization of antibody species specificity, generation of genetically encoded small antibody fragments, and conversion of single chain antibody fragments (e.g. scFv) into full-length, bivalent antibodies. This study focuses on antibodies directed to epitopes important for mitosis and kinetochore function; however, the methods and reagents described here are applicable to antibodies and antibody fragments for use in any field.


Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3414
Author(s):  
Sarah Nasreen Schmidt ◽  
Wilfried Reichardt ◽  
Beat A. Kaufmann ◽  
Carolin Wadle ◽  
Dominik von Elverfeldt ◽  
...  

Previous mouse studies have shown the increased presence of platelets in the myocardium during early stages of myocarditis and their selective detection by MRI. Here, we aimed to depict early myocarditis using molecular contrast-enhanced ultrasound of activated platelets, and to evaluate the impact of a P2Y12 receptor platelet inhibition. Experimental autoimmune myocarditis was induced in BALB/c mice by subcutaneous injection of porcine cardiac myosin and complete Freund adjuvant (CFA). Activated platelets were targeted with microbubbles (MB) coupled to a single-chain antibody that binds to the “ligand-induced binding sites” of the GPIIb/IIIa-receptor (=LIBS-MB). Alongside myocarditis induction, a group of mice received a daily dose of 100 g prasugrel for 1 month. Mice injected with myosin and CFA had a significantly deteriorated ejection fraction and histological inflammation on day 28 compared to mice only injected with myosin. Platelets infiltrated the myocardium before reduction in ejection fraction could be detected by echocardiography. No selective binding of the LIBS-MB contrast agent could be detected by either ultrasound or histology. Prasugrel therapy preserved ejection fraction and significantly reduced platelet aggregates in the myocardium compared to mice without prasugrel therapy. Therefore, P2Y12 inhibition could be a promising early therapeutic target in myocarditis, requiring further investigation.


2021 ◽  
Author(s):  
Michael J. Robertson ◽  
Feng He ◽  
Justin G. Meyerowitz ◽  
Alpay B. Seven ◽  
Ouliana Panova ◽  
...  

Cryogenic electron microscopy (cryo-EM) has widened the field of structure-based drug discovery by allowing for routine determination of membrane protein structures previously intractable. However, despite representing one of the largest classes of therapeutic targets, most inactive-state G protein-coupled receptors (GPCRs) have remained inaccessible for cryo-EM because their small size and membrane-embedded nature impedes projection alignment for high-resolution map reconstructions. Here we demonstrate that a camelid single-chain antibody (nanobody) recognizing a grafted intracellular loop can be used to obtain cryo-EM structures of different inactive-state GPCRs at resolutions comparable or better than those obtained by X-ray crystallography. Using this approach, we obtained the structure of human neurotensin 1 receptor (NTSR1) bound to antagonist SR48692, of μ-opioid receptor (MOR) bound to the clinical antagonist alvimopan, as well as the structure of the previously uncharacterized somatostatin receptor 2 (SSTR2) in the apo state; each of these structures yields novel insights into ligand binding and specificity. We expect this rapid, straightforward approach to facilitate the broad structural exploration of GPCR inactive states without the need for extensive engineering and crystallization.


Viruses ◽  
2021 ◽  
Vol 13 (11) ◽  
pp. 2201
Author(s):  
Tyng Hwey Tan ◽  
Elizabeth Patton ◽  
Carol A. Munro ◽  
Dora E. Corzo-Leon ◽  
Andrew J. Porter ◽  
...  

ORF3a has been identified as a viroporin of SARS-CoV-2 and is known to be involved in various pathophysiological activities including disturbance of cellular calcium homeostasis, inflammasome activation, apoptosis induction and disruption of autophagy. ORF3a-targeting antibodies may specifically and favorably modulate these viroporin-dependent pathological activities. However, suitable viroporin-targeting antibodies are difficult to generate because of the well-recognized technical challenge associated with isolating antibodies to complex transmembrane proteins. Here we exploited a naïve human single chain antibody phage display library, to isolate binders against carefully chosen ORF3a recombinant epitopes located towards the extracellular N terminal and cytosolic C terminal domains of the protein using peptide antigens. These binders were subjected to further characterization using enzyme-linked immunosorbent assays and surface plasmon resonance analysis to assess their binding affinities to the target epitopes. Binding to full-length ORF3a protein was evaluated by western blot and fluorescent microscopy using ORF3a transfected cells and SARS-CoV-2 infected cells. Co-localization analysis was also performed to evaluate the “pairing potential” of the selected binders as possible alternative diagnostic or prognostic biomarkers for COVID-19 infections. Both ORF3a N and C termini, epitope-specific monoclonal antibodies were identified in our study. Whilst the linear nature of peptides might not always represent their native conformations in the context of full protein, with carefully designed selection protocols, we have been successful in isolating anti-ORF3a binders capable of recognising regions of the transmembrane protein that are exposed either on the “inside” or “outside” of the infected cell. Their therapeutic potential will be discussed.


2021 ◽  
Author(s):  
Zerong Chen ◽  
Haibo Zhang ◽  
Jialiang Hui ◽  
Yaodong Jiang ◽  
Zehai Huang ◽  
...  

Abstract Background T cell immunoglobulin-3(Tim-3) is an immune checkpoint molecule; Tim-3 antibody is suitable for treating malignant renal tumours. However, Tim-3 antibody drugs are expensive, which limits their application. To overcome the disadvantages of expensive immunotherapeutic drugs, Lactococcus lactis was used as the host bacteria to express Tim-3 single-chain antibody in the intestine, and its promotion of the mouse immune system and inhibitory effects on the transplanted tumour of kidney cancer in mice were tested. Methods Molecular cloning technology was used to construct plasmids pLAN-CTB-Tim3scFv and pLAN-Tim3scFv, which were transformed into Lactococcus lactis. The expression of the transformed bacteria was analysed using western blotting, and the immune activity of secreted proteins of the transformed bacteria was detected using ELISA in vitro. A subcutaneous transplanted tumour model of renal adenocarcinoma was constructed in RAG mice, and the promoting effect of transforming bacteria on the activation of mouse spleen lymphocytes, and its inhibitory effect on transplanted tumours in mice was analysed. Results (1) Transformed Lactococcus lactis NZ -CTB-Tim3scFv and NZ -Tim3scFv, which secrete CTB-Tim3scFv and Tim3scFv single-chain antibodies, were successfully constructed. (2) CTB-Tim3scFv secreted by NZ-CTB-Tim3scFv transformed bacteria showed immunological activity. (3) Compared with the NZ-Tim3scFv and NZ-Vector groups, the characteristics of the subgroups of splenic lymphocytes in the NZ-CTB-Tim3scFv group had a higher proportion of CD3+CD4+, CD3+CD8a+, and CD3+CD69+ cells. Ki67 and CD31 expression in the NZ-CTB-Tim3scFv group was significantly reduced. The tumour volume of the NZ-CTB-Tim3scFv group increased the least, and was statistically different from that of the other two groups. Conclusions The Tim-3 single-chain antibody gene was successfully constructed and transformed in Lactococcus lactis. After feeding mice NZ-CTB-Tim3scFv transforming Lactobacillus, the CTB-Tim3scFv secreted by the transforming Lactobacillus promoted the proliferation and activation of spleen lymphocytes and inhibited volume growth, cell proliferation, and angiogenesis of the tumour in mice. In summary, apply transgenic lactobacillus secreting CTB-Tim-3scFv, to perform its role in anti-tumor immunotherapy through oral approach, is low cost and convenient. It is expected to become a new way of immunotherapy for renal cell carcinoma.


Molecules ◽  
2021 ◽  
Vol 26 (21) ◽  
pp. 6436
Author(s):  
Kanasap Kaewchim ◽  
Kittirat Glab-ampai ◽  
Kodchakorn Mahasongkram ◽  
Monrat Chulanetra ◽  
Watee Seesuay ◽  
...  

Proviral integration site of Moloney virus-2 (PIM2) is overexpressed in multiple human cancer cells and high level is related to poor prognosis; thus, PIM2 kinase is a rational target of anti-cancer therapeutics. Several chemical inhibitors targeting PIMs/PIM2 or their downstream signaling molecules have been developed for treatment of different cancers. However, their off-target toxicity is common in clinical trials, so they could not be advanced to official approval for clinical application. Here, we produced human single-chain antibody fragments (HuscFvs) to PIM2 by using phage display library, which was constructed in a way that a portion of phages in the library carried HuscFvs against human own proteins on their surface with the respective antibody genes in the phage genome. Bacterial derived-recombinant PIM2 (rPIM2) was used as an antigenic bait to fish out the rPIM2-bound phages from the library. Three E. coli clones transfected with the HuscFv genes derived from the rPIM2-bound phages expressed HuscFvs that bound also to native PIM2 from cancer cells. The HuscFvs presumptively interact with the PIM2 at the ATP binding pocket and kinase active loop. They were as effective as small chemical drug inhibitor (AZD1208, which is an ATP competitive inhibitor of all PIM isoforms for ex vivo use) in inhibiting PIM kinase activity. The HuscFvs should be engineered into a cell-penetrating format and tested further towards clinical application as a novel and safe pan-anti-cancer therapeutics.


2021 ◽  
Vol 99 (Supplement_3) ◽  
pp. 265-266
Author(s):  
Mauro Venturini ◽  
Demian Bellido ◽  
Josefina Baztarrica ◽  
Lucia Rocha ◽  
Viviana Parreño ◽  
...  

Abstract Bovine viral diarrhea virus (BVDV) infects ruminants, with a worldwide distribution, the virus causes a broad spectrum of clinical diseases and economic losses. Vaccination against BVDV is an important component of prevention and control programs. Currently, only modified live vaccines (MLV) and inactivated vaccines are used. Both have historical disadvantages; MLV in terms of safety and inactivated vaccines in terms of immunogenicity. We have previously reported the development and efficacy trials of the first targeted subunit vaccine against BVDV. The core of the vaccine is a fusion of the BVDV structural protein, E2, to a single-chain antibody, APCH, together termed, APCH-E2. The APCH antibody targets the E2 antigen to the MHC-II present on antigen-presenting cells. The goal of this work was to evaluate passive immunity through colostrum and active immunity of calves immunized with the novel vaccine. 24 A. angus heifers were divided into two groups, 12 immunized with the vaccine and 12 received placebo, in the last trimester of gestation. Serum samples from calves were taken at day 30 of age and analyzed by competitive ELISA. In the vaccinated group, 92% of the d30 old calves maintained medium (25%) or high (67%) antibody levels against BVDV, while 50% of the animals in the control group presented low antibody level (Pearson’s chi-squared p:0,07) (Table 1). At 5 months of age, calves of both groups were immunized with the targeted vaccine. Thirty days later, 96% of the calves had medium or high antibody levels against BVDV, which was independent of their respective heifer vaccine status. These results confirm the new targeted subunit vaccine against BVDV is safe and efficacious to be used in pregnant cattle and can passively immunize newborn calves. They also show that these maternal antibodies do not interfere with the active immunization of calves.


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