scholarly journals Shelf-Life Extension of Fc-Fused Single Chain Fragment Variable Antibodies by Lyophilization

2021 ◽  
Vol 11 ◽  
Author(s):  
Kai-Thomas Schneider ◽  
Toni Kirmann ◽  
Esther Veronika Wenzel ◽  
Jan-Hendrik Grosch ◽  
Saskia Polten ◽  
...  
Keyword(s):  
Phage Display ◽  
Shelf Life ◽  
Neutralizing Antibodies ◽  
Binding Activity ◽  
Life Extension ◽  
Market Demand ◽  
Single Chain ◽  
Single Chain Fv ◽  
The Stability

Generation of sequence defined antibodies from universal libraries by phage display has been established over the past three decades as a robust method to cope with the increasing market demand in therapy, diagnostics and research. For applications requiring the bivalent antigen binding and an Fc part for detection, phage display generated single chain Fv (scFv) antibody fragments can rapidly be genetically fused to the Fc moiety of an IgG for the production in eukaryotic cells of antibodies with IgG-like properties. In contrast to conversion of scFv into IgG format, the conversion to scFv-Fc requires only a single cloning step, and provides significantly higher yields in transient cell culture production than IgG. ScFv-Fcs can be effective as neutralizing antibodies in vivo against a panel of pathogens and toxins. However, different scFv fragments are more heterologous in respect of stability than Fab fragments. While some scFv fragments can be made extremely stable, this may change due to few mutations, and is not predictable from the sequence of a newly selected antibody. To mitigate the necessity to assess the stability for every scFv-Fc antibody, we developed a generic lyophilization protocol to improve their shelf life. We compared long-term stability and binding activity of phage display-derived antibodies in the scFv-Fc and IgG format, either stored in liquid or lyophilized state. Conversion of scFv-Fcs into the full IgG format reduced protein degradation and aggregation, but in some cases compromised binding activity. Comparably to IgG conversion, lyophilization of scFv-Fc resulted in the preservation of the antibodies’ initial properties after storage, without any drop in affinity for any of the tested antibody clones.

scholarly journals Screening of potent neutralizing antibodies against SARS-CoV-2 using convalescent patients-derived phage-display libraries

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Yongbing Pan ◽  
Jianhui Du ◽  
Jia Liu ◽  
Hai Wu ◽  
Fang Gui ◽  
...  
Keyword(s):  
Phage Display ◽  
Neutralizing Antibodies ◽  
Variable Region ◽  
Neutralizing Antibody ◽  
Chain Gene ◽  
Prophylactic Efficacy ◽  
Heavy Chains ◽  
Effective Interventions ◽  
Phage Display Libraries

AbstractAs the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to threaten public health worldwide, the development of effective interventions is urgently needed. Neutralizing antibodies (nAbs) have great potential for the prevention and treatment of SARS-CoV-2 infection. In this study, ten nAbs were isolated from two phage-display immune libraries constructed from the pooled PBMCs of eight COVID-19 convalescent patients. Eight of them, consisting of heavy chains encoded by the immunoglobulin heavy-chain gene-variable region (IGHV)3-66 or IGHV3-53 genes, recognized the same epitope on the receptor-binding domain (RBD), while the remaining two bound to different epitopes. Among the ten antibodies, 2B11 exhibited the highest affinity and neutralization potency against the original wild-type (WT) SARS-CoV-2 virus (KD = 4.76 nM for the S1 protein, IC50 = 6 ng/mL for pseudoviruses, and IC50 = 1 ng/mL for authentic viruses), and potent neutralizing ability against B.1.1.7 pseudoviruses. Furthermore, 1E10, targeting a distinct epitope on RBD, exhibited different neutralization efficiency against WT SARS-CoV-2 and its variants B.1.1.7, B.1.351, and P.1. The crystal structure of the 2B11–RBD complexes revealed that the epitope of 2B11 highly overlaps with the ACE2-binding site. The in vivo experiment of 2B11 using AdV5-hACE2-transduced mice showed encouraging therapeutic and prophylactic efficacy against SARS-CoV-2. Taken together, our results suggest that the highly potent SARS-CoV-2-neutralizing antibody, 2B11, could be used against the WT SARS-CoV-2 and B.1.1.7 variant, or in combination with a different epitope-targeted neutralizing antibody, such as 1E10, against SARS-CoV-2 variants.

scholarly journals Characterization of LL25X, a Newly Isolated Anti-SIV Monoclonal Antibody, to Determine Binding Specificity

Elements ◽  
2017 ◽  
Vol 13 (1) ◽  
Author(s):  
Zackary Tajin Park
Keyword(s):  
Phage Display ◽  
Mammalian Cells ◽  
Expression Vector ◽  
Restriction Enzymes ◽  
Phage Display Library ◽  
Single Chain ◽  
Phage Display Technology ◽  
Mammalian Expression ◽  
Mammalian Expression Vector ◽  
Single Chain Fv

A phage display library was previously constructed from an SIV-infected rhesus macaque. Several single chain Fv (scFv), including SU24, SU343 and LL25X, were selected using phage display technology. Sequences corresponding to SU24, SU343 and LL25X were optimized for expression in a mammalian system and commercially synthesized. SU24 and SU343 had previously been cloned into a mammalian expression vector. In this study, we aimed to characterize the specificity of SU24, SU343, and LL25X.. The codon-optimized version of the scFv LL25X gene sequence was cloned into a mammalian expression vector (pCEP4).  LL25X DNA was amplified by PCR, and the PCR product and mammalian expression vector were both digested with KpnI/SapI restriction enzymes. Digested fragments were purified, and the fragments were ligated using T4DNA ligase. E. coli cells were transformed with the ligation reaction. Single colonies were selected on LB agar plates containing the selective antibiotic (ampicillin). Positive colonies were identified after DNA mini-preparation and test-digestion with KpnI and SapI. Sanger sequencing confirmed cloning results and DNA sequence accuracy. Following transfection of mammalian cells (293T), LL25X-Fc cells, and purifying our protein, the binding of LL25X-Fc to the SIV gp140 envelope protein was confirmed via ELISA and Western Blotting.

scholarly journals Human C5-specific single-chain variable fragment ameliorates brain injury in a model of NMOSD

2019 ◽  
Vol 6 (3) ◽  
pp. e561 ◽  
Author(s):  
Wenli Zhu ◽  
Zhen Wang ◽  
Suying Hu ◽  
Ye Gong ◽  
Yuanchu Liu ◽  
...  
Keyword(s):  
Mouse Model ◽  
Phage Display ◽  
Membrane Attack Complex ◽  
Single Chain ◽  
Inflammatory Infiltration ◽  
Transfected Cells ◽  
Complement Dependent Cytotoxicity ◽  
Test Specificity

ObjectiveUsing phage display, we sought to screen single-chain variable fragments (scFvs) against complement C5 to treat neuromyelitis optica spectrum disorder (NMOSD).MethodsAfter 5 rounds of phage display, we isolated individual clones and identified phage clones specifically binding to C5 using ELISA. Using aquaporin-4 (AQP4)-transfected cells in vitro, we confirmed whether these scFvs prevented complement-dependent cytotoxicity (CDC) caused by the serum of patients with NMOSD and human complement (hC). We selected an NMOSD mouse model, in which intracerebral NMOSD immunoglobulin G (IgG) and hC injections induce NMOSD-like lesions in vivo.ResultsWe obtained scFvs to test specificity and blocking efficiency. The scFv C5B3 neutralized C5 in the complement activation pathway, which prevented AQP4-IgG–mediated CDC in AQP4-transfected cells. In an NMOSD mouse model, C5B3 prevented AQP4 and astrocyte loss, decreased demyelination, and reduced inflammatory infiltration and membrane attack complex formation in lesions.ConclusionsWe used phage display to screen C5B3 against C5, which was effective in inhibiting cytotoxicity in vitro and preventing CNS pathology in vivo.

scholarly journals Engineered Fragments of the PSMA-Specific 5D3 Antibody and Their Functional Characterization

2020 ◽  
Vol 21 (18) ◽  
pp. 6672
Author(s):  
Zora Novakova ◽  
Nikola Belousova ◽  
Catherine A. Foss ◽  
Barbora Havlinova ◽  
Marketa Gresova ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Xenograft Model ◽  
In Vitro Assays ◽  
Experimental Therapy ◽  
Single Chain ◽  
Single Chain Fv ◽  
Murine Xenograft Model ◽  
Single Target

Prostate-Specific Membrane Antigen (PSMA) is an established biomarker for the imaging and experimental therapy of prostate cancer (PCa), as it is strongly upregulated in high-grade primary, androgen-independent, and metastatic lesions. Here, we report on the development and functional characterization of recombinant single-chain Fv (scFv) and Fab fragments derived from the 5D3 PSMA-specific monoclonal antibody (mAb). These fragments were engineered, heterologously expressed in insect S2 cells, and purified to homogeneity with yields up to 20 mg/L. In vitro assays including ELISA, immunofluorescence and flow cytometry, revealed that the fragments retain the nanomolar affinity and single target specificity of the parent 5D3 antibody. Importantly, using a murine xenograft model of PCa, we verified the suitability of fluorescently labeled fragments for in vivo imaging of PSMA-positive tumors and compared their pharmacokinetics and tissue distribution to the parent mAb. Collectively, our data provide an experimental basis for the further development of 5D3 recombinant fragments for future clinical use.

scholarly journals Expression of Engineered Antibodies in Plants: A Possible Tool for Spiroplasma and Phytoplasma Disease Control

1998 ◽  
Vol 88 (12) ◽  
pp. 1367-1371 ◽  
Author(s):  
Y. D. Chen ◽  
T. A. Chen
Keyword(s):  
Cell Line ◽  
Pathogen Resistance ◽  
Single Chain ◽  
Contributing Factors ◽  
Factors Affecting ◽  
Transgenic Cell ◽  
Single Chain Fv ◽  
Scfv Gene ◽  
A Cell

In an attempt to develop maize plants with resistance to corn stunt spi-roplasma (CSS), a single-chain Fv fragment (scFv) gene that was constructed from antibodies with strong inhibitory activity against CSS, was expressed in a cell line of maize. However, plants regenerated from this transgenic cell line showed no distinct resistance to CSS infection under the greenhouse conditions. The affinity and functionality of scFv in vivo and the locations of CSS and expressed scFv in maize could be a part of the contributing factors affecting this result. Progress of expressing antibodies in plants for plant pathogen resistance is also discussed.

HM1.24/CD317-directed immunotoxin to eliminate malignant plasma cells in vitro and in vivo.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 8604-8604
Author(s):  
Martin Gramatzki ◽  
Matthias Staudinger ◽  
Pia Glorius ◽  
Katja Klausz ◽  
Christian Kellner ◽  
...  
Keyword(s):  
Mouse Model ◽  
Plasma Cells ◽  
Xenograft Model ◽  
Clinical Activity ◽  
Target Structure ◽  
Single Chain ◽  
Pseudomonas Exotoxin A ◽  
Single Chain Fv

8604 Background: Targeted immunotherapy, based on antibodies against tumor-associated antigens, is a promising approach for the treatment of multiple myeloma (MM). Recently, antibody-based strategies delivering a toxic payload have documented impressive clinical activity in hematological malignancies. In particular, surface molecules overexpressed on malignant plasma cells and efficiently internalized represent promising targets for developing myeloma-directed immunoconstructs. Here, the identification of CD317 (HM1.24) as a potent target structure and the characterization of a novel CD317-directed single-chain immunotoxin, HM1.24-ETA', is described. Methods: Using a novel screening tool, a panel of antibodies against MM-associated antigens was evaluated for their ability to mediate antigen-dependent delivery of a truncated version of Pseudomonas exotoxin A (ETA’) to MM cells. HM1.24-ETA' was generated by genetic fusion of a CD317-specific single-chain Fv antibody and ETA'. The anti-myeloma activity of the E. coli-expressed immunotoxin was evaluated in vitro and in a xenograft mouse model. Results: By screening a panel of antibodies including CD38, CS1, IL-6R, CD138 and CD317, CD317 was identified as a suitable receptor to deliver ETA’ to MM cells. The subsequently designed recombinant HM1.24-ETA' immunotoxin efficiently inhibited growth of MM cell lines with halfmaximal growth inhibition at concentrations of less than 1 nM. Antigen-specific MM cell killing occurred via induction of apoptosis. The proliferation of IL-6 dependent INA-6 cells was completely inhibited by HM1.24-ETA' even in the presence of bone marrow stromal cells that otherwise strongly support tumor cell growth. Importantly, HM1.24-ETA' strongly triggered apoptosis (up to 80%) in freshly isolated tumor cells from 7 out of 7 MM patients. In a xenograft SCID mouse model, establishment of INA-6 plasma cell tumors was efficiently abrogated by treatment with HM1.24-ETA' immunotoxin (p < 0.04). Conclusions: The HM1.24-ETA' immunotoxin in vitro and in the preclinical xenograft model in vivo demonstrates that the CD317 antigen may represent a promising target structure for immunotherapy of MM using immunoconjugates with toxic payloads.

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