scholarly journals Peptides derived from phage display libraries as potential neutralizers of Shiga toxin-induced cytotoxicity in vitro and in vivo

2014 ◽  
Vol 116 (5) ◽  
pp. 1322-1333 ◽  
Author(s):  
R.A. Bernedo-Navarro ◽  
M.M. Miyachiro ◽  
M.J. da Silva ◽  
C.F. Reis ◽  
R.A. Conceição ◽  
...  
Keyword(s):  
Phage Display ◽  
Shiga Toxin ◽  
Phage Display Libraries

scholarly journals Developing Recombinant Antibodies by Phage Display Against Infectious Diseases and Toxins for Diagnostics and Therapy

2021 ◽  
Vol 11 ◽  
Author(s):  
Kristian Daniel Ralph Roth ◽  
Esther Veronika Wenzel ◽  
Maximilian Ruschig ◽  
Stephan Steinke ◽  
Nora Langreder ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Infectious Diseases ◽  
Phage Display ◽  
Antibody Fragment ◽  
Recombinant Antibodies ◽  
Phage Display Libraries ◽  
Antibody Phage ◽  
Antibody Phage Display

Antibodies are essential molecules for diagnosis and treatment of diseases caused by pathogens and their toxins. Antibodies were integrated in our medical repertoire against infectious diseases more than hundred years ago by using animal sera to treat tetanus and diphtheria. In these days, most developed therapeutic antibodies target cancer or autoimmune diseases. The COVID-19 pandemic was a reminder about the importance of antibodies for therapy against infectious diseases. While monoclonal antibodies could be generated by hybridoma technology since the 70ies of the former century, nowadays antibody phage display, among other display technologies, is robustly established to discover new human monoclonal antibodies. Phage display is an in vitro technology which confers the potential for generating antibodies from universal libraries against any conceivable molecule of sufficient size and omits the limitations of the immune systems. If convalescent patients or immunized/infected animals are available, it is possible to construct immune phage display libraries to select in vivo affinity-matured antibodies. A further advantage is the availability of the DNA sequence encoding the phage displayed antibody fragment, which is packaged in the phage particles. Therefore, the selected antibody fragments can be rapidly further engineered in any needed antibody format according to the requirements of the final application. In this review, we present an overview of phage display derived recombinant antibodies against bacterial, viral and eukaryotic pathogens, as well as microbial toxins, intended for diagnostic and therapeutic applications.

scholarly journals A Novel ICAM-1/CD54 Antibody Identified By Phage Display with Potent Pre-Clinical Anti-Myeloma Activity

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2095-2095
Author(s):  
Katja Klausz ◽  
Renate Burger ◽  
Christian Kellner ◽  
Matthias Peipp ◽  
Martin Gramatzki
Keyword(s):  
T Lymphocytes ◽  
Phage Display ◽  
Nk Cells ◽  
Cell Lines ◽  
Plasma Cell ◽  
Healthy Individuals ◽  
In Vivo Models ◽  
Phage Display Libraries

Abstract Introduction: Despite novel agents in multiple myeloma (MM) a substantial number of patients is still in need of better disease control. Monoclonal antibodies provide therapeutic options in other tumor entities but in MM no antibody has been approved yet. Two antibodies currently evaluated namely Daratumumab (CD38) and Elotuzumab (CD319), are ahead and achieved phase 3 clinical trials, but other antigens on myeloma cells could also be promising targets. Here, a cell-based phage display screening strategy is presented that identified a novel intercellular adhesion molecule-1 (ICAM-1)/CD54 antibody suitable for myeloma immunotherapy. Methods: Synthetic human single-chain fragment variable (scFv) phage display libraries were pre-absorbed with granulocytes and T lymphocytes of healthy individuals and subsequently panned with plasma cell lines INA-6 and JK-6L. Phage binding characteristics were evaluated by cellular ELISA and flow cytometry. The coding sequence of the leading candidate MSH-TP15 was used to generate scFv-Fc fusion proteins and fully human IgG1 antibodies with wild type or modified Fc domains for functional in vitro and in vivo testing. Results: Panning of human phage display libraries with a newly established subtractive cellular screening approach resulted in phages with preferential binding to MM cells. Based on its reactivity with MM cell lines and patient-derived CD138+ malignant plasma cells from bone marrow, pleural effusion and peripheral blood, the leading candidate MSH-TP15 was selected. Notably, MSH-TP15 showed no or only marginal binding to B and T lymphocytes, NK cells, monocytes and granulocytes of healthy individuals and MM patients. Recent studies identified ICAM-1/CD54 as the antigen recognized by MSH-TP15. The antibody alone was not capable to significantly inhibit growth of plasma cell lines in vitro, however induced antibody-dependent cell-mediated cytotoxicity (ADCC), an important killing mechanism of many therapeutic antibodies. An Fc-engineered antibody variant of MSH-TP15 triggered dose-dependent ADCC against INA-6, L363, RPMI-8226, U266 and MM1.S plasma cell lines and freshly isolated tumor cells using blood mononuclear cells and NK cells of healthy donors as effector cells. In contrast, complement-dependent cytotoxicity was not induced. Importantly, in vivo studies revealed that MSH-TP15 completely prevented tumor engraftment in the INA-6 myeloma xenograft model. Conclusions: The cell-based screening of phage display libraries led to the identification of a novel ICAM-1/CD54 antibody that is effective in antibody-mediated cellular cytotoxicity of MM cells in vitro and completely prevented myeloma tumor engraftment in vivo. The relevant mechanisms of action of MSH-TP15 will be defined with different antibody variants and in vivo models to further evaluate its potential for an antibody-based treatment approach of MM. Disclosures No relevant conflicts of interest to declare.

scholarly journals Selection and Characterization of YKL-40-Targeting Monoclonal Antibodies from Human Synthetic Fab Phage Display Libraries

2020 ◽  
Vol 21 (17) ◽  
pp. 6354
Author(s):  
Kyungjae Kang ◽  
Kicheon Kim ◽  
Se-Ra Lee ◽  
Yoonji Kim ◽  
Joo Eon Lee ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Phage Display ◽  
Cancer Therapeutics ◽  
Cell Types ◽  
Migration Assay ◽  
Synthetic Antibody ◽  
Phage Display Libraries ◽  
Anti Cancer

YKL-40, also known as chitinase-3-like 1 (CHI3L1), is a glycoprotein that is expressed and secreted by various cell types, including cancers and macrophages. Due to its implications for and upregulation in a variety of diseases, including inflammatory conditions, fibrotic disorders, and tumor growth, YKL-40 has been considered as a significant therapeutic biomarker. Here, we used a phage display to develop novel monoclonal antibodies (mAbs) targeting human YKL-40 (hYKL-40). Human synthetic antibody phage display libraries were panned against a recombinant hYKL-40 protein, yielding seven unique Fabs (Antigen-binding fragment), of which two Fabs (H1 and H2) were non-aggregating and thermally stable (75.5 °C and 76.5 °C, respectively) and had high apparent affinities (KD = 2.3 nM and 4.0 nM, respectively). Reformatting the Fabs into IgGs (Immunoglobulin Gs) increased their apparent affinities (notably, for H1 and H2, KD = 0.5 nM and 0.3 nM, respectively), presumably due to the effects of avidity, with little change to their non-aggregation property. The six anti-hYKL-40 IgGs were analyzed using a trans-well migration assay in vitro, revealing that three clones (H1, H2, and H4) were notably effective in reducing cell migration from both A549 and H460 lung cancer cell lines. The three clones were further analyzed in an in vivo animal test that assessed their anti-cancer activities, demonstrating that the tumor area and the number of tumor nodules were significantly reduced in the lung tissues treated with H1 (IgG). Given its high affinity and desirable properties, we expect that the H1 anti-hYKL-40 mAb will be a suitable candidate for developing anti-cancer therapeutics.

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 Evaluation of Fab and F(ab′)2 Fragments and Isotype Variants of a Recombinant Human Monoclonal Antibody against Shiga Toxin 2

2010 ◽  
Vol 78 (3) ◽  
pp. 1376-1382 ◽  
Author(s):  
Donna E. Akiyoshi ◽  
Abhineet S. Sheoran ◽  
Curtis M. Rich ◽  
L. Richard ◽  
Susan Chapman-Bonofiglio ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Shiga Toxin ◽  
Chinese Hamster Ovary Cells ◽  
Human Monoclonal Antibody ◽  
Chinese Hamster ◽  
The Body ◽  
Neutralization Activity ◽  
Shiga Toxin 2

ABSTRACT 5C12 HuMAb is a human monoclonal antibody against the A subunit of Shiga toxin 2 (Stx2). We have previously shown that 5C12 HuMAb effectively neutralizes the cytotoxic effects of this toxin by redirecting its transport within the cell and also by neutralizing the toxin's ability to inhibit protein synthesis. The 5C12 HuMAb and its recombinant IgG1 version protect mice at a dose of 0.6 μg against a lethal challenge of Stx2. The contribution of the Fc region to this observed neutralization activity of the 5C12 antibody against Stx2 was investigated in this study. Using recombinant DNA technology, 5C12 isotype variants (IgG1, IgG2, IgG3, and IgG4) and antibody fragments [Fab, F(ab′)2] were expressed in Chinese hamster ovary cells and evaluated in vitro and in vivo. All four 5C12 isotype variants showed protection in vitro, with the IgG3 and IgG4 variants showing the highest protection in vivo. The Fab and F(ab′)2 fragments also showed protection in vitro but no protection in the mouse toxicity model. Similar results were obtained for a second HuMAb (5H8) against the B subunit of Stx2. The data suggest the importance of the Fc region for neutralization activity, but it is not clear if this is related to the stability of the full-length antibody or if the Fc region is required for effective elimination of the toxin from the body.

scholarly journals Tumor-Targeting Peptides Search Strategy for the Delivery of Therapeutic and Diagnostic Molecules to Tumor Cells

2020 ◽  
Vol 22 (1) ◽  
pp. 314
Author(s):  
Maria D. Dmitrieva ◽  
Anna A. Voitova ◽  
Maya A. Dymova ◽  
Vladimir A. Richter ◽  
Elena V. Kuligina
Keyword(s):  
Phage Display ◽  
Cell Sorting ◽  
Targeted Delivery ◽  
Tumor Targeting ◽  
Search Strategy ◽  
Tumor Therapy ◽  
Therapeutic Agents ◽  
Phage Libraries

Background: The combination of the unique properties of cancer cells makes it possible to find specific ligands that interact directly with the tumor, and to conduct targeted tumor therapy. Phage display is one of the most common methods for searching for specific ligands. Bacteriophages display peptides, and the peptides themselves can be used as targeting molecules for the delivery of diagnostic and therapeutic agents. Phage display can be performed both in vitro and in vivo. Moreover, it is possible to carry out the phage display on cells pre-enriched for a certain tumor marker, for example, CD44 and CD133. Methods: For this work we used several methods, such as phage display, sequencing, cell sorting, immunocytochemistry, phage titration. Results: We performed phage display using different screening systems (in vitro and in vivo), different phage libraries (Ph.D-7, Ph.D-12, Ph.D-C7C) on CD44+/CD133+ and without enrichment U-87 MG cells. The binding efficiency of bacteriophages displayed tumor-targeting peptides on U-87 MG cells was compared in vitro. We also conducted a comparative analysis in vivo of the specificity of the accumulation of selected bacteriophages in the tumor and in the control organs (liver, brain, kidney and lungs). Conclusions: The screening in vivo of linear phage peptide libraries for glioblastoma was the most effective strategy for obtaining tumor-targeting peptides providing targeted delivery of diagnostic and therapeutic agents to glioblastoma.

scholarly journals Human Recombinant Fab Fragment Neutralizes Shiga Toxin Type 2 Cytotoxic Effects in vitro and in vivo

Toxins ◽  
2018 ◽  
Vol 10 (12) ◽  
pp. 508 ◽  
Author(s):  
Daniela Luz ◽  
Maria Amaral ◽  
Flavia Sacerdoti ◽  
Alan Bernal ◽  
Wagner Quintilio ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Lethal Dose ◽  
Dependent Manner ◽  
Fab Fragment ◽  
Cell Viability Assay ◽  
Cytotoxic Effects ◽  
Morphological Alterations ◽  
Viability Assay

Shiga toxin (Stx) producing Escherichia coli (STEC) is responsible for causing hemolytic uremic syndrome (HUS), a life-threatening thrombotic microangiopathy characterized by thrombocytopenia, hemolytic anemia, and acute renal failure after bacterially induced hemorrhagic diarrhea. Until now, there has been neither an effective treatment nor method of prevention for the deleterious effects caused by Stx intoxication. Antibodies are well recognized as affinity components of therapeutic drugs; thus, a previously obtained recombinant human FabC11:Stx2 fragment was used to neutralize Stx2 in vitro in a Vero cell viability assay. Herein, we demonstrated that this fragment neutralized, in a dose-dependent manner, the cytotoxic effects of Stx2 on human glomerular endothelial cells, on human proximal tubular epithelial cells, and prevented the morphological alterations induced by Stx2. FabC11:Stx2 protected mice from a lethal dose of Stx2 by toxin-antibody pre-incubation. Altogether, our results show the ability of a new encouraging molecule to prevent Stx-intoxication symptoms during STEC infection.

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 A Toxic Environment: a Growing Understanding of How Microbial Communities Affect Escherichia coli O157:H7 Shiga Toxin Expression

2020 ◽  
Vol 86 (24) ◽  
Author(s):  
Erin M. Nawrocki ◽  
Hillary M. Mosso ◽  
Edward G. Dudley
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Microbial Interactions ◽  
Severe Illness ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Lambdoid Prophage

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) strains, including E. coli O157:H7, cause severe illness in humans due to the production of Shiga toxin (Stx) and other virulence factors. Because Stx is coregulated with lambdoid prophage induction, its expression is especially susceptible to environmental cues. Infections with Stx-producing E. coli can be difficult to model due to the wide range of disease outcomes: some infections are relatively mild, while others have serious complications. Probiotic organisms, members of the gut microbiome, and organic acids can depress Stx production, in many cases by inhibiting the growth of EHEC strains. On the other hand, the factors currently known to amplify Stx act via their effect on the stx-converting phage. Here, we characterize two interactive mechanisms that increase Stx production by O157:H7 strains: first, direct interactions with phage-susceptible E. coli, and second, indirect amplification by secreted factors. Infection of susceptible strains by the stx-converting phage can expand the Stx-producing population in a human or animal host, and phage infection has been shown to modulate virulence in vitro and in vivo. Acellular factors, particularly colicins and microcins, can kill O157:H7 cells but may also trigger Stx expression in the process. Colicins, microcins, and other bacteriocins have diverse cellular targets, and many such molecules remain uncharacterized. The identification of additional Stx-amplifying microbial interactions will improve our understanding of E. coli O157:H7 infections and help elucidate the intricate regulation of pathogenicity in EHEC strains.

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