A Recombinant Human Monoclonal Antibody to Human Metapneumovirus Fusion Protein That Neutralizes Virus In Vitro and Is Effective Therapeutically In Vivo

ABSTRACT Human metapneumovirus (hMPV) is a recently discovered paramyxovirus that is a major cause of lower-respiratory-tract disease. hMPV is associated with more severe disease in infants and persons with underlying medical conditions. Animal studies have shown that the hMPV fusion (F) protein alone is capable of inducing protective immunity. Here, we report the use of phage display technology to generate a fully human monoclonal antibody fragment (Fab) with biological activity against hMPV. Phage antibody libraries prepared from human donor tissues were selected against recombinant hMPV F protein with multiple rounds of panning. Recombinant Fabs then were expressed in bacteria, and supernatants were screened by enzyme-linked immunosorbent assay and immunofluorescent assays. A number of Fabs that bound to hMPV F were isolated, and several of these exhibited neutralizing activity in vitro. Fab DS7 neutralized the parent strain of hMPV with a 60% plaque reduction activity of 1.1 μg/ml and bound to hMPV F with an affinity of 9.8 ×10−10 M, as measured by surface plasmon resonance. To test the in vivo activity of Fab DS7, groups of cotton rats were infected with hMPV and given Fab intranasally 3 days after infection. Nasal turbinates and lungs were harvested on day 4 postinfection and virus titers determined. Animals treated with Fab DS7 exhibited a >1,500-fold reduction in viral titer in the lungs, with a modest 4-fold reduction in the nasal tissues. There was a dose-response relationship between the dose of DS7 and virus titer. Human Fab DS7 may have prophylactic or therapeutic potential against severe hMPV infection.

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A Novel Mouse Monoclonal Antibody C42 against C-Terminal Peptide of Alpha-1-Antitrypsin

International Journal of Molecular Sciences ◽

10.3390/ijms22042141 ◽

2021 ◽

Vol 22 (4) ◽

pp. 2141

Author(s):

Srinu Tumpara ◽

Elena Korenbaum ◽

Mark Kühnel ◽

Danny Jonigk ◽

Beata Olejnicka ◽

...

Keyword(s):

Monoclonal Antibody ◽

Flow Cytometry ◽

Western Blotting ◽

Complex Mixture ◽

Immunoglobulin M ◽

Confocal Laser ◽

Enzyme Linked Immunosorbent Assay ◽

Dot Blot

The C-terminal-fragments of alpha1-antitrypsin (AAT) have been identified and their diverse biological roles have been reported in vitro and in vivo. These findings prompted us to develop a monoclonal antibody that specifically recognizes C-36 peptide (corresponding to residues 359–394) resulting from the protease-associated cleavage of AAT. The C-36-targeting mouse monoclonal Immunoglobulin M (IgM) antibody (containing κ light chains, clone C42) was generated and enzyme-linked immunosorbent assay (ELISA)-tested by Davids Biotechnologie GmbH, Germany. Here, we addressed the effectiveness of the novel C42 antibody in different immunoassay formats, such as dot- and Western blotting, confocal laser microscopy, and flow cytometry. According to the dot-blot results, our novel C42 antibody detects the C-36 peptide at a range of 0.1–0.05 µg and shows no cross-reactivity with native, polymerized, or oxidized forms of full-length AAT, the AAT-elastase complex mixture, as well as with shorter C-terminal fragments of AAT. However, the C42 antibody does not detect denatured peptide in SDS-PAGE/Western blotting assays. On the other hand, our C42 antibody, unconjugated as well as conjugated to DyLight488 fluorophore, when applied for immunofluorescence microscopy and flow cytometry assays, specifically detected the C-36 peptide in human blood cells. Altogether, we demonstrate that our novel C42 antibody successfully recognizes the C-36 peptide of AAT in a number of immunoassays and has potential to become an important tool in AAT-related studies.

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

Infection and Immunity ◽

10.1128/iai.00867-09 ◽

2010 ◽

Vol 78 (3) ◽

pp. 1376-1382 ◽

Cited By ~ 17

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.

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Modulation of the Lung Inflammatory Response to Serotype 8 Pneumococcal Infection by a Human Immunoglobulin M Monoclonal Antibody to Serotype 8 Capsular Polysaccharide

Infection and Immunity ◽

10.1128/iai.73.8.4530-4538.2005 ◽

2005 ◽

Vol 73 (8) ◽

pp. 4530-4538 ◽

Cited By ~ 26

Author(s):

Tamika Burns ◽

Maria Abadi ◽

Liise-anne Pirofski

Keyword(s):

Monoclonal Antibody ◽

Inflammatory Response ◽

Capsular Polysaccharide ◽

Human Monoclonal Antibody ◽

Pneumococcal Infection ◽

Immunoglobulin M ◽

Enzyme Linked Immunosorbent Assay ◽

Necrosis Factor Alpha ◽

Monocyte Chemoattractant Protein 1

ABSTRACT The human monoclonal antibody to serotype 8 pneumococcal capsular polysaccharide D11 [immunoglobulin M(κ)] protects wild-type and complement component 4 knockout (C4 KO) mice against lethal intratracheal challenge with serotype 8 pneumococcus, but it does not promote polymorphonuclear leukocyte (PMN)-mediated pneumococcal killing in vitro. In this study, we investigated the effect of D11 on the blood and lung bacterial burdens and the serum and lung expression of inflammatory chemokines and cytokines in an intratracheal challenge model with serotype 8 pneumococcus in C4 KO mice. Pneumococcus was not detected in the blood of D11-treated mice, whereas control mice had high-grade bacteremia with >107 CFU. Control mice had a >5-log increase in lung CFU and D11-treated mice manifested a nearly 3-log increase in lung CFU compared to the original inoculum 24 h after infection. Serum and lung levels of soluble macrophage inflammatory protein 2 (MIP-2) and interleulin-6 (IL-6) as measured by an enzyme-linked immunosorbent assay were lower in D11-treated mice than in control mice 24 h after infection. Real-time PCR was performed to examine lung mRNA chemokine and cytokine expression. The results showed that D11-treated mice had significantly less gamma interferon, MIP-2, IL-12, monocyte chemoattractant protein 1/JE, and tumor necrosis factor alpha expression than control mice 24 h after infection. Histopathology and immunohistochemical staining of lung tissues revealed that D11-treated mice had less inflammation, fewer PMNs, and less myeloperoxidase staining than control mice 24 h after infection. These findings suggest that the efficacy of certain serotype-specific antibodies against pneumococcal pneumonia could be associated with modulation of the lung inflammatory response and a reduction in host damage.

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In vivo and in vitro studies with a novel human monoclonal antibody against tetanus neurotoxin

Toxicon ◽

10.1016/j.toxicon.2013.08.032 ◽

2013 ◽

Vol 75 ◽

pp. 216

Author(s):

E. Girard ◽

P. Villeneuve ◽

V. Devos ◽

A.S. Dezetter ◽

A. Fontayne ◽

...

Keyword(s):

Monoclonal Antibody ◽

Human Monoclonal Antibody ◽

In Vitro Studies ◽

Tetanus Neurotoxin

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Supression of FLT3-Expressing Leukemia by a Monoclonal Antibody-Auristatin Conjugate.

Blood ◽

10.1182/blood.v104.11.1788.1788 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1788-1788

Author(s):

Yiwen Li ◽

Hongli Li ◽

Mei-Nai Wang ◽

Rajiv Bassi ◽

Dale Ludwig ◽

...

Keyword(s):

Monoclonal Antibody ◽

Human Monoclonal Antibody ◽

Flow Cytometric Analysis ◽

Myelogenous Leukemia ◽

Leukemia Cells ◽

Human Leukemia ◽

Lymphoid Leukemia ◽

Juxtamembrane Region

Abstract The receptor tyrosine kinase FLT3 is overexpressed in blasts of ~90% of acute myelogenous leukemia (AML) and the majority of B-lymphoid leukemia patients. Internal tandem duplications (ITDs) in the juxtamembrane region and point mutations in the kinase domain of FLT3 are found in ~37% of AML patients and are associated with a poor prognosis. We have recently developed a fully human monoclonal antibody (IMC-EB10) which binds with high affinity to FLT3 receptor on human leukemia cells. In the present study, a novel auristatin conjugate of the anti-FLT3 antibody (EB10-MMAF) was prepared using a dipeptide linker that allows for drug release inside the lysosomes of antigen-positive cells. The MMAF conjugates were stable in buffers and plasma. EB10-MMAF (drug/antibody raito = 8) was highly potent, and selectively inhibited the growth of FLT3-expressing leukemia cells with an IC50 of 0.19 nM and 0.08 nM for MV4;11 and BaF3-ITD cells (both positive for FLT3-ITD), 1.11 nM, 6.18 nM and 1.82 nM for REH , EOL-1, EM3 cells (all three positive for wild-type FLT3), and 135 nM for JM1 (negative for FLT3). An MMAF conjugate with a control antibody was not active in these cell lines (IC50s > 5.9 uM). Flow cytometric analysis with annexin V indicated that EB10-MMAF treatment induced apoptosis of leukemia cells in vitro. In vivo treatment with EB10-MMAF strongly inhibited leukemia growth and prolonged survival of mice in both EOL-1 and BaF3-ITD leukemia models. In summary, immunoconjugates composed of a fully human anti-FLT3 antibody and a potent auristatin drug may provide a valuable therapeutic approach for AML and other FLT3-positive leukemias.

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Escape from Human Monoclonal Antibody Neutralization Affects In Vitro and In Vivo Fitness of Severe Acute Respiratory Syndrome Coronavirus

The Journal of Infectious Diseases ◽

10.1086/651022 ◽

2010 ◽

Vol 201 (6) ◽

pp. 946-955 ◽

Cited By ~ 64

Author(s):

Barry Rockx ◽

Eric Donaldson ◽

Matthew Frieman ◽

Timothy Sheahan ◽

Davide Corti ◽

...

Keyword(s):

Monoclonal Antibody ◽

Severe Acute Respiratory Syndrome ◽

Human Monoclonal Antibody ◽

Antibody Neutralization

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A Human monoclonal antibody targeting scavenger receptor class B type I precludes hepatitis C virus infection and viral spread in vitro and in vivo

Hepatology ◽

10.1002/hep.24692 ◽

2011 ◽

Vol 55 (2) ◽

pp. 364-372 ◽

Cited By ~ 97

Author(s):

Philip Meuleman ◽

Maria Teresa Catanese ◽

Lieven Verhoye ◽

Isabelle Desombere ◽

Ali Farhoudi ◽

...

Keyword(s):

Monoclonal Antibody ◽

Human Monoclonal Antibody ◽

Scavenger Receptor ◽

Type I ◽

Hepatitis C Virus Infection ◽

Viral Spread ◽

Scavenger Receptor Class ◽

Class B

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Heterosubtypic Protection Conferred by the Human Monoclonal Antibody PN-SIA28 against Influenza A Virus Lethal Infections in Mice

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00118-15 ◽

2015 ◽

Vol 59 (5) ◽

pp. 2647-2653 ◽

Cited By ~ 1

Author(s):

Miguel Retamal ◽

Yacine Abed ◽

Chantal Rhéaume ◽

Francesca Cappelletti ◽

Nicola Clementi ◽

...

Keyword(s):

Monoclonal Antibody ◽

Body Weight ◽

Influenza Virus ◽

Influenza A ◽

Human Monoclonal Antibody ◽

Influenza A Viruses ◽

Virus Challenge ◽

Influenza A H1n1

ABSTRACTPN-SIA28 is a human monoclonal antibody (Hu-MAb) targeting highly conserved epitopes within the stem portion of the influenza virus hemagglutinin (HA) (N. Clementi, et al, PLoS One 6:e28001, 2011,http://dx.doi.org/10.1371/journal.pone.0028001). Previousin vitrostudies demonstrated PN-SIA28 neutralizing activities against phylogenetically divergent influenza A subtypes. In this study, the protective activity of PN-SIA28 was evaluated in mice inoculated with lethal influenza A/WSN/33 (H1N1), A/Quebec/144147/09 (H1N1)pdm09, and A/Victoria/3/75 (H3N2) viruses. At 24 h postinoculation (p.i.), animals received PN-SIA28 intraperitoneally (1 or 10 mg/kg of body weight) or 10 mg/kg of unrelated Hu-MAb (mock). Body weight loss and mortality rate (MR) were recorded for 14 days postinfection (p.i.). Lung viral titers (LVT) were determined at day 5 p.i. In A/WSN/33 (H1N1)-infected groups, all untreated and mock-receiving mice died, whereas MRs of 87.5% and 25% were observed in mice that received PN-SIA28 1 and 10 mg/kg, respectively. In influenza A(H1N1) pdm09-infected groups, an MR of 75% was recorded for untreated and mock-treated groups, whereas the PN-SIA28 1-mg/kg and 10-mg/kg groups had rates of 62.5% and 0%, respectively. In A/Victoria/3/75 (H3N2)-infected animals, untreated and mock-treated animals had MRs of 37.5% and 25%, respectively, and no mortalities were recorded after PN-SIA28 treatments. Accordingly, PN-SIA28 treatments significantly reduced weight losses and resulted in a ≥1-log reduction in LVT compared to the control in all infection groups. This study confirms that antibodies targeting highly conserved epitopes in the influenza HA stem region, like PN-SIA28, not only neutralize influenza A viruses of clinically relevant subtypesin vitrobut also, more importantly, protect from a lethal influenza virus challengein vivo.

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Monoclonal Antibody-Based Screening Assay for Factor Inhibiting Hypoxia-Inducible Factor Inhibitors

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057108318800 ◽

2008 ◽

Vol 13 (6) ◽

pp. 494-503 ◽

Cited By ~ 23

Author(s):

Sang-Hyeup Lee ◽

Jeong Hee Moon ◽

Eun Ah Cho ◽

Seong-Eon Ryu ◽

Myung Kyu Lee

Keyword(s):

Monoclonal Antibody ◽

High Throughput Screening ◽

Transcriptional Activity ◽

Hypoxia Inducible Factor ◽

Enzyme Linked Immunosorbent Assay ◽

Sensitive Assay ◽

Screening Assay ◽

Hypoxic Conditions

The factor-inhibiting hypoxia-inducible factor (FIH) hydroxylates the asparagine 803 (Asn803) residue of the hypoxia-inducible factor 1α (HIF-1α), and the modification abrogates the transcriptional activity of HIF-1α. Because FIH is more active on HIF-1α than prolyl hydroxylase domain proteins under hypoxic conditions, its inhibitors have potential to be developed as anti-ischemic drugs targeting normal cells stressed by hypoxia. In this study, the authors developed the first monoclonal antibody, SHN-HIF1α, specifically to Asn803 hydroxylated HIF-1α and a sensitive assay system for FIH inhibitors using the monoclonal antibody (Mab). SHN-HIF1α showed 740 times higher affinity to the Asn803 hydroxylated HIF-1α peptide than the unmodified one. An enzyme-linked immunosorbent assay—based system using SHN-HIF1α displayed at least 30 times more sensitivity than previous methods for screening FIH inhibitors and was easily applicable to develop a high-throughput screening system. SHN-HIF1α also showed an Asn803 hydroxylation-dependent specificity to HIF-1α in cells. Taken together, the results suggest that it may be used to analyze the in vivo and in vitro activities of FIH inhibitors. ( Journal of Biomolecular Screening 2008:494-503)

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