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Construction of a human monoclonal antibody against bFGF for suppression of NSCLC.

ABSTRACT: Compelling evidence implicates that overexpression of basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor 1 (FGFR1) in non-small cell lung cancer (NSCLC) drives tumor progression, can serve as prognostic biomarkers or therapeutic targets for NSCLC patients. But at present, we still lack of effective drugs for bFGF. The preparation of monoclonal antibodies against bFGF or to understand its mechanism of action is urgently need. Previously, we used hybridoma technology to produce a murine anti-bFGF monoclonal antibody (E12). However, E12 carries risks of heterogeneity and immunogenicity. In the present work, we produced three humanized variants (H1L1, H2L2 and H3L3) based on E12 by substituting residues in or near the complementarity-determining region (CDR). In addition, we thoroughly explored VH/VL domain combinations to simulate full-length IgG1 antibodies using computational protein design. H3L3 was selected for further study, as it demonstrated the best humanization and strongest affinity for bFGF. Specially, humanization of H3L3's light chain and heavy chain were 100% and 98.89%, respectively. The FGF2 neutralizing effect of H3L3 were confirmed by ELISA. We also found that H3L3 can effectively suppress the growth and angiogenesis of cancer through reduce the phosphorylation of AKT and MAPK. Moreover, H3L3 dramatically reduced tumor size and micro-vessel density in nude mice. Altogether, our study demonstrates that H3L3 exerts anti-tumor effects by impeding NSCLC development.

SUBMITTER: Wang S

PROVIDER: S-EPMC5995934 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Construction and sequencing analysis of scFv antibody fragment derived from monoclonal antibody against norfloxacin (Nor155).

Project description:Norfloxacin belongs to the group of fluoroquinolone antibiotics which has been approved for treatment in animals. However, its residues in animal products can pose adverse side effects to consumer. Therefore, detection of the residue in different food matrices must be concerned. In this study, a single chain variable fragment (scFv) that recognizes norfloxacin antibiotic was constructed. The cDNA was synthesized from total RNA of hybridoma cells against norfloxacin. Genes encoding VH and VL regions of monoclonal antibody against norfloxacin (Nor155) were amplified and size of VH and VL fragments was 402 bp and 363 bp, respectively. The scFv of Nor155 was constructed by an addition of (Gly4Ser)3 as a linker between VH and VL regions and subcloned into pPICZ?A, an expression vector of Pichia pastoris. The sequence of scFv Nor155 (GenBank No. AJG06891.1) was confirmed by sequencing analysis. The complementarity determining regions (CDR) I, II, and III of VH and VL were specified by Kabat method. The obtained recombinant plasmid will be useful for production of scFv antibody against norfloxacin in P. pastoris and further engineer scFv antibody against fluoroquinolone antibiotics.

| S-EPMC6296615 | biostudies-literature

Production of Monoclonal Antibody against Human Nestin.

Project description:We have employed a peptide-based antibody generation protocol for producing antibody against human nestin. Using a 12-mer synthetic peptide from repetitive region of human nestin protein devoid of any N- or O-glyco-sylation sequences, we generated a mouse monoclonal antibody capable of recognizing human, mouse, bovine, and rat nestin. A wide variety of nestin proteins ranging from 140-250 kDa was detected by this antibody. This antibody is highly specific and functional in applications such as ELISA, flow cytometry, immunocytochemistry, and Western blot assays.

| S-EPMC3558152 | biostudies-literature

A monoclonal antibody against human MUDENG protein.

Project description:MUDENG (mu-2-related death-inducing gene, MuD) encodes a predicted ∼54-kDa protein in humans, considered to be involved in trafficking proteins from endosomes toward other membranous compartments as well as in inducing cell death. Here we report on the generation of a mouse monoclonal antibody (MAb) against the middle domain of human (h) MuD. This IgG sub 1 MAb, named M3H9, recognizes residues 244-326 in the middle domain of the MuD protein. Thus, the MuD proteins expressed in an astroglioma cell line and primary astrocytes can be detected by the M3H9 MAb. We showed that M3H9 MAb can be useful in enzyme-linked immunosorbent assay (ELISA) and immunoblot experiments. In addition, M3H9 MAb can detect the expression of the MuD protein in formalin-fixed, paraffin-embedded mouse ovary and uterus tissues. These results indicate that the MuD MAb M3H9 could be useful as a new biomarker of hereditary spastic paraplegia and other related diseases.

| S-EPMC3733323 | biostudies-literature

A broadly neutralizing human monoclonal antibody is effective against H7N9.

Project description:Emerging strains of influenza represent a significant public health threat with potential pandemic consequences. Of particular concern are the recently emerged H7N9 strains which cause pneumonia with acute respiratory distress syndrome. Estimates are that nearly 80% of hospitalized patients with H7N9 have received intensive care unit support. VIS410, a human antibody, targets a unique conserved epitope on influenza A. We evaluated the efficacy of VIS410 for neutralization of group 2 influenza strains, including H3N2 and H7N9 strains in vitro and in vivo. VIS410, administered at 50 mg/kg, protected DBA mice infected with A/Anhui/2013 (H7N9), resulting in significant survival benefit upon single-dose (-24 h) or double-dose (-12 h, +48 h) administration (P < 0.001). A single dose of VIS410 at 50 mg/kg (-12 h) combined with oseltamivir at 50 mg/kg (-12 h, twice daily for 7 d) in C57BL/6 mice infected with A/Shanghai 2/2013 (H7N9) resulted in significant decreased lung viral load (P = 0.002) and decreased lung cytokine responses for nine of the 11 cytokines measured. Based on these results, we find that VIS410 may be effective either as monotherapy or combined with antivirals in treating H7N9 disease, as well as disease from other influenza strains.

| S-EPMC4568252 | biostudies-literature

Monoclonal antibody against the N-terminal end of human plasma fibronectin.

Project description:Purified human plasma fibronectin was digested with cathepsin G and the degradation products were tested for reactivity towards a monoclonal antibody. In an immunoblotting assay, after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the digestion products, the 85 000-Mr and 72 000-Mr gelatin- and heparin-binding fragments as well as the N-terminal 30 000-Mr heparin-binding fragment reacted with the antibody, whereas the 64 000-Mr gelatin- and heparin-binding fragment did not. In enzyme immunoassay the antibody reacted with intact fibronectin and the 30 000-Mr fragment but not with a 40 000-Mr gelatin-binding fragment. The alignment of the binding domains in these fragments and in the intact molecule [Vartio (1982) Eur. J. Biochem. 123, 223-233] localizes the antigenic determinant to the 21 000 Da N-terminal Staphylococcus aureus-binding region of fibronectin.

| S-EPMC1152374 | biostudies-other

Human-monoclonal-antibody therapy protects nonhuman primates against advanced Lassa fever.

Project description:There are no approved treatments for Lassa fever, which is endemic to the same regions of West Africa that were recently devastated by Ebola. Here we show that a combination of human monoclonal antibodies that cross-react with the glycoproteins of all four clades of Lassa virus is able to rescue 100% of cynomolgus macaques when treatment is initiated at advanced stages of disease, including up to 8 d after challenge.

| S-EPMC5719877 | biostudies-literature

A human monoclonal antibody with neutralizing activity against highly divergent influenza subtypes.

Project description:The interest in broad-range anti-influenza A monoclonal antibodies (mAbs) has recently been strengthened by the identification of anti-hemagglutinin (HA) mAbs endowed with heterosubtypic neutralizing activity to be used in the design of "universal" prophylactic or therapeutic tools. However, the majority of the single mAbs described to date do not bind and neutralize viral isolates belonging to highly divergent subtypes clustering into the two different HA-based influenza phylogenetic groups: the group 1 including, among others, subtypes H1, H2, H5 and H9 and the group 2 including, among others, H3 subtype. Here, we describe a human mAb, named PN-SIA28, capable of binding and neutralizing all tested isolates belonging to phylogenetic group 1, including H1N1, H2N2, H5N1 and H9N2 subtypes and several isolates belonging to group 2, including H3N2 isolates from the first period of the 1968 pandemic. Therefore, PN-SIA28 is capable of neutralizing isolates belonging to subtypes responsible of all the reported pandemics, as well as other subtypes with pandemic potential. The region recognized by PN-SIA28 has been identified on the stem region of HA and includes residues highly conserved among the different influenza subtypes. A deep characterization of PN-SIA28 features may represent a useful help in the improvement of available anti-influenza therapeutic strategies and can provide new tools for the development of universal vaccinal strategies.

| S-EPMC3230632 | biostudies-literature

Monoclonal antibody therapies against SARS-CoV-2.

Project description:Monoclonal antibodies (mAbs) targeting the spike protein of SARS-CoV-2 have been widely used in the ongoing COVID-19 pandemic. In this paper, we review the properties of mAbs and their effect as therapeutics in the pandemic, including structural classification, outcomes in clinical trials that led to the authorisation of mAbs, and baseline and treatment-emergent immune escape. We show how the omicron (B.1.1.529) variant of concern has reset treatment strategies so far, discuss future developments that could lead to improved outcomes, and report the intrinsic limitations of using mAbs as therapeutic agents.

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Isolation of a human monoclonal antibody with strong neutralizing activity against diphtheria toxin.

Project description:We isolated a human monoclonal antibody against diphtheria toxin (DT). It bound to fragment B with a binding activity (Kd) of 3.01 nM. The neutralizing activity assayed by the rabbit skin test was estimated to be 73,600 IU/g. This could be used as a therapeutic drug against DT in place of the traditional equine sera.

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A Human Monoclonal Antibody against Hepatitis B Surface Antigen with Potent Neutralizing Activity.

Project description:We describe the production and characterization of human monoclonal antibodies (mAb) specific for the major hepatitis B virus (HBV) S protein. The mAbs, two IgG1? and one IgG1?, were secreted by B-cell clones obtained from peripheral blood mononuclear cells (PBMC) of one person convalescent from acute hepatitis B and one vaccinated individual. The former recognized a denaturation-insensitive epitope within the p24 protein whereas the latter recognized a denaturation-sensitive, conformational epitope located within the HBsAg common "a" determinant. This mAb, denominated ADRI-2F3, displayed a very high protective titer of over 43,000 IU/mg mAb and showed an extremely potent neutralizing activity in the in vitro model of HBV infection using primary hepatocytes from Tupaia belangeri as target. Recombinant variable heavy and light domain sequences derived from mAb ADRI-2F3 were cloned into eukaryotic expression vectors and showed identical fine specificity and 1 log10 higher titer than the original IgG1?. It is envisaged that such mAb will be able to efficiently prevent HBV reinfection after liver transplantation for end-stage chronic HBV infection or infection after needle-stick exposure, providing an unlimited source of valuable protective anti-HBs antibody.

| S-EPMC4414269 | biostudies-literature

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