Humanization and mammalian expression of a murine monoclonal antibody against Venezuelan equine encephalitis virus

Hu, Weigang; Chau, Damon; Wu, Josh; Jager, Scott; Nagata, Les P.

doi:10.1016/j.vaccine.2007.01.034

Cited by 23 publications

(13 citation statements)

References 12 publications

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“…The humanization of murine antibodies, however, has not been reported yet. Different approaches have been developed to humanize murine mAbs [25], [28], [34], [35], [36]. Despite the development of molecular display technologies and transgenic animals for the generation of fully human antibodies, CDR grafting to transfer murine CDRs onto the human antibody FRs, remains an attractive and proven strategy for overcoming therapeutic deficiencies of murine antibodies.…”

Section: Discussionmentioning

confidence: 99%

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Humanization and Characterization of an Anti-Ricin Neutralization Monoclonal Antibody

Yin

Chau

et al. 2012

PLoS ONE

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Ricin is regarded as a high terrorist risk for the public due to its high toxicity and ease of production. Currently, there is no therapeutic or vaccine available against ricin. D9, a murine monoclonal antibody developed previously in our laboratory, can strongly neutralize ricin and is therefore a good candidate for humanization. Humanization of D9 variable regions was achieved by a complementarity-determining region grafting approach. The humanized D9 (hD9) variable regions were further grafted onto human heavy and light chain constant regions to assemble the complete antibody gene. A foot-and-mouth-disease virus-derived 2A self-processing sequence was introduced between heavy and light chain DNA sequences to cleave the recombinant protein into a functional full-length antibody molecule from a single open reading frame driven by a single promoter in an adenoviral vector. After expression in mammalian cells and purification, the hD9 was demonstrated to have equimolar expression of the full-length antibody heavy and light chains. More importantly, the hD9 exhibited high affinity to ricin with KD of 1.63 nM, comparable to its parental murine D9 (2.55 nM). In a mouse model, intraperitoneal (i.p.) administration of hD9, at a low dose of 5 µg per mouse, 4 hours after the i.p. challenge with 5×LD50 ricin was found to rescue 100% of the mice. In addition, administered 6 hours post-challenge, hD9 could still rescue 50% of the mice. The hD9 has the potential to be used for prophylactic or therapeutic purposes against ricin poisoning.

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Section: Discussionmentioning

confidence: 99%

“…The humanization of D9 Fv was done by CDR-grafting as described previously with minor modification [28]. Briefly, D9 CDR canonical structures were determined based on identification of unique residues both in CDRs and FRs using the online free program “AbCheck - Antibody Sequence Test” (http://www.bioinf.org.uk/abs/seqtest.html).…”

Section: Methodsmentioning

confidence: 99%

Humanization and Characterization of an Anti-Ricin Neutralization Monoclonal Antibody

Yin

Chau

et al. 2012

PLoS ONE

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“…The linker can be either proteases, such as furin or a self-cleavage sequence, such as 2A. Although the furin linker has worked effectively in a human Fab construct with producing balanced Fd and LC [33], it has not worked well in our full antibody construct [34]. One possible reason is the furin linker sequence in a full antibody structure is probably inaccessible to the furin.…”

Section: Discussionmentioning

confidence: 99%

Generation of a Recombinant Full-Length Human Antibody Binding to Botulinum Neurotoxin A

Jager

Chau

et al. 2009

Appl Biochem Biotechnol

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In order to develop a recombinant full-length human anti-botulinum neurotoxin A (BoNT/A) antibody, human peripheral blood mononuclear cells (PBMC) were collected from three healthy volunteers and induced for BoNT/A-specific immune response by in vitro immunization. The genes encoding human Fd fragment, consisting of antibody heavy chain variable region and constant region 1 with the genes encoding antibody light chain, were cloned from the immunized PBMC. Afterwards, one combinatory human antigen-binding fragment (Fab) library was constructed using a lambda phage vector system. The size of the constructed library was approximately 10(5) Escherichia coli transformants. After screening the library by BoNT/A antigen using a plaque lifting with immunostaining approach, 55 clones were identified as positive. The Fab gene of the most reactive clone exhibiting particularly strong BoNT/A binding signal was further subcloned into a full-length human IgG1 antibody gene template in an adenoviral expression vector, in which the heavy and light chains were linked by a foot-and-mouth-disease virus-derived 2A self-cleavage peptide under a single promoter. After the full-length human IgG1 was expressed in mammalian cells and purified with protein L column, sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the heavy and light chains of the antibody were cleaved completely. The affinity expressed as the dissociation constant (K(d)) for the recombinant human antibody to bind to BoNT/A was determined by indirect enzyme-linked immunosorbent assay and results confirmed that the recombinant full-length human antibody retained BoNT/A-binding specificity with K(d) value of 10(-7) M.

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“…A single open reading frame, encoding the full‐length, humanized anti‐VEEV antibody, was previously cloned into a recombinant adenovirus serotype 5 vector (pAd5‐anti‐VEEV [44]) using the pAd‐Easy system (Stratagene, Mississauga, ON, Canada, http://www.agilent.com). Custom good laboratory practice‐grade pAd5‐anti‐VEEV was produced by Norgen Biotek Corp. (Thorold, ON, Canada, http://www.norgenbiotek.com).…”

Section: Methodsmentioning

confidence: 99%

“…More recently, the live‐attenuated vaccine V3526 was shown to be unsafe for use in humans [40] despite its success in rodent and nonhuman primate animal models of VEEV infection [41–43]. To address the current lack of licensed medical countermeasures to combat VEEV infection, we previously developed a humanized antibody that neutralizes VEEV [44, 45]. This antibody fully protects mice from a lethal dose of VEEV when administered 24 hours before, or after, exposure [45].…”

Section: Introductionmentioning

confidence: 99%

Engineered Mesenchymal Cells Improve Passive Immune Protection Against Lethal Venezuelan Equine Encephalitis Virus Exposure

Braid

Davies

et al. 2016

Stem Cells Translational Medicine

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Direct injection of monoclonal antibodies (mAbs) is an important strategy to immediately protect the recipient from a pathogen. This strategy is critical during natural outbreaks or after the intentional release of bio-weapons. Vaccines require weeks to become effective, which is not practical for first responders immediately deployed to an infected region. However, mAb recipients often require booster shots to maintain protection, which is expensive and impractical once the first responders have been deployed. The present study has shown, for the first time, that mesenchymal stromal cells are effective gene delivery vehicles that can significantly improve mAb-mediated immune protection in a single, intramuscular dose of engineered cells. Such a cell-based delivery system can provide extended life-saving protection in the event of exposure to biological threats using a more practical, single-dose regimen.

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Humanization and mammalian expression of a murine monoclonal antibody against Venezuelan equine encephalitis virus

Cited by 23 publications

References 12 publications

Humanization and Characterization of an Anti-Ricin Neutralization Monoclonal Antibody

Humanization and Characterization of an Anti-Ricin Neutralization Monoclonal Antibody

Generation of a Recombinant Full-Length Human Antibody Binding to Botulinum Neurotoxin A

Engineered Mesenchymal Cells Improve Passive Immune Protection Against Lethal Venezuelan Equine Encephalitis Virus Exposure

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