Development of an EGFRvIII specific recombinant antibody

BACKGROUND: EGF receptor variant III (EGFRvIII) is the most common variant of the EGF receptor observed in human tumors. It results from the in frame deletion of exons 2-7 and the generation of a novel glycine residue at the junction of exons 1 and 8. This novel juxtaposition of amino acids within t...

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Autores principales: Gupta, Puja, Han, Shuang-Yin, Holgado-Madruga, Marina, Mitra, Siddhartha S, Li, Gordon, Nitta, Ryan T, Wong, Albert J
Formato: Texto
Lenguaje:English
Publicado: BioMed Central 2010
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2959087/
https://www.ncbi.nlm.nih.gov/pubmed/20925961
http://dx.doi.org/10.1186/1472-6750-10-72
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author Gupta, Puja
Han, Shuang-Yin
Holgado-Madruga, Marina
Mitra, Siddhartha S
Li, Gordon
Nitta, Ryan T
Wong, Albert J
author_facet Gupta, Puja
Han, Shuang-Yin
Holgado-Madruga, Marina
Mitra, Siddhartha S
Li, Gordon
Nitta, Ryan T
Wong, Albert J
author_sort Gupta, Puja
collection PubMed
description BACKGROUND: EGF receptor variant III (EGFRvIII) is the most common variant of the EGF receptor observed in human tumors. It results from the in frame deletion of exons 2-7 and the generation of a novel glycine residue at the junction of exons 1 and 8. This novel juxtaposition of amino acids within the extra-cellular domain of the EGF receptor creates a tumor specific and immunogenic epitope. EGFRvIII expression has been seen in many tumor types including glioblastoma multiforme (GBM), breast adenocarcinoma, non-small cell lung carcinoma, ovarian adenocarcinoma and prostate cancer, but has been rarely observed in normal tissue. Because this variant is tumor specific and highly immunogenic, it can be used for both a diagnostic marker as well as a target for immunotherapy. Unfortunately many of the monoclonal and polyclonal antibodies directed against EGFRvIII have cross reactivity to wild type EGFR or other non-specific proteins. Furthermore, a monoclonal antibody to EGFRvIII is not readily available to the scientific community. RESULTS: In this study, we have developed a recombinant antibody that is specific for EGFRvIII, has little cross reactivity for the wild type receptor, and which can be easily produced. We initially designed a recombinant antibody with two anti-EGFRvIII single chain Fv's linked together and a human IgG1 Fc component. To enhance the specificity of this antibody for EGFRvIII, we mutated tyrosine H59 of the CDRH2 domain and tyrosine H105 of the CDRH3 domain to phenylalanine for both the anti-EGFRvIII sequence inserts. This mutated recombinant antibody, called RAb(DMvIII), specifically detects EGFRvIII expression in EGFRvIII expressing cell lines as well as in EGFRvIII expressing GBM primary tissue by western blot, immunohistochemistry (IHC) and immunofluorescence (IF) and FACS analysis. It does not recognize wild type EGFR in any of these assays. The affinity of this antibody for EGFRvIII peptide is 1.7 × 10(7 )M(-1 )as determined by enzyme-linked immunosorbent assay (ELISA). CONCLUSION: This recombinant antibody thus holds great potential to be used as a research reagent and diagnostic tool in research laboratories and clinics because of its high quality, easy viability and unique versatility. This antibody is also a strong candidate to be investigated for further in vivo therapeutic studies.
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spelling pubmed-29590872010-10-22 Development of an EGFRvIII specific recombinant antibody Gupta, Puja Han, Shuang-Yin Holgado-Madruga, Marina Mitra, Siddhartha S Li, Gordon Nitta, Ryan T Wong, Albert J BMC Biotechnol Research Article BACKGROUND: EGF receptor variant III (EGFRvIII) is the most common variant of the EGF receptor observed in human tumors. It results from the in frame deletion of exons 2-7 and the generation of a novel glycine residue at the junction of exons 1 and 8. This novel juxtaposition of amino acids within the extra-cellular domain of the EGF receptor creates a tumor specific and immunogenic epitope. EGFRvIII expression has been seen in many tumor types including glioblastoma multiforme (GBM), breast adenocarcinoma, non-small cell lung carcinoma, ovarian adenocarcinoma and prostate cancer, but has been rarely observed in normal tissue. Because this variant is tumor specific and highly immunogenic, it can be used for both a diagnostic marker as well as a target for immunotherapy. Unfortunately many of the monoclonal and polyclonal antibodies directed against EGFRvIII have cross reactivity to wild type EGFR or other non-specific proteins. Furthermore, a monoclonal antibody to EGFRvIII is not readily available to the scientific community. RESULTS: In this study, we have developed a recombinant antibody that is specific for EGFRvIII, has little cross reactivity for the wild type receptor, and which can be easily produced. We initially designed a recombinant antibody with two anti-EGFRvIII single chain Fv's linked together and a human IgG1 Fc component. To enhance the specificity of this antibody for EGFRvIII, we mutated tyrosine H59 of the CDRH2 domain and tyrosine H105 of the CDRH3 domain to phenylalanine for both the anti-EGFRvIII sequence inserts. This mutated recombinant antibody, called RAb(DMvIII), specifically detects EGFRvIII expression in EGFRvIII expressing cell lines as well as in EGFRvIII expressing GBM primary tissue by western blot, immunohistochemistry (IHC) and immunofluorescence (IF) and FACS analysis. It does not recognize wild type EGFR in any of these assays. The affinity of this antibody for EGFRvIII peptide is 1.7 × 10(7 )M(-1 )as determined by enzyme-linked immunosorbent assay (ELISA). CONCLUSION: This recombinant antibody thus holds great potential to be used as a research reagent and diagnostic tool in research laboratories and clinics because of its high quality, easy viability and unique versatility. This antibody is also a strong candidate to be investigated for further in vivo therapeutic studies. BioMed Central 2010-10-07 /pmc/articles/PMC2959087/ /pubmed/20925961 http://dx.doi.org/10.1186/1472-6750-10-72 Text en Copyright ©2010 Gupta et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Gupta, Puja
Han, Shuang-Yin
Holgado-Madruga, Marina
Mitra, Siddhartha S
Li, Gordon
Nitta, Ryan T
Wong, Albert J
Development of an EGFRvIII specific recombinant antibody
title Development of an EGFRvIII specific recombinant antibody
title_full Development of an EGFRvIII specific recombinant antibody
title_fullStr Development of an EGFRvIII specific recombinant antibody
title_full_unstemmed Development of an EGFRvIII specific recombinant antibody
title_short Development of an EGFRvIII specific recombinant antibody
title_sort development of an egfrviii specific recombinant antibody
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2959087/
https://www.ncbi.nlm.nih.gov/pubmed/20925961
http://dx.doi.org/10.1186/1472-6750-10-72
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