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Development of an EGFRvIII specific recombinant antibody.

Gupta P, Han SY, Holgado-Madruga M, Mitra SS, Li G, Nitta RT, Wong AJ - BMC Biotechnol. (2010)

Bottom Line: 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.It does not recognize wild type EGFR in any of these assays.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Brain Tumor Research Laboratories, Department of Neurosurgery, and Program in Cancer Biology, Stanford University Medical Center, Stanford, CA 94305, USA. ajwong@stanford.edu

ABSTRACT

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 × 10⁷ 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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Detection of EGFRvIII by RAbDMvIII Immunohistochemistry. (A-C) Immunohistochemical staining of U87vIII & A431 mouse tumor xenografts and (D-G) Glioblastoma section. A) Staining of A431 and U87vIII with polyclonal EGFRvIII antibody. B) RAbDMvIII antibody staining, C) RAbCD133antibody staining. D) Glioblastoma 08-023 staining with RAbDMvIII. E) Glioblastoma 08-023 staining with polyclonal EGFRvIII ab. F) Glioblastoma 04-34497 stained with polyclonal EGFRvIII antibody and G) RAbDMvIII staining of 04-34497.
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Figure 6: Detection of EGFRvIII by RAbDMvIII Immunohistochemistry. (A-C) Immunohistochemical staining of U87vIII & A431 mouse tumor xenografts and (D-G) Glioblastoma section. A) Staining of A431 and U87vIII with polyclonal EGFRvIII antibody. B) RAbDMvIII antibody staining, C) RAbCD133antibody staining. D) Glioblastoma 08-023 staining with RAbDMvIII. E) Glioblastoma 08-023 staining with polyclonal EGFRvIII ab. F) Glioblastoma 04-34497 stained with polyclonal EGFRvIII antibody and G) RAbDMvIII staining of 04-34497.

Mentions: Immunohistochemistry (IHC) is another sensitive biochemical test to monitor for the expression of EGFRvIII. To determine if the RAbDMvIII antibody could be used for IHC, we used mouse tumor xenografts from HC2 and A431 cells (Figure 6A-C) and GBM cases (Figure 6D-G). The tumor xenografts contain both HC2 tissue (top) and A431 tissue (bottom). RAbDMvIII shows strong positivity to the HC2 section which is comparable in intensity to slides stained with the positive control anti-EGFRvIII polyclonal antibody (Figure 6A &6B). In addition, the RAbDMvIII antibody did not stain the A431 tissue sections (Figure 6A). The recombinant antibody RAbCD133 was used as a negative antibody control (Figure 6C). We extended our findings to two GBM cases, one positive for EGFRvIII (08023) and one negative (04-34497). Consistent with the mouse tumor xenografts, the RAbDMvIII antibody only stained the GBM positive for EGFRvIII and had no cross reactivity to the negative GBM case (Figure 6D &6F). As a control, we also stained these sections with affinity purified polyclonal anti-EGFRvIII and observed similar results (Figure 6E and 6G).


Development of an EGFRvIII specific recombinant antibody.

Gupta P, Han SY, Holgado-Madruga M, Mitra SS, Li G, Nitta RT, Wong AJ - BMC Biotechnol. (2010)

Detection of EGFRvIII by RAbDMvIII Immunohistochemistry. (A-C) Immunohistochemical staining of U87vIII & A431 mouse tumor xenografts and (D-G) Glioblastoma section. A) Staining of A431 and U87vIII with polyclonal EGFRvIII antibody. B) RAbDMvIII antibody staining, C) RAbCD133antibody staining. D) Glioblastoma 08-023 staining with RAbDMvIII. E) Glioblastoma 08-023 staining with polyclonal EGFRvIII ab. F) Glioblastoma 04-34497 stained with polyclonal EGFRvIII antibody and G) RAbDMvIII staining of 04-34497.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2959087&req=5

Figure 6: Detection of EGFRvIII by RAbDMvIII Immunohistochemistry. (A-C) Immunohistochemical staining of U87vIII & A431 mouse tumor xenografts and (D-G) Glioblastoma section. A) Staining of A431 and U87vIII with polyclonal EGFRvIII antibody. B) RAbDMvIII antibody staining, C) RAbCD133antibody staining. D) Glioblastoma 08-023 staining with RAbDMvIII. E) Glioblastoma 08-023 staining with polyclonal EGFRvIII ab. F) Glioblastoma 04-34497 stained with polyclonal EGFRvIII antibody and G) RAbDMvIII staining of 04-34497.
Mentions: Immunohistochemistry (IHC) is another sensitive biochemical test to monitor for the expression of EGFRvIII. To determine if the RAbDMvIII antibody could be used for IHC, we used mouse tumor xenografts from HC2 and A431 cells (Figure 6A-C) and GBM cases (Figure 6D-G). The tumor xenografts contain both HC2 tissue (top) and A431 tissue (bottom). RAbDMvIII shows strong positivity to the HC2 section which is comparable in intensity to slides stained with the positive control anti-EGFRvIII polyclonal antibody (Figure 6A &6B). In addition, the RAbDMvIII antibody did not stain the A431 tissue sections (Figure 6A). The recombinant antibody RAbCD133 was used as a negative antibody control (Figure 6C). We extended our findings to two GBM cases, one positive for EGFRvIII (08023) and one negative (04-34497). Consistent with the mouse tumor xenografts, the RAbDMvIII antibody only stained the GBM positive for EGFRvIII and had no cross reactivity to the negative GBM case (Figure 6D &6F). As a control, we also stained these sections with affinity purified polyclonal anti-EGFRvIII and observed similar results (Figure 6E and 6G).

Bottom Line: 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.It does not recognize wild type EGFR in any of these assays.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Brain Tumor Research Laboratories, Department of Neurosurgery, and Program in Cancer Biology, Stanford University Medical Center, Stanford, CA 94305, USA. ajwong@stanford.edu

ABSTRACT

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 × 10⁷ 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.

Show MeSH
Related in: MedlinePlus