Limits...
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.

Show MeSH

Related in: MedlinePlus

Affinity determination of RAbDMvIII by Saturation ELISA. Calibration curve of the binding of RAbDMvIII and RAbvIII recombinant antibodies to coated EGFRvIII in the ELISA. A) The concentration range for RAbDMvIII and RAbvIII was 20 to 0.02 ug/ml (x axis) and the absorbance was read at 450 nm (Y axis). The assay was analyzed twice. B) Saturation ELISA shows the Scatchard plot of the binding of RAbDMvIII to EGFRvIII. The resulting data is plotted as ratio of α/c (y axis) as function of alpha (x axis) where α is the fraction of bound antibody sites and c is the concentration of free antigen. The slope is -K which is the affinity constant of antibody and that is 1.7 × 107 liters/mole (M-1).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2959087&req=5

Figure 3: Affinity determination of RAbDMvIII by Saturation ELISA. Calibration curve of the binding of RAbDMvIII and RAbvIII recombinant antibodies to coated EGFRvIII in the ELISA. A) The concentration range for RAbDMvIII and RAbvIII was 20 to 0.02 ug/ml (x axis) and the absorbance was read at 450 nm (Y axis). The assay was analyzed twice. B) Saturation ELISA shows the Scatchard plot of the binding of RAbDMvIII to EGFRvIII. The resulting data is plotted as ratio of α/c (y axis) as function of alpha (x axis) where α is the fraction of bound antibody sites and c is the concentration of free antigen. The slope is -K which is the affinity constant of antibody and that is 1.7 × 107 liters/mole (M-1).

Mentions: To determine the affinity constant of the antibody, we used the method described by Friguet et al. (1984) [33]. It is necessary that the concentration of the antibody used in the ELISA should be equal to or less than the value of dissociation constant of the antibody. Thus a preliminary ELISA was performed to deduce the dissociation constant of the antibody. From the preliminary ELISA, the dissociation constant of RAbvIII and RAbDMvIII is calculated to be approximately 1.7 μM and 886 nM, respectively, against EGFRvIII peptide (Figure 3A). This result suggests that RAbDMvIII did not lose its affinity after mutation as RAbDMvIII has a lower dissociation constant than RAbvIII.


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)

Affinity determination of RAbDMvIII by Saturation ELISA. Calibration curve of the binding of RAbDMvIII and RAbvIII recombinant antibodies to coated EGFRvIII in the ELISA. A) The concentration range for RAbDMvIII and RAbvIII was 20 to 0.02 ug/ml (x axis) and the absorbance was read at 450 nm (Y axis). The assay was analyzed twice. B) Saturation ELISA shows the Scatchard plot of the binding of RAbDMvIII to EGFRvIII. The resulting data is plotted as ratio of α/c (y axis) as function of alpha (x axis) where α is the fraction of bound antibody sites and c is the concentration of free antigen. The slope is -K which is the affinity constant of antibody and that is 1.7 × 107 liters/mole (M-1).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Affinity determination of RAbDMvIII by Saturation ELISA. Calibration curve of the binding of RAbDMvIII and RAbvIII recombinant antibodies to coated EGFRvIII in the ELISA. A) The concentration range for RAbDMvIII and RAbvIII was 20 to 0.02 ug/ml (x axis) and the absorbance was read at 450 nm (Y axis). The assay was analyzed twice. B) Saturation ELISA shows the Scatchard plot of the binding of RAbDMvIII to EGFRvIII. The resulting data is plotted as ratio of α/c (y axis) as function of alpha (x axis) where α is the fraction of bound antibody sites and c is the concentration of free antigen. The slope is -K which is the affinity constant of antibody and that is 1.7 × 107 liters/mole (M-1).
Mentions: To determine the affinity constant of the antibody, we used the method described by Friguet et al. (1984) [33]. It is necessary that the concentration of the antibody used in the ELISA should be equal to or less than the value of dissociation constant of the antibody. Thus a preliminary ELISA was performed to deduce the dissociation constant of the antibody. From the preliminary ELISA, the dissociation constant of RAbvIII and RAbDMvIII is calculated to be approximately 1.7 μM and 886 nM, respectively, against EGFRvIII peptide (Figure 3A). This result suggests that RAbDMvIII did not lose its affinity after mutation as RAbDMvIII has a lower dissociation constant than RAbvIII.

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