Preparation Of Fab Fragments Derived From Anti-EGFRv? Monoclonal Antibody And Its Radioimmunoimaging In Tumor-bearing Nude Mice

As one of the most crucial epidermal growth factor receptor?EGFR?variants,EGFRv? can be detected in various tumors but rarely in normal tissues,making it an ideal target for prognosis diagnosis or immune therapy.Recently,our team has developed a novel anti-EGFRv? monoclonal antibody?mAb?,4G1,with high specificity and affinity for EGFRv?.Radiolabeled 4G1 has been validated as a promising molecular probe to detect EGFRv? expression in tumors by single-photon emission computed tomography/computed tomography imaging.To overcome shortcomings associated with the whole antibody,including long-term retention,circulation and enhanced permeability and retention effects,the Fab fragment of 4G1 was generated,labeled with ¹³¹I and evaluated in vitro and in vivo to test its potential application in molecular imaging.Objective:To prepare Fab fragments derived from the anti-EGFRv? monoclonal antibody 4G1 and to evaluate its radioimmunoimaging potential after labeling with ¹³¹ I.Part I:Preparation and identification of Fab fragments derived from anti-EGFRv? monoclonalMethods:Intact mAb 4G1 was first digested by immobilized ficin and then purified through a protein A column to generate the Fab fragment.Next,sodium dodecyl sulfate-polyacrylamide gel electrophoresis?SDS-PAGE?was perpormed to detect the molecular weight.Western blot,fluorescence assay,and flow cytometry were performed to detect the specifity.Enzyme-linked immunosorbent assay were performed to detect affinity of Fab.Parallel studies were also performed with intact 4G1 as a positive control.Results:The molecular weight of Fab was determined to be about40kDa by SDS-PAGE.Western blot results showed that Fab and 4G1specifically recognized EGFRv ? protein expressed in F98_npEGFRv? cells at145 kDa,but could not recognize the wild type EGFR protein expressed in F98_EGFR cells.Flow cytometry results showed that the binding rates of 4G1and Fab to F98_{np EGFRv?} cells were 99.6%and 59.9%,respectively.The binding rates of 4G1 and Fab to F98_EGFR cells were 2.80%and 2.06%,respectively.Immunofluorescence results showed that Fab and 4G1 can specifically bind to F98_npEGFRv? cells and can hardly bind to F98_EGFR cells.The Kd values of Fab and 4G1 were?9.40±0.89?nmol/L and?0.29±0.03?nmol/L,respectively,and the Fab affinity was lower than that of parent antibody 4G1.Part II:Radiolabeling and Identification of Fab fragments derived from an anti-EGFRv? monoclonalMethods:The Fab and 4G1 were labeled with ¹³¹I by chloramine-T method.The labeling rate were determined by paper chromatography.Then the labeling product was immediately separated and purified by PD midiTrap G-25 column.The radiochemical purity were determined by paper chromatography.Results:The labeling rates of ¹³¹I for Fab and 4G1 were?86.63±2.39?%and?90.04±3.11?%,respectively.The radiochemical purities were?97.71±0.28?%and?96.59±1.25?%.Part ?:Comparative analysis of biodistribution and radioimmunoimaging of ¹³¹I-Fab in tumor-bearing nude miceMethods:F98_npEGFRv? cells?overexpressing EGFRv? protein?were injected subcutaneously into the right hind limb of a 4-week-old female BALB/c nude mice to construct a nude mouse model bearing an EGFRv?-positive tumor.When the tumor volume reached approximately500 mm³,¹³¹I-Fab or ¹³¹I-4G1 were injected into the tumor-bearing nude mice through the tail vein and biodistribution studies were performed at 2 h,4 h,24 h,48 h;when the tumor volume reached 1000 mm³,¹³¹I-Fab or¹³¹I-4G1 were injected into the tumor-bearing nude mice through the tail vein and radioimmunoimaging was performed at 2 h,4 h,24 h,and 48 h.Results:Tumor uptake of ¹³¹I-Fab was?7.50±0.77?%ID/g at 2 h,?3.99±0.4?%ID/g at 4 h,?0.91±0.09?%ID/g at 24 h,and?0.4±0.03?%ID/g at48 h.Tumor uptake of ¹³¹I-4G1 was?14.09±2.59?%ID/g at 2 h,?17.50±4.05?%ID/g at 4 h,?10.53±1.06?%ID/g at 24 h,and?9.08±0.60?%ID/g at 48 h.Tumor uptake of ¹³¹I-Fab was decreased at each time point compared to ¹³¹I-4G1,but it was cleared faster in normal organs..Radioimmunoimages showed that ¹³¹I-Fab was clearly visualized at 2 h after injection and metabolized faster than ¹³¹I-4G1.Conclusion:¹³¹I-labeled Fab fragment derived from an anti-EGFRv? antibody is expected to be a radioimmunological agent for EGFRv?-positive tumors detection.