Anti-PEG antibody for the quantification and targeting of PEGylated compounds and affinity-evolution by somatic hypermutaiton

• Main Authors: Yu-Cheng Su, 蘇昱誠
• Other Authors: Steve Roffler
• Format: Others
• Language: en_US
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/03316727046853384847

博士 === 國立陽明大學 === 微生物及免疫學研究所 === 101 === PEGylation is a mature technology for modification of compounds to improve their pharmacokinetic index. To successfully launch the PEGylated compounds to the clinic, the development of accurate methods for the quantitative analysis of pharmacokinetic parameters in animals and patients is very important. In this study, we produced two second-generation monoclonal antibodies (mAbs) to PEG (AGP4, IgM and 3.3, IgG) that specifically bind to the PEG backbone with more sensitive measurement for PEGylated compounds. A sandwich ELISA in which AGP403.3-biotin were employed as the capture/detection antibodies for sensitive quantitative measurement of several PEGyalted compounds. The assay also tolerated the presence of human serum and free PEG molecules. Since more and more PEGylated nanodevices are also employed in nanotechnology, we sought to enhance the affinity of anti-PEG antibodies by a directed protein evolution system based on the AID-mediated SHM. In the second part, a controllable mouse AID (loxP-mAID) gene cassette was staly expressed in anti-PEG hybridoma cells. The expression of AID is down-regulated in differentiated plasma cells and hybridomas. Many studies showed that forced expression of AID allowed protein evolution in several cell lines. Therefore, we expected that the forced expression of mAID in anti-PEG hybridomas could introduce mutation at V region of Ig genes and then allow antibody maturation in vitro. In addition, many PEGylated drugs have been approved by FDA, to investigate whether anti-PEG antibodies can serve as an adaptor for universal tumor-specific delivery of PEGylated compounds, especially PEG-nanoparticles. In the third part, we constructed bispecific antibodies composed of the anti-PEG antibody (E11) fused to single chain disulfide-stabilized variable fragments (scdsFv) that recognize human epidermal growth factor receptor (EGFR) (11F8 scdsFv), human HER2/neu (human C6ML3-9 scdsFv) and TAG-72 tumor antigens (humanized CC49 scdsFv). The anti-PEG portion of the bispecific antibody binds to the backbone of PEG chains present on the surface of the nanoparticles whereas the other arm of the antibody confers tissue targeting selectivity. Dual-binding specificities to PEG and tumor-associated antigens of these bispecific antibodies were demonstrated by using ELISA and FACS. These bispecific antibodies may assist the specific uptake of PEGylated lipoDox and Doxisome to cancer cells.