Construction Of Long-term/Targeting Protein Pharmaceuticals Through Molecular Modification Technology

Molecule modification technology is a method of chemical conjugation of therapeutic proteins and antibodies produced by biological methods to solve or alleviate the problems in application process. Wherein, the protein PEGylation and antibody drug conjugates technology are two important aspects of molecular modification technology. On the basis of previous studies, we have explored innovative development of N-terminal hydrazide PEGylation and antibody fragments drug conjugates (AFDC) strategy, constructing long-term/targeting pharmaceuticals.PEGylation, including non-specific and site-directed ways, is a well-established and validated strategy to increase the stability, in vivo plasma retention time, and efficacy of protein pharmaceutics come together with the reduction of immunogenicity and hydrophobicity. Site-directed conjugation by PEG-aldehyde is the most widely used method for N-terminal modification, however, the generation of multi-modified products is inevitable due to lysine chemistry which always leads to difficulties in purification and quantification procedure. In this study, we developed a specific PEGylation strategy through the periodation of N-terminus of interferon beta-1b (IFN-β-1b) followed by the coupling of PEG-hydrazide. The prolonged elimination half-life and significantly diminished immunogenicity of PEG hydrazide-modified protein indicated an effective process in improving the properties of pharmacology and immunogenicity of IFN-β-1b. We further conducted comparisons on selectivity, velocity, yield and pharmacokinetics of the two methods. Results demonstrated that the hydrazide-based conjugation was a highly specific coupling reaction that only produced homogeneous N-terminal mono-PEGylated conjugate, while heterogeneous multi-modified products were generated in aldehyde-based process. In addition, fairly higher PEGylation yield was presented in the hydrazide conjugation compared with that of aldehyde strategy. These investigations supplied a practical approach for site specific modification of proteins with N-terminal serine or threonine to achieve improved homogeneity of conjugates as well as enhanced pharmacological properties.Conventional preparation strategies for antibody-drug conjugates (ADCs) result in heterogeneous products with various molecular size and species. In this study, we developed a homogenous preparation strategy by site-specific conjugation of the anticancer drug with antibody fragment. The model drug doxorubicin (DOX) was coupled to the Fab’ fragment of anti-CD20 IgG at its permissive sites through a heterotelechelic PEG linker, generating an antibody fragment-drug conjugate (AFDC). Anti-CD20 IgG was digested and reduced specifically with β-mercaptoethylamine to generate Fab’fragment with two free mercapto groups in its hinge region. Meanwhile, DOX was conjugated with a-succinimidylsuccinate ω-maleimide polyethylene glycol (NHS-PEG-MAL) to form MAL-PEG-DOX, which was subsequently linked to the free mercapto containing Fab’ fragment to form a Fab’-PEG-DOX conjugate. The dual site-specific bioconjugation was achieved through the combination of highly selective reduction of IgG and the introduction of heterotelechelic PEG linker. The resulting AFDC provides an utterly homogeneous product, with a definite ratio of one fragment with two drugs. Laser confocal microscopy and cell ELISA revealed that the AFDC could accumulate in the antigen-positive Daudi tumor cell. In addition, the Fab’-PEG-DOX retained appreciable targeting ability and improved anti-tumor activity, demonstrating excellent therapeutic effect on lymphoma mice model for better cure rate and significantly reduced side effects. The results in this study show that site-specific reduction of IgG and conjugation to the resulting Fab’ fragment is an applicable and efficient strategy for preparing antibody drug conjugates with definite composition to achieve improved antitumor activity.