An Fc Domain Protein–Small Molecule Conjugate as an Enhanced Immunomodulator

expressed protein ligation;conjugation;half-life;not listed

Chiang, Meng-JungCole, Philip A. ;

Johns Hopkins University

关键词: expressed protein ligation;    conjugation;    half-life;    not listed;   

Others  :  https://jscholarship.library.jhu.edu/bitstream/handle/1774.2/37161/CHIANG-DISSERTATION-2014.pdf?sequence=1&isAllowed=y

瑞士|英语

来源: JOHNS HOPKINS DSpace Repository

【 摘 要 】

Small molecules as well as protein drugs have demonstrated tremendous success in clinical applications. Despite this success, their off-target effects diminish their pharmacologic value. Decades ago, innovations in molecular biology and recombinant DNA technology have generated a new revolution of protein-based therapeutics, with treatments applicable to a wide range of diseases including cancers, cardiovascular disorders, and autoimmune diseases. Protein-based drug therapy has shown the ability to achieve high potency and specificity towards cell surface. However, protein drugs usually lack the homogenicity and precise chemical synthetic manipulation, which are characteristic of small molecules. Chemical modification of proteins has historically suffered from a lack of site-specificity, uneven stoichiometry and the challenge of general applicability. In an effort to take full advantage of the best aspects of both protein drug and small molecule drug fields, this thesis research focuses on the development of a protein-small molecule conjugate, which we hope will be a precise and effective therapeutic in targeting immune-related diseases.Activation of adenosine 2a receptor (A2AR) suppresses the immune system, especially in T cells. A2AR has thus been identified as a potential target to treat inflammation and autoimmune diseases. A2AR agonists usually suffer from short half-lives and high toxicity in central nervous system and cardiac tissues. Here we have tethered CGS 21680 (CGS), a potent but short-lived synthetic A2AR agonist in vivo, to the immunoglobulin Fc domain using expressed protein ligation (EPL)　secreted from Sf9 cell, to form the bivalent protein-small molecule conjugate Fc-CGS. Fc-CGS is stabilized by glycosylation and forms a dimer. By conjugating CGS to the Fc domain, not only is CGS half-life increased, but also its potency and specificity by Fc/FcγRI binding. In cell-based assays, Fc-CGS can trigger cAMP production intracellularly, has lower IC50 than CGS in suppressing IL-2 production at longer time points, stable in both cell culture medium and mouse serum. In vivo, we used a pneumonitis disease mouse model, to show that administering Fc-CGS prolonged survival rates. Moreover, Fc-CGS can be detected at the area of inflammatory pulmonary tissue by immunohistochemistry staining 18 days post intraperitoneal injection.Taken together, our data suggest Fc-CGS shows enhanced pharmacokinetic and pharmacodynamic performance. The conjugation approach EPL can be a novel technique to generate protein-small molecule bivalent conjugate for pharmacological development in the future.