양친성 링커 합성 및 세포독성 약물과의 생물결합을 통한 신규 항체-약물 접합체 개발

Abstract

Antibody drug conjugate (ADC) is a new class of antibody therapeutics. In ADC, antibody acts as a homing molecule to target cancer cell. After penetrating into tumor cell, a drug molecule is released to deteriorate tumor cell. For an effective ADC, linker hydrophilicity can play a key role in potency, stability and efficacy. It can determine DAR (drug-to-antibody ratio), influence aggregation, and affect penetration into tumor cells. Many linkers are hydrophobic, and when they are usually conjugated with hydrophobic drugs, ADCs tend to form aggregates and show faster clearance rate in systemic circulation. We developed an amphiphilic oxime linker by conjugating aldehyde group of D-glucuronic acid with amino oxy group of amino maleimide. This conjugation was carried out in an acetate buffer (pH 4.5) at room temperature for overnight. The reaction mixture was fed to a C18 RP-HPLC system and the oxime linker peak fraction was isolated for ESI-MS analysis. The result showed the linker MW of 388 Da, identical to the theoretical MW. Next, DM1 (N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine), a cytotoxic drug with a thiol group, was conjugated with maleimide group in the oxime linker. The oxime linker-DM1 complex was fed to the same RP-HPLC system to fraction collect the DM1-linker peak, which was then evaporated to remove acetonitrile and to reduce the volume. DM1 hydrophobicity was reduced as demonstrated by a peak shift in the RP-HPLC chromatogram as well as a reduction in the calculated Clog P value from 4.18 (DM1) to 2.17 (oxime-DM1 complex). The NHS-activated oxime-DM1 complex was randomly conjugated to lysine residues of an antibody (Herceptin®) through amine coupling. Through HIC (butyl)-HPLC and LC-QTOF analysis, we can find that synthesized ADCs are homogeneous in its molecular entity and four linker-drug molecules are conjugated to one antibody molecule. In cell cytotoxicity assay, the ADC shows more efficient antiproliferation activity than Herceptin®. Improved hydrophilicity of the oxime linker would allow higher DAR and improve the ADC’s penetration into breast tumor cells (SK-BR-3). In conclusion, the present study suggests that ADCs using the amphiphilic carbohydrate oxime linker induces lower aggregation and high tumor penetration.