Forsythe Neil L, Maynard Heather D
Department of Chemistry and Biochemistry and California NanoSystems Institute, 607 Charles E. Young Drive East, University of California, Los Angeles, CA 90095-1569.
Polym Chem. 2021 Mar 7;12(9):1217-1223. doi: 10.1039/d0py01579b. Epub 2021 Feb 11.
Antibodies and antigen binding fragments (FABs) are widely used as therapeutics and conjugated polymers can enhance the properties of these important biomolecules. However, limitations to the selectivity and stability of current conjugation methodologies can inhibit the exploration of new antibody-polymer conjugates. Herein, we describe a new strategy for the synthesis of these conjugates that forms a stable thioether bond and can be directly incorporated into an atom transfer radical polymerization (ATRP) initiator. Specifically, a bis-sulfone alkyl bromide initiator was synthesized and utilized in the activators generated by electron transfer (AGET) ATRP of ethylene glycol methacrylate and trehalose methacrylate to form the respective polymers. The trehalose polymer was then irreversibly inserted into the disulfide bonds of Herceptin and Herceptin FAB after mild reduction to form the conjugates with quantitative conversions as verified by Western Blot and mass spectrometry after cleavage of the polymer. The binding of the Herceptin and Herceptin Fab conjugates to the receptor was investigated by indirect ELISA (enzyme-linked immunosorbent assay) and the EC50's were 0.90 and 2.74 nM, respectively, compared to Herceptin (0.26 nM) and the Fab (0.56 nM). The conjugates were subjected to heating studies at a constant 75 °C, the temperature determined in a heat ramp to be the threshold of stability for the antibody and FAB; the trehalose polymer was found to considerably increase the thermal stability of both Herceptin and Herceptin Fab. This work provides a new way to prepare polymer-antibody/Fab conjugates utilizing bis-sulfone end groups installed by atom transfer radical polymerization of the functionalized initiators and a way to stabilize these important molecules by conjugation to trehalose polymers.
抗体和抗原结合片段（FABs）被广泛用作治疗剂，共轭聚合物可以增强这些重要生物分子的性能。然而，当前共轭方法的选择性和稳定性的局限性可能会阻碍新型抗体-聚合物共轭物的探索。在此，我们描述了一种合成这些共轭物的新策略，该策略形成稳定的硫醚键，并且可以直接并入原子转移自由基聚合（ATRP）引发剂中。具体而言，合成了一种双砜烷基溴引发剂，并将其用于甲基丙烯酸乙二醇酯和甲基丙烯酸海藻糖的电子转移活化剂生成的电子转移（AGET）ATRP中，以形成各自的聚合物。然后，在温和还原后，将海藻糖聚合物不可逆地插入赫赛汀和赫赛汀FAB的二硫键中，以形成共轭物，如通过聚合物裂解后的蛋白质免疫印迹和质谱法所验证的那样，具有定量转化率。通过间接ELISA（酶联免疫吸附测定）研究了赫赛汀和赫赛汀Fab共轭物与受体的结合，与赫赛汀（0.26 nM）和Fab（0.56 nM）相比，其EC50分别为0.90和2.74 nM。对共轭物在75°C恒定温度下进行加热研究，该温度在热梯度中确定为抗体和FAB的稳定性阈值；发现海藻糖聚合物可大大提高赫赛汀和赫赛汀Fab的热稳定性。这项工作提供了一种利用功能化引发剂的原子转移自由基聚合安装的双砜端基制备聚合物-抗体/Fab共轭物的新方法，以及一种通过与海藻糖聚合物共轭来稳定这些重要分子的方法。
