Omelyanenko V, Kopecková P, Gentry C, Shiah J G, Kopecek J
Department of Pharmaceutics, University of Utah, Salt Lake City, Utah 84112, USA.
J Drug Target. 1996;3(5):357-73. doi: 10.3109/10611869608996827.
The influence of different methods of binding the OV-TL16 antibody and its Fab' fragment to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer--drug (adriamycin [ADR] or meso chlorin e6 mono(N-2-aminoethylamide) (Mce6)) conjugates on the affinity of conjugates to an ovarian carcinoma (OVCAR-3) cell associated antigen was investigated. The binding of the antibody to HPMA copolymer--drug (ADR or Mce6) conjugates via amino groups resulted in conjugates which were heterogeneous in their antigen binding. Coupling, the HPMA copolymer--Mce6 conjugate to the carbohydrate region of the antibody resulted in conjugates with a more homogeneous distribution of affinity constants than conjugates prepared by linking the antibody to the polymer via amino groups. However, both methods resulted in a decrease in the affinity constant compared to the native antibody. Conjugates prepared with the Fab' frgment of the OV-TL16 antibody demonstrated a more homogenous affinity than either conjugate prepared with the whole antibody. To verify the hypothesis that the changes in the binding affinity and homogeneity are a consequence of conformational changes in the antibody structure, a series of physiocochemical methods were employed to characterize the conjugates. The excitation energy transfer between OV-TL16 antibody and drugs (ADR and Mce6) and the spectral properties of Mce6 were used to monitor the interactions between the antibody and drugs. The quenching of the intrinsic fluorescence of the antibody was also employed to study its conformational changes. An attempt has been made to correlate the biorecognition at the cellular surface with the interactions of drug with the antibody molecule and with the changes in antibody conformation.
研究了将OV-TL16抗体及其Fab'片段与N-(2-羟丙基)甲基丙烯酰胺(HPMA)共聚物-药物(阿霉素[ADR]或中氯e6单(N-2-氨基乙酰胺)(Mce6))缀合物结合的不同方法对缀合物与卵巢癌(OVCAR-3)细胞相关抗原亲和力的影响。抗体通过氨基与HPMA共聚物-药物(ADR或Mce6)缀合物的结合导致缀合物在抗原结合方面存在异质性。将HPMA共聚物-Mce6缀合物偶联到抗体的碳水化合物区域，得到的缀合物的亲和常数分布比通过氨基将抗体连接到聚合物上制备的缀合物更均匀。然而，与天然抗体相比，这两种方法都导致亲和常数降低。用OV-TL16抗体的Fab'片段制备的缀合物比用完整抗体制备的任何一种缀合物都表现出更均匀的亲和力。为了验证结合亲和力和均匀性的变化是抗体结构构象变化的结果这一假设，采用了一系列物理化学方法来表征缀合物。利用OV-TL16抗体与药物(ADR和Mce6)之间的激发能量转移以及Mce6的光谱特性来监测抗体与药物之间的相互作用。还利用抗体固有荧光的猝灭来研究其构象变化。已尝试将细胞表面的生物识别与药物与抗体分子的相互作用以及抗体构象的变化联系起来。
