Impact of Drug Conjugation and Loading on Target Antigen Binding and Cytotoxicity in Cysteine Antibody-Drug Conjugates
Impact of Drug Conjugation and Loading on Target Antigen Binding and Cytotoxicity in Cysteine Antibody-Drug Conjugates
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DOI：
10.1021/acs.molpharmaceut.0c00873
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发表时间：
2021-01-20
影响因子：
4.9
通讯作者：
Meyer, Debra M.
中科院分区：
文献类型：
--
作者：
Nadkarni, Durgesh, V;Lee, Jamie;Meyer, Debra M.
抗体-药物偶联物（adc）由靶标特异性抗体组成，该抗体通过连接物与药物共价偶联。adc被设计用于递送细胞毒性药物（有效载荷），特别是对癌细胞，同时最小化全身毒性。传统的半胱氨酸偶联通常导致ADC分子的形成，其中包含2、4、6和8种载药物种的异质混合物。混合物的药抗比（DAR）代表这些物种的加权平均值。在本报告中，我们研究了有效负载的疏水性和总体药物负载对ADC物种的体外结合和细胞毒性的影响。采用半胱氨酸偶联法制备了几种adc。从ADC混合物中纯化出具有不同DAR值的ADC物种，并用标准分析技术对其进行表征。使用免疫测定法，酶联免疫吸附试验（ELISA）评估这些ADC物种的靶抗原结合。使用基于细胞的细胞毒性试验评估效力。这些结构-功能研究有助于更好地了解影响ADC物种体外靶结合和细胞毒性的因素。ELISA结果显示，与未偶联抗体或DAR接近4的异质参考ADC相比，含有高DAR的疏水有效载荷的ADC物种具有较低的靶标结合。在类似的实验条件下，与亲水性有效载荷共轭的adc对目标结合没有显着影响。在基于细胞的细胞毒性试验中，ADC物种的细胞毒性随药物负荷水平的增加而增加。
Antibody-drug conjugates (ADCs) consist of a target-specific antibody that is covalently conjugated to a drug via a linker. ADCs are designed to deliver cytotoxic drugs (payloads), specifically to cancer cells, while minimizing systemic toxicity. Conventional cysteine conjugation typically results in the formation of ADC molecules containing a heterogeneous mixture of 2, 4, 6, and 8 drug-loaded species. The drug-to-antibody ratio (DAR) of the mixture represents the weighted average of these species. In this report, we have investigated the impact of the hydrophobicity of payloads and the overall drug loading on the in vitro binding and cytotoxicity of ADC species. Several ADCs were prepared by conventional cysteine conjugation using different payloads. ADC species with different DAR values were purified from the ADC mixture and characterized by standard analytical techniques. These ADC species were evaluated for target antigen binding using an immunoassay, enzyme-linked immunosorbent assay (ELISA). The potency was assessed using a cell-based cytotoxicity assay. These structure-function studies lead to a better understanding of factors that impact the in vitro target binding and cytotoxicity of ADC species. ADC species containing hydrophobic payloads with high DAR were found to have lower target binding by ELISA compared to that of the unconjugated antibody or the heterogeneous reference ADC with DAR similar to 4. Under similar assay conditions, the ADCs conjugated to hydrophilic payloads did not show a significant impact on the target binding. The cytotoxic potency of ADC species increased with increasing level of drug loading in the cell-based cytotoxicity assay.