Target Discovery Division, A&G Pharmaceutical, Inc., Columbia, Maryland, United States of America.
Department of Pharmaceutical Sciences, University of Maryland, Baltimore School of Pharmacy, Baltimore, Maryland, United States of America.
PLoS One. 2021 Jan 27;16(1):e0246197. doi: 10.1371/journal.pone.0246197. eCollection 2021.
Antibody-drug conjugates (ADC) are effective antibody-based therapeutics for hematopoietic and lymphoid tumors. However, there is need to identify new targets for ADCs, particularly for solid tumors and cancers with unmet needs. From a hybridoma library developed against cancer cells, we selected the mouse monoclonal antibody 33B7, which was able to bind to, and internalize, cancer cell lines. This antibody was used for identification of the target by immunoprecipitation and mass spectrometric analysis, followed by target validation. After target validation, 33B7 binding and target positivity were tested by flow cytometry and western blot analysis in several cancer cell lines. The ability of 33B7 conjugated to saporin to inhibit in vitro proliferation of PTFRN positive cell lines was investigated, as well as the 33B7 ADC in vivo effect on tumor growth in athymic mice. All flow cytometry and in vitro internalization assays were analyzed for statistical significance using a Welsh's T-test. Animal studies were analyzed using Two-Way Analysis of Variance (ANOVA) utilizing post-hoc Bonferroni analysis, and/or Mixed Effects analysis. The 33B7 cell surface target was identified as Prostaglandin F2 Receptor Negative Regulator (PTGFRN), a transmembrane protein in the Tetraspanin family. This target was confirmed by showing that PTGFRN-expressing cells bound and internalized 33B7, compared to PTGFRN negative cells. Cells able to bind 33B7 were PTGFRN-positive by Western blot analysis. In vitro treatment PTGFRN-positive cancer cell lines with the 33B7-saporin ADC inhibited their proliferation in a dose-dependent fashion. 33B7 conjugated to saporin was also able to block tumor growth in vivo in mouse xenografts when compared to a control ADC. These findings show that screening antibody libraries for internalizing antibodies in cancer cell lines is a good approach to identify new cancer targets for ADC development. These results suggest PTGFRN is a possible therapeutic target via antibody-based approach for certain cancers.
抗体药物偶联物（ADC）是治疗血液系统和淋巴系统肿瘤的有效抗体药物。然而，需要为 ADC 确定新的靶点，特别是针对实体瘤和有未满足需求的癌症。我们从针对癌细胞的杂交瘤文库中筛选出能够与癌细胞结合并内化的小鼠单克隆抗体 33B7。该抗体用于通过免疫沉淀和质谱分析鉴定靶标，然后进行靶标验证。靶标验证后，通过流式细胞术和 Western blot 分析在几种癌细胞系中测试 33B7 与 SAP 的结合和靶阳性。还研究了 33B7 与 SAP 缀合以抑制 PTFRN 阳性细胞系体外增殖的能力，以及 33B7 ADC 对裸鼠肿瘤生长的体内作用。所有流式细胞术和体外内化实验均使用 Welsh's T 检验进行统计学意义分析。动物研究使用 Two-Way Analysis of Variance (ANOVA) 进行分析，利用事后 Bonferroni 分析和/或混合效应分析。33B7 细胞表面靶标被鉴定为前列腺素 F2 受体阴性调节剂（PTGFRN），一种四跨膜蛋白家族中的跨膜蛋白。通过显示与 PTGFRN 阴性细胞相比，表达 PTGFRN 的细胞结合并内化 33B7，证实了该靶标。Western blot 分析显示能够结合 33B7 的细胞为 PTGFRN 阳性。用 33B7-saporin ADC 体外处理 PTGFRN 阳性癌细胞系可剂量依赖性地抑制其增殖。与对照 ADC 相比，33B7 缀合的 SAP 也能够阻止体内异种移植小鼠肿瘤的生长。这些发现表明，在癌细胞系中筛选内化抗体的抗体文库是鉴定 ADC 开发新癌症靶点的一种很好的方法。这些结果表明，PTGFRN 是通过抗体为基础的方法治疗某些癌症的潜在治疗靶标。
