PO.CL06.03 · 临床研究
表面CADM1（细胞黏附分子1）丰度可预测CADM1-GGFG-Exatecan ADC在骨肉瘤中的载荷递送及体外疗效，有助于优选候选药物
Surface CADM1 (Cell Adhesion Molecule 1) abundance predicts payload delivery and in-vitro efficacy of CADM1-GGFG-Exatecan ADC in osteosarcoma, aiding optimal candidate selection
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摘要 Abstract
中文摘要
背景：骨肉瘤的ADC开发因缺乏有效、可靶向的表面抗原而受阻。我们此前将CADM1这一细胞表面黏附分子鉴定为一个有前景的治疗靶点，并生成了三种CADM1导向的ADC（CADM1-Tesirine、CADM1-PEG-Exatecan和CADM1-GGFG-Exatecan），它们具有不同的连接子-载荷组合。为选择用于临床转化的最佳候选药物，我们在多种体外模型中评估了靶点结合及靶点依赖性的细胞毒性。
方法：采用共聚焦显微镜观察ADC经细胞各区室的转运及其随时间的溶酶体定位。使用CRISPR/Cas9生成CADM1敲除（KO）骨肉瘤细胞系（SaOS2和OS31），并通过流式细胞术、Western印迹及质谱（MS）验证。通过MS定量细胞表面CADM1水平，并与IncuCyte平台上实时活细胞成像测得的ADC细胞毒性（IC50值）相关联。在九种骨肉瘤细胞系中进行Spearman相关分析，以评估三种构建体的CADM1表达与ADC效力之间的关系。
结果：共聚焦时间进程成像显示ADC从质膜（30分钟）转运至溶酶体（6小时）及胞质弥散（24小时），而在CADM1-KO细胞中不存在此现象，证实了靶点介导的内化及溶酶体加工。在SaOS2和OS31骨肉瘤细胞系中实现了CRISPR/Cas9介导的CADM1敲除。通过流式细胞术、Western印迹及质谱证实了CADM1表达的缺失，显示表面蛋白完全耗竭。CADM1缺失显著降低了ADC细胞毒性（SaOS2 CADM1-KO中IC50 = 77.41 nM，而SaOS2中为0.014 nM），证实了靶点特异性活性。在九种骨肉瘤模型中，CADM1表面丰度与药物效力密切相关，尤以CADM1-GGFG-Exatecan最为显著（Spearman ρ = -0.98，p = 0.001）。这些数据将CADM1鉴定为ADC疗效的决定因素，并凸显了其他ADC组分的贡献。
结论：这些数据证明了所生成的CADM1靶向ADC具有强效的、靶点依赖性的活性，验证了CADM1作为合理治疗靶点的地位。其中，CADM1-GGFG-Exatecan在IC50与靶点表达之间显示出最强的相关性，支持其选择性的细胞毒性递送。体内疗效及毒理学研究正在进行中，以最终确定候选药物的选择。
查看英文原文 English abstract
Background: ADC development in osteosarcoma has been hindered by the absence of effective, targetable surface antigens. We previously identified CADM1, a cell-surface adhesion molecule, as a promising therapeutic target and generated three CADM1-directed ADCs (CADM1-Tesirine, CADM1-PEG-Exatecan, and CADM1-GGFG-Exatecan) with distinct linker-payload combinations. To select the optimal candidate for clinical translation, we evaluated target engagement and target-dependent cytotoxicity across multiple in vitro models.
Methods: Confocal microscopy was used to visualize ADC trafficking through cellular compartments and its lysosomal localization over time. CADM1 knockout (KO) osteosarcoma cell lines (SaOS2 and OS31) were generated using CRISPR/Cas9 and validated by flow cytometry, Western blotting, and mass spectrometry (MS). Cell-surface CADM1 levels were quantified by MS and correlated with ADC cytotoxicity (IC₅₀ values) measured by real-time live-cell imaging on the IncuCyte platform. Spearman correlation analyses across nine osteosarcoma cell lines assessed the relationship between CADM1 expression and ADC potency for all three constructs.
Results: Confocal time-course imaging showed ADC trafficking from the plasma membrane (30 min) to lysosomes (6 h) and cytoplasmic dispersion (24 h), absent in CADM1-KO cells, confirming target-mediated internalization and lysosomal processing. CRISPR/Cas9-mediated knockout of CADM1 was achieved in SaOS2 and OS31 osteosarcoma cell lines. Loss of CADM1 expression was confirmed by flow cytometry, Western blotting, and mass spectrometry, showing complete depletion of surface protein. CADM1 loss markedly reduced ADC cytotoxicity (IC₅₀ = 77.41 nM in SaOS2 CADM1-KO vs 0.014 nM in SaOS2), confirming on-target activity. Across nine osteosarcoma models, CADM1 surface abundance strongly correlated with drug potency, most notably for CADM1-GGFG-Exatecan (Spearman ρ = -0.98, p = 0.001). These data identify CADM1 as a determinant of ADC efficacy and highlight the contribution of other ADC components.
Conclusions: These data demonstrate potent, target-dependent activity of the generated CADM1-targeted ADCs, validating CADM1 as a rational therapeutic target. Among them, CADM1-GGFG-Exatecan showed the strongest correlation between IC₅₀ and target expression, supporting selective cytotoxic delivery. In vivo efficacy and toxicology studies are ongoing to finalize candidate selection.
利益披露 Disclosure
C. Longo, None..
A. Bahadir, None..
Y. Yanhua, None..
Z. Xu, None.