肿瘤细胞治疗研究
英文原题：Discovery and preclinical development of a therapeutically active nanobody-based chimeric antigen receptor targeting human CD22.
靶向 B 细胞限制性抗原 CD19、CD20 或 CD22 的嵌合抗原受体（CAR）T 细胞疗法可对部分 B 细胞恶性肿瘤产生强效临床缓解，但复发仍然常见。
靶向B细胞限制性抗原CD19、CD20或CD22的嵌合抗原受体（CAR）T细胞疗法可使部分B细胞恶性肿瘤患者获得强效临床应答，但复发仍很常见。骆驼科单域抗体（sdAb，又称纳米抗体）比多数CAR使用的单链可变片段（scFv）更小、更简单且更易重组，但已报道的sdAb-CAR较少。因此，我们旨在识别一种具有治疗活性、靶向人CD22的sdAb-CAR。用CD22蛋白免疫成年美洲驼，构建sdAb-cDNA文库并进行噬菌体淘选，获得20余种表位和结合特性各异的sdAb。在Jurkat细胞中表达CD22-sdAb-CAR后，观察到不同程度的CD22特异性反应性，但其与抗体亲和力无相关性。将CD28跨膜结构改为CD8设计，可提高体外CAR持久性和表达。不同候选CD22-sdAb-CAR体外诱导的CD22依赖性CAR-T扩增相似，但只有靶向膜近端表位的CD22-sdAb-CAR能激活直接细胞毒性杀伤，并在淋巴瘤异种移植模型中延长生存。基于盲法异种移植研究中改善的生存结局，研究选定一种领先的CD22sdCAR-T；在高剂量实验中，其完全应答率与基准短连接肽m971-scFv CAR-T相当。最后，免疫组化和流式细胞术证实该候选CD22-sdAb具有组织和细胞层面的特异性。本文完整报告了一种新型CD22sdCAR疗法的临床前开发。
Chimeric antigen receptor (CAR) T cell therapies targeting B cell-restricted antigens CD19, CD20, or CD22 can produce potent clinical responses for some B cell malignancies, but relapse remains common. Camelid single-domain antibodies (sdAbs or nanobodies) are smaller, simpler, and easier to recombine than single-chain variable fragments (scFvs) used in most CARs, but fewer sdAb-CARs have been reported. Thus, we sought to identify a therapeutically active sdAb-CAR targeting human CD22. Immunization of an adult Llama glama with CD22 protein, sdAb-cDNA library construction, and phage panning yielded >20 sdAbs with diverse epitope and binding properties. Expressing CD22-sdAb-CAR in Jurkat cells drove varying CD22-specific reactivity not correlated with antibody affinity. Changing CD28- to CD8-transmembrane design increased CAR persistence and expression in vitro . CD22-sdAb-CAR candidates showed similar CD22-dependent CAR-T expansion in vitro, although only membrane-proximal epitope targeting CD22-sdAb-CARs activated direct cytolytic killing and extended survival in a lymphoma xenograft model. Based on enhanced survival in blinded xenograft studies, a lead CD22sdCAR-T was selected, achieving comparable complete responses to a benchmark short linker m971-scFv CAR-T in high-dose experiments. Finally, immunohistochemistry and flow cytometry confirm tissue and cellular-level specificity of the lead CD22-sdAb. This presents a complete report on preclinical development of a novel CD22sdCAR therapeutic.
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