靶向FcRL5 的CAR-T联合sPD1-Ig过表达对多发性骨髓瘤的治疗作用及机制研究

Identification of novel chimeric antigen receptor-expressing T cell (CAR-T) targets for multiple myeloma (MM) is needed considering that the antigen targets available have some limitation. FcRL5 is overexpressed on the surface of myeloma cells but absent in essential normal cells. Our preliminary data showed that second generation of FcRL5-redirected CAR-T cells efficiently lysed MM cells with surface FcRL5 expression but spared essential normal cells in vitro, suggesting that FcRL5 may be a candidate target. In addition, the immune inhibitory molecule PD-1 was found to be upregulated in FcRL5-specific CAR-T cells upon stimulation with MM cells. Based on these preliminary data, we propose a new therapeutic approach for MM by combining FcRL5-specific CAR-T cells with blockade of PD-1 signaling. In this project, we will firstly evaluate the anti-MM efficacy and the safety of FcRL5-specific CAR-T cells，and then investigate the impact on the effect of FcRL5-specific CAR-T cells mediated by blockade of PD-1/PD-L1 signaling through overexpression of soluble PD1-Ig, and also attempt to explore the molecular mechanism responsible for the action of PD-1/PD-L1 signaling on FcRL5-specific CAR-T cells. The outcome of our project will validate the anti-myeloma efficacy of FcRL5-redirected CAR-T cells in combination with sPD-1-Ig overexpression and unravel the mechanism of action; and its clinical translation will provide a novel therapeutic strategy for relapsed and refractory MM.

多发性骨髓瘤的现有CAR-T靶标均有不足之处，因此需要探寻新靶标。FcRL5在骨髓瘤细胞表面过度表达，但不表达于重要的正常细胞。我们的预实验结果显示二代FcRL5 CAR-T在体外显著杀伤骨髓瘤细胞但不损伤重要的正常细胞，提示FcRL5可作为潜在的靶标。同时，我们发现骨髓瘤细胞能诱导FcRL5 CAR-T上调免疫抑制分子PD-1。基于这些，我们提出FcRL5 CAR-T联合PD-1/PD-L1信号阻断的新方案。本项目首先评估FcRL5 作为CAR-T靶标的有效性和安全性；进一步明确以共表达sPD-1-Ig的新策略阻断PD-1/PD-L1信号对FcRL5 CAR-T抗骨髓瘤作用的影响，并探寻PD-1/PD-L1信号影响FcRL5 CAR-T效应的分子机制。本项目的实施能明确FcRL5 CAR-T联合sPD1-Ig过表达治疗骨髓瘤的效应及机制，其临床转化将为复发难治性骨髓瘤提供新的治疗策略。

本项目基于嵌合抗原受体修饰的T细胞（CAR-T）疗法治疗骨髓瘤的现状以及前期实验结果提出“FcRL5 CAR-T联合sPD1-Ig过表达是治疗骨髓瘤的新策略”这一科学假设。本项目首先从有效性和安全性角度研究FCRL5是否适合作为CAR-T治疗骨髓瘤的新靶标，进一步测试通过过表达sPD1-Ig 的方式阻断PDL1/PD1信号对FCRL5 CAR-T抗骨髓瘤效应的影响，最后探索了PD-1基因敲低影响FcRL5 CAR-T细胞抗骨髓瘤作用的分子机制。我们首先发现FCRL5在骨髓瘤患者的肿瘤细胞表面高度表达且其表达水平与BCMA表达无明显相关性。然后我们将构建的CAR表达载体导入T细胞后成功制备出FCRL5 CAR-T细胞，发现该CAR-T细胞在体外有效杀伤表达FCRL5的骨髓瘤细胞，并分泌多种细胞因子和增殖，同时骨髓瘤患者自体来源的FCRL5 CAR-T细胞也能有效识别和杀伤BCMA阳性或者低表达的自体肿瘤细胞。FCRL5 CAR-T细胞在多种免疫缺陷小鼠荷瘤模型包括BCMA抗原逃逸模型中表现出明显的抗骨髓瘤效应。我们还构建了双特异性FCRL5/BCMA 双特异性CAR并证实双特异性CAR-T细胞可以识别单独表达BCMA或者FCRL5的肿瘤细胞，在安全性方面，FCRL5在正常细胞的表达仅限于B细胞，FCRL5 CAR-T对B细胞仅有微弱的杀伤作用，同时引入icaspase-9自杀基因可提升安全性。另外，我们发现体内存在的两种可溶性形式的FCRL5蛋白对FCRL5 CAR-T的体外效应功能具有不同的影响。过表达sPD1-Ig 显著提升了FCRL5 CAR-T的体内外抗肿瘤效应。在分子机制研究方面，我们发现在CAR-T细胞中敲低PD-1基因可以下调与免疫耗竭相关的核受体NR4A1的表达水平。该项目的研究结果为过继性输注携带sPD1-Ig的FcRL5 CAR-T治疗复发难治性骨髓瘤提供了理论和实验学依据，具有潜在的临床转化前景。