肿瘤细胞治疗研究
英文原题：Nanoscale, antigen encounter-dependent, IL-12 delivery by CAR T cells plus PD-L1 blockade for cancer treatment.
RB-312 是首个临床相关的产品，整合了 CRISPRa 系统，实现非基因编辑且可逆地上调内源性基因表达，从而促进 CAR-T 细胞的持久性并增强对表达 HER2 肿瘤的疗效。
背景：过去十年，用于治疗血液系统恶性肿瘤的嵌合抗原受体（CAR）T细胞疗法取得巨大进展。但要进一步提高疗效并降低毒性，仍需解决关键局限，以确保CAR-T细胞持续存留、迁移至肿瘤部位、抵抗恶劣的肿瘤微环境（TME），并将毒性控制在局部。最后一点可通过肿瘤抗原识别后按环境条件释放因子实现，这些因子能够将免疫抑制性TME转变为有利于免疫排斥的环境。方法：我们采用CRISPR激活（CRISPRa）系统构建靶向HER2的CAR-T细胞（RB-312），通过对IL-12两个内源亚基p35和p40进行条件性转录，诱导IL-12异二聚体表达。该回路由两个慢病毒构建体组成。第一个构建体（HER2-TEV）表达抗人表皮生长因子受体2（HER2）CAR单链可变片段（scFv），并含CD28和CD3ζ共刺激结构域；其与烟草蚀刻病毒（TEV）蛋白酶及分别靶向白细胞介素（IL）-12A和IL12B转录起始位点（TSS）的两条单导RNA（sgRNA）相连。第二个构建体（LdCV）编码T细胞活化连接蛋白（LAT），并通过TEV可切割序列（TCS）与核酸酶失活的化脓链球菌Cas9（dCas9）-VP64-p65-Rta（VPR）融合。CAR活化使HER2-TEV靠近LdCV，进而释放dCas9并使其进入细胞核。该条件性回路可诱导IL-12/p70异二聚体的条件性、可逆表达。我们在体外将RB-312与缺少IL-12 sgRNA的对照（cRB-312）及传统HER2 CAR（convCAR）比较。结果：可诱导CRISPRa系统激活内源性IL-12表达，增强体外继发性干扰素（IFN）生成、细胞毒性和CAR-T细胞增殖，并延长体内持续存留；与传统CAR-T产品相比，该系统对HER2阳性FaDu口咽癌细胞生长的抑制更强。外周循环中未检测到系统性IL-12。此外，联合程序性死亡配体1（PD-L1）阻断显示出强劲协同效应。结论：RB-312是首个具有临床相关性的产品，采用非基因编辑CRISPRa系统可逆上调内源基因表达，从而促进CAR-T细胞持续存留，并增强其对HER2表达肿瘤的疗效。自分泌、可逆且局部微尺度的IL-12生成可降低因子逸出肿瘤部位及全身毒性的风险。
BACKGROUND: Chimeric antigen receptor (CAR)-T cell therapies for the treatment of hematological malignancies experienced tremendous progress in the last decade. However, essential limitations need to be addressed to further improve efficacy and reduce toxicity to assure CAR-T cell persistence, trafficking to the tumor site, resistance to an hostile tumor microenvironment (TME), and containment of toxicity restricting production of powerful but potentially toxic bioproducts to the TME; the last could be achieved through contextual release upon tumor antigen encounter of factors capable of converting an immune suppressive TME into one conducive to immune rejection. METHODS: We created an HER2-targeting CAR-T (RB-312) using a clustered regularly interspaced short palindromic repeats (CRISPR) activation (CRISPRa) system, which induces the expression of the IL-12 heterodimer via conditional transcription of its two endogenous subunits p35 and p40. This circuit includes two lentiviral constructs. The first one (HER2-TEV) expresses an anti-human epidermal growth factor receptor 2 (HER2) CAR single chain variable fragment (scFv), with CD28 and CD3z co-stimulatory domains linked to the tobacco etch virus (TEV) protease and two single guide RNAs (sgRNA) targeting the interleukin (IL)-12A and IL12B transcription start site (TSS), respectively. The second construct (LdCV) encodes linker for activation of T cells (LAT) fused to nuclease-deactivated Streptococcus Pyogenes Cas9 (dCas9)-VP64-p65-Rta (VPR) via a TEV-cleavable sequence (TCS). Activation of the CAR brings HER2-TEV in close proximity to LdCV releasing dCas9 for nuclear localization. This conditional circuit leads to conditional and reversible induction of the IL-12/p70 heterodimer. RB-312 was compared in vitro to controls (cRB-312), lacking the IL-12 sgRNAs and conventional HER2 CAR (convCAR). RESULTS: The inducible CRISPRa system activated endogenous IL-12 expression resulting in enhanced secondary interferon (FN)- production, cytotoxicity, and CAR-T proliferation in vitro, prolonged in vivo persistence and greater suppression of HER2 + FaDu oropharyngeal cancer cell growth compared to the conventional CAR-T cell product. No systemic IL-12 was detected in the peripheral circulation. Moreover, the combination with programmed death ligand (PD-L1) blockade demonstrated robust synergistic effects. CONCLUSIONS: RB-312, the first clinically relevant product incorporating a CRISPRa system with non-gene editing and reversible upregulation of endogenous gene expression that promotes CAR-T cells persistence and effectiveness against HER2-expressing tumors. The autocrine effects of reversible, nanoscale IL-12 production limits the risk of off-tumor leakage and systemic toxicity.
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