靶向PD-L1胶质瘤免疫PET显像及放射免疫治疗研究

Glioblastoma (GBM) is the deadliest and most common form of glioma (WHO grade IV). The current standard of care (SOC): maximal safe surgical resection, fractionated radiation, and systemic temozolamide chemotherapy can only offer patients a median survival of 14.6 months after diagnosis and a 5-year survival rate of 0.05–4.7%. Patient treatment failure is attributed to the aggressive diffuse infiltration observed at the tumor margins and GBM cellular heterogeneity allowing resistance to radiotherapy and the cytotoxic agents...During the recently years, much progress has been made in refining immunotherapeutic approaches for the treatment of GBM, and it is likely that soon, immunotherapy will be included in SOC for GBM. Of them, The immune checkpoint, programmed cell death protein-1/ programmed cell death ligand-1 (PD-1/PD-L1) pathway has emerged as a critical target for cancer immunotherapy, and monoclonal antibodies that block either side of this inhibitory interaction have demonstrated impressive activity across a broad set of cancer subtypes including glioma. It was reported that targeting PD-L1 blockade showed durable tumor regression with less grade 3 or 4 adverse events compared with anti-CTLA-4 and anti-PD-1 blockade. Moreover, Patients with tumors expressing PD-L1 are most likely to respond to anti-PD-1/PD-L1 treatment...In our previous study, KN035, an anti-PD-L1 heavy chain antibody selected from a large camel naïve phage display nanobody library, was labeled with radionuclide 89Zr through conjugated to pisothiocyanatobenzyl-desferrioxamine (Df-Bz-NCS). The resulting probe, 89Zr-Df-KN035, showed a high affinity with PD-L1 positive cell lines, human lung adenocarcinoma cell line A549. Serial immnuo-PET imaging revealed that A549 tumors xenografts were clearly visible since 24h to 120h post-injection of 89Zr-Df-KN035, while markedly reduced accumulations of 89Zr-Df-KN035 were noticed in blocking group. ..Given the promising results of 89Zr-Df-KN035, the aim of our study is at first to develop a novel immune-PET technique using 89Zr-Df-KN035 to noninvasively determine tumor PD-L1 expression in vivo, which could help to select the patients with glioma who are most likely to benefit from anti-PD-1/PD-L1 treatment and monitor PD-L1 expression response to therapy. On this basis, to further investigate the PD-L1 targeting radioimmunotherapy（RIT）for glioma with 177Lu-DOTA-KN035 in vitro and in nude mice model bearing glioma, which could be an avenue to overcome the residue or recurrence post-surgery in patients with glioma leading to prolong the patient’s overall survival (OS). ..In this study, on one hand, 89Zr-Df-KN035 will be sequentially evaluated by in vitro cell uptake and blocking studies using PD-L1 positive glioma cell lines (for example, human GMB cell line LN229). Next, serial immnuo-PET and biodistribution studies will be performed in nude mice bearing LN229 xenografts subcutaneously and orthotopicly to evaluate in vivo performance of 89Zr-Df-KN035. On the other hand, a novel radiopharmcieutic for RIT, 177Lu-DOTA-KN035, will be developed through conjugated to p-SCN-Bn-DOTA. After evaluated by in vitro cell uptake and blocking studies, biodistribution, radiation absorbed dose, the maximal tolerated dose (MTD) and the optimum dose will be studied in nude mice model. Finally，RIT response will be assessed in nude mice bearing LN229 xenografts subcutaneously and orthotopicly...All in all, motivated by the breaking through of anti PD-1/PD-L1 tumor immunotherapy，We aim to develop both immuno-PET imaging and RIT targeting PD-L1 for theranostic and precise therapy in glioma, which might be extended to the diagnosis and treatment of other tumors with PD-L1 positive expression.

针对胶质瘤临床治疗困境，基于免疫治疗有望加入胶质瘤治疗的SOC、程序性死亡蛋白配体1（PD-L1）胶质瘤高表达及本组前期靶向PD-L1免疫PET显像研究，拟选用PD-L1纳米抗体KN035，一方面构建分子影像探针89Zr-Df-KN035，通过体外研究、体内生物学分布、荷人胶质瘤皮下及原位裸鼠模型活体免疫PET显像鉴定该探针的稳定性、特异性及亲和力，评价其用于胶质瘤PD-L1表达显像的可行性和效能，并监测PD-L1抗体的治疗；另一方面制备RIT药物177Lu-DOTA-KN035，应用于荷人胶质瘤裸鼠模型，研究其体内分布、辐射吸收剂量、最大耐受剂量和量效时效关系，评估其治疗效果。旨在活体评估胶质瘤的PD-L1的表达、预测免疫治疗的反应概率、决策免疫治疗、监测治疗反应，对胶质瘤术后残余病灶及复发病灶进行靶向放射免疫治疗，实现诊疗一体化及精准治疗，并推广至其他高表达PD-L1的肿瘤的诊治。

肿瘤PD-L1的高表达水平与抗PD-1/PD-L1免疫治疗的疗效密切相关。本项目构建了一种新型靶向PD-L1的免疫PET探针89Zr-Df-KN035，利用荷人胶质瘤裸鼠模型及健康食蟹猴，通过免疫PET显像活体评价该探针用于PD-L1表达显像的可行性和效能，研究其体内分布，以期筛选抗PD-1/PD-L1免疫治疗的获益人群。荷胶质瘤系列免疫PET显像显示肿瘤自89Zr-Df-KN035尾静脉注射后6h即开始显影，24h显影清晰，直至120h仍显影清晰，KN035封闭对照组肿瘤摄取明显降低。体外生物分布结果显示 24h和120h的肿瘤/肌肉摄取比值良好，分别为5.64±0.65和7.70±1.37。IHC验证了肿瘤PD-L1的高表达。健康食蟹猴系列免疫PET显像显示该探针体内经肝肾代谢，生物分布稳定，淋巴结、脾和唾液腺等器官有轻度放射性摄取，余本底分布较低。上述结果提示89Zr-Df-KN035有望作为靶向PD-L1的分子探针，活体无创地监测肿瘤PD-L1的表达水平。基于EGFR-TKIs可下调PD-L1表达的相关报道，本项目进一步通过89Zr-Df-KN035 PET显像活体监测荷肺腺癌A549移植瘤裸鼠EGFR-TKI药物（吉非替尼）治疗后PD-L1表达水平的变化。治疗后，高剂量组肿瘤体积较高剂量对照组增长缓慢（p＜0.05），而肿瘤摄取较对照组下降明显（p＜0.05），IHC证实高剂量组PD-L1表达比高剂量对照组降低（p＜0.05）；低剂量组肿瘤体积与低剂量对照组无统计学差异（p＞0.05），肿瘤摄取和对照组较治疗前均明显下降（p＜0.05），但肿瘤摄取变化与对照组无统计学差异（p＞0.05），IHC证实低剂量组与对照组PD-L1表达均降低（HE示肿瘤坏死明显），且无统计学差异（p＞0.05）。上述结果证实EGFR-TKI治疗后小鼠肿瘤摄取值的变化与PD-L1表达的变化相关，为靶向PD-L1免疫PET显像用于监测肿瘤治疗过程中PD-L1表达的变化提供了依据，更有助于肿瘤治疗决策及优化肿瘤免疫治疗。此外，本项目还制备了靶向FAP的核素治疗药物177Lu-DOTA-FAPI-04，初步探索放射性核素靶向治疗药物的质控和显像结果。

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