Gene Expression profile for the characterization of PD-L1 blockade in melanoma model. Gene Expression profile for the characterization of PD-L1 blockade in melanoma model

Only a subset of patients responds to immune checkpoint blockade in melanoma. A preclinical model recapitulating the clinical activity of ICB would provide a valuable platform for mechanistic studies. We used melanoma tumors arising from an Hgftg;Cdk4R24C/R24C genetically engineered mouse (GEM) model to evaluate the efficacy of an anti-mouse PD-L1 antibody similar to the anti-human PD-L1 antibodies durvalumab and atezolizumab. Consistent with clinical observations for ICB in melanoma, anti-PD-L1 treatment elicited complete and durable response in a subset of melanoma-bearing mice. We also observed tumor growth delay or regression followed by recurrence. For early treatment assessment, we analyzed gene expression profiles, T cell infiltration, and T cell receptor (TCR) signatures in regressing tumors compared to tumors exhibiting no response to anti-PD-L1 treatment. We found that CD8+ T cell tumor infiltration corresponded to response to treatment, and that anti-PD-L1 gene signature response indicated an increase in antigen processing and presentation, cytokine-cytokine receptor interaction, and natural killer cell-mediated cytotoxicity. TCR sequence data suggest that an anti-PD-L1-mediated melanoma regression response requires not only an expansion of the TCR repertoire that is unique to individual mice, but also tumor access to the appropriate TCRs. Thus, this melanoma model recapitulated the variable response to ICB observed in patients and exhibited biomarkers that differentiate between early response and resistance to treatment, providing a valuable platform for prediction of successful immunotherapy. Overall design: Mice were treated with anti-mouse PD-L1 mIgG1 D265A (clone 80; AstraZeneca Cat# AB740080, Batch# SP15-221) in PBS at 10mg/kg or 20mg/kg intraperitoneally twice weekly for a maximum of 6 doses over 3 weeks in the initial efficacy study, or over a longer period to assess tumor response as indicated in the text. For each study the vehicle control group received PBS (10ml/kg) and an additional antibody control group received mouse IgG1 D265A (AstraZeneca clone: Cat# NIP228, Batch# SP16-017) in PBS at 10 or 20mg/kg. Tumor growth was monitored by caliper measurement. Tumors were harvested from treated mice at several different endpoints (delayed growth, stabilization, regression) or when they reached a maximum tumor volume of 2000 mm3. At necropsy, tumors were divided into sections for fixation or flash-freezing. Blood samples were taken from each mouse prior to treatment and at the time of tumor harvest. To evaluate response in biomarker studies (Study 2 or 3), tumors from mice treated with anti-PD-L1 were harvested after 2-12 doses based on regression in tumor volume after two consecutive measurements (Responders). Anti-PD-L1-treated tumors that exhibited growth progression were harvested at matching timepoints (Non-responders). Tumors that regressed to below measurable volumes after initial treatment but regrew after 4-10 doses of anti-PD-L1 were also harvested (Relapse). PBS- and control IgG-treated tumors were also harvested at multiple time points for comparison. Additionally, any mice that did not experience initial tumor growth, but developed tumors after anti-PD-L1 treatment were designated as Growth Delay. Any tumor that decreased in volume by 5% or more was designated as a Responder. Within the Responder group, tumors with no volume change between 2 consecutive measurements were designated as growth stabilization.

黑色素瘤患者中仅部分人群可从免疫检查点阻断（immune checkpoint blockade, ICB）治疗中获益。能够复现ICB临床疗效的临床前模型，将为机制研究提供极具价值的研究平台。我们使用源自Hgftg;Cdk4R24C/R24C基因工程小鼠（genetically engineered mouse, GEM）模型的黑色素瘤肿瘤，评估了与度伐利尤单抗（durvalumab）、阿替利珠单抗（atezolizumab）同类的抗小鼠PD-L1抗体的疗效。与黑色素瘤ICB治疗的临床观察一致，抗PD-L1治疗可在部分荷瘤小鼠中诱导完全且持久的应答。我们还观察到肿瘤生长延迟、消退后复发的现象。为开展早期治疗评估，我们对比分析了对抗PD-L1治疗无应答的肿瘤与出现消退的肿瘤的基因表达谱、T细胞浸润情况及T细胞受体（T cell receptor, TCR）特征。研究发现，CD8+ T细胞肿瘤浸润与治疗应答相关，且抗PD-L1基因特征应答提示抗原加工呈递、细胞因子-细胞因子受体相互作用及自然杀伤细胞介导的细胞毒性通路的活性上调。TCR测序数据表明，抗PD-L1介导的黑色素瘤消退应答，不仅需要个体小鼠特异性TCR库的扩增，还需要肿瘤能够接触到适配的TCR。综上，该黑色素瘤模型复现了患者中观察到的ICB应答异质性，并可鉴定区分治疗早期应答与耐药性的生物标志物，为预测免疫治疗的成功性提供了极具价值的研究平台。

总体实验设计：
初始疗效研究中，小鼠经腹腔注射给予抗小鼠PD-L1 mIgG1 D265A（克隆号80；阿斯利康（AstraZeneca）货号AB740080，批次SP15-221），浓度为10mg/kg或20mg/kg，每周给药两次，3周内最多给药6次；也可延长给药周期以评估肿瘤应答，具体如文中所述。每组实验的溶剂对照组给予PBS（10ml/kg），额外设置的抗体对照组给予小鼠IgG1 D265A（阿斯利康（AstraZeneca）克隆：货号NIP228，批次SP16-017），浓度为10或20mg/kg，溶于PBS中。通过卡尺测量监测肿瘤生长。在多个不同终点（生长延迟、病灶稳定、肿瘤消退）或当肿瘤体积达到2000mm³最大限值时，处死处理组小鼠并收获肿瘤。剖检时，将肿瘤分为若干部分，分别用于固定或快速冷冻。每只小鼠在治疗前及肿瘤收获时采集血液样本。
针对生物标志物研究（研究2或3）的应答评估：对于抗PD-L1治疗组的小鼠，在连续两次测量后肿瘤体积出现消退（应答者Responders）时，于给药2-12次后收获肿瘤。抗PD-L1治疗后肿瘤出现生长进展的样本，在匹配时间点收获（无应答者Non-responders）。初始治疗后肿瘤消退至可测量体积以下，但经4-10次抗PD-L1治疗后复发的肿瘤，同样予以收获（复发者Relapse）。同时收获PBS及对照IgG治疗组的肿瘤，于多个时间点采样以作对照。此外，初始未出现肿瘤生长，但经抗PD-L1治疗后长出肿瘤的小鼠，被归类为生长延迟组（Growth Delay）。肿瘤体积缩小5%及以上者被归类为应答者。在应答者组中，连续两次测量后体积无变化的肿瘤被归类为生长稳定组。