PLT012, a humanized CD36-blocking antibody, is effective for unleashing anti-tumor immunity against liver cancer and liver metastasis

Tumor cells develop various strategies to evade immune surveillance, one of which is the modulation of the metabolic state of the tumor microenvironment (TME). In response to metabolic stress in the TME, several tumor-infiltrating immune subsets upregulate CD36 to take up lipids. This leads to impaired anti-tumor immunity, as intratumoral regulatory T cells (Tregs) exhibit increased survival and suppressive activity, while CD8+ T cells become more susceptible to ferroptosis and exhaustion. Here, we develop a humanized anti-CD36 IgG4 antibody, PLT012, against the lipid-binding domain of CD36 with excellent safety and favorable pharmacokinetic features in mice and cynomolgus monkey. PLT012 alone or in combination with PD-L1 blockade or standard-of-care immunotherapy results in robust anti-tumor immunity in both immunotherapy-sensitive and -resistant hepatocellular carcinomas (HCCs). Notably, PLT012 also reprograms immune landscape of human HCC ex vivo. Our findings provide proof-of-concept evidence that PLT012 effectively reprograms anti-tumor immunity in HCC, positioning it as a first-in-class immunotherapy targeting CD36. To investigate the immune response to PLT012 treatment, we isolated CD45+ tumor-infiltrating cells from liver tumors and subjected them to single-cell RNA sequencing (scRNA-seq). Liver tissues were finely minced and enzymatically digested in RPMI medium containing 2% FBS, 1% penicillin-streptomycin, DNase I (1 µg/mL), and collagenase (0.5 mg/mL) at 37°C for 40 minutes. The digested suspension was filtered through a 70-μm strainer, treated with RBC Lysing Buffer, and washed with FACS buffer. Tumor-infiltrating leukocytes were enriched using Percoll density gradient centrifugation (800g, 20 minutes, room temperature). CD45+ cells were then isolated for scRNA-seq analysis, comparing transcriptional profiles between PLT012-treated and control groups.

肿瘤细胞可演化出多种策略以逃避免疫监视，其中之一便是调控肿瘤微环境（tumor microenvironment, TME）的代谢状态。为响应肿瘤微环境中的代谢应激，多种肿瘤浸润免疫亚群会上调CD36的表达以摄取脂质。这一过程会导致抗肿瘤免疫功能受损：瘤内调节性T细胞（regulatory T cells, Tregs）的存活与免疫抑制活性增强，而CD8+ T细胞则更易发生铁死亡（ferroptosis）与功能耗竭。

本研究开发了一款靶向CD36脂质结合结构域的人源化抗CD36 IgG4抗体PLT012，该抗体在小鼠与食蟹猴体内均展现出优异的安全性与良好的药代动力学特性。单独使用PLT012，或联合PD-L1阻断疗法与标准护理免疫疗法，均可在免疫治疗敏感型与耐药型肝细胞癌（hepatocellular carcinomas, HCCs）模型中诱导强效的抗肿瘤免疫应答。值得注意的是，PLT012还可在体外重塑人肝细胞癌的免疫图谱。本研究结果为PLT012可有效重塑肝细胞癌中的抗肿瘤免疫提供了概念验证证据，使其成为首款靶向CD36的同类首创免疫治疗药物。

为探究PLT012治疗引发的免疫应答，我们从肝肿瘤组织中分离出CD45+肿瘤浸润细胞，并对其进行单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）。将肝组织充分剪碎后，于含2%胎牛血清（fetal bovine serum, FBS）、1%青霉素-链霉素、脱氧核糖核酸酶I（DNase I，1 µg/mL）与胶原酶（collagenase，0.5 mg/mL）的RPMI培养基中，于37℃条件下酶解40分钟。将酶解后的细胞悬液通过70μm滤网过滤，使用红细胞裂解液（RBC Lysing Buffer）处理后，用流式细胞术缓冲液（FACS buffer）洗涤。采用Percoll密度梯度离心（800g，20分钟，室温）富集肿瘤浸润白细胞。随后分离CD45+细胞用于scRNA-seq分析，比较PLT012处理组与对照组的转录组表达谱差异。