背景:双特异性抗体由于其在血液恶性肿瘤中令人印象深刻的初步功效,近年来引起了广泛关注。对于实体瘤,然而,主要障碍是抑制性肿瘤微环境,这有效地阻碍了浸润T细胞的激活。在这里,我们设计了一种对PD-L1和CD137具有高结合亲和力的双特异性抗体AP203,并评估了其安全性和抗肿瘤功效,并探讨了其作用机制。
方法:从OmniMab噬菌粒文库中筛选针对PD-L1和CD137的最佳抗体结合物。使用酶联免疫吸附测定(ELISA)和生物层干涉法(BLI)评估构建的AP203的结合亲和力。使用同种异体混合淋巴细胞反应(MLR)评估T细胞刺激能力,抗原特异性召回反应,并与PD-L1表达细胞共培养。使用两种具有肿瘤浸润淋巴细胞(TIL)概况的肿瘤异种移植人源化小鼠模型评估体内抗肿瘤功效。使用人PBMC的体外细胞因子释放测定法检查AP203的可能毒性。
结果:AP203同时靶向PD-L1和共刺激CD137,在T细胞活化方面比单独或联合使用的亲本抗体引起更好的激动作用,增强记忆回忆反应,克服Treg介导的免疫抑制(P<0.05)。通过将T细胞与表达PD-L1的细胞共培养,进一步证明了AP203的激动活性是PD-L1依赖性的。使用免疫缺陷或免疫活性小鼠的体内动物研究均显示出优于组合的亲本抗体的剂量相关抗肿瘤功效(P<0.05)。相应地,AP203显著增加肿瘤浸润CD8+T细胞,而CD4+T细胞减少,以及Treg细胞(P<0.05),导致CD8+/CD4+比率的剂量依赖性增加。此外,可溶性或固定化的AP203均不诱导人PBMC产生炎性细胞因子。
结论:AP203不仅通过阻断PD-1/PD-L1抑制信号而发挥有效的抗肿瘤活性,而且还通过激活效应T细胞中的CD137共刺激信号传导,从而抵消Treg介导的免疫抑制。基于有希望的临床前结果,AP203应该是临床治疗实体瘤的合适候选者。
Bispecific antibody has garnered considerable attention in the recent years due to its impressive preliminary efficacy in hematological malignancies. For solid tumors, however, the main hindrance is the suppressive tumor microenvironment, which effectively impedes the activation of infiltrating T cells. Herein, we designed a bispecific antibody AP203 with high binding affinity to PD-L1 and
CD137 and assessed its safety and anti-tumor efficacy, as well as explored the mechanism of action.
The optimal antibody binders against PD-L1 and
CD137 were screened from the OmniMab phagemid library. The binding affinity of the constructed AP203 were evaluated using enzyme-linked immunosorbent assay (ELISA) and biolayer interferometry (BLI). T-cell stimulatory capacity was assessed using the allogeneic mixed lymphocyte reaction (MLR), antigen-specific recall response, and coculture with PD-L1-expressing cells. In vivo antitumor efficacy was evaluated using two models of tumor-xenografted humanized mice with profiling of tumor infiltrating lymphocytes (TILs). The possible toxicity of AP203 was examined using in vitro cytokine release assay by human PBMCs.
AP203, which simultaneously targeted PD-L1 and costimulatory
CD137, elicit superior agonistic effects over parental antibodies alone or in combination in terms of T cell activation, enhanced memory recall responses, and overcoming Treg-mediated immunosuppression (P < 0.05). The agonistic activity of AP203 was further demonstrated PD-L1-dependent by coculturing T cells with PD-L1-expressing cells. In vivo animal studies using immunodeficient or immunocompetent mice both showed a dose-related antitumor efficacy superior to parental antibodies in combination (P < 0.05). Correspondingly, AP203 significantly increased tumor infiltrating CD8 + T cells, while decreased CD4 + T cells, as well as Treg cells (P < 0.05), resulting in a dose-dependent increase in the CD8 + /CD4 + ratio. Moreover, either soluble or immobilized AP203 did not induce the production of inflammatory cytokines by human PBMCs.
AP203 exerts potent antitumor activity not only by blocking PD-1/PD-L1 inhibitory signaling, but also by activating
CD137 costimulatory signaling in effector T cells that consequently counteracts Treg-mediated immunosuppression. Based on promising preclinical results, AP203 should be a suitable candidate for clinical treatment of solid tumors.