Department of Geriatrics, The First Affiliated Hospital of University of Science and Technology of China, Gerontology Institute of Anhui Province, Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei, 230001, Anhui, China.
Hefei HankeMab Biotechnology Limited, Hefei, 230088, Anhui, China.
Cell Mol Biol Lett. 2023 May 31;28(1):47. doi: 10.1186/s11658-023-00461-w.
Resistance to immune checkpoint inhibitor (ICI) therapy narrows the efficacy of cancer immunotherapy. Although 4-1BB is a promising drug target as a costimulatory molecule of immune cells, no 4-1BB agonist has been given clinical approval because of severe liver toxicity or limited efficacy. Therefore, a safe and efficient immunostimulatory molecule is urgently needed for cancer immunotherapy.
HK010 was generated by antibody engineering, and the Fab/antigen complex structure was analyzed using crystallography. The affinity and activity of HK010 were detected by multiple in vitro bioassays, including enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), flow cytometry, and luciferase-reporter assays. Humanized mice bearing human PD-L1-expressing MC38 (MC38/hPDL1) or CT26 (CT26/hPDL1) tumor transplants were established to assess the in vivo antitumor activity of HK010. The pharmacokinetics (PK) and toxicity of HK010 were evaluated in cynomolgus monkeys.
HK010 was generated as an Fc-muted immunoglobulin (Ig)G4 PD-L1x4-1BB bispecific antibody (BsAb) with a distinguished Fab/antigen complex structure, and maintained a high affinity for human PD-L1 (KD: 2.27 nM) and low affinity for human 4-1BB (KD: 493 nM) to achieve potent PD-1/PD-L1 blockade and appropriate 4-1BB agonism. HK010 exhibited synergistic antitumor activity by blocking the PD-1/PD-L1 signaling pathway and stimulating the 4-1BB signaling pathway simultaneously, and being strictly dependent on the PD-L1 receptor in vitro and in vivo. In particular, when the dose was decreased to 0.3 mg/kg, HK010 still showed a strong antitumor effect in a humanized mouse model bearing MC38/hPDL1 tumors. Strikingly, HK010 treatment enhanced antitumor immunity and induced durable antigen-specific immune memory to prevent rechallenged tumor growth by recruiting CD8+ T cells and other lymphocytes into tumor tissue and activating tumor-infiltrating lymphocytes. Moreover, HK010 not only did not induce nonspecific production of proinflammatory cytokines but was also observed to be well tolerated in cynomolgus monkeys in 5 week repeated-dose (5, 15, or 50 mg/kg) and single-dose (75 or 150 mg/kg) toxicity studies.
We generated an Fc-muted anti-PD-L1x4-1BB BsAb, HK010, with a distinguished structural interaction with PD-L1 and 4-1BB that exhibits a synergistic antitumor effect by blocking the PD-1/PD-L1 signaling pathway and stimulating the 4-1BB signaling pathway simultaneously. It is strictly dependent on the PD-L1 receptor with no systemic toxicity, which may offer a new option for cancer immunotherapy.
免疫检查点抑制剂（ICI）治疗的耐药性缩小了癌症免疫疗法的疗效。虽然 4-1BB 作为免疫细胞的共刺激分子是一种很有前途的药物靶点，但由于严重的肝毒性或有限的疗效，没有 4-1BB 激动剂获得临床批准。因此，癌症免疫疗法迫切需要一种安全有效的免疫刺激分子。
HK010 通过抗体工程生成，并用结晶学分析 Fab/抗原复合物结构。通过多种体外生物测定法，包括酶联免疫吸附试验（ELISA）、表面等离子体共振（SPR）、流式细胞术和荧光素酶报告基因测定法，检测 HK010 的亲和力和活性。建立了携带人 PD-L1 表达的 MC38（MC38/hPDL1）或 CT26（CT26/hPDL1）肿瘤移植的人源化小鼠，以评估 HK010 的体内抗肿瘤活性。在食蟹猴中评估了 HK010 的药代动力学（PK）和毒性。
HK010 作为一种 Fc 修饰的 IgG4 PD-L1x4-1BB 双特异性抗体（BsAb）生成，具有独特的 Fab/抗原复合物结构，对人 PD-L1 保持高亲和力（KD：2.27 nM）和对人 4-1BB 的低亲和力（KD：493 nM），从而实现有效的 PD-1/PD-L1 阻断和适当的 4-1BB 激动作用。HK010 通过同时阻断 PD-1/PD-L1 信号通路和刺激 4-1BB 信号通路，表现出协同的抗肿瘤活性，并且在体外和体内严格依赖 PD-L1 受体。特别是，当剂量降低至 0.3 mg/kg 时，HK010 仍在携带 MC38/hPDL1 肿瘤的人源化小鼠模型中表现出强烈的抗肿瘤作用。引人注目的是，HK010 通过将 CD8+T 细胞和其他淋巴细胞募集到肿瘤组织中并激活肿瘤浸润淋巴细胞，不仅增强了抗肿瘤免疫，还诱导了持久的抗原特异性免疫记忆，从而防止了复发性肿瘤的生长。此外，在食蟹猴中进行的 5 周重复剂量（5、15 或 50mg/kg）和单次剂量（75 或 150mg/kg）毒性研究中，HK010 不仅没有诱导非特异性产生促炎细胞因子，而且也被观察到具有良好的耐受性。
我们生成了一种 Fc 修饰的抗 PD-L1x4-1BB BsAb，HK010，与 PD-L1 和 4-1BB 具有独特的结构相互作用，通过同时阻断 PD-1/PD-L1 信号通路和刺激 4-1BB 信号通路，表现出协同的抗肿瘤作用。它严格依赖 PD-L1 受体，没有全身毒性，这可能为癌症免疫疗法提供新的选择。
