双特异性抗体介导的 NKG2D-CAR 自然杀伤细胞重定向促进双重靶向并增强抗肿瘤活性。

BACKGROUND: Natural killer group 2D (NKG2D) is an activating receptor of natural killer (NK) cells and other lymphocytes that mediates lysis of malignant cells through recognition of stress-induced ligands such as MICA and MICB. Such ligands are broadly expressed by cancer cells of various origins and serve as targets for adoptive immunotherapy with effector cells endogenously expressing NKG2D or carrying an NKG2D-based chimeric antigen receptor (CAR). However, shedding or downregulation of NKG2D ligands (NKG2DL) can prevent NKG2D activation, resulting in escape of cancer cells from NKG2D-dependent immune surveillance. METHODS: To enable tumor-specific targeting of NKG2D-expressing effector cells independent of membrane-anchored NKG2DLs, we generated a homodimeric recombinant antibody which harbors an N-terminal single-chain fragment variable (scFv) antibody domain for binding to NKG2D, linked via a human IgG Fc region to a second C-terminal scFv antibody domain for recognition of the tumor-associated antigen ErbB2 (HER2). The ability of this molecule, termed NKAB-ErbB2, to redirect NKG2D-expressing effector cells to ErbB2-positive tumor cells of different origins was investigated using peripheral blood mononuclear cells, ex vivo expanded NK cells, and NK and T cells engineered with an NKG2D-based chimeric receptor. RESULTS: On its own, bispecific NKAB-ErbB2 increased lysis of ErbB2-positive breast carcinoma cells by peripheral blood-derived NK cells endogenously expressing NKG2D more effectively than an ErbB2-specific IgG mini-antibody able to induce antibody-dependent cell-mediated cytotoxicity via activation of CD16. Furthermore, NKAB-ErbB2 synergized with NK-92 cells or primary T cells engineered to express an NKG2D-CD3ζ chimeric antigen receptor (NKAR), leading to targeted cell killing and greatly enhanced antitumor activity, which remained unaffected by soluble MICA known as an inhibitor of NKG2D-mediated natural cytotoxicity. In an immunocompetent mouse glioblastoma model mimicking low or absent NKG2DL expression, the combination of NKAR-NK-92 cells and NKAB-ErbB2 effectively suppressed outgrowth of ErbB2-positive tumors, resulting in treatment-induced endogenous antitumor immunity and cures in the majority of animals. CONCLUSIONS: Our results demonstrate that combining an NKAB antibody with effector cells expressing an activating NKAR receptor represents a powerful and versatile approach to simultaneously enhance tumor antigen-specific as well as NKG2D-CAR and natural NKG2D-mediated cytotoxicity, which may be particularly useful to target tumors with heterogeneous target antigen expression.

背景：自然杀伤细胞群 2D（NKG2D）是自然杀伤（NK）细胞和其他淋巴细胞的一种激活受体，通过识别应激诱导的配体（如 MICA 和 MICB）介导恶性细胞的裂解。此类配体广泛表达于各种来源的癌细胞中，可作为通过内源性表达 NKG2D 或携带基于 NKG2D 的嵌合抗原受体（CAR）的效应细胞进行过继免疫治疗的靶点。然而，NKG2D 配体（NKG2DL）的脱落或下调会阻止 NKG2D 的激活，导致癌细胞逃避 NKG2D 依赖性免疫监视。

方法：为了实现独立于膜锚定 NKG2DL 的表达 NKG2D 的效应细胞的肿瘤特异性靶向，我们生成了一种同二聚体重组抗体，该抗体通过人 IgG Fc 区连接一个 N 端单链片段可变（scFv）抗体结构域，用于结合 NKG2D，通过一个 C 端 scFv 抗体结构域用于识别肿瘤相关抗原 ErbB2（HER2）。使用外周血单核细胞、体外扩增的 NK 细胞以及用基于 NKG2D 的嵌合受体工程改造的 NK 和 T 细胞，研究了这种称为 NKAB-ErbB2 的分子将表达 NKG2D 的效应细胞重新导向不同来源的 ErbB2 阳性肿瘤细胞的能力。

结果：单独使用时，双特异性 NKAB-ErbB2 比能够通过激活 CD16 诱导抗体依赖性细胞介导的细胞毒性的针对 ErbB2 的 IgG 迷你抗体更有效地增加内源性表达 NKG2D 的外周血来源 NK 细胞对 ErbB2 阳性乳腺癌细胞的裂解。此外，NKAB-ErbB2 与 NK-92 细胞或表达 NKG2D-CD3ζ 嵌合抗原受体（NKAR）的原代 T 细胞协同作用，导致靶向细胞杀伤和大大增强的抗肿瘤活性，而可溶性 MICA（一种已知的抑制 NKG2D 介导的自然细胞毒性的抑制剂）对其无影响。在模拟低表达或不存在 NKG2DL 的免疫活性的小鼠脑胶质瘤模型中，NKAR-NK-92 细胞与 NKAB-ErbB2 的联合有效地抑制了 ErbB2 阳性肿瘤的生长，导致治疗诱导的内源性抗肿瘤免疫和大多数动物的治愈。

结论：我们的研究结果表明，将 NKAB 抗体与表达激活的 NKAR 受体的效应细胞相结合，是一种同时增强肿瘤抗原特异性以及 NKG2D-CAR 和天然 NKG2D 介导的细胞毒性的强大而通用的方法，这可能特别有用以靶向具有异质靶抗原表达的肿瘤。