Optimizing NK Cell-Engaging Bispecific Antibody Therapy Targeting CD33

摘要 CD33 靶向疗法长期以来一直致力于治疗急性髓系白血病 (AML)。一些人的生存时间更长 使用抗体-药物偶联物吉妥珠单抗奥佐米星 (GO) 治疗的患者验证了这种方法，但是 许多 CD33 AML 患者并未从 GO 中受益。这促使我们努力开发更好的 CD33- 定向治疗，包括 T 细胞接合双特异性抗体 (BsAb)。近日，多家代理商 进入早期临床测试，初步结果表明有一定的功效，但也显示出很大的毒性 与 T 细胞激活相关的细胞因子释放综合征。因此，人们越来越关注探索 其他免疫细胞的效用，例如自然杀伤 (NK) 细胞（例如通过 CD16 的参与），这可能 规避这些限制。如何优化 CD33/CD16 定向治疗尚不清楚。作为一项限制 CD33 靶向治疗中，CD33 抗体（包括 GO）通常识别免疫优势表位 在膜远端 V 形域内。在我们对 CD33V-set/CD3 BsAb 和人工 然而，我们观察到 CD33 蛋白通过近膜增强 T 细胞介导的细胞毒性。 CD33 的结合。因此，我们产生了一系列针对近膜 C2 组的抗体 CD33 的结构域，识别所有天然存在的 CD33 变体（即“CD33PAN 抗体”）作为基础 用于新疗法。我们之前的研究也表明T细胞导向的BsAbs的抗肿瘤功效 被抑制性 T 细胞辅助受体信号传导消除，例如通过 PD-L1/PD-1 轴。我们进一步证明了 使用配对的 BsAb 结合并阻断这种抑制信号，同时提供共- 对 T 细胞的刺激（例如通过交联 CD28）可以将细胞抑制转化为激活，并显着 提高 T 细胞接合 BsAb 的功效。迄今为止，NK受体的激活和抑制作用及其作用 相应的配体作为基于 BsAb 的 NK 细胞疗法的调节剂尚未得到评估。基于我们的 通过 T 细胞接合 BsAb 的研究，我们假设 CD33/CD16 定向的 NK 细胞接合疗法可以 通过 CD33 的近膜靶向进行优化。我们还预测激活和抑制 NK 细胞配体 调节 CD33/CD16 BsAb 的抗肿瘤功效，从而降低对 CD33/CD16 的耐药性 BsAb 可以通过与结合/阻断抑制性 NK 细胞配体并刺激 NK 细胞配体的 BsAb 配对来逆转。 激活 NK 细胞受体。我们将在严格控制的体外和体内临床前研究中测试这些假设。 完成拟议的研究后，我们期望我们能够对如何 CD33/CD16 NK 细胞参与疗法的抗 AML 功效可以最大化。我们的工作预计 具有重要的积极影响，因为它可能构成优化 NK 细胞参与疗法的基础 患有 AML 和其他 CD33 疾病的患者，其结果仍然不令人满意。

ABSTRACT CD33-targeted therapies have long been pursued in acute myeloid leukemia (AML). Longer survival of some patients treated with the antibody-drug conjugate gemtuzumab ozogamicin (GO) validates this approach, but many patients with CD33+ AML do not benefit from GO. This has prompted efforts to develop better CD33- directed therapeutics, including T cell engaging bispecific antibodies (BsAbs). Several agents have recently entered early phase clinical testing, with initial results indicating some efficacy but also substantial toxicities from cytokine-release syndrome associated with T cell activation. There is therefore increasing focus on exploring the utility of other immune cells such as natural killer (NK) cells (e.g. via engagement of CD16), which might circumvent these limitations. How CD33/CD16-directed therapy can be optimized is unknown. As one limitation of CD33-targeted therapy, CD33 antibodies (including GO) typically recognize immune-dominant epitope(s) within the membrane-distal V-set domain. In our preliminary studies with CD33V-set/CD3 BsAbs and artificial CD33 proteins, however, we have observed enhanced T cell-mediated cytotoxicity with membrane-proximal binding of CD33. We have therefore generated a series of antibodies against the membrane-proximal C2-set domain of CD33 that recognize all naturally occurring variants of CD33 (i.e. are “CD33PAN antibodies”) as basis for novel therapeutics. Our previous studies have also shown that the anti-tumor efficacy of T cell-directed BsAbs is abrogated by inhibitory T cell co-receptor signaling, e.g. via the PD-L1/PD-1 axis. We further demonstrated that the use of a paired BsAb which binds and blocks such an inhibitory signal while, in turn, providing co- stimulation to T cells (e.g. via cross-linking CD28) can convert cellular inhibition into activation and dramatically increase the efficacy of T cell engaging BsAbs. So far, the role of activating and inhibitory NK receptors and their corresponding ligands as modulators of BsAb-based NK cell therapy has not been evaluated. Based on our studies with T cell-engaging BsAbs, we hypothesize CD33/CD16-directed NK cell engaging therapy can be optimized by membrane-proximal targeting of CD33. We also predict that activating and inhibitory NK cell ligands modulate the anti-tumor efficacy of CD33/CD16 BsAbs and that, consequently, the resistance to CD33/CD16 BsAbs can be reversed by pairing with a BsAb that binds/blocks an inhibitory NK cell ligand and stimulates an activating NK cell receptor. We will test these hypotheses in well-controlled preclinical studies in vitro and in vivo. Upon completion of the proposed research, it is our expectation that we have gained critical insight into how the anti-AML efficacy of CD33/CD16 NK cell-engaging therapeutics can be maximized. Our work is anticipated to have an important positive impact because it may form the basis for optimized NK cell engaging therapeutics for patients with AML and other CD33+ disorders, for which outcomes continue to be unsatisfactory.