Optimizing NK Cell-Engaging Bispecific Antibody Therapy Targeting CD33

摘要 CD33靶向治疗急性髓系白血病(AML)由来已久。一些人的生存时间更长 接受抗体-药物结合物吉妥珠单抗(GO)治疗的患者证实了这一方法，但 许多CD33 AML患者不能从GO中受益。这促使人们努力开发更好的CD33- 定向治疗，包括T细胞结合双特异性抗体(BsAbs)。几个特工最近 进入早期临床测试，初步结果显示有一定的疗效，但也有很大的毒性。 与T细胞活化相关的细胞因子释放综合征。因此，越来越多的人关注探索 利用其他免疫细胞，如自然杀伤(NK)细胞(例如，通过参与CD16)，这可能 绕过这些限制。CD33/CD16指导的治疗如何优化尚不清楚。作为一个限制 在CD33靶向治疗中，CD33抗体(包括GO)通常识别免疫优势表位(S) 在膜-远端V-set结构域内。在我们对CD33V-SET/CD3 BsAbs和人工的初步研究中 然而，我们观察到CD33蛋白通过膜近端增强了T细胞介导的细胞毒作用 CD33的结合。因此，我们产生了一系列抗膜近端C2-SET的抗体 CD33的结构域，可识别CD33的所有自然产生的变体(即CD33PAN抗体)作为基础 寻找新的治疗方法。我们以前的研究也表明，T细胞导向的BsAbs的抗肿瘤效果 被抑制性T细胞共受体信号所抑制，例如通过PD-L1/PD-1轴。我们进一步展示了 使用成对的BsAb结合和阻断这样的抑制信号，同时又提供共同的- 对T细胞的刺激(例如，通过交联型CD28)可以将细胞抑制转化为激活，并显著 提高T细胞与BsAbs的结合效率。到目前为止，激活和抑制NK受体的作用及其机制 相应的配体作为基于BsAb的NK细胞治疗的调节剂还没有得到评估。基于我们的 对于T细胞结合BsAbs的研究，我们假设CD33/CD16导向的NK细胞结合治疗可以 通过CD33的膜-近端靶向进行优化。我们还预测，激活和抑制NK细胞配体 调节CD33/CD16BsAbs的抗肿瘤效应及对CD33/CD16的耐药性 BsAb可以通过与BsAb配对来逆转，BsAb结合/阻断抑制性NK细胞配体并刺激 激活NK细胞受体。我们将在良好控制的体外和体内临床前研究中检验这些假说。 在完成建议的研究后，我们期望能深入了解 CD33/CD16 NK细胞结合疗法可以最大限度地发挥抗AML作用。我们的工作预计将 有重要的积极影响，因为它可能形成优化的NK细胞参与治疗的基础 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.