Phase 1 Evaluation of Enhanced Natural Killer Cells as a Treatment Strategy in Non-Small cell Lung Cancer Patients Refractory to PD-1/PD-L1 Immune Checkpoint Inhibitors

项目摘要：肺癌(LC)是导致男性和女性癌症死亡的最常见原因， 据估计，2020年美国将出现228,820例新病例和135,720例死亡。近几年来 抑制免疫检查点，如程序性细胞死亡-1(PD-1)和程序性细胞死亡配体- 1(PD-L1)，已被证明为LC患者提供生存益处。然而，大多数患者 要么表现出原发耐药性，要么经历肿瘤复发并死于其疾病。 我们的团队已经证明，癌细胞上调自然杀伤(NK)细胞上的PD-L1，免疫细胞 可以在不需要嵌合抗原受体或先前抗原暴露的情况下靶向恶性肿瘤，并且 不需要与受者的人类白细胞抗原相匹配即可发挥潜在的活性。上调PD- L1组NK细胞功能增强。此外，PD-L1抑制剂阿达唑单抗(AZ)导致 增强白血病细胞对PD-L1表达缺失的髓系白血病的杀伤作用 选择性细胞因子(IL-12、IL-15和IL-18)与AZ联合应用显示显著改善 即使在肿瘤组织中没有PD-L1表达的情况下也能存活。 我们能够表达截短的上皮生长因子受体标记的可溶性IL-15(sIL-15)。 脐带血NK细胞体外培养，同时上调NK细胞内源性PD-L1的表达。这些 与未转导的NK细胞相比，转导的NK细胞具有30%以上的抗原特异性肿瘤杀伤活性 并显示对A549非小细胞肺癌(NSCLC)细胞的细胞毒作用。人A549非小细胞肺癌 随后将细胞注射到非同基因小鼠中，然后用这些“增强的”细胞进行治疗。 封锁血液中的CB-NK细胞。与模拟转导NK细胞或表达NK细胞的治疗相比 SIL-15(sIL-15-NK)，但在没有体外激活的情况下，增强的CB NK细胞导致 肿瘤体积。我们还进行了这种方法的安全性/毒性体内研究，并与单独使用AZ进行了比较。 我们的数据表明，抗PD-L1单抗治疗PD-L1阴性具有独特的治疗作用 癌症，通过PD-L1(+)NK细胞发挥作用。这一活性是独立于PD-1活性实现的，并且在 NK激活细胞因子的存在。我们假设细胞因子“增强”的NK细胞将提供临床 有益于非小细胞肺癌患者，并将进一步增强这种方法的抗肿瘤活性 亚洲区的行政管理。为了检验这一假设并证明这一策略的安全性，我们建议在 体内安全性和有效性研究，随后是一项第一阶段研究，在该研究中，CB NK细胞，转基因到 注射sIL-15，然后在存在IL-2、IL-18和IL-12的情况下进行体外扩增 或单独或联合AZ，在淋巴细胞耗尽后用于其肿瘤的NSCLC患者 曾在PD-1/PD-L1抑制剂治疗期间或治疗后取得进展。

PROJECT SUMMARY: Lung cancer (LC), the most frequent cause of cancer deaths for men and women, is estimated to lead to 228,820 new cases and 135,720 deaths in the United States in 2020. In recent years inhibition of immune checkpoints, such as programmed cell death-1 (PD-1) and programmed cell death ligand- 1 (PD-L1), has been shown to provide survival benefits to patients with LC. However, most patients demonstrate either primary resistance or experience tumor recurrence and die of their disease. Our group has demonstrated that cancer cells upregulate PD-L1 on natural killer (NK) cells, immune cells that can target malignancies without the necessity of chimeric antigen receptors or prior antigen exposure and do not require matching to recipient's human leukocyte antigen for potential activity. Upregulation of PD- L1resulted in enhanced NK-cell function. Furthermore, the PD-L1 inhibitor atezolizumab (AZ) resulted in enhanced leukemic cell killing against myeloid leukemia lacking PD-L1 expression, and mice treated with selective cytokines (IL-12, IL-15, and IL-18) in combination with AZ showed a significant improvement in survival even in the absence of PD-L1 expression in their tumor tissue. We were able to express soluble IL-15 (sIL-15) tagged with a truncated epithelial growth factor receptor in umbilical cord NK cells in vitro, while upregulating endogenous PD-L1 expression on the NK cells. These transduced NK cells maintained greater than 30% antigen-specific tumor lysis compared to mock-transduced NK cells and demonstrated cytotoxicity against A549 Non-Small-Cell LC (NSCLC) cells. Human A549 NSCLC cells were subsequently injected in non-syngeneic mice and followed with treatment with these “enhanced” cordon blood CB NK cells. In comparison to treatment with mock-transduced NK cells, or NK cells expressing sIL-15 (sIL-15-NK) but without ex vivo activation, the enhanced CB NK cells induced substantial reduction in tumor volume. We also performed safety/toxicity in vivo studies of this approach and compared with AZ alone. Our data suggest that anti-PD-L1 mAb therapy has a unique therapeutic role in treating PD-L1 negative cancer, acting through PD-L1(+) NK cells. This activity is achieved independent of PD-1 activity and in the presence of NK-activating cytokines. We hypothesize that cytokine “enhanced” NK cells will provide clinical benefit to NSCLC patients and that the antitumor activity of this approach will be further enhanced by co- administration of AZ. To test this hypothesis and document the safety of this strategy, we propose additional in vivo safety and efficacy studies followed by a phase 1 study in which CB NK cells, genetically modified to express sIL-15, followed by ex-vivo expansion in the presence of IL-2, IL-18, and IL-12 will be administered either by themselves or combined with AZ, following lymphocyte depletion, to NSCLC patients whose tumor has previously progressed on or after treatment with PD-1/PD-L1 inhibitors.