Cancer Immunotherapeutic Potential of NKTT320, a Novel, Invariant, Natural Killer T Cell-Activating, Humanized Monoclonal Antibody
新型、不变、自然杀伤 T 细胞激活、人源化单克隆抗体 NKTT320 的肿瘤免疫治疗潜力
- 作者列表："Nishant P. Patel","Peng Guan","Devika Bahal","Tanwir Hashem","Felix Scheuplein","Robert Schaub","Kim E. Nichols","Rupali Das
<b>: </b>Invariant natural killer T cells (iNKTs) directly kill tumor cells and trans-activate the anti-tumor functions of dendritic cells (DC), natural killer (NK) cells, and T and B cells. As such, iNKTs serve as a powerful tool for use in cell-based cancer immunotherapy. iNKT cell activation commonly requires engagement of the invariant T cell receptor (iTCR) by CD1d presenting glycolipid antigens. However, transformed cells often down-regulate CD1d expression, which results in a reduction of iNKT cell anti-tumor functions. One approach to circumvent this critical barrier to iNKT cell activation is to develop an agonistic antibody that binds directly to the iTCR without the requirement for CD1d-mediated antigen presentation. To this end, we have characterized the iNKT cell stimulatory properties of NKTT320, a novel, recombinant, humanized, monoclonal antibody that binds selectively and with high affinity to human iTCRs. Strikingly, immobilized NKTT320 mediated robust iNKT cell activation (upregulation of CD25 and CD69) and proliferation (carboxyfluorescein succinimidyl ester (CFSE) dilution), as well as Th1 and Th2 cytokine production. Additionally, iNKTs stimulated by plate-bound NKTT320 exhibited increased intracellular levels of granzyme B and degranulation (exposure of CD107 on the cell surface). Furthermore, both soluble and immobilized NKTT320 induced iNKT cell-mediated activation of bystander immune cells, suggesting that this novel anti-iTCR antibody facilitates both direct and indirect iNKT cell cytotoxicity. These studies are significant, as they provide a framework by which iNKT cell anti-cancer functions could be enhanced for therapeutic purposes.
<B>: </b> 不变自然杀伤 T 细胞 (inkt) 直接杀伤肿瘤细胞，反式激活树突状细胞 (DC) 的抗肿瘤功能,自然杀伤 (NK) 细胞，以及 T 和 B 细胞。因此，iNKTs 作为一种强有力的工具用于基于细胞的癌症免疫治疗。INKT 细胞活化通常需要通过 CD1d 呈递糖脂抗原来参与不变的 T 细胞受体 (iTCR)。然而，转化细胞经常下调 CD1d 表达，导致 iNKT 细胞抗肿瘤功能降低。规避 iNKT 细胞活化这一关键屏障的一种方法是开发一种直接与 iTCR 结合的激动性抗体，而不需要 CD1d-mediated 抗原呈递。为此，我们对 NKTT320 的 iNKT 细胞刺激特性进行了表征，NKTT320 是一种新型的、重组的、人源化的单克隆抗体，选择性地与人 iTCRs 高亲和力结合。引人注目的是，固定化 NKTT320 介导了稳健的 iNKT 细胞活化 (上调 CD25 和 CD69) 和增殖 (羧基荧光素琥珀酰亚胺酯 (CFSE) 稀释)，以及 Th1 和 Th2 细胞因子的产生。此外，平板结合 NKTT320 刺激的 iNKTs 表现出颗粒酶 B 和脱颗粒的细胞内水平增加 (CD107 在细胞表面暴露)。此外，可溶性和固定化 NKTT320 均诱导 iNKT 细胞介导的旁观者免疫细胞活化，提示这种新型抗 iTCR 抗体同时促进直接和间接 iNKT 细胞毒性。这些研究具有重要意义，因为它们提供了一个框架，通过这个框架可以增强 iNKT 细胞的抗癌功能以达到治疗目的。
METHODS::Macrophages (MΦ) play a critical role in tumor growth, immunosuppression and inhibition of adaptive immune responses in cancer. Hence, targeting signaling pathways in MΦs that promote tumor immunosuppression will provide therapeutic benefit. PI3Kγ has been recently established by our group and others as a novel immuno-oncology target. Herein, we report that a macrophage Syk-PI3K axis drives polarization of immunosuppressive MΦs which establish an immunosuppressive tumor microenvironment in in vivo syngeneic tumor models. Genetic or pharmacological inhibition of Syk and/or PI3Kγ in MΦs promotes a pro-inflammatory MΦphenotype, restores CD8+ T cell activity, destabilizes HIF under hypoxia, and stimulates an antitumor immune response. Assay for Transposase-accessible Chromatin using Sequencing (ATAC-seq) analyses on the bone marrow derived macrophages (BMDMs) show that inhibition of Syk kinase promotes activation and binding of NF-κB motif in SykMC-KO BMDMs, thus stimulating immunostimulatory transcriptional programming in MΦs to suppress tumor growth. Finally, we have developed in silico the "first in class" dual Syk/PI3K inhibitor, SRX3207, for the combinatorial inhibition of Syk and PI3K in one small molecule. This chemotype demonstrates efficacy in multiple tumor models and represents a novel combinatorial approach to activate antitumor immunity.
METHODS::Dysfunction of invariant natural killer T (iNKT) cells in tumor microenvironment hinders their anti-tumor efficacy, and the underlying mechanisms remain unclear. Here we report that iNKT cells increase lipid biosynthesis after activation, and that is promoted by PPARγ and PLZF synergically through enhancing transcription of Srebf1. Among those lipids, cholesterol is required for the optimal IFN-γ production from iNKT cells. Lactic acid in tumor microenvironment reduces expression of PPARγ in intratumoral iNKT cells and consequently diminishes their cholesterol synthesis and IFN-γ production. Importantly, PPARγ agonist pioglitazone, a thiazolidinedione drug for type 2 diabetes, successfully restores IFN-γ production in tumor-infiltrating iNKT cells from both human patients and mouse models. Combination of pioglitazone and alpha-galactosylceramide treatments significantly enhances iNKT cell-mediated anti-tumor immune responses and prolongs survival of tumor-bearing mice. Our studies provide a strategy to augment the anti-tumor efficacy of iNKT cell-based immunotherapies via promoting their lipid biosynthesis.
METHODS::Near-infrared photoimmunotherapy (NIR-PIT) is a newly developed and selective cancer treatment that induces necrotic and immunogenic cell death and utilizes a monoclonal antibody conjugated to a photo-absorber dye, IR700DX, activated by NIR light. Although CD44 is surface cancer marker associated with drug resistance, anti-CD44-IR700 NIR-PIT results in inhibited cell growth and prolonged survival in multiple tumor types. Meanwhile, anti-CD25-IR700-targeted NIR-PIT has been reported to achieve selective and local depletion of FOXP3+CD25+CD4+ regulatory T cells (Tregs), which are primary immunosuppressive cells in the tumor microenvironment (TME), resulting in activation of local antitumor immunity. Combined NIR-PIT with CD44- and CD25-targeted agents has the potential to directly eliminate tumor cells and also amplify the immune response by removing FOXP3+CD25+CD4+ Tregs from the TME. We investigated the difference in therapeutic effects of CD44-targeted NIR-PIT alone, CD25-targeted NIR-PIT alone, and the combination of CD44- and CD25-targeted NIR-PIT in several syngeneic tumor models, including MC38-luc, LL/2, and MOC1. The combined NIR-PIT showed significant tumor growth inhibition and prolonged survival compared with CD44-targeted NIR-PIT alone in all tumor models and showed prolonged survival compared with CD25-targeted NIR-PIT alone in MC38-luc and LL/2 tumors. Combined CD44/CD25 NIR-PIT also resulted in some complete remissions, whereas this was not achieved with either type of NIR-PIT alone. Therefore, combined NIR-PIT simultaneously targeting cancer antigens and immunosuppressive cells in the TME may be more effective than either type of NIR-PIT alone and may have potential to induce prolonged immune responses in treated tumors.