Background Lectin-like transcript 1 (LLT1/CLEC2D) interaction with CD161 on NK cells facilitates tumor immune escape.1, 2, 3 Others and we have shown that anti LLT1 antibody disrupts LLT1-CD161 interaction to release the break on NK cells, activate NK cells, improve immune cell infiltration and enhance tumor cell cytotoxicity.4, 5, 6 Present work describes a novel anti LLT1 antibody ZM008, it’s mode of action, GLP safety and toxicity studies, in vivo and ex vivo anti-tumor effects.
Methods Cytokine release and immune cell activation were monitored using human PBMC. PC3 xenografted HuNOG-EXL mice were treated with ZM008 to monitor anti-tumor effects and tumor IHC was performed to study the TME. 4 weeks repeat dose GLP study in Cynomolgus monkey was conducted to determine safety and toxicity of ZM008 at 10-125mg/kg dosing. 3D ex vivo culture of patient biopsies from NSCLC and muscle invasive bladder cancer (MIBC) were used to determine efficacy of ZM008.
Results ZM008 induced CD69 activation and perforin/granzyme B expression on cytotoxic CD8+ T cells and CD16+ NK cells and promoted release of IFNγ and TNFα from immune cells. In HuNOG-EXL-PC3 xenograft mice, ZM008 treatment resulted in ~48% tumor growth reduction and significant tumor infiltration of CD8+ T cells, CD56+ NK cells and NKG2D+ cells was observed. Additionally, low Ki67 and high caspase 3 expression after ZM008 treatment indicates antitumor effects (figure 1). ZM008 exposure was maintained over the treatment period with no mortality or toxicity and was well tolerated in 4 weeks repeat intravenous dosing in Cynomolgus monkeys. No ZM008 related gross pathological findings, organ weight changes or histologic lesions were observed. 3D tumoroid culture from biopsies of NSCLC and MIBC patients were treated with ZM008 monotherapy and Pembrolizumab combination therapy revealed >50% reduction of tumoroids. High-content imaging of the tumoroid clearly shows disintegration of TME and infiltration of immune cells (figure 2).
Conclusions This is the first report describing anti LLT1 antibody efficacy in patient biopsy samples. Tumoroids derived from lung cancer and bladder cancer biopsies revealed significant growth reduction and immune cell infiltration with ZM008 treatment. ZM008 disrupts LLT1-CD161 pathway and transforms the TME to immune responsive “Hot” tumor by activation of immune cells, cytokine secretion, granzyme B/perforin release, and tumor cytotoxicity. GLP safety and toxicity studies in Cynomolgus monkey support future clinical application of ZM008. Overall, the data suggests ZM008 drug product is poised to initiate phase 1 trial (monotherapy and combination with Pembrolizumab) in multiple solid cancers.
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Ethics Approval Ethics Approval of preclinical study: This study was approved by the Institutional Animal Ethics Committee IAEC Protocol Approval No: SYNGENE/IAEC/1140/02-2020. Institutional Animal Care and Use Committee (IACUC) responsible for the Testing Facility’s compliance with applicable laws and regulations concerning the humane care and use of laboratory animals.
Ethics Approval for GLP safety and toxicity study: “This study is approved by Altasciences Preclinical Services Institutional Animal Care and Use Committee (IACUC protocol number – 162822-01)
Ethics Approval for Ex Vivo study: ”This study was approved by the National Bioethics Committee in Romania, approval number 9S/4 from 25.11.2019.”
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