Abrogation of natural killer cell check point pathway LLT1/CD161 by novel anti LLT1 antibody ZM008 and its therapeutic applications in solid cancers.
Authors: Ghosh Maloy , Bhattacharjee Sanghamitra , Tiwari Anurag , Dubey Ashvini Kumar , Manjunath Yogendra , Lahiri Pallavi , Horowitz Amir 
Published: June 2022

e14503

Background: LLT1/CD161 interaction plays important roles in cancer immune surveillance, fine tuning of NK cells responsiveness and down regulating effector functions. LLT1 gene expression was confirmed on multiple tumors as well as on various infiltrating immune cells. Therefore, it is relevant to develop therapeutic monoclonal antibody against LLT1 that could potentiate ADCC, ADCP, CDC, bispecific, CAR-T/NK functions to eliminate tumor cells. In this work, we have described discovery of novel human anti-LLT1 mAb ZM008, NK cells activation and anti-tumor function of ZM008. A Phase 1 open label, multicenter dose escalation clinical trial is planned in patients with advanced solid tumors to evaluate the safety and tolerability of ZM008, as monotherapy and in combination with Pembrolizumab. Methods: LLT1 gene expression was analyzed using TCGA database for 33 cancer indications. 60 Prostate cancer FFPE samples from Mt. Sinai hospital biorepository, NY were tested. Multiple in vitro studies were conducted to demonstrate the mode of action of ZM008 and efficacy was determined in HuNOG-EXL animals with PC3 xenograft. Cynomolgus monkeys were dosed with ZM008 (QW, 4 weeks) to understand the safety, pharmacokinetics, and toxicokinetics. Results: TCGA analysis revealed differential LLT1 gene expression in advance stages of NSCLC, TNBC, HNSCC, PRAD and urothelial cancers and high correlation with immune checkpoint pathways. HALO® data analysis of PRAD FFPE samples have shown distinct and strong signal on tumor cells with ZM008. Cell based assays with ZM008 revealed activation of CD69, NKG2D, CD107 and IFNγ on human NK cells leading to significant cytotoxicity of PC3 (MHC+ve) and K562 (MHC-ve) tumor cells. In vivo tumor growth inhibition was achieved at 10 mg/kg ZM008 treatment in HuNOG-PC3 xenograft tumor model. Anti-tumor function of ZM008 was demonstrated by high infiltration of CD8+ T cells and CD56+ NK cells in the TME. Cynomolgus study suggests ZM008 was well tolerated at 10 mg/kg and upto 125 mg/kg dosing with no mortality or adverse reactions. Half-life of ZM008 was determined up to 259 hours. Conclusions: This study for the first time revealed intense staining of LLT1 protein in high Gleason score prostate cancer, demonstrating therapeutic potential of ZM008 in solid cancers not responsive to current I/O checkpoint therapies. In vitro and in vivo experiments have shown human NK cells activation resulting in significant tumor growth reduction. CD56+ NK and CD8+ T cells infiltrations indicate transformation of “Cold tumors” to “Hot tumors”. Targeting LLT1/CD161 interaction in TME could stimulate robust NK and T cell mediated anti-tumor checkpoint functions. The ZM008 drug product is safe and well tolerated in Cynomolgus monkey safety and toxicity studies paving the way for the open-label, multicenter, Phase 1 dose-escalation study with ZM008. .