Development of Novel Immunoconjugates Activating NK Cells for Targeted Immunotherapy

Background: NK cells are crucial effectors in the immune response against viral diseases and cancer. Specifically in HIV-1 and pancreatic ductal adenocarcinoma (PDAC), their functions are suppressed leading to impaired cytotoxicity towards the target. To restore their functions, Natural Killer (NK) cell immunotherapy, encompassing various approaches such as IL-15-based therapy and NK cell engagers (NKCE), has emerged as an attractive therapeutic strategy.
Methods: We designed several immunotherapeutic constructs aimed to stimulate NK cell functions and specifically tailored for the disease context. Natural killer activating Multimeric immunotherapeutic compleX (NaMiX) is a multimer of IL-15/IL-15Rα associated with single chain variable fragments (scFvs) targeting NK cell receptors. We designed different formats of NaMiX targeted against NKG2A and KIR2DL to tackle HIV-1 infection, and against NKp46 to tackle PDAC. Additionally, we also developed a trispecific killer engager (TriKE) targeting NKG2D, NKp30 and CEA to enhance the interactions between NK cells and PDAC cells. We characterized the structure of our immunoconjugates and evaluated their ability to stimulate NK cell functions towards the desired target in vitro. We then investigated their therapeutic potential in humanized mice models.
Results: All NaMiX formats stimulated the activation, degranulation and cytotoxic activity of NK cells through the pSTAT5 pathway. NaMiX tackled against HIV-1 increased the cytotoxicity of PBMCs from healthy donors and people living with HIV (PLHIV) against ACH-2 cells, a T cell line latently infected with HIV, but also against Raji cells, a NK cell-resistant cancer cell line. In humanized mice infected with HIV and under combination antiretroviral therapy (cART), NaMiX stimulated the development of functional cytotoxic NK cells, and tended to decrease total HIV-1 DNA in human CD45+ cells from the lung and the bone marrow. In PDAC models, NaMiX enhanced NK cell cytotoxicity against pancreatic cancer cells, both in 2D and in 3D spheroids, and against organoids derived from PDAC patients. In humanized mice bearing a subcutaneous pancreatic xenograft, TriKE tended to delay xenograft growth without NK cell activation while NaMiX enhanced the development of cytotoxic lymphocytes and the infiltration of NK cells within the xenograft.
Conclusions: NaMiX is a promising therapeutic strategy, both against HIV-1 and hostile solid tumors like PDAC. In PDAC, combining NK cell activation and enhanced interactions with pancreatic cancer cells through NK cell engagers showed potential, although optimizations of our model and immunoconjugates are required to reach in vivo efficacy. Therefore, enhancing NK cell recruitment with allogenic NK cell administration and incorporating features in our immunoconjugates to tackle disease-specific challenges such as viral reservoirs or tumor microenvironment (TME) inhibitors, represent relevant future directions.