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Development of bispecific CAR-NK cells targeting tumor cells and cancer-associated fibroblasts to modulate NK-specific signaling and overcome the tumor microenvironment
Abstract No. : 33

Category

Engineered innate immune cell therapy (e.g., NK,NKT, macrophage,γδT, etc.)
Chimeric antigen receptors (CARs) have shown promising outcomes for the treatment of blood cancers. Unfortunately, their efficacy for treating solid tumors, particularly pancreatic adenocarcinoma (PDAC), has been limited due to various challenges. The immune-suppressive tumor microenvironment (TME) in PDAC, is a major barrier, characterized by low infiltration and exhausted status of immune cells. Cancer-associated fibroblasts (CAFs), a key component of the TME, significantly contribute to immune suppression. To overcome this, we design bispecific CAR-NK (BiCAR-NK) cells targeting both tumor cells and CAFs to modulate the TME and enhance efficacy. BiCAR-NK cells recognize the tumor antigen mesothelin (MSLN), a well-known tumor-associated antigen in various solid cancers, and the CAFs antigen endoglin (ENG), which is highly expressed in the CAFs. We construct BiCAR-NK cells with different linkers between two single chain variable fragments(scFvs) and varied the order of scFvs. BiCAR3, designed as αENG-Linker2-αMSLN shows high cytotoxicity against MSLN- or ENG-expressing target cells. We further evaluate killing activity of BiCAR-NK cells using TME-mimetic 3D multi-cancer spheroids overlaid with pancreatic stellate cells. Consistent with the observations in the BiCAR3-mediated cancer killing in a 2D system, BiCAR3 exhibits efficient cytotoxicity in a 3D system. Additionally, we develop an NK-specific CAR construct in BiCAR3 to improve persistence. We newly discover that BiCAR3 bearing co-domain4 has enhanced serial and cross-serial killing activity compared to 2nd generation CAR construct(4-1BB-CD3ζ), which is commonly used in CAR-T/NK constructs. Notably, cytotoxicity against ENG is significantly increased. This BiCAR-NK cells with co-domain4 may serve as a potential therapeutic option for solid tumors.
#CAR-NK
#Bispecific-CAR
#Tumor microenvironment (TME)
#Cancer-associated fibroblasts (CAFs)
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