Frizzled-7-targeting antibody-derived bifunctional protein retargets NK cells against triple-negative breast cancer cells via MICA-NKG2D axis.

Background: Hypoxia is associated with the evasion of triple-negative breast cancer (TNBC) from immune surveillance. Hypoxia increases the subpopulation of putative TNBC stem-like cells (TNBCSCs) through activating Wnt/β-Catenin signaling. The shedding of MHC class I-related chain A (MICA) is particularly noteworthy in cancer stem cells (CSCs), promoting the resistance of CSCs to natural killer (NK) cell cytotoxicity. To reestablish MICA/NKG2D-mediated immunosurveillance, we proposed the design of a fusion protein (SHH002-hu1-MICA) which consists of Frizzled-7 (Fzd7)-targeting antibody and MICA, serving as an engager retargeting NK cells against TNBCs, especially TNBCSCs.

Methods: Opal multicolor immunohistochemistry staining was used to validate the expression of membrane MICA (mMICA) and existence of NK cells in TNBC tumors; flow cytometry (FCM) assay was used to detect the expression of Fzd7/mMICA on TNBCs. Biolayer interferometry (BLI) and surface plasmon resonance (SPR) assays were executed to assess the affinity of SHH002-hu1-MICA towards rhFzd7/rhNKG2D; near-infrared imaging assay was used to evaluate the targeting capability. A cytotoxicity assay was conducted to assess the effects of SHH002-hu1-MICA on NK cell-mediated killing of TNBCs, and FCM assay to analyze the effects of SHH002-hu1-MICA on the degranulation of NK cells. Finally, TNBC cell-line-derived xenografts were established to evaluate the anti-tumor activities of SHH002-hu1-MICA in vivo.

Results: The expression of mMICA is significantly downregulated in hypoxic TNBCs and TNBCSCs, leading to the evasion of immune surveillance exerted by NK cells. The expression of Fzd7 is significantly upregulated in TNBCSCs and exhibits a negative correlation with the expression of mMICA and infiltration level of NK cells. On accurate assembly, SHH002-hu1-MICA shows a strong affinity for rhFzd7/rhNKG2D, specifically targets TNBC tumor tissues, and disrupts Wnt/β-Catenin signaling. SHH002-hu1-MICA significantly enhances the cytotoxicity of NK cells against hypoxic TNBCs and TNBCSCs by inducing the degranulation of NK cells and promotes the infiltration of NK cells in CD44 regions within TNBC xenograft tumors, exhibiting superior anti-tumor activities than SHH002-hu1.

Conclusions: SHH002-hu1-MICA maintains the targeting property of SHH002-hu1, successfully activates and retargets NK cells against TNBCs, especially TNBCSCs, exhibiting superior antitumor activities than SHH002-hu1. SHH002-hu1-MICA represents a promising new engager for NK cell-based immunotherapy for TNBC.