# Engineering Bi-Specific CAR-NK Cells to Restore Antibody-Dependent Cellular Cytotoxicity in Solid Tumors

## TL;DR

This study shows how engineered CAR-NK cells can overcome tumor-induced immune suppression and restore antibody-dependent cell toxicity in solid tumors.

## Contribution

A novel bi-specific CAR-NK cell design combining tumor targeting and CD16 arming to restore ADCC in TGF-β-rich tumor environments.

## Key findings

- TGF-β impairs NK cell function by downregulating CD16 and NKG2D and increasing exhaustion markers.
- Bi-specific CAR-NK cells with scFv and non-cleavable CD16 domains showed ADCC synergy in tumoroids despite TGF-β exposure.
- The TGF-β/SMAD2 axis is identified as a central driver of NK cell dysfunction in ovarian cancer.

## Abstract

What are the main findings?
Impairment of NK cells under Tumor microenvironment (TME): Chronic exposure to Transforming growth factor-beta (TGF-β) induces a sustained dysfunctional NK cell phenotype characterized by the downregulation of key activating receptors (CD16 and NKG2D) and a profound inability to infiltrate 3D tumor structures.Dual-Targeting Potency: Developed a novel CAR-NK cell architecture incorporating both a tumor-specific scFv (e.g., targeting FRα) and a non-cleavable CD16 domain, which demonstrated ADCC-mediated synergistic cytotoxicity in vitro and tumoroids even when exposed to high concentrations of TGF-β.

Impairment of NK cells under Tumor microenvironment (TME): Chronic exposure to Transforming growth factor-beta (TGF-β) induces a sustained dysfunctional NK cell phenotype characterized by the downregulation of key activating receptors (CD16 and NKG2D) and a profound inability to infiltrate 3D tumor structures.

Dual-Targeting Potency: Developed a novel CAR-NK cell architecture incorporating both a tumor-specific scFv (e.g., targeting FRα) and a non-cleavable CD16 domain, which demonstrated ADCC-mediated synergistic cytotoxicity in vitro and tumoroids even when exposed to high concentrations of TGF-β.

What are the implications of the main findings?
Versatility of CD16 Arming in CAR-NK: The study demonstrates that bi-specific CAR-NK platform can restore the synergy between cellular immunotherapy and monoclonal antibodies, potentially expanding the therapeutic window and preventing antigen escape through ADCC.Clinical Translation: These advances provide a compelling proof of concept for next-generation CAR-NK therapies engineered to function effectively within highly suppressive solid tumor environments.

Versatility of CD16 Arming in CAR-NK: The study demonstrates that bi-specific CAR-NK platform can restore the synergy between cellular immunotherapy and monoclonal antibodies, potentially expanding the therapeutic window and preventing antigen escape through ADCC.

Clinical Translation: These advances provide a compelling proof of concept for next-generation CAR-NK therapies engineered to function effectively within highly suppressive solid tumor environments.

Natural Killer (NK) cell-based immunotherapy relies on CD16-mediated Antibody-Dependent Cellular Cytotoxicity (ADCC), yet the ovarian tumor microenvironment (TME) severely compromises this function via Transforming Growth Factor-beta (TGF-β). This study investigated the molecular mechanisms driving this suppression and evaluated a bi-specific Chimeric Antigen Receptor (CAR) strategy to overcome this hurdle. Primary PBNK cells exposed to TGF-β showed sustained canonical SMAD2 phosphorylation, accompanied by a marked reduction in activating receptors such as CD16 and NKG2D and an increase in exhaustion markers such as PD-1. Functionally, these phenotypic alterations led to failed infiltration and cytotoxicity in vitro and within ovarian cancer-derived spheroids. To overcome this limitation, we engineered NK-92 cells with a bi-specific CAR-targeting Folate Receptor Alpha (FRα) and CD16. While TGF-β typically impairs NK cell function, our armed CAR-NK cells successfully infiltrated tumoroids and synergized with Trastuzumab to induce potent ADCC-mediated lysis. Our findings define the TGF-β/SMAD2 axis as a central driver of NK cell dysfunction in ovarian cancer and demonstrate that bi-specific CAR-NK platforms offer a robust therapeutic solution to bypass TME-induced suppression and restore antibody-mediated tumor suppression.