Improving effector cells for CAR-NK immunotherapy against colorectal cancer

Chimeric Antigen Receptor (CAR)-based immunotherapy targeting carcinoembryonic antigen (CEA) represents a promising strategy for the treatment of colorectal cancer (CRC), a leading cause of cancer-related mortality worldwide. With respect to CAR-T cells, whose activity against solid tumors is limited by significant side effects, CAR-NK cells represent a potentially safer alternative, offering reduced toxicity while maintaining effective anti-tumor activity. We therefore worked at developing and optimizing CEA-specific CAR-NK cells against CRC, along three research lines: (i) selection and validation of a reference CEA CAR in the NK-92 model; (ii) further engineering of CEA.CAR-NK-92 cells with interleukins, and (iii) exploration of additional NK effector types. To select the reference CEA CAR construct, second generation CARs based on two scFvs with different affinities (hMN-14 and BW431/26, respectively higher and lower affinity) were constructed. Stable NK-92 clones were generated for each construct (higher affinity: NK-92.F3 clone; lower affinity: NK-92.G8 clone) and functionally characterized. In vitro , the NK-92.F3 clone exhibited stronger killing activity against CEA-positive CRC cells. In vivo , in NOD/Scid mice bearing CEA+ CRC xenografts, the two clones displayed comparable activity, both significantly reducing tumor progression and confirming their therapeutic potential. Considering the higher affinity and the better in vitro performances, the hMN-14-based CAR was selected as the reference CAR for subsequent evolutions of the NK platform. To enhance CAR-NK-92 performances, wild-type NK-92 and NK-92.F3 cells were engineered to constitutively express different interleukins. Functional assays demonstrated that specific interleukin expression markedly increased cytotoxicity and activation, highlighting cytokine engineering as an effective strategy to potentiate CAR-NK activity. To overcome the major intrinsic limitation of NK-92 cells, i.e. the need for irradiation before infusion, we investigated induced pluripotent stem cell (iPSC)-derived NK cells (iNKs) as a renewable and clinically scalable alternative NK source. Four iPSC-to-NK differentiation protocols were compared, three based on 3D embryoid body intermediates and one on 2D monolayer. Of these, one 3D and the 2D protocol proved more reliable. iPSC cells were then transduced with the reference CEA CAR, cloned, differentiated into NK and validated for functionality and cytotoxic performances. In conclusion, this work identified a robust reference CEA-CAR construct and established two complementary strategies, cytokine engineering to enhance effector function and iPSC-derived NK cell generation to overcome NK-92 limitations, providing a foundation for the development of safer, scalable, and next-generation CAR-NK therapies for colorectal cancer.