Background Adoptive immunotherapy based on chimeric antigen receptor (CAR)-T cells has led to successful treatment of different hematological malignancies. However, it remains extremely challenging for solid tumors, mostly because of “on-target off-tumour” toxicity, as observed in the case of anti-HER2 CAR-T treatment of HER2-amplified colorectal cancer (HER2_amp CRC).¹ To enable adoptive immunotherapy against HER2_amp CRC, we therefore considered a combinatorial strategy based on the synNotch-based artificial regulatory network. A synthetic Notch receptor including anti-HER2 scFv as extracellular domain and the GAL4-VP64 artificial transcription factor as intracellular domain was employed.² Engagement of the anti-HER2 domain by target cells drives GAL4-VP64 cleavage and translocation to the nucleus, leading to the expression of a CAR under a GAL4-responsive promoter. In this way, only cells co-expressing both HER2 and the CAR target are killed. As a CRC-specific CAR target we selected CEA, product of the CEACAM5 gene. CEA expression is restricted to the digestive tract and is increased in cancer.

Methods For the generation of HER2-synNotch CEA-CAR effectors, we chose the natural killer cell line NK-92 and transduced it with two lentiviral vectors, encoding respectively the HER2 synNotch and the second-generation anti-CEA CAR with CD28 costimulatory domain.Transduced cells were repeatedly sorted in the ON and OFF state to select those with the best CAR induction after synNotch engagement; cloning of sorted cells led to identification of an optimally responsive clone (clone 5F).

Results In vitro, the 5F clone showed no basal CEA-CAR expression and massive induction in the presence of HER2_amp cancer cells. It also displayed selective cytotoxicity against HER2_amp/CEA_pos CRC cells, with minimal killing activity against HER2_amp/CEA_neg breast cancer cells, or against CRC cells expressing CEA but without HER2 amplification. In vitro 3D models highlighted better recruitment and infiltration by clone 5F respect to NK-92 WT cells, only of HER2_amp organoids. In vivo, the clone 5F significantly impaired tumor growth in two different HER2_amp CRC models.

Conclusions The observed selective efficacy both in vitro and in vivo of the HER2-synNotch/CEA-CAR approach opens a perspective for possible clinical applications for HER2_amp CRC displaying primary or secondary resistance to HER2/EGFR blockade.

References

1. Morgan RA, Yang JC, Kitano M, Dudley ME, Laurencot CM, Rosenberg SA. Case Report of a Serious Adverse Event Following the Administration of T Cells Transduced With a Chimeric Antigen Receptor Recognizing ErbB2. Mol. Ther.2010; 18:843-851.

2. Roybal KT, Williams JZ, Morsut L, Rupp LJ, Kolinko I, Choe JH, Walker WJ, McNally KA, Lim WA. Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors. Cell. 2016; 167:419-432.

Ethics Approval All the experiments were performed in compliance with institutional guidelines under the Ethical committee of Candiolo Cancer Institute consensus and under the Italian Ministry of Health approved protocol (Authorization n°225/2021-PR).