The CD64/CD28/CD3 ζ chimeric receptor reprograms T-cell metabolism and promotes T-cell persistence and immune functions while triggering antibody-independent and antibody-dependent cytotoxicity
Authors: Caratelli Sara , De Paolis Francesca , Silvestris Domenico Alessandro , Baldari Silvia , Salvatori Illari , Tullo Apollonia , Lanzilli Giulia , Gurtner Aymone , Ferri Alberto , Valle Cristiana , Padovani Simona , Cesarini Valeriana , Sconocchia Tommaso , Cifaldi Loredana , Arriga Roberto , Spagnoli Giulio Cesare , Ferrone Soldano , Venditti Adriano , Rossi Piero , Pesole Graziano , Toietta Gabriele , Sconocchia Giuseppe 
Published: December 2024

Background
Recent studies have shown that CD32/CD8a/CD28/CD3ζ chimeric receptor cells directly kill breast cancer cells, suggesting the existence of cell surface myeloid FcγR alternative ligands (ALs). Here, we investigated the metabolism, ALs, cytotoxicity, and immunoregulatory functions of CD64/CD28/CD3ζ in colorectal cancer (CRC) and squamous cell carcinoma of the head and neck.
Methods
The CD64/CD28/CD3ζ -SFG retroviral vector was used to produce viruses for T-cell transduction. T-cell expansion and differentiation were monitored via flow cytometry. Gene expression was assessed by RNA-seq. Bioenergetics were documented on a Seahorse extracellular flux analyzer. CD64/CD28/CD3ζ polarization was identified via confocal microscopy. Cytotoxicity was determined by MTT assay and bioluminescent imaging. Tridimensional antitumor activity of CD64/CD28/CD3ζ T cells was achieved by utilizing HCT116-GFP 3-D spheroids via the IncuCyte S3 Live-Cell Analysis system. The intraperitoneal distribution and antitumor activity of NIR-CD64/CD28/CD3ζ and NIR-nontransduced T cells were investigated in CB17-SCID mice bearing subcutaneous FaDu Luc + cells by bioluminescent and fluorescent imaging. IFNγ was assessed by ELISA.
Results
Compared to CD16/CD8a/CD28/CD3ζ T cells, CD32/CD8a/CD28/CD3ζ T cells, and nontransduced T cells, CD64/CD28/CD3ζ T cells exhibited the highest levels of cell expansion and persistence capacity. A total of 235 genes linked to cell division and 52 genes related to glycolysis were overexpressed. The glycolytic phenotype was confirmed by functional in vitro studies accompanied by preferential T-cell effector memory differentiation. Upon CD64/CD28/CD3ζ T-cell conjugation with CRC cells, CD64/CD28/CD3ζ cells polarize at immunological synapses, leading to CRC cell death. CD64/CD28/CD3ζ T cells kill SCCHN cells, and in combination with the anti-B7-H3 mAb (376.96) or anti-EGFR mAb, these cells trigger ADCC in vitro under 2D and 3D conditions. The 376.96 mAb combined with CD64/CD28/CD3ζ T cells had anti-SCCHN activity in vivo. In addition, they induce the upregulation of PD-L1 and HLA-DR expression on cancer cells via IFNγ. PD-L1 upregulation resulted in the generation of ADCC, which enhanced direct cytotoxicity. These findings indicate that, despite the glycolytic phenotype, these cells mediate long-lasting HLA-independent cytotoxicity and ADCC in CRC and SCCHN cells.
Conclusions
CD64/CD28/CD3ζ T cells could significantly impact the rational design of personalized studies to treat CRC and HNSCC and the identification of novel FcγR ALs in cancer and healthy cells.