Functions And Molecular Mechanisms Of Epidermal Growth Factor Receptor-targeted Chimeric Antigen Receptor-modified T Cells In Triple-negative Breast Cancer

Triple-negative breast cancer(TNBC) is well known for its strong invasiveness,rapid recurrence and poor prognosis.Immunotherapy,including chimeric antigen receptor-modified T(CAR-T) cells,has emerged as a promising tool to treat TNBC.The identification of a specific target tumor antigen and the design of an effective CAR are among the many challenges of CAR-T therapy.First,we reported that epidermal growth factor receptor(EGFR)is highly expressed in TNBC.Therefore,we consequently designed an optimal third generation of CAR targeting EGFR constituted by a signal peptide(Spl) of the IL-2 receptor,an anti-EGFR scFv from cetuximab(a monoclonal antibody used in the clinic),a spacer(IgG1 Fc or hinge),a CD28 transmembrane domain and CD28,human 4-1BB and CD3ζ intracellular signalling domains.The efficacy of primary T lymphocytes infected with EGFR CAR lentivirus(EGFR CAR-T)against TNBC was evaluated both in vitro and in vivo.The functional status of activated EGFR-CAR T cells and signalling pathways activated in tumor and EGFR CAR-T cells were revealed by RNA sequencing analysis.Mechanistically,in vitro stimulation with TNBC cells induced the expansion of na(?)ve-associated EGFR CAR-T cells and enhanced their persistence.Furthermore,EGFR CAR-T cells activated the interferony,granzyme-perforin-PARP and Fas-FADD-caspase signalling pathways in TNBC cells.Finally,we demonstrate that third-generation EGFR CAR-T cells exerted potent and specific suppression of TNBC cell growth in vitro,whereas limited cytotoxicity was observed towards normal breast epithelial cells or estrogen receptor-positive breast cancer cells.This capability was further demonstrated in vivo in a xenograft mouse model,with minimal off-tumor cytotoxicity.In conclusion,we demonstrate that EGFR is a relevant immunotherapeutic target in TNBC,and EGFR CAR-T exhibits potent and specific antitumor activity against TNBC,suggesting the potential of this third-generation EGFR CAR-T as an immunotherapy tool to treat TNBC in the clinic.