Targeted Inhibition of Colorectal Carcinoma Using a Designed CEA-Binding Protein to Deliver p53 Protein and TCF/LEF Transcription Factor Decoy DNA

Colorectal carcinoma (CRC) is characterized by mutations in p53 and the Wnt signaling pathway, and immunotherapy has shown limited efficacy in microsatellite-stable CRC. Here, CEABP1, a binding protein for the CRC biomarker carcinoembryonic antigen (CEA), was designed de novo through the AI-based computational generation methods RFDiffusion/ProteinMPNN and stringent in silico selection, for targeted delivery of purified p53 protein and transcription factor T-cell factor (TCF)/lymphoid enhancer-binding factor (LEF) transcription factor decoy (TFD) DNA into CRC cells. The cell-penetrating peptide (CPP) p28 was employed to deliver the p28-p53-CEABP1 protein, which significantly enhanced p53's inhibition of CRC cell proliferation and xenograft tumor growth. Codelivery of the p14ARF protein together with p53 prolonged the effective antitumor duration of p53. In addition, the DNA binding domain of Max was fused with CPP and CEABP1 to deliver TCF/LEF TFD DNA, comprising concatenated consensus binding motifs for TCF/LEF and Max, into CRC cells to inhibit Wnt target gene transcription, leading to marked suppression of CRC cell proliferation and xenograft tumor growth. These findings paved the way for the development of precision anticancer therapeutics using designed binding proteins of tumor biomarkers for targeted delivery of tumor suppressor proteins and TFD DNA.