The Cysteine-Containing Cell-Penetrating Peptide AP Enables Efficient Macromolecule Delivery to T Cells and Controls Autoimmune Encephalomyelitis

Abstract

T cells are key immune cells involved in the pathogenesis of several diseases, rendering them important therapeutic targets. Although drug delivery to T cells is the subject of continuous research, it remains challenging to deliver drugs to primary T cells. Here, we used a peptide-based drug delivery system, AP, which was previously developed as a transdermal delivery peptide, to modulate T cell function. We first identified that AP-conjugated enhanced green fluorescent protein (EGFP) was efficiently delivered to non-phagocytic human T cells. We also confirmed that a nine-amino acid sequence with one cysteine residue was the optimal sequence for protein delivery to T cells. Next, we identified the biodistribution of AP-dTomato protein in vivo after systemic administration, and transduced it to various tissues, such as the spleen, liver, intestines, and even to the brain across the blood–brain barrier. Next, to confirm AP-based T cell regulation, we synthesized the AP-conjugated cytoplasmic domain of CTLA-4, AP-ctCTLA-4 peptide. AP-ctCTLA-4 reduced IL-17A expression under Th17 differentiation conditions in vitro and ameliorated experimental autoimmune encephalomyelitis, with decreased numbers of pathogenic IL-17A+GM-CSF+ CD4 T cells. These results collectively suggest the AP peptide can be used for the successful intracellular regulation of T cell function, especially in the CNS.

Cell-Penetrating Peptides-Mediated Therapeutic Agents Delivery into the Central Nervous System

“…They are crucial immune cells involved in the pathogenesis of many diseases, rendering them an essential therapeutic target. Kim et al 86 utilized a unique transdermal delivery peptide called AP, consisting of nine amino acids, which was derived from the human astrotactin-1 protein. The purpose of this peptide was to specifically target T cells with a T cell modulatory protein, namely ctCTLA-4.…”

Current understanding of CTLA-4: from mechanism to autoimmune diseases

“…A study reported that CTLA-4 signaling peptide can induce Treg cells and inhibit the activity of MS ( 131 ) ( Figure 3C ). Furthermore, a cysteine-containing cell-penetrating peptide (AP)-conjugated CTLA-4 cytoplasmic domain (AP-ctCTLA-4) peptide attenuated the activity of EAE by inhibiting IL-17A expression and reducing the number of pathogenic IL-17A + GM-CSF + CD4 T cells ( 132 ). The results suggest that CTLA-4 is a target for the treatment of MS.…”

The Role of Cytotoxic T-Lymphocyte Antigen 4 in the Pathogenesis of Multiple Sclerosis

“…Moreover, Kim et al have identified another interesting approach [ 110 ]. They synthesized an AP-ctCTLA-4 peptide to modulate the T cell function in a mouse model of EAE, and they showed that AP-ctCTLA-4 decreased IL-17A expression under Th17 differentiation conditions in vitro and improved EAE, with reduced numbers of pathogenic IL-17A + GM-CSF + CD4 T cells.…”

“…They synthesized an AP-ctCTLA-4 peptide to modulate the T cell function in a mouse model of EAE, and they showed that AP-ctCTLA-4 decreased IL-17A expression under Th17 differentiation conditions in vitro and improved EAE, with reduced numbers of pathogenic IL-17A + GM-CSF + CD4 T cells. Both male and female animals were used for the experimental procedures [ 110 ].…”