Assistant Professor Boston University Boston, Massachusetts, United States
Introduction: : Cancer cells evade detection and attack by the host immune system through various mechanisms, including the recruitment of regulatory T cells (Tregs) that inactivate other immune cells. Indeed, a high ratio of tumor-infiltrating Tregs:CD8+ T cells is associated with poor clinical outcomes.[1] Current immunotherapies targeting Tregs, such as monoclonal antibodies and small molecules, traditionally work to deplete the number of Tregs. However, this can lead to autoimmunity. Thus, a promising alternative is to functionally inactivate Tregs without depleting their numbers. Forkhead box protein 3 (Foxp3) is a Treg transcription factor integral to their suppressive function and can be targeted using P60, a 15-mer cell-penetrating peptide.[2] However, peptides free in solution have limited efficacy due to degradation and poor biodistribution, necessitating high dosages which can cause off-target toxicity. To overcome these limitations, we harness metal-organic framework (MOF) nanostructures, highly porous and modular nanomaterials that can protect biomolecular cargo and elongate release.[3] This enables the delivery of lower doses of P60 to achieve the same therapeutic effect. Herein, we explore the ability to restore antitumor activity by inactivating Tregs using MOF-mediated P60 delivery.
Materials and
Methods: : Peptide P60 (MW: 1994.3 g/mol) was encapsulated into zirconium-based 300-400 nm MOF with a 3.3 nm pore diameter (P60@MOF). Encapsulation efficiency was quantified using electrospray ionization mass spectrometry. Release of P60 was measured using SDS PAGE. Foxp3-luciferase Jurkat reporter cells were used to determine P60 inhibition of Foxp3, which was induced by phorbol 12-myristate 13-acetate (PMA) and ionomycin stimulation. Cells were treated with 10 µM by P60 for 24 h, after which luciferase luminescence was measured. MOF-cell interactions were visualized using confocal microscopy, using a cell membrane stain and the MOF’s inherent fluorescence.
Results, Conclusions, and Discussions:: P60 was successfully encapsulated into MOFs with an encapsulation efficiency ca. 98% and a loading of 0.5 mg/mg MOF. P60 exhibited ca. 30% release from the MOF after 24 h and sustained release up to 1 week ex cellulo. When treated with reporter cells to isolate the mechanistic Foxp3 inhibition due to MOF-mediated P60 delivery, we observed that P60@MOF significantly reduced luciferase signal (associated with Foxp3 expression) by 40%. Notably, this significant decrease was not observed by reporter cells treated with free P60 peptide at the same concentration. Incubation of the MOF with isolated murine CD4+ T cells illustrated clear co-localization of the MOF with the T cell surface. We further observed elevated antigen-specific pro-inflammatory cytokine secretion from splenocytes treated in vivo with P60@MOF compared to P60 or saline, as measured via an ELISpot ex vivo. This supports that targeted Treg inhibition via P60 allows for increased cytotoxic T cell activity upon ex vivo antigen stimulation. Harnessing the MOF architecture to stably deliver P60 offers the opportunity to enhance antitumor activity through Treg modulation without long-term autoimmune effects.
Acknowledgements and/or References (Optional): : 1. Tanaka, A; Sakaguchi, S. Eur. J. Immun. 2019, 49:1140-1146. 2. Lozano, T, et al. Biomaterials. 2016, 91:73-80. 3. Teplensky MH, et al. Chem. 2019, 5:2926-2941.
