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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering - Poster Session D

Poster V12 - A CATCH supramolecular peptide assembly induces cross presentation in dendritic cells

Friday, October 25, 2024

3:30 PM – 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

Stephen Michel, BS

Graduate Student
University of Florida, United States

Primary Investigator(s)

BK

Benjamin G. Keselowsky

Professor
University of Florida, United States

Last Author(s)

Gregory A. Hudalla, PhD (he/him/his)

Associate Professor
University of Florida
Gainesville, Florida, United States

Co-Author(s)

ES

Elias Sayour

Professor
University of Florida, United States

Introduction: The incorporation of antigenic peptides into supramolecular delivery systems can be leveraged to induce potent T cell mediated immune responses [1]. The induction of a cytotoxic T cell response requires a system that can enter cells and undergo endosomal escape, delivering antigen for an MHC Class I (MHCI) mediated CD8 T cell response. Here we present a charge-complementary peptide system, CATCH [2], as a potent tool for inducing cross presentation in dendritic cells. The mixing of CATCH- and a molar excess of CATCH+ molecules in the presence of an intermolecular crowder, polyethylene glycol (PEG), produces a nano-scale CATCH granule system. These granules enable transfection of cells with co-assembled fusion peptides or proteins in the granule. Granules for antigen presentation were generated by co-assembling CATCH+ peptides linked to OT-I restricted ovalbumin peptide (SIINFEKL) (CATCH+SIINFEKL) and CATCH- peptides recombinantly linked to green fluorescent protein (GFP) (CATCH-GFP). These antigen presentation granules demonstrate significant cross-presentation in a mouse dendritic cell line (DC2.4).

References:
1. Chesson, C. B., Huelsmann, E. J., Lacek, A. T., Kohlhapp, F. J., Webb, M. F., Nabatiyan, A., Zloza, A., & Rudra, J. S. (2014). Antigenic peptide nanofibers elicit adjuvant-free CD8⁺ T cell responses. Vaccine, 32(10), 1174–1180. https://doi.org/10.1016/j.vaccine.2013.11.047
2. Seroski, Dillon & Restuccia, Antonietta & Sorrentino, Anthony & Knox, Kevin & Hagen, Stephen & Hudalla, Gregory. (2016). Co-Assembly Tags Based on Charge Complementarity (CATCH) for Installing Functional Protein Ligands into Supramolecular Biomaterials. Cellular and Molecular Bioengineering. 9. 10.1007/s12195-016-0459-2.

Materials and

Methods: SIINFEKL and CATCH+SIINFEKL were produced by peptide synthesis. CATCH-GFP was recombinantly expressed in E. coli and purified.

DC2.4s were plated and incubated for 24 hours before granule treatment. CATCH granules were prepared by mixing CATCH-GFP with a molar excess of CATCH+SIINFEKL and PEG in PBS and immediately added to cells. After 24 hours, cells were harvested, counted, and stained for flow cytometry. A positive control was prepared by pulsing DC2.4s with SIINFEKL peptide 2 hours before staining. Flow cytometry was performed on an Aurora 5L Spectral Cytometer.

Results, Conclusions, and Discussions:

Results:
DC2.4 cells treated with CATCH granules displayed significantly greater signal for both MHCI-SIINFEKL and GFP in comparison to negative controls (Figures 1 and 2). The geometric mean of cells positive for MHCI-SIINFEKL complexes after CATCH granule treatment was 90% of the positive SIINFEKL pulsed control. The treatment of cells with an equivalent amount of either CATCH+, CATCH-, or PEG alone did not result in a significant change in the signal of either MHCI-SIINFEKL or GFP in comparison to an untreated group.

Discussion and

Conclusions:
The high signal of GFP in only the full CATCH-granule formulation shows that granules are being internalized by DC2.4s and that the full formulation is necessary for internalization. Further, the presence of significant MHCI-SIINFEKL presentation as well as the existence of a double positive population of MHCI-SIINFEKL and GFP cells (Figure 3) indicates that this granule system is inducing cross presentation and endosomal escape. The results of this study show that the CATCH granule system is a potent tool for inducing cross presentation in dendritic cells. This cross presentation would be necessary for the induction of a cytotoxic T cell response and demonstrates the promise of the CATCH granule system as a cancer vaccine platform.

Acknowledgements (Optional):