Checkpoint blockades and adoptive T cell therapy have highlighted recent breakthroughs in cancer immunotherapy, but clinically effective cancer vaccines have yet to be achieved. By leveraging biomaterials for the delivery of antigen and adjuvant it is possible to fine-tune an anticancer immune response. Nanoparticles have demonstrated tremendous potential for the enhancement of immunotherapies by promoting increased antigen uptake, programming accumulation in the lymph nodes, and decreasing clearance. Here we describe the use of a diblock consisting of reslin-like polypetides (RLPs), hydrophobic repetitive proteins, and elastin-like polypeptides (ELPs), hydrophilic repetitive protein biopolymers for a cancer vaccine consisting of antigen and negatively charged adjuvants, such as CpG. RLP-ELP diblocks, a novel class of amphiphilic diblock polypeptides, are a strong choice for such as they provide a robust platform for creating micelles of a given size and morphology. Here, we created a library of Ova-RLP-ELP-K12 constructs and successfully expressed and purified Ova-RLP140-ELP180-K12 and Ova-RLP180-ELP1160-K12. We have shown the binding of Ova-RLP140-ELP180-K12 to CpG and the protein’s self-assembling behavior. Further characterization of different RLP-ELP variants will be used to determine the fusion that is the ideal candidate for a nanoparticle-based vaccine platform.

Future directions

● Assess the antigen presentation of Ova-RLP1-40-ELP1-80-K12 by performing flow cytometry. Verify that the Ova peptide is being properly cross-presented.

● Test the activity of CpG in the Ova-RLP1-40-ELP1-80-K12:CpG complex using a HEK-Blue TLR9 Assay● Confirm CpG uptake using confocal imaging.● Further characterize the self-assembly of Ova-RLP1-40-ELP1-80-K12 with CpG using cyroTEM.● Finish cloning remaining constructs in Ova-RLP-ELP-K12 library.● Express and purify remaining Ova-RLP-ELP-K12 constructs.● Characterize the remaining Ova-RLP-ELP-K12 constructs (with and without CpG) using UV-Vis spectroscopy,

dynamic light scattering, and cryoTEM. Perform additional assays on viable vaccine candidates.● Determine ideal candidate for nanoparticle-based vaccine platform.● Perform initial animal study to assess therapeutic potential.

Conclusion

This project has ultimately resulted in the creation of a library of novel Ova-RLP-ELP-K12 fusions. Ova-RLP140-ELP180-K12 and Ova-RLP180-ELP1160-K12 were successfully expressed and purified via ITC purification, as verified by SDS-page analysis. A gel shift assay confirms the ability of the Ova-RLP140-ELP180-K12 to electrostatically bind the adjuvant. DLS results confirmed self-assembly formation. CryoTEM shows the formation of both spherical and wormlike micelles, in addition to larger, zipper-like structures in the presence of CpG. Cary Scans indicate that this fusion did not exhibit the steep, reversible thermoresponsive behavior that is characteristic of ELPs. Instead the samples without the adjuvant only experienced a slight increase in absorbance as the temperature was temperature. Additionally, the samples with the CpG showed an overall decrease in absorbance as temperature increased. It is possible that the additional negative charge (or the neutralization of the polylysine tail) altered micelle formation, or that the micelles are inverting. Finally, the activity of Ova-RLP140-ELP180-K12 with CpG was found to be superior to that of the E4-60-K12 with CpG. This suggests that the self-assembly of the protein enhances the activity of the adjuvant, perhaps by increasing cellular uptake. Recommendations for future research include additional characterization with cryo-TEM to explore factors such as N:P and time on the self-assembling behavior of Ova-RLP140-ELP180-K12:CpG. Additionally, the cross presentation of Ova must be verified with flow cytometry, and the uptake of CpG uptake must be confirmed with confocal imaging. The activity of CpG should be verified with the HEK-BlueTM TLR9 Assay using the proper protein control. Finally, the expression, purification and characterization of other Ova-RLP-ELP-K12 fusions is recommended to determine the construct (micelle morphology, size) that is optimal for vaccine development.

Recombinant Synthesis Of Micelle-Forming Diblock Polypeptides For Cancer ImmunotherapyStephanie Zelenetz, Ashutosh Chilkoti

Acknowledgements

Thank you to my supervisor Dr. Ashutosh Chilkoti for the opportunity to pursue research in your laboratory. Thank you to my mentor, Garrett Kelly, for the support and direction. Thank you to Dr. Michael Dzuricky for the RLP sequences, and for conducting cyroTEM. Thank you to the Grand Challenge Scholars Program Committee for the opportunity to complete such a fulfilling project, and the resources that have enriched this experience. Finally, thank you to Dr. Schaad for the guidance, and for the providing me with flexibility needed to pursue a project so meaningful to me.