(M1230-01-04) Engineering ‘Eat Me’ Signals for LNPs to Enhance mRNA Cancer Vaccination

Purpose: Effective mRNA delivery remains a critical challenge in nucleic acid therapeutics, particularly for cancer vaccination. This study aims to engineer peptide-lipid conjugates that leverage the "Eat Me" signal to enhance lipid nanoparticle (LNP)-mediated mRNA delivery for colon cancer immunotherapy. By systematically identifying and optimizing calreticulin-derived peptide fragments, we designed a strategy to selectively promote immune-targeted uptake of LNP-mRNA, improving antigen presentation and therapeutic efficacy. This approach provides a novel framework for developing next-generation LNP-based mRNA cancer vaccines with enhanced immune activation and delivery precision.

Methods: Calreticulin-derived peptide candidates were selected based on their compatibility with LNP formulation and their ability to enhance immune-targeted mRNA delivery. Peptide-lipid conjugates were synthesized using azide-alkyne click chemistry and incorporated into lipid nanoparticles (LNPs) via microfluidic-based assembly. Peptide-functionalized LNPs were first screened in DC2.4 dendritic cells using Fluc-mRNA transfection efficiency. Antigen presentation activity was then evaluated using B3Z reporter cells, which measure MHC-I presentation of SIINFEKL peptides. Findings were further validated using confocal microscopy to assess intracellular mRNA trafficking and endosomal escape. For in vivo evaluation, a colon cancer CT26.WT^OVA+ model was established in gender-balanced BALB/c mice. Anti-OVA total IgG, IgG1, and IgG2a titers were quantified by ELISA, while splenic memory cell populations were analyzed via flow cytometry. Antigen-specific immune responses were assessed using IFN-γ ELISPOT to quantify T-cell activation.

Results: Calreticulin peptide-functionalized LNPs exhibited a uniform size distribution (100 ± 30 nm), low polydispersity (PDI < 0.2), and high mRNA encapsulation efficiency ( >90%), comparable to unmodified LNPs. Fluc-mRNA screening in DC2.4 dendritic cells demonstrated that specific calreticulin-derived peptide-LNPs enhanced mRNA uptake and translation compared to standard LNPs. B3Z reporter assays confirmed significantly increased antigen presentation from DC2.4 cells, particularly in LNPs functionalized with optimized peptide fragments. Confocal microscopy studies further validated improved intracellular mRNA localization and enhanced endosomal escape in peptide-modified LNPs. In the CT26.WT-OVA colon cancer tumor model, mice vaccinated with peptide-functionalized LNP-mRNA-OVA exhibited a more balanced IgG2a/IgG1 (Th1/Th2) immune response than standard LNPs, as measured by ELISA. Flow cytometry analysis of splenic memory cell populations indicated enhanced immune priming. IFN-γ ELISPOT assays on splenocytes confirmed a notable increase in antigen-specific IFN-γ responses. Additionally, peptide-functionalized LNPs provided stronger tumor protection in male subjects compared to standard LNP-mRNA vaccination. These findings suggest that calreticulin-derived dendritic cell-targeted peptide-functionalization of LNPs enhances mRNA delivery, improves antigen presentation, and modulates immune responses, offering a promising approach for next-generation mRNA-based cancer vaccines.

Conclusion: This study demonstrates that calreticulin-derived peptide-functionalization of LNPs enhances mRNA delivery, antigen presentation, and immune activation, providing a targeted approach for improving mRNA-based cancer vaccines. The peptide-modified LNPs not only facilitated more efficient mRNA uptake and translation in dendritic cells but also promoted a balanced Th1/Th2 immune response and stronger tumor protection, particularly in male subjects. These findings highlight the potential of peptide-functionalized LNPs as a next-generation platform for enhancing mRNA vaccine efficacy, paving the way for further clinical translation in cancer immunotherapy.

References: [1] Mei, K.-C.; Thota, N.; Wei, P.-S.; Yi, B.; Bonacquisti, E.; Nguyen, J. Calreticulin P-domain-derived “eat-me” peptides for enhancing liposomal uptake in dendritic cells. International Journal of Pharmaceutics, 2024, doi: 10.1016/j.ijpharm.2024.123844.
[2] Wei, P.-S.; Thota, N.; John, G.; Chang, E.; Lee, S.; Wang, Y.; Ma, Z.; Tsai, Y.-H.; Mei, K.-C. Enhancing RNA-lipid nanoparticle delivery: organ- and cell-specificity and barcoding strategies, Journal of Controlled Release, 2024, 375, 366-388, doi: 10.1016/j.jconrel.2024.08.030
[3] Mei, K.-C., Wei, P.-S. Peptide-Lipid Conjugates for Enhanced Nucleic Acid Delivery and Methods of Making and Using Same. U.S. Patent Application No. 63/745,773 (2025)

Acknowledgements: This work was supported by the Binghamton University-SUNY faculty start-up fund and the Maximizing Investigators' Research Award (MIRA) (R35) for Early Stage Investigators (ESIs) from the NIGMS, NIH (Award# R35GM155362). A U.S. patent application has been filed for this technology: Mei, K.-C., Wei, P.-S. Peptide-Lipid Conjugates for Enhanced Nucleic Acid Delivery and Methods of Making and Using Same. U.S. Patent Application No. 63/745,773 (2025).


Fig. 1. Design and Synthesis of Calreticulin-Derived Peptide-Lipid Conjugates for LNP Functionalization. (A) Structural mapping of calreticulin (CRT) highlighting selected peptide regions (CRT_P3, CRT_P4, CRT_P5, CRT_P6) based on their amphipathic properties and putative "Eat Me" signal contribution. (B) Amino acid sequences, isoelectric points (pI), net charges at pH 7, and GRAVY scores of the selected CRT-derived peptides. (C) Schematic representation of the azide-alkyne click chemistry reaction used to conjugate CRT peptides to 16:0 DPPE, forming peptide-lipid conjugates for LNP incorporation. (D) Chemical structures of the synthesized peptide-lipid conjugates and corresponding MALDI-TOF mass spectrometry spectra confirming successful conjugation and purity of CRT_P3, CRT_P4, CRT_P5, and CRT_P6 modifications.


Fig. 2. Characterization and Functional Evaluation of Peptide-Functionalized LNPs for mRNA Delivery. (A) Formulation composition of standard LNPs and peptide-functionalized LNPs (CRT_P3/4/5/6-LNPs), showing incorporation of DSPE-CRT conjugates into the LNP lipid matrix. (B) Microfluidic-based LNP assembly for mRNA encapsulation, followed by in-line dilution and dialysis for purification. (C) Physicochemical characterization of peptide-functionalized LNPs, including particle size, zeta potential, polydispersity index (PDI), and mRNA encapsulation efficiency, showing comparable properties to standard LNPs. CRT peptide-LNP formulations did not alter the apparent pKa of the LNP significantly. (D) Luciferase mRNA transfection assay in DC2.4 dendritic cells, comparing different ionizable lipid formulations (SM-102, ALC-0315, MC3) and demonstrating enhanced mRNA translation efficiency with specific CRT-functionalized LNPs. (E) Comparison of Fluc-mRNA bioluminescence intensity in DC2.4 cells transfected with peptide-functionalized LNPs versus scrambled peptide controls, confirming sequence-dependent enhancement of mRNA translation. (F) Hemolysis-based membrane disruption study for evaluating LNP and cell membrane interaction at pH 7.4 (physiological), pH 6.5 (early endosome), and pH 5.5 (late endosome) environments.


Fig. 3 mRNA Antigen Presentation and Immunogenicity of Peptide-Functionalized LNPs in Dendritic Cells and Tumor Vaccination Models. (A) Confocal microscopy analysis of DC2.4 dendritic cells treated with GFP-OVA mRNA-loaded LNPs, demonstrating lysosomal entrapment vs. cytosolic escape of mRNA. CRT_P3-LNPs show enhanced endosomal escape and cytosolic localization, facilitating antigen expression. (B) Antigen presentation efficiency of peptide-functionalized LNPs, assessed via B3Z reporter assay, indicating CRT_P3-LNPs promote higher MHC-I antigen presentation compared to standard LNPs. (C) In vivo vaccination and tumor challenge model, where BALB/c mice received OVA-mRNA-LNPs via intramuscular (I.M.) injection, followed by tumor inoculation and immune profiling at 18-21 days post-vaccination. (D) Tumor growth curves comparing different LNP formulations in gender-balanced, male, and female groups, highlighting improved tumor suppression in males with CRT-functionalized LNPs. (E) Flow cytometry analysis of splenic central memory T cells (TCM, CD44+CD62Lhi) and effector memory T cells (TEM, CD44+CD62Llo), showing CRT_P3/P5-LNPs drive enhanced memory T-cell formation. (F) Serum antibody responses (ELISA) measuring total anti-OVA IgG and IgG subclass (IgG1, IgG2a) titers, confirming CRT_P3/5-LNPs induce balanced Th1/Th2 responses. (G) IFN-γ ELISPOT assay on splenocytes from vaccinated mice, demonstrating increased antigen-specific T-cell responses with CRT-functionalized LNPs, particularly in male subjects.