Engineering Virus-Mimicking Protein Nanoparticles for Cancer Immunotherapy.

摘要

The immune system represents a powerful resource for the eradication of cancer. To harness the full potential of this sophisticated network to overcome the low immunogenicity of tumor cells, a sufficiently strong cytotoxic CD8 T cell (CTL)-mediated adaptive immune response is required, which is partly orchestrated by the professional antigen presenting cells of the innate immune system, most notably the dendritic cell (DC). Protein nanoparticles represent a potentially exceptional vaccination platform for cancer, as they have the ability to mimic viral infections, which are known potent inducers of CTL immunity.;We have been exploring the E2 protein nanoparticle as a delivery platform for antigens and immune-stimulating compounds. The E2 nanoparticle was successfully packaged internally with endolysosomal-releasable immune-activating DNA (CpG) and surface functionalized with MHC I-restricted peptide epitopes. The virus-mimicking nanoparticle induced DC activation at a 25-fold lower concentration compared to free CpG and induced a 3-fold increase in cross-presentation of attached epitopes, compared to free forms of peptides or activators. Furthermore, we demonstrated that co-delivery of melanoma-associated epitopes and immune-activating CpG with E2 enhanced antigen specific CTL proliferation index by 1.5-fold with a concomitant 5-fold increase in IFN-gamma cytokine secretion, compared to unbound peptide and CpG. Remarkably, a single immunization with the multifunctional E2 nanoparticle increased the frequency of melanoma-specific CTL in vivo (120-fold increase in the lymph node and 30-fold increase in the spleen) and the CTL generated showed approximately three times the lytic capacity toward a gp100-expressing melanoma cell line, compared to unbound peptide and CpG immunization.;We were also able to tune cellular and immunological interactions toward the E2 nanoparticle with surface display of poly(ethylene glycol) polymers, where PEGylation through various methods (native surface amines or a recombinantly introduced cysteine) was shown to decrease cell uptake by greater than 50% of both mouse and human cell lines. PEGylation was also shown to mediate moderate increases in complement activation (∼35% C5a production, compared to a known activator), a humoral innate immune mechanism, whereas E2 itself did not cause complement activation. Surface display of CpG on PEGylated E2 nanoparticles was shown to specifically increase cellular uptake by antigen presenting cells.;Fluorescently-labelled E2 was shown to preferentially drain to the lymph nodes after subcutaneous administration, and surface PEGylation allowed further diffusion to more distal locations and blood draining organs. In contrast, surface display of CpG caused increased proximal lymph node accumulation, and demonstrated superior retention, with ∼ 10-fold increase in LN fluorescence after 48 hours over the other nanoparticles. Within the lymph nodes, ∼50% of the nanoparticles were associated with antigen presenting cells, including dendritic cells.;Altogether, our results demonstrate the potential of the E2 protein nanoparticle as a versatile virus-mimicking immunomodulatory cancer vaccine platform. We have developed a nanoparticle biomaterial for DC-targeting, lymph node retention, and superior induction of CTL-mediated responses against cancer.