Polyanhydride nanoparticles stabilize pancreatic cancer antigen MUC4β

Abstract

Pancreatic cancer (PC) is one of the most lethal malignancies and represents an increasing and challenging threat, especially with an aging population. The identification of immunogenic PC-specific upregulated antigens and an enhanced understanding of the immunosuppressive tumor microenvironment have provided opportunities to enable the immune system to recognize cancer cells. Due to its differential upregulation and functional role in PC, the transmembrane mucin MUC4 is an attractive target for immunotherapy. In the current study we characterized the antigen stability, antigenicity and release kinetics of a MUC4β-nanovaccine to guide further optimization and, in vivo evaluation. Amphiphilic polyanhydride copolymers based on 20 mol % 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane and 80 mol % 1,6-bis(p-carboxyphenoxy)hexane were used to synthesize nanoparticles. Structurally stable MUC4β protein was released from the particles in a sustained manner and characterized by gel electrophoresis and fluorescence spectroscopy. Modest levels of protein degradation were observed upon release. The released protein was also analyzed by MUC4β-specific monoclonal antibodies using ELISA and showed no significant loss of epitope availability. Further, mice immunized with multiple formulations of combination vaccines containing MUC4β-loaded nanoparticles generated MUC4β-specific antibody responses. These results indicate that polyanhydride nanoparticles are viable MUC4β vaccine carriers, laying the foundation for evaluation of this platform for PC immunotherapy.

Keywords: MUC4; antigenicity; immunogenicity; nanoparticle; pancreatic cancer; polyanhydride; protein stability.

FIGURE 1

Nanoparticle synthesis and sustained release of MUC4β antigen. (a) Scanning electron microscopy image showing representative 2% MUC4β-loaded 20:80 CPTEG:CPH polyanhydride nanoparticles. Scale bar: 1 μm. (b) Release profile of MUC4β from 20:80 CPTEG:CPH polyanhydride nanoparticles for 30 days. Error bars represent SD. Three independent replicates were analyzed in the release studies. CPH, 1,3-bis (p-carboxyphenoxy)hexane; CPTEG, 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane

FIGURE 2

Analysis of MUC4β structural stability. (a) SDS-PAGE of MUC4β released from nanoparticles. Lanes represent (1) molecular weight standard ladder; (2) released MUC4β; and (3) control MUC4β with 10 μg in each lane. The arrow indicates the location of the main MUC4β protein band. (b) Fluorescence spectroscopy of MUC4β released from nanoparticles. SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis

FIGURE 3

MUC4β antigen epitope availability (represented as an OD@405 nm) upon recognition by distinct anti-MUC4β mAbs. Indirect ELISA was used to assess the reactivity of each mAb against MUC4β released from the nanoparticles. No significant differences relative to the control MUC4β (at the same MUC4β coating concentration) were observed with mAbs 6E8 and E9. Reactivity of anti-MUC4α mAb 8G7 was not significantly different from the no-antigen-coating control. Error bars represent SEM. No significance was observed in comparison to the corresponding control (using p < 0.05). mAbs, monoclonal antibodies

FIGURE 4

Anti-MUC4 immune response from sera of mice vaccinated with three MUC4β-based vaccine formulations. (a) Indirect ELISA using MUC4β coated plates. (b) Indirect ELISA using MUC16-coated plates. Significant differences were observed depending on formulation and regimen. Reactivity of CDN + NP combination formulations was significantly higher than that provided by either CDN or NP alone. Reactivity after second immunization is significantly higher than that of the pre-immune control and after single immunization. For both MUC4β- and MUC16-coated plates, OD values at 1:6400 dilution were used to generate the plots. Data were not normalized. Error bars represent SEM. * indicates statistical significance (p < 0.05) between different vaccine formulations. *** indicates statistical significance (p < 0.05) between from secondary immunization to primary immunization and unimmunized group. CDN, cyclic dinucleotide

FIGURE 5

Relative reactivities of different MUC4β nanovaccine elicited antisera to full length human MUC4 and cleaved MUC4β fragment. Western blotting analysis of protein lysates (80 μg) from three different cell lines resolved either on 2% SDS-agarose gels (upper panel) or 10% SDS PAGE gels (lower panel) and passively transferred to the PVDF membrane and probed with the sera from mice immunized with the indicated formulations. Anti-MUC4β mAb 6E8 was used as a positive control. The arrow indicates the interface of stacking and resolving gels. The molecular weight markers are indicated on the left. PVDF, polyvinylidene difluoride; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis