Protein-based vaccines have significant potential as infectious disease and anticancer therapeutics, but clinical impact has been limited in some applications by their ability to generate a coordinated cellular immune response. Here, a pH-responsive carrier incorporating poly(propylacrylic acid) (PPAA was evaluated to test whether improved cytosolic delivery of a protein antigen could enhance CD8+ cytotoxic lymphocyte generation and prophylactic tumor vaccine responses. PPAA was directly conjugated to the model ovalbumin antigen via reducible disulfide linkages and was also tested in a particulate formulation after condensation with the cationic poly(dimethylaminoethyl methacrylate) (PDMAEMA). Intracellular trafficking studies revealed that both PPAA-containing formulations were stably internalized compared to control conjugates and evaded exocytotic pathways, leading to increased intracellular accumulation and potential access to the cytosolic MHC-1 antigen presentation pathway. In an EG.7-OVA mouse tumor protection model, both PPAA-containing carriers robustly inhibited tumor growth and led to an approximately 3.5 fold increase in the longevity of tumor free survival relative to controls. Mechanistically this response was attributed to the 8-fold increase in production of ovalbuminspecific CD8+ T-lymphocytes and an 11-fold increase in production of anti-ovalbumin IgG. Significantly, this is one of the first demonstrated examples of in vivo immunotherapeutic efficacy using soluble protein-polymer conjugates. These results suggest that carriers enhancing cytosolic delivery of protein antigens could lead to more robust CD8+ T-cell response and demonstrate the potential of pH-responsive PPAA-based carriers for therapeutic vaccine applications.

Protein-based vaccines play an important role in controlling infectious disease but their full clinical impact has been limited by their inability to generate a coordinated cellular CD4+ and CD8+ immune response. Vaccines that better deliver antigens to the cytosolic MHC1 display system could in principle provide a better coordinated response. Here, controlled radical polymerization was employed to prepare a diblock copolymer containing an endosomal releasing segment based on poly(propylacrylic acid) (poly(PAA)) and a hydrophilic segment containing thiol-reactive disulfide moieties for antigen conjugation. Propylacrylic acid (PAA) was polymerized in the presence of a trithiocarbonate based RAFT chain transfer agent (CTA). The resultant poly(PAA) was then employed as a macroCTA in the copolymerization of pyridyl disulfide methacrylamide (PDSMA) (thiol-reactive monomer) and N,Ndimethylacrylamide (DMA) as a hydrophilic comonomer. Copolymer compositions and molecular weights were determined via 1 H NMR spectroscopy and size exclusion chromatography. Native polyacrylamide gel electrophoresis (PAGE) showed a complete disappearance of the bands corresponding to free thiolated ovalbumin after conjugation to the polymer at pyridyl disulfide to thiol ratios as low as 2.5. The ability of the poly[(PAA)-b-(DMA) co (PDSMA)]-ovalbumin conjugates to activate CTLs was evaluated in vivo, tumor protection using the EG7 tumor protection model. Tumors were visible in the PBS and free ovalbumin immunized mice by day 7 but were not visible in the polyPAA-ovalbumin conjugate immunized mice until day 18. The mice immunized with the polyPAAovalbumin conjugate had a survival rate at day 21 of 100% versus 20% for PBS and 40% for ovalbumin immunized mice.

Here we describe a new graft copolymer architecture of poly(propylacrylic acid) (polyPAA) that displays potent pH-dependent, membrane-destabilizing activity and in addition is shown to enhance protein blood circulation kinetics. PolyPAA containing a single telechelic alkyne functionality was prepared via reversible addition-fragmentation chain transfer (RAFT) polymerization with an alkyne-functional chain transfer agent (CTA) and coupled to RAFT polymerized poly(azidopropyl methacrylate) (polyAPMA) through azide-alkyne [3+2] Huisgen cycloaddition. The graft copolymers become membrane destabilizing at endosomal pH values and are active at significantly lower concentrations than the linear polyPAA. A biotin terminated polyPAA graft copolymer was prepared by grafting PAA onto polyAPMA polymerized with a biotin functional RAFT CTA. The blood circulation time and biodistribution of tritium labeled avidin conjugated to the polyPAA graft copolymer was characterized along with a clinically utilized 40 kDa branched polyethylene glycol (PEG) also possessing biotin functionalization. The linear and graft polyPAA increase the area under the curve (AUC) over avidin alone by 9 and 12 times, respectively. Furthermore, polyPAA graft copolymer conjugates accumulated in tumor tissue significantly more than the linear polyPAA and the branched PEG conjugates. The collective data presented in this report indicate that the polyPAA graft copolymers exhibit robust pH-dependent, membrane-destabilizing activity, low cytotoxicity and significantly enhance blood circulation time and tumor accumulation.