Elsevier

Journal of Controlled Release

Volume 267, 10 December 2017, Pages 181-190
Journal of Controlled Release

A PEGylated hyaluronic acid conjugate for targeted cancer immunotherapy

https://doi.org/10.1016/j.jconrel.2017.08.032Get rights and content

Abstract

The cell-free approach to foreignizing tumor cells with non-self antigens has received increasing attention as a method to induce cytotoxic T lymphocyte (CTL)-mediated immunological rejection of tumors, because the clinical translation of the conventional CTL-based cancer immunotherapies has been limited by a complicated manufacturing process and autotransplantation. In this study, we prepared matrix metalloproteinase 9 (MMP9)-responsive polymeric conjugates consisting of PEGylated hyaluronic acid (HA) as the targeting moiety and ovalbumin (OVA) as the model foreign antigen. The MMP9-cleavable linker was introduced between PEG and the HA backbone to facilitate the detachment of the PEG corona from the conjugate at the tumor site. From the in vitro cellular uptake study, it was revealed that the conjugate was effectively taken up by the CD44-expressing TC-1 cancer cells in the presence of MMP9 via receptor-mediated endocytosis. When the conjugate was systemically administered into the tumor-bearing mice with endogenous OVA-specific CTLs, the tumor growth was markedly inhibited, which was attributed to the significant antigen presentation on the tumor cells. Overall, the MMP9-responsive conjugates bearing foreign antigens might have the potential as an alternative to CTL-based cancer immunotherapeutics.

Introduction

CD8+ cytotoxic T lymphocytes (CTLs) have been explored as a tool to eliminate target cells with antigenic peptides presented by surface major histocompatibility complex (MHC) class I molecules [1], [2], [3]. Therefore, CTL-based cancer immunotherapies such as adoptive cell therapy (ACT) and engineered chimeric antigen receptor (CAR) T cell therapy have shown the potential to treat cancer without side effects [4], [5], [6], [7]. In the clinic, these therapies have exhibited statistically significant improvement over conventional chemotherapy and radiotherapy by avoiding non-specific cell death [8], [9], [10]. Although ACT and CAR T cell therapies are effective strategies to overwhelm cancer, they require a high cost, a complicated manufacturing process and autografting, limiting their extended applications to cancer patients [11], [12], [13]. Besides, most existing approaches for CTL activation focus on delivering tumor antigen to antigen-presenting cells not tumors. In this regard, the innovative cell-free approach to foreignizing tumor cells via site-specific non-self antigen delivery can be an option to induce the CTL-mediated immunological rejection of tumors. Recently, we reported on a hyaluronic acid (HA)-based polymeric conjugate as a potential immunotherapeutic agent to foreignize cancer cells by delivering the antigen via receptor-mediated endocytosis [14]. Owing to its specific binding affinity to the HA receptor (CD44), the conjugate was selectively taken up by the cancer cells, resulting in high antitumor efficacy after its systemic administration into the tumor-bearing mice [15]. The conjugate, however, exhibited significant accumulation in the liver. This might be due to its uptake by phagocytic cells in the reticuloendothelial system and by liver sinusoidal endothelial cells expressing the other HA receptor (stabilin-2), and thus requires a strategy to minimize the liver accumulation of the conjugate [16], [17].

In recent years, stimuli-sensitive drug delivery systems have emerged to treat intractable diseases, since they release the drug at the target site by changing their physicochemical properties in response to external (changes in temperature, magnetic force or light) or internal (variations in pH, enzyme concentration or redox potential) stimuli [18], [19], [20], [21], [22], [23], [24], [25]. The relevant microenvironmental characteristics to differentiate malignancy from the normal tissues include acidic pH, high levels of specific enzymes such as matrix metalloproteinase (MMP) and hypoxia [26], [27], [28], [29]. In particular, MMPs play important roles in tumor initiation, progression and metastasis while their levels are proportional to the malignancy of the tumor [30], [31]. Therefore, significant efforts have been made to harness MMPs as the keys to developing nanomedicines for cancer imaging and therapy [32], [33], [34].

In an attempt to foreignize the cancer cells effectively, we herein prepared an HA-based polymeric conjugate grafted with poly(ethylene glycol) (PEG) through an MMP9-cleavable linker (Fig. 1). The PEGylated HA derivative was then chemically attached to ovalbumin (OVA), which was chosen as the model foreign antigen. Since the resulting conjugate had a PEG corona that could be detached in the presence of MMP9, the HA of the conjugate could be exposed only at the tumor site, facilitating its cellular uptake via receptor-mediated endocytosis. Consequently, foreignized cancer cells may present antigenic epitopes on the MHC class I molecules, resulting in the immunological rejection by CTLs. To test this hypothesis, we investigated the antigen presentation capability, tumor-homing ability and therapeutic efficacy of the conjugate in vivo.

Section snippets

Materials

HA (MW = 3.5 × 104 g/mol) was purchased from Lifecore Biomedical Inc. (Chaska, MN, USA). Monomethoxy polyethylene glycol amine (PEG-NH2, MW = 5 × 103 g/mol) was bought from Laysan Bio Inc. (Arab, AL, USA). The OVA257–264 peptide (purity > 95%) and 9-fluorenylmethoxycarbonyl (Fmoc)-PLGLWADR peptide (pep; purity > 95%) were obtained by custom synthesis from Peptron (Daejeon, Korea); their purity was verified by high performance liquid chromatography. Sodium cyanoborohydride (NaBH3CN),

Results and discussion

For the CTL-mediated immunological rejection of tumors by foreignization, it is essential to develop carriers that can effectively deliver the antigens to the cancer cells. Of various biocompatible polymers, PEG has been extensively investigated as a drug carrier component, because it improves the solubility of poorly water-soluble drugs and allows for prolonged blood circulation of the drug [37]. In this study, PEG was introduced via the MMP9-cleavable linker as the corona of the HA-OVA

Conclusion

Inducing a sustained and robust CD8+ T cell response is necessary for therapeutic intervention in chronic infectious diseases and cancer. Although ACT is an effective strategy to treat such intractable diseases, its high cost and complicated process have limited its long term benefit to patients. Our findings in this study suggested that an alternative therapeutic strategy, a tumor-targetable conjugate with a foreign antigen, could be a cost-effective and innovative agent to induce the

Acknowledgements

This research was supported by funding from the National Research Foundation of Korea (20100027955, 2015R1A2A2A05001390, 2013M3A9D3045881, and 2014R1A2A1A10054205).

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    These authors contributed equally to this paper.

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