Analysis of Anti Pancreatic Cancer Vaccine Candidates

Pancreatic Cancer (PC) arises from exocrine pancreatic ducts and is expected to claim 44330 lives in United States by 2018. PC is forecasted to become the second leading cause of cancer-related death in United States by 2030 (Rahib et. al 2014). PC has a poor survival outcome with overall survival of patients being no more than 8%, due to the limited success rate of different treatment modalities for PC like surgery, chemotherapy, and radiation (Seigel 2018, Sideras 2014). Furthermore, PC has an elaborate immunosuppressive tumor microenvironment comprising of desmoplasia, immune-suppressive cells {like regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs)}, and anti-inflammatory cytokine milieu (Banerjee K. 2017)

Because of complex tumor microenvironment, the PC tumors are refractory to systemically delivered therapies. Therapies including radiotherapy, are more palliative than curative. The widely used gemcitabine, nab-paclitaxel, Fluorouracil (5-FU), and Folfirinox (more effective in metastatic PC cases) alone or in combination improves the patient’s survival at early stages (Garrido-Laguna 2015, Passero FC 2017); however, the overall survival is completely stage and case dependent (Bilimoria KY Cancer 2007). In addition, the high level of therapy-induced toxicity and immunosuppression in PC patients further aggravates the quality of PC patients’ life.Recent studies have shown that immunotherapy strategies like cancer vaccines might be useful in overcoming the above challenges by breaking the tolerance, immunosuppression and therefore, improving the overall survival and quality of life (Delitto D 2016, Okamoto M 2016).

However, in contrast to the great achievement of vaccines to eradicate various infectious diseases, the development of anti-cancer vaccine is rather more arduous due to suboptimal immune response against tumor-associated antigens (TAAs), which are associated with self-tolerance, tumor heterogeneity, and variable host responsiveness in most of the malignancies including PC. Optimizing candidates for cancer vaccines face major challenge of self-tolerance because majority of antigens behave as “self” due to immunological ignorance by the host immune system (Delitto D 2016).

Mucins (MUC) are high molecular glycoproteins that are overexpressed and have oncogenic functions in PC pathogenesis. MUC family members have emerged as tumor associated antigens (TAAs) for PC and are currently being exploited for cancer immunotherapy. Mucin1 (MUC1) has been one of the well-studied target for PC vaccine studies (Torres M.P. 2012). MUC1 peptides and glycopeptides vaccine studies have showed the potential of reprogramming immune system against mucins and generate anti-tumor responses in various malignancies (Glaffig, Stergiou et al. 2017, Liu, Wang et al. 2017, Scheikl-Gatard, Tosch et al. 2017, Stergiou, Glaffig et al. 2017, Tosch, Bastien et al. 2017, Ramanathan RK 2005). But MUC1 vaccines have achieved limited clinical success in PC patients (Rong Y 2012, Kondo H 2008, Ramanathan 2005). Unlike MUC1, Mucin4 (MUC4) is undetectable in normal pancreatic tissue but its expression gradually increases with PC disease progression and pathogenesis (Singh AP 2004).

Altered glycosylation, epitope multiplicity in tandem-repeat region, and oncogenic role in PC pathogenesis, make MUC4 an attractive target for immunotherapy of PC (Shailendra 2017). MUC4 gets putatively cleaved at Gly-AspPro-His (GDPH) site in an autocatalytic manner into two subunits: a large N-terminal MUC4α and a smaller membrane tethered MUC4β. Membrane tethered MUC4β region has 3 EGF-like domains that interacts with HER-2 and promotes cell proliferation. Further due to the transmembrane region present in MUC4β subunit, its easy to target this subunit since post-cleavage MUC4β will be still present on the cell surface of PC tumor cells. Previous studies with MUC4 peptides have demonstrated generation of MUC4 specific cytotoxic T-cells (CTLs) in vitro but with increased surface expression of MUC4, those CTLs died in a MUC4 mediated contact- dependent manner thus making the therapy ineffective (Zhu Y 2014). In another study mice immunized with MUC4 glycopeptides mixed with tetanus toxoin (Ttox) induced strong immune responses and predominantly produced IgG1 antibodies (Cai Hu 2015).

These studies suggests that targeting MUC4 with a proper vaccine generation strategy could lead to effective therapy. However, studies have not been investigated for type as well as quality of immune responses elicited by full length MUC4 or its subunit proteins (subunit-α and subunit-β). Most mucin based vaccine studies have utilized peptides for vaccine formulations that doesn’t capture the entire immunogenic epitopes present on original proteins. Vaccination with entire proteins or protein fragments could enhance anti-tumor cellular and humoral responses against these TAAs (Banerjee K 2017). The high molecular weight of MUC4 poses major challenges in its purification and maintaining its native antigenicity, thus hindering proper immunological evaluation of MUC4 for testing its potential as vaccine candidate till date.

Furthermore, the overall immune response depends on frequency of immunodominant epitopes in the protein, antigen presentation, as well as on vaccine formulation and its release kinetics in vivo.

One of the major challenges of protein encapsulation is to ensure that the protein stability is protected from physiological environment and its released in sustained release kinetics depending on the therapeutic needs (Bilati U. 2005 Drug Deliv Tech). Amphiphilic polyanhydride nanoparticles can stabilize protein structure in their neutral pH core and release protein in a sustained manner by surface erosion mechanism which conserves the protein activity (Narasimhan B. 2007 Biomaterials). Narasimhan et. al developed a novel amphiphilic polyanhydride system comprising of copolymers of the anhydride monomers, 1,6-bis(p-carboxyphenoxy) hexane (CPH) and 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane (CPTEG) containing oligomeric ethylene glycol, thus creating an environment to stabilize proteins as well as modulate protein surface erosion kinetics and corresponding immune responses by changing CPH to CPTEG ratios (Narasimhan B. 2007 Biomaterials, Balaji J Biomed Mater Res, 76A (1) (2006)).

Encapsulation of proteins into biodegradable CPTEG:CPH polymers enhances the epitope repertoire that are limited in peptide vaccines. Furthermore, antigen presenting cells (APCs) like dendritic cells and macrophages, efficiently uptake varying chemistries of these polyanhydride nanoparticles leading to upregulation of surface activation markers like major histocompatibility complexes class I & II (MHC-I & MHC-II), co-activating ligands (CD86, CD40), and secretion of cytokines (). Immunization of mice with OVA-loaded 50:50 CPTEG:CPH nanoparticles demonstrates generation of anti-OVA antibodies (both IgG2a and IgG1) (Salem AK Acta Biomater. 2013).

In a similar study gp41-54Q-GHC-loaded 20:80 CPTEG:CPH nanoparticles immunized mice results in formation of germinal centers, antigen specific antibody responses and isotype switching compared to antigen mixed with alum (a potent adjuvant), thus suggesting that sustained release of antigens provided by these polyanhydride nanoparticles formulations induces antigen-specific immune responses and can have overall therapeutic advantages (Narasimhan B. J Biomed Nanotechnol. 2016). In present study, we encapsulated endotoxin free recombinant human MUC4β in 20:80 CPTEG:CPH nanoparticles (MUC4 nanovaccine) and demonstrate the immunogenicity of MUC4 nanovaccine.

Importantly, we emphasize on nano-vaccine formulation, its release kinetics, and its immunological activity in terms of antigen presentation and humoral responses. We also present here the cytokine profile of DCs after antigen pulsing to analyze the type of immune response generated by this nano-formulation. Interestingly, MUC4β is immunologically active and profoundly activates the in vitro matured dendritic cells eliciting more Th1 type of immune response. The MUC4β-encapsulated nanovaccine elicits better immune response when analyzed for antigen presentation and Th1 cytokines. We further observe anti-MUC4β antibodies in serum of immunized mice and isotype analysis shows that MUC4 nanovaccine immunized mice generated more Th1 IgG2b antibodies over Th2 IgG1 antibodies that has not been reported in previous MUC4 based vaccine studies.

Therefore, polyanhydride based nano vaccine loaded with recombinant human MUC4β would be better platform for analysis of MUC4 based vaccine in pancreatic cancer. Future studies are warranted to validate these findings in MUC4 transgenic pancreatic cancer mice model.