Polymer-Based DNA Delivery Systems for Cancer Immunotherapy

10.1.1 Cancer Immunotherapy

The field of immunotherapy aims at activating a patient’s own immune system against a host disease. Immunotherapy includes the investigation of different mechanisms leading to activation of the immune response, and the development of methods to control such responses in a desired manner so as to combat disease, either as a single strategy or in combination with other treatments. In the specific case of cancer immunotherapy, it is speculated that the in vivo activity of endogenous immune cells would be far more sensitive and specific than would any exogenous treatment [8]. Anti-cancer vaccines have, thus been designed to induce both tumor-specific effector T cells and tumor-specific memory T-cells [4]. Such anti-cancer vaccination, it is hoped, will induce regression of established tumors, as well as prevent the onset of secondary tumors and metastases, possibly by addressing the issue of minimal residual disease (MRD). In this case, sparse cancerous cells that evaded irradiation or chemotherapy will be detected and destroyed by the immune system cells that, unlike the temporary exogenous treatment, are present in the body at all times. A major requirement for an efficient anti-cancer vaccination is the generation of a cytotoxic T-lymphocyte (CTL) response, but manipulation of the other arms of the immune response, mainly CD4⁺ T helper cells, is critical for a robust and long lasting CTL response [9, 10, 11]. CTL response generation depends on antigen (Ag) presentation on MHC to naïve DC8⁺ T cells. The poor Ag presentation activity demonstrated by tumor cells [12] highlights the need for professional APCs to generate the desired CTL response. Among the several types of APCs in the body, DCs are the most suitable for this purpose.

10.2.1 Dendritic Cell Biology

In 2011, Ralph Steinman was awarded the Nobel Prize for his 1973 discovery of DCs. DCs are a family of professional APCs. As the most powerful APCs, they are equipped with specialized machinery that enables them to regulate the initiation of a primary immune response [13, 14, 15]. Originally derived from bone marrow, DCs are seeded in all tissues, where they sample their environment in an attempt to detect tissue damage, pathogen entry, inflammation and malignantly transformed cells by taking up particles and molecules, processing them into short peptides and presenting the resulting peptides on MHC molecules. DCs use different routes to capture Ags, including phagocytosis, micro- and macropinocytosis, and receptor-mediated endocytosis [9, 15, 16]. Importantly, receptor-mediated endocytosis can be exploited for the targeting of TAAs into DCs.

Immature DCs in peripheral tissues are characterized by high Ag capture activity, low surface MHC levels and co-stimulatory molecules, as well as a tendency to induce tolerance to self-antigens. Upon recognition of “danger” signals, DCs undergo a maturation process characterized by a reduction of Ag capture activity and an increase of MHC and co-stimulatory molecule expression on the cell surface [17]. Mature DCs migrate to adjacent lymph nodes, where they present antigenic peptides to naïve T-cells [4]. Ag presentation, co-stimulatory molecules expression, and the secretion of an appropriate set of cytokines and chemokines by DCs facilitate the differentiation of naïve CD4⁺ and CD8⁺ T-cells into effector T-helper or T-cytotoxic cells, respectively [18, 19]. DCs can, thus, simultaneously activate both a CTL response to directly kill pathogens or affected cells and a T-helper response to recruit and enhance the activity of other immune cells in the right context. This unique ability of DCs to activate the various arms of the immune system to achieve a powerful and efficient immune response makes them the most promising candidates for immunotherapeutic manipulations.

10.2.2 Antigen Presentation Pathways in Dendritic Cells

Three distinct pathways are used by different cell types for the presentation of antigens, namely MHC class I and class II antigen presentation, and cross-presentation.

10.2.2.1 MHC Class I Antigen Presentation

This pathway is present in all nucleated cells, and is used to present endogenous antigens derived from the nucleus or the cytoplasm. MHC class I antigen presentation can, therefore, report intracellular bacterial or viral infections, as well as malignant transformation in the presenting cell. The resulting intracellular products are degraded in the cytoplasm by the proteasome, translocated into the endoplasmic reticulum (ER) via the transporter associated with antigen presentation (TAP), and loaded on newly formed MHC class I molecules. The MHC class I/peptide complexes are then exported along the constitutive secretory pathway to the cell membrane, where they are presented to naïve CD8⁺ T-cells. The interaction between MHC class I/peptide complexes on DCs and T-cell receptors (TCR) on CD8⁺ T-cells induce the differentiation of naïve CD8⁺ T-cells into cytotoxic T-cells that are capable of killing the infected or transformed cells (Fig. 10.1) [14, 18, 20].

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10.2.2.2 MHC Class II Antigen Presentation

This pathway is restricted to APCs, including macrophages (MΦs), DCs and B lymphocytes, and is used to present exogenous antigens. The exogenous antigens are taken up into these cells by endocytosis, where they are found in early endosomes, a mildly acidic cell compartment containing a small amount of proteases. Early endosomes develop into late endosomes that are more acidic and present higher proteolytic activity, which process the captured Ag into short peptides suitable for MHC presentation [21]. The α and β chains of the MHC class II molecule are assembled in the ER and are associated with the Invariant chain (Ii) that occupies the peptide-binding groove of the complex. The MHC class II molecule is then transported to the late endosome, also referred to as the MHC class II compartment, where, following partial digestion of Ii by cathepsins, it finally binds the exogenous peptide. The MHC class II/peptide complexes are transported to the cell membrane for peptide presentation to CD4⁺ T-cells, and induction of T-helper response to support and enhance the activity of other immune cells (Fig. 10.2) [18].

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10.2.3 The Role of DCs in Anti-Tumor CTL-Response Generation

As mentioned earlier, anti-cancer immunity requires the generation of a specific anti-tumor CTL response that, in turn, requires proper antigen presentation. As part of the many techniques tumor cells use to avoid or weaken the immune system, they can substantially reduce the expression of MHC class I/peptide complexes on their cell surface. Since tumor cells are, thus, poor Ag-presenting cells, cross-presentation of tumor antigens by DCs is essential for the generation of tumor-specific CTL responses [4]. In addition, given the unique ability of DCs to initiate and orchestrate the various arms of the immune system, including recruitment and activation of macrophages, natural killer (NK) cells, T-helper cells and B-cells, a strong and comprehensive anti-cancer immune response can hopefully be achieved by inducing such cells to present an appropriate tumor antigen [10].

10.3.1 DC Immunization

DC immunization requires that an appropriate antigen be presented to DCs in a proper manner and in the right immune context so as to allow Ag uptake, processing and presentation on MHC molecules, parallel to DC maturation and T-cell priming. The first method developed for DC immunization involved the use of whole tumor cells (WTC) or tumor cell lysates, either alone, mixed with an adjuvant, or genetically modified to express an adjuvant. The major advantage of this method is that no Ag identification is required, and multiple Ags are being delivered simultaneously. Nevertheless, all WTC vaccine methods showed limited efficiency in clinical trials, probably due to insufficient interaction between the tumor antigens and the DCs [25]. Pulsing DCs with antigenic proteins or peptides corresponds to another approach. Protein-pulsed DCs are capable of presenting Ags on both MHC class I and MHC class II molecules, with a long half-life of MHC class I presentation and no HLA restriction (i.e., no need for patient selection), but they require an appropriate processing of the protein by DCs, which may be difficult to achieve. Peptide-pulsed DCs, on the other hand, require no processing. Still, they show limited MHC class II presentation, a short half-life of MHC class I presentation, and a HLA restriction. Specific or total tumor mRNA can be processed and presented on both MHC class I and class II molecules. This, however, requires mRNA extraction from a tumor sample and is, thus, patient-specific [15, 26]. Lately, the delivery of tumor Ag-encoding plasmid DNA has emerged as a promising method for DC immunization [19, 27].

10.3.2 DNA Vaccines

DNA vaccines are DCs that were genetically modified to express TAAs. DNA vaccines are advantageous for several reasons. They enable the presentation of multiple epitopes of full-length TAAs on MHC class I and class II molecules, and since processing occurs within the cell, they are not HLA-restricted. In addition, efficient gene transfer allows for a continuous supply of peptides in the modified DC [10]. Genetic modification of DCs can be achieved either by ex vivo gene delivery methods, including use of a gene gun, electroporation, ultrasound and microinjection, or by in vivo approaches, including naked DNA delivery, and viral or synthetic vectors [8, 27]. Ex vivo delivery offers many advantages. The extracted cells are cultured and undergo maturation under controlled conditions and in the absence of inhibitory signals provided by the tumor cells, such that maturation status can be determined before re-administration. Since transfection occurs in culture, high specificity is achieved as only the selected cells are transfected. Nevertheless, ex vivo gene delivery carries substantial limitations. The process is laborious, costly and time-consuming [12]. Patients must undergo cytopheresis, followed by culturing and maturation of the acquired cells, steps that must be performed for each patient separately. Reproducibility is very low and different quality control methods are used. Thus, only a limited number of patients would be expected to benefit from this approach. Hence, ex vivo gene delivery is not likely to become widely marketable. Moreover, transfected mature DCs can show poor distribution from the injection site and ex vivo maturation can impair DC trafficking to lymph nodes, a critical requirement for cross-priming [28, 29]. In contrast, in vivo gene delivery may be expected to become “off the shelf” therapy. As a single product suitable for all patients, in vivo gene delivery can be produced on a large scale with lower costs. Simple and uniform manufacturing and product control procedures will enable reproducibility and control over product quality. In addition, DCs can be targeted at different sites and in their natural environment, thereby not impairing their natural course of maturation and activation [25, 29]. For these reasons, in vivo gene delivery is considered by many to be the best strategy for Ag delivery into DCs [8, 10, 19].

10.3.3 In Vivo Gene Delivery

In vivo gene carriers must meet important requirements. First, they should be able to incorporate their plasmid DNA cargo into the core of the nanoparticle and be stable enough to carry such cargo in the circulation and protect it from degradation. Second, they should be able to selectively target the desired cell type and be properly internalized. Third, they must facilitate escape from the endosome, cytoplasm trafficking, nuclear transport and DNA unpacking [30]. To date, the most efficient gene delivery systems are viral vectors. Using small amounts of DNA, viral vectors can induce high transfection efficiency and stable, long-term gene expression. Unfortunately, viral vectors possess some serious safety issues, including toxicity, immunogenicity and oncogenicity, with numerous clinical trials having been terminated because of this [15, 31, 32]. Restricted gene size is another major limitation of viral vectors. Synthetic vectors correspond to cationic lipids or cationic polymers, respectively termed lipoplexes or polyplexes, which electrostatically bind the negatively charged DNA. The main drawback of such vectors is their low transfection efficiency, as compared to viral vectors. Yet, synthetic vectors are simple, safe, easy to manufacture on a large scale and can carry plasmids of unrestricted size. Moreover, they can be easily modified to possess desirable properties, including targetability, serum stability, reactivity to external signals and an ability to stimulate an immune response. In fact, cationic polymers themselves may provide immune stimulation and mimic nuclear localization signals (NLS), facilitating nuclear transport of their cargo [9, 30].

10.4.1 Poly(l-Lysine)

Poly(l-lysine) (PLL) was the first cationic polymer developed for gene delivery. PLL is a linear polypeptide presenting l-lysine residues in repeat units (Fig. 10.3c). This first generation cationic polymer bears ɛ-amino groups that electrostatically bind the negatively charged nucleic acids, and interact with the negatively charged cell membrane. The large number of active functional amine groups allows for easy modification with targeting ligands [34]. However, the transfection efficiency of PLL is very low because it cannot mediate escape from the endosomal compartment and release into the cytosol. To overcome this barrier, PLL is usually used in combination with chloroquine, although use of this agent is limited due to its cytotoxicity [35]. Another disadvantage of PLL polyplexes is that transfection efficiency is significantly influenced by serum, probably due to the rapid binding to negatively-charged serum components [36]. Furthermore, in vivo applications of PLL polyplexes are complicated by a high level of cytotoxicity [37] and lack of in vivo stability [38, 39]. Because of low transfection efficiency, imidazole groups (pKa ~ 6.5) were introduced into PLL to improve buffer capacity, thereby, enhancing transfection efficiency [40]. PLL with high imidazole content mediated high gene transfection efficiency, with gene expression levels close to those of PEI (discussed below), and exhibited low cytotoxicity. The PEGylation of PLL can also improve its cytotoxicity profile [36].

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Finally, whereas it has been found that PLL/DNA complexes can be taken up by DCs, PLL/DNA did not alter DC phenotype through surface marker expression [41]. Complexation of plasmid DNA encoding for chicken egg ovalbumin (OVA) with PLL-coated polystyrene (PS) particles induced high levels of CD8+ T-cells as well as OVA-specific antibodies in C57BL/6 mice, and further inhibited tumor growth after challenge with OVA expressing tumor cells [42]. PLL-based microspheres displaying mannan or mannoside-modified surfaces (for targeting the mannose receptor, discussed below) were readily phagocytosed by both DCs and macrophages, however neither surface-assembled mannan- nor mannoside-modified microspheres could stimulate DC maturation [43]. Thus, despite the promising results shown in early studies, PLL is not likely to find clinical applications [30].

10.4.2 Poly(ethylenimine)

Poly(ethylenimine) (PEI) is a second generation and one of the most useful polycations for gene delivery [44]. Linear PEI has only secondary amino groups that are almost all protonated under physiological conditions. Branched PEI presents not only primary and secondary amines but also tertiary amines. As such, only about two-thirds of the amino groups in branched PEI are protonable under physiological conditions. The transfection efficiency of PEIs depends on the molecule weight, the PEI nitrogen/DNA phosphate charge ratio (N/P) and the cell type. As every third position on the polymer backbone is occupied by a protonable amino group (Fig. 10.3a), its cationic charge density is very high, allowing for condensation of the negatively charged DNA. Under physiological conditions, only about 20 % of the PEI nitrogen atoms are protonated, leaving the other 80 % to facilitate the important step of endosomal escape by the so-called “proton sponge effect” [30, 45, 46]. Thus, PEI is the most popular and most effective polymeric transfection reagent cited to date [47]. However, the positive charge of PEI/DNA polyplexes cause some serious problems, including adsorption to cells and negatively charged blood components, recognition by the immune system components, resulting in rapid clearance from the circulation, and cytotoxicity to non-target cells [44, 48]. To overcome these limitations, PEI has been conjugated to hydrophilic polymers (i.e., poly(ethylene glycol) (PEG) [49], or hyaluronic acid (HA)) of different molecular weights [50]. PEG is wildly used in drug and gene delivery systems to shield charged, immunogenic or toxic segments, resulting in less toxic “stealth” particles that can evade the immune system and, thus, avoid rapid clearance from the circulation [51, 52]. The conjugation of PEG to PEI was previously shown to increase polyplex solubility and serum stability and reduce cytotoxicity by shielding the high positive charge of PEI [49]. PEGylated PEI, however, showed lower transfection efficiency, as compared to PEI/DNA complexes, and also lacked cell-specificity [53]. Ligation of PEI/DNA complexes to molecules targeting DC uptake receptors can significantly increase transfection efficiency.

Approaches for targeting DC receptors have generally involved either natural receptor ligands or the use of antibodies raised against specific receptors. Targeting DC cell surface receptors may also provide cell activation signals [4, 54]. Different DC receptors have been used to facilitate targeted delivery of gene and drug carriers into DCs, including β-integrins (CD11b and CD11c) [55], CD40 [56] and the Fc receptor [57], but the most studied proteins for this purpose are members of the C-type lectin receptor (CLR) family [58]. CLR is a family of receptors sharing structural homology in their carbohydrate recognition domain (CRD), where specific sugar residues are bound in a calcium-dependent manner. One of the CLRs, the mannose receptor (MR, CD206), is the most widely used receptor for targeting DCs, using both mannose and mannan [59]. While several studies demonstrated that mannosylated-PEI conjugates are effective in gene delivery via MR [60, 61, 62], their transfection potential for primary human and mouse DC was found to be rather low [60]. This might be due to the low affinity of carbohydrate ligands to their receptors, which is usually in the low millimolar range [63], thus, limiting in vivo transfection efficiency. The eight adjacent CRDs in MR may help to increase the binding affinity and specificity of polyplexes containing mannosylated glycans in a multivalent display [59, 64]. We have recently described the design of multivalent mannosylated PEI/DNA complexes bearing mono- and trivalent mannose as a ligand for targeting MR-positive DCs [65]. Complexes bearing mono- and trivalent mannose (Man-PEG-b-PEI/DNA and Man₃-PEG-b-PEI/DNA, respectively) were safe and demonstrated significantly higher in vitro transfection efficiency in DCs. The mannosylated complexes were injected into the tail base of C57/BL6 mice, and 24 h post-injection, the percentages of GFP positive cells in the entire cell population and in the CD11c + cells taken from the inguinal lymph nodes were measured. When examining the entire cells population extracted from the lymph node, only PEI/DNA complexes showed detectable GFP activity, but no significant change was observed between the treatments (Fig. 10.4a). However, Man₃-PEG-b-PEI/DNA was significantly more efficient in transfecting CD11c+ DCs collected from inguinal lymph nodes, as compared to polyplexes prepared with PEG-b-PEI/DNA or PEI/DNA (Fig. 10.4b).

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One challenge of targeting the MR, as holds true for all of the other above-mentioned receptors, is that they are not exclusively expressed on DCs, and are also on several other cell types [57]. MR, for example, is also expressed on monocytes, subsets of endothelial cells and tumor-associated macrophages [9]. Furthermore, the synthesis of carbohydrate ligands and analogs, especially multivalent or complex carbohydrates, often requires many time-consuming, low yielding steps [66]. The stereochemistry of such carbohydrate ligands is difficult to control, and the products are difficult to purify. A frequently used alternative for carbohydrate ligands are antibodies raised against DC receptors (i.e., integrin CD11c/CD18 [55], Fc receptors [67], DEC-205 [7], DC-SIGN [68] and MR [69]), but these, even when humanized, may still elicit adverse immune responses that decrease the efficiency of treatment and induce auto-immune side-effects [70].

A DC3-nona-arginine fusion peptide (DC3-9dR) that binds nucleic acids by electrostatic interactions was previously exploited for the specific delivery of siRNA to DCs in vitro and in vivo [78] and was also used to silence immunosuppressive molecules in DCs so as to induce strong human T-cell immune responses [79]. The DC3 targeting peptide (Table 10.1) was recently studied by our group to mediate the specific delivery of PEGylated-PEI/DNA polyplexes into DCs [80]. Polyplexes show significant transfection efficiency in DCs when decorated with DC3 peptide but not in endothelial cells. The transfection efficiency observed was higher than that of PEI/DNA, signifying the potential use of DC3-bearing polyplexes for immunotherapy via DCs.

10.4.3 Polyamidoamine (PAMAM) Dendrimers

Polyamidoamine (PAMAM) dendrimers are hyper-branched, symmetrical, flexible and monodisperse polymeric molecules (Fig. 10.3d). Szoka et al. firstly investigated PAMAM cascade polymers as non-viral gene delivery vectors [81]. These polymers have an ammonia initiator core and amido-amine repeat units of different generations (G2–G10). Their stepwise synthesis results in products that are uniform in terms of structure and size [82] and bear a well-defined number of primary and tertiary amines. The surface primary amines are protonated, resulting in an extremely positively charged surface that enables DNA binding in stable complexes, and interaction with the cell membrane. The tertiary amines in the interior of the polyplexes are protonable, endowing the dendrimers with pH buffering capacity that enables endosomal escape of the polyplexes following cellular uptake [83]. All these properties, in addition to their non-immunogenic nature, make PAMAM dendrimers an alternative to the highly efficient and highly immunogenic viral vectors. However, a major drawback of PAMAM dendrimers is that low generation dendrimers (G5 or lower) show poor transfection efficiencies, while dendrimers of high generation (G6 and higher) are efficient transfection agents, yet possess serious cytotoxicity [82]. In addition, synthesis of high generation dendrimers is a high cost, labor-consuming process that last several days, making them less likely to be designed for large scale production [83].

Mannosylated G4-PAMAM dendrimers conjugated to OVA specifically targeted DCs and induced cross-presentation in vivo [84]. Moreover, pre-immunization with mannosylated PAMAM-OVA leads to delayed onset of B16-OVA melanoma development, slower kinetics of tumor growth and increased survival of OVA-immunized mice. G4-PAMAM dendrimers bearing DC-SIGN ligand in multivalent presentation also achieved efficient DC targeting properties, however, did not affect DCs maturation [20]. It has been postulated that mannosylated dendrimers, as opposed to DC-SIGN-modified dendrimers, may trigger not only DC-SIGN, but also other mannose-specific CLRs that contributes to DC maturation and activation. G5-PAMAM dendrimers conjugated to MHC class II-targeting peptide (PADRE, Table 10.1) and surface-loaded DNA have been shown to effectively transfect murine and human APCs in vitro [85]. When applied subcutaneously, this conjugate preferentially transfected DCs in draining lymph nodes, promoted generation of high affinity T-cells, and elicited rejection of established B16 tumors.

10.4.4 Chitosan

Chitosan is a linear polysaccharide (Fig. 10.3e) obtained by deacetylation of its parent polymer chitin, a compound that is widely distributed in nature. Chitosan is composed of randomly distributed ß-(1-4)-linked d-glucosamines (deacetylated units) and N-acetyl-d-glucosamines (acetylated units) and has an apparent pKa value of 6.5 [86]. Chitosan is a biodegradable and biocompatible polymer, with low or no immunogenicity and antibacterial activity [86, 87]. It was previously shown to be non-toxic in both test animals [88] and humans [89]. Due to these properties, chitosan is attractive for drug and gene delivery. The high density of positive charges along the polymeric chain contributes to the condensation of the negatively charged DNA, and more frequently, siRNA molecules, into compact structures, thus protecting them from degradation by blood nucleases, promoting their cellular uptake [90]. Chitosan also possess adhesive properties by interacting with glycoproteins in the mucus [91], and is thus used in muco-adhesive drug delivery systems as an adsorption enhancer [92]. DNA plasmid incorporated into chitosan nanoparticles was able to induce DCs maturation and increase IFN-γ secretion from T cells after pulmonary mucosal immunization [93]. However, unless modified, chitosan has low solubility at physiological pH, which significantly limits its use in many applications [87, 92]. Chemical modifications of chitosan have been performed to improve its solubility at physiological pH, including PEGylation or quaternization of the amine groups of chitosan. However, the transfection efficiency of chitosan-based derivatives reported so far is generally not superior to that of PEI [94].

Various DC-targeted chitosan-based DNA delivery formulations has been designed to overcome the major obstacles facing the clinical development of chitosan, namely, the lack of cell-specificity and low transfection efficiency. Mannosylated chitosan/DNA complexes were more efficient in transfecting DCs, as compared to water-soluble chitosan/DNA, and induced better INF-γ production from DCs [95]. Mannosylated-chitosan-entrapping PEI/HBV-DNA complexes induced significantly enhanced serum antibody production and CTL levels after intramuscular immunization [96]. Biotinylated chitosan nanoparticles were modified with bifunctional fusion protein (bfFp) vectors for achieving DC-selective targeting. bfFp is a recombinant fusion protein consisting of truncated core-streptavidin fused to an anti-DEC-205 single chain antibody (scFv). Intranasal administration of plasmid DNA-loaded bfFp/chitosan nanoparticles, along with anti-CD40 DC maturation stimuli, enhanced the amount of mucosal IgAs, as well as systemic IgGs, against nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) [97]. Finally, to improve low transfection efficiency, chitosan-linked-PEI/DNA complexes have been designed, and showed high transfection efficiency and low cytotoxicity towards DCs [98]. Vaccination with DCs transfected with chitosan-linked-PEI/DNA encoding gp100 (melanoma-associated antigen) slightly improved resistance to the B16BL6 melanoma challenge.

10.4.5 Micro- and Nano-Particulate DNA Delivery Vectors

Polymeric particulates have been shown to be efficient in delivering plasmid DNA into APCs (reviewed in [99, 100, 101]). A key advantage of particulate vectors relative to other non-viral gene delivery systems is their superior in vivo stability. The principal types of polymers studied in this context include those made of poly(lactide) (PLA; reviewed in [99]), poly(lactide-co-glycolide) (PLGA; reviewed in [99, 100, 102]), polyorthoesters [103], polystyrene (PS) [104, 105] and poly(ε-caprolactone) [106]. Of these, PLGA has been studied most extensively in terms of its capacity to stimulate APCs.