Advanced Strategies in Immune Modulation of Cancer Using Lipid-Based Nanoparticles

Immunotherapy has a great potential in advancing cancer treatment, especially in light of recent discoveries and therapeutic interventions that lead to complete response in specific subgroups of melanoma patients. By using the body’s own immune system, it is possible not only to specifically target and eliminate cancer cells while leaving healthy cells unharmed but also to elicit long-term protective response. Despite the promise, current immunotherapy is limited and fails in addressing all tumor types. This is probably due to the fact that a single treatment strategy is not sufficient in overcoming the complex antitumor immunity. The use of nanoparticle-based system for immunotherapy is a promising strategy that can simultaneously target multiple pathways with the same kinetics to enhance antitumor response. Here, we will highlight the recent advances in the field of cancer immunotherapy that utilize lipid-based nanoparticles as delivery vehicles and address the ongoing challenges and potential opportunities.

Immunotherapy has a great potential in advancing cancer treatment, especially in light of recent discoveries and therapeutic interventions that lead to complete response in specific subgroups of melanoma patients. By using the body's own immune system, it is possible not only to specifically target and eliminate cancer cells while leaving healthy cells unharmed but also to elicit long-term protective response. Despite the promise, current immunotherapy is limited and fails in addressing all tumor types. This is probably due to the fact that a single treatment strategy is not sufficient in overcoming the complex antitumor immunity. The use of nanoparticle-based system for immunotherapy is a promising strategy that can simultaneously target multiple pathways with the same kinetics to enhance antitumor response. Here, we will highlight the recent advances in the field of cancer immunotherapy that utilize lipid-based nanoparticles as delivery vehicles and address the ongoing challenges and potential opportunities.
Keywords: lipid nanoparticles, cancer immunotherapy, sirNA therapeutic, tumor microenvironment, cancer vaccines iNtrODUctiON tumor immunology Hundred years after Paul Ehrlich coined the term "magic bullet, " it is well established that the immune system can be utilized in the epic battle against cancer. Immune cells possess a unique ability to distinguish between cancer and normal cells with reliance on specific expression of cell-surface tumor-associated antigens (TAA) or self-antigens. Due to this ability, immune cells can act as "killing bullets" that are able to specifically eliminate tumors cells.
The development of a tumor is a dynamic process whereby the immune system not only protects against cancer development but also shapes the character of emerging tumors, a process named "Cancer Immunoediting. " This complex process can be divided to three phases according to the nature of the interaction of tumor cells with the immune system: elimination, equilibrium, and escape (1).
In the elimination phase, both the innate and adaptive immune systems detect and destroy early tumors (2). Tumor cells express TAA-derived peptides in context of MHC molecules that are recognized by TAA-specific-cytotoxic CD8 + T-lymphocytes (CTL). Activated CTL and natural killer (NK) cells directly induce tumor cell apoptosis and, in addition, release IFN-γ that mediates inhibition of tumor-cell proliferation and angiogenesis. Antigen-presenting cells (APCs), LNP Based Immunotherapy for Cancer Frontiers in Immunology | www.frontiersin.org February 2017 | Volume 8 | Article 69 such as tumor-associated macrophages (TAMs) and dendritic cells (DC), take up TAAs from tumor cells debris and present their peptides in the context of MHC to TAA-specific CD8 + and CD4 + T-lymphocytes. Activated CD4 + T-lymphocytes produce inflammatory cytokines such as, IFN-γ and TNF-α, that can both suppress tumor survival and upregulate the expression of MHC molecules on the surface of tumor cells. This upregulation facilitates the recognition of cancer cells by CTL, which mediates their killing mechanism (1, 3). The equilibrium phase is a balance phase upon which tumor progression is still controlled by the immune system; however, sporadic tumor cells manage to survive immune destruction (1,4).
In the escape phase, tumors manage to evade immune surveillance, begin to grow progressively and establish an immunosuppressive tumor microenvironment (TME) (1,5,6). In this phase, tumor cells acquire the ability to escape from host immune response through different strategies; first, tumor cells downregulate MHC expression, thus preventing recognition and attack by CTL (7). Second, tumor cells upregulate their expression of immune checkpoints proteins, such as PD-L1 and CD80/ CD86, which ligate the negative co-stimulators PD1 and CTLA4 on the T-lymphocyte's membrane. This results in attenuation of T-lymphocytes activation (8,9). Furthermore, tumor cells induce the infiltration of other cell types (fibroblasts, endothelial, and immune cells) and instruct them to establish TME that promotes tumor progression (10). For example, infiltration of regulatory T-cells (Tregs) into the TME inhibits CTL (11). Finally, tumor cells encourage cells in TME to release anti-inflammatory cytokines, such as IL-10 and TGF-β. These cytokines suppress the ability of resident immune cells to act against tumor cells, prevent the recruitment of CTL and NK cells, and, therefore, promote tumor growth (12).
In the past few years, the rapidly advancing field of cancer immunotherapy has produced several new opportunities for treating cancer: either by stimulating the activities of specific components of the immune system or by counteracting immunosuppressive signals produced by tumor cells. Efficient antitumor immunotherapy can be achieved by combining delivery of TAAs to APC along with removing tumor-derived negative regulators of immune cell activation (13,14). Therefore, delivery of immunomodulatory molecules to the appropriate cells is crucial for the successful development of cell-based antitumor immunotherapy.

Nanotechnology-Based therapies
Nanotechnology affords a unique opportunity to deliver therapeutic molecules to specific cells and simultaneously attack several biological avenues promoting tumor eradication (15). A variety of delivery platform vehicles are under development to target immune cells, such as antibodies (16), polymers (17), aptamers (18), and lipid nanoparticles (LNPs) (19,20). In comparison to other NPs, LNPs have attractive biological properties, which include general biocompatibility, biodegradability, and the ability to entrap both hydrophilic and hydrophobic drugs (15,21). Multiple properties of LNPs can be altered via different lipid composition and ratios or by surface chemistry, including their size, charge, and surface functionality (22). LNPs serve as carriers for a variety of therapeutic molecules: from nucleic acid to proteins, small molecules and chemotherapy drugs, and combinations of the aforementioned agents (22)(23)(24). LNPs shelter their cargo from clearance in the surrounding biological milieu, increasing the half-life in circulation, minimizing systemic toxicity, and hence enabling a wide therapeutic window (15,22,25). In addition, LNPs can promote delivery of their cargo directly to specific immune cell, such as APC or T-lymphocytes (20,26,27). Here, we review the latest developments in nanoparticle-based cancer immunotherapy, centering on LNPs.

tHe eFFects OF tHe PHYsicOcHeMicAL PrOPerties OF LNPs ON iMMUNe MODULAtiON
The physicochemical characteristics of LNPs are crucial for their fate and performance following administration. Several parameters were examined including nanoparticle size, shape, surface charge, lipid composition, and aggregation. Size is one of the major causes for immune stimulation by LNPs. This can be related to the fact that these nanoparticles fall within the size range of pathogens, such as viruses and small bacteria (28). Size has a significant impact on blood circulation half-life as an optimal vesicle should be large enough to avoid renal clearance but small enough to avoid clearance via the mononuclear phagocytic system (15). The size of NPs should be also adjusted in accordance to the route of administration and delivery purposes. For example, it was shown that, following intravenous administration, 25 nm size NPs are significantly more efficient than 100 nm-sized NPs, as these smaller nanoparticles are better delivered to the lymph nodes (29). In addition, size of below 100 nm enables NPs to utilize the enhanced permeability and retention effect characteristic of solid tumors. Below 100 nm, NPs can reach solid tumors by extravasation via the leaky tumor vasculature and accumulate due to poor lymphatic drainage (15).
The shape and curvature of NPs can also affect immune activation as it was shown that oblate-shaped NPs have a lower macrophage uptake in comparison to spherical nanoparticles and, therefore, longer circulation time and altered biodistribution (30).
Surface features of LNPs have also been thoroughly investigated. Surface charge is another major contributor of immune activation, as positively charged LNPs can better interact with the negatively charged mucosal surface. This results in better uptake of positively charged LNPs by cells in comparison to their negatively charged or neutral counterparts (31). However, positively charged LNPs have toxic effects including induction of pro-inflammatory response (32). Surface charge (both negative and positive) and high membrane cholesterol content are also related to complement activation by liposomes (28). Contrarily, the presence of phosphatidylserine (PS) has an antiinflammatory effect probably due to specific recognition by PS receptors (33,34). Surface functionalization has been widely used to improve LNPs performance. One of the main causes for the immune recognition of LNPs is the absence of self-discriminating molecules (such as complement control proteins) on the bilayer membrane, which protect "self " cells from attack by the complement system (28). Among the most common of these are hydrophilic moieties, such as polyethylene glycol (PEG), which enables prolonged blood circulation time and improved biodistribution by protecting NPs from opsonization (15). Despite the multiple advantages, functionalizion with PEG can induce complement activation (5,35,36). In addition, subsequent injections of PEGylated liposomes can result in their accelerated blood clearance, probably due to transient IgM production (37). Another option for hydrophilic surface coating is polysaccharides. These naturally occurring molecules are a great alternative as they are characterized by low toxicity, low immunogenicity, biocompatibility, stability, low cost, cryoprotection, and availability of reactive sites for chemical modification (38). A specific example is hyaluronan (HA), which is also characterized by bioadhesive properties as part of the extracellular matrix. In addition, HA is the major ligand for CD44 and CD168 receptors and, therefore, suitable for specific targeting to cells, such as those in tumors that overexpress these receptors (39)(40)(41)(42).
Additional surface functionalization includes ligands that enable specific cell targeting, such as antibodies, peptides, and aptamers (15). However, despite the advantage, LNPs decorated with proteins or peptides can elicit an unwanted immune response, cross link receptors, and generate an outside-in signaling cascade (43)(44)(45).
There are additional factors that induce immune activation of LNPs; among these are inhomogeneity of size or shape, multilamellar structure, endotoxin contamination, aggregation, and the presence of non-encapsulated drugs that can bind and aggregate lipids. Ultimately, it seems that the longest circulation time is obtained using LNPs that better mimic natural membranes. Therefore, there are increasing efforts to create LNPs using natural materials and even natural membrane-derived LNPs. Recently, Rodriguez et al. have shown that surface modification of LNPs with a "self " peptide segment of CD47 resulted in reduced blood clearance by macrophages and, therefore, longer circulation time (46,47).
The vast progress in the field of drug delivery enables tailoring specific NPs for a particular location (TME, lymph nodes) and cell type while avoiding unwanted non-specific immune responses and toxicities as detailed below.

LNP-BAseD iMMUNOtHerAPies immunomodulation of APcs
Therapeutic anticancer vaccination is a strategy that attempts to improve host immunity to cancer by upregulating host immune response against TAAs. Tumor vaccines deliver the antigen and/ or adjuvant to APCs for the activation of both humoral and cellular immunity as presented in Figure 1.
NP delivery can significantly boost immunogenicity of tumor antigens by co-delivery of antigens and adjuvants to the same location (48). One such platform is interbilayer-crosslinked multilamellar vesicles (ICMVs), which entrap high amounts of protein antigens in the vesicle core and lipid-based adjuvant in the vesicle walls (49). The authors found that ICMVs elicited robust antibody titers greater than simple liposomes or multi lamellar vesicles of identical lipid compositions. These synthetic vesicles triggered steadily increasing antibody production and CTL responses following repeated administrations.
Several NP-based vaccine formulations have been developed to deliver antigens specifically to APCs, especially to DCs and TAMs. Recently Others have shown targeting of DCs using NPs decorated by specific anti DC antibodies. For example, Macho-Fernandez et al. (52) used NPs generated from poly lactic-co-glycolic acid (PLGA), coated with lipid-PEG and decorated by Abs recognizing DEC205, a cell-surface receptor that is expressed on spleen and lymph node CD8 + DCs. Using these NPs, they have demonstrated that co-delivery of an agonist (a-GalCer) and a protein antigen (ovalbumin) to CD8 + DCs triggers optimal humoral and CTL responses. In addition, this platform promotes potent antitumor responses in a B16F10 murine melanoma tumor model.

immunomodulation of tumor cells and tMe
Over time, cancer cells can develop phenotypes that are less immunogenic in order to escape immune surveillance. One strategy used by cancer cells is an elevated expression of self-markers to avoid immune recognition by professional phagocytes. An example of using this strategy is the over expression of CD47 on the cancer cell surface, which labels the cells with the "self " marker and correlates with poor clinical prognosis (53)(54)(55). Yang et al. developed a systemic delivery strategy based on CD47 siRNA encapsulated in HA-coated LNPs, which led to an efficient knockdown of CD47 in cancer cells. Decreased expression of CD47 eventually led to growth inhibition of melanoma tumors and suppressed lung metastasis in a B16F10 murine melanoma tumor model (56).
Additional strategy used by cancer cells to avoid immune recognition is secretion of anti-inflammatory cytokines. Antiinflammatory cytokines prevent the recruitment of immune cells to the TME and suppress the ability of resident immune cells to act against tumor cells. In order to block these complex interactions, immune modulation based on targeting both the tumor cells and DCs was developed by Xu et al. (57). They delivered a combination of NPs: liposome-protamine-HA NPs encapsulated with siRNA against the immune-suppressive cytokine TGF-β, and mannose-modified lipid-calcium-phosphate NPs encapsulated with tumor antigen and adjuvant. TGF-β downregulation boosted the vaccine efficacy and inhibited tumor growth, as a result of increased levels of tumor infiltrating CD8 + T-lymphocytes and decreased level of Tregs.
A liposomal polymeric gel system developed by Park et al., which combines the delivery of TGF-β inhibitor and the proinflammatory cytokine IL-2, is another example for a complex approach of immunomodulation (Figure 2) (58). IL-2 was shown to enhance NK and CTL activity against several cancer types; however, this ability is hampered due to the secretion of anti-inflammatory agents, such as TGF-β by tumor cells (59). The authors showed a significant reduction of tumor growth in vivo and an increased immune response.
Another example of combining several immunotherapy strategies was recently presented by Moynihan et al. (61). The authors used the amphiphilic vaccine platform containing a tumor antigen and adjuvant conjugated to albumin binding lipid. This enables utilizing the "albumin hitchhiking" strategy upon which binding with albumin directly targets molecules to lymphatics and draining LNs, thereby allowing them to accumulate in APCs. This platform was combined with systemic administration of tumor antigen-specific antibodies, IL-2, and anti-PD-1 antibodies. The authors showed recruitment of both innate and adaptive immune cells that led to elimination of large established tumor burdens in syngeneic and genetically engineered murine tumor models. The treatment also elicited long protective T cell memory response.

seQUeNtiAL treAtMeNt OF cHeMOtHerAPY FOLLOWeD BY iMMUNOtHerAPY
A combined chemo-immunotherapeutic approach may be beneficial for the efficient elimination of cancer. Chemotherapy eliminates tumor cells, causing the cancer to shrink. As a result, tumor-derived antigens, such as peptides or proteins isolated from the tumor cells, can be efficiently internalized by APCs, thereby increasing the antitumor immunity of CTL. Lu et al. (62) developed cisplatin LNPs and CpG-encapsulated liposomes for treatment of melanoma. Such combination therapy established strong synergistic effects, both on the apoptotic level and subsequent abrogation of tumor growth. Heo et al. (63) sequentially subjected tumor-bearing mice to chemotherapy consisting of pacitaxel dissolved in HA in addition to immunotherapy using CpG ODNs and IL-10 siRNA incorporated into PLGA NPs. The sequential treatment with chemotherapeutic drugs followed by a combined immunostimulation strategy resulted in a synergistic effect against solid tumors.
A recent study by He et al. designed coordination polymer LNPs, which combines two therapeutic modalities, chemotherapy and photodynamic therapy, to elicit antitumor immunity (64). These LNPs kill cancer cells by inducing apoptosis and necrosis, stimulating host immune system, and causing an acute inflammation and leukocyte infiltration to the tumors; all of which increase the presentation of tumor-derived antigens to T cells. Combining polymer NPs treatment with PD-L1 checkpoint blockade therapy led to the regression of the primary tumors treated locally with irradiation, and also resulted in the regression of the distant tumors in bilateral syngeneic mouse tumor models of CT26 and MC38.

cONcLUsiON AND FUtUre OUtLOOK
Cancer immunotherapy has been getting a lot of attention in the past few years and for many good reasons. There are multiple benefits for immunotherapy in comparison to conventional medicine, as the former treatment modality enables utilization of the immune system to eliminate tumor cells, while leaving healthy cells untouched. As our understanding of the complex interplay between cancer cells and the immune system deepens, the potential for achieving efficient therapeutics grows. Advances in research in the past few years have provided a solid basis for the development of several therapeutic strategies on the basis of targeting specific pathways and checkpoints. Further understanding of the immunosuppressive TME and antitumor immunity is the key for successful treatments and avoiding unfavorable outcome such as induction of autoimmunity.
NP-based immunotherapy provides multiple advantages upon administration of immune modulators since it enables targeted delivery both locally and temporally, therefore enhancing the effectiveness and reducing toxicity. It also enables transport of several compounds simultaneously and delivery of RNAi-based therapeutics, significantly increasing the therapeutic index.
A successful immunotherapy requires both innate and adoptive responses. Tumor cells utilize several mechanisms to escape immune recognition and induce immune-suppressive TME; thus, tackling only one pillar would not be sufficient. This may be responsible for the limited clinical achievements of current immunotherapy. Indeed, recent treatments combining several immune effectors were reported to be synergistic. The identification of the optimal combination of NPs and immune modulators for the appropriate TME is also crucial, and further development may be aided by the use of computerized biology. Improvement of currently used murine tumor models to appropriately reflect the complex TME is also required. Many reports are treating early-stage tumors in which the immune-suppressive TME is not completely developed. In addition, direct manipulation of tumor-residing lymphocytes has not yet been achieved and the appropriate delivery system is still an unmet need. This is especially significant in light of the recently approved anti T-lymphocyte treatments (anti-CTLA-4 and anti-PD-1 antibodies) that emphasize the potential of T-lymphocyte modulation for immunotherapy. Currently, lymphocyte manipulation can be achieved indirectly via specific modulators; and direct targeting of specific subsets of APCs has already been demonstrated. Some reports even show this simply by controlling lipid composition or charge, avoiding the use of targeting moieties altogether. The effectiveness of such untargeted systems in humans still has yet to be determined. To date, impressive therapeutic effects have been achieved upon using adoptive cell transfer therapy; however, this approach is not feasible for large-scale treatment. Therefore, the optimal NP-based system should include the appropriate immune mediator combination that would elicit highly effective and wholly endogenous response.