The clinical usage of immunotherapy for tumor treatment has been hindered by evidence of dose-limiting toxicities, observed in pre-clinical animal studies and early human clinical trials [12
]. Finding an appropriate dosing regimen that can balance between stimulating an anti-tumor immune response and avoiding off-target inflammatory effects has proven to be challenging for a number of immunomodulatory agonists. For example, the co-administration of interleukin-12 (IL-12) and IL-18 therapy causes a fatal inflammatory response in mice [12
], while the systemic toxicity of recombinant IL-2 therapy at high doses in humans is well established [10
]. Immuno-agonistic antibodies that remain in the process of clinical testing include anti-CD40 and anti-CTLA-4, both of which have demonstrated anti-tumor efficacy simultaneous with dose-limiting systemic side effects [13
]. Nevertheless, the therapeutic potency of such immunotherapeutics has been well established, suggesting that developing a strategy for the restricted delivery of these compounds could substantially improve their prospects for clinical translation.
Other previous studies have attempted to address the issue of minimizing systemic side effects of immunostimulatory therapy. In one recent study, Ahonen et al [39
] found paradoxically that the hepatotoxic effects of intravenous anti-CD40 therapy could be greatly reduced or even eliminated by the systemic co-adminstration of a TLR7 agonist. The authors were not able to determine specific cellular or molecular mechanisms by which the combined therapy could result in reduced toxicity. Based on our own data, this reduction of toxicity is not universal to the combination of all TLR agonists with anti-CD40 therapy, since the addition of CpG (TLR9 agonist) to soluble anti-CD40 therapy in the current study significantly increased symptoms of systemic inflammation (, ), rather than decreasing them. The use of targeting motifs to enhance the specific localization of immunostimulatory ligands at tumor sites represents another possible strategy for reducing the off-target inflammatory effects of systemically administered immunotherapy. In two separate studies, Hamzah et al described the use of fusion peptide-targeted anti-CD40 + IL-2 [22
], or surface peptide-targeted liposomes encapsulating CpG [74
]. Although both methods succeeded in increasing the localization of therapy to the tumor site and greatly improved the anti-tumor response relative to non-targeted therapy, neither strategy was able to eliminate systemic exposure to the immuno-stimulatory agonists. Targeted delivery of anti-CD40 + IL-2 still resulted in elevated serum levels of hepatic ALT enzyme and the inflammatory cytokine TNF-α, while targeted delivery of CpG-liposomes could not prevent non-specific scavenging by the reticulo-endothelial system (RES), as indicated by substantial particle uptake in the spleen. Similarly, Johnson et al [48
] studied the intravenous administration of a tumor antigen-targeted antibody-IL-2 fusion protein, and found that less than 5% of the injected dose actually reached the tumor following i.v. delivery, confirming that the use of tumor-specific antibody targeting is not sufficient to abrogate systemic circulation and exposure. In another recent study, Dominguez et al [75
] used a peptide-specific antibody (anti-neu) to target anti-CD40-delivering PLA nanoparticles to solid tumors following intravenous administration. Although the authors demonstrated that anti-neu targeting improved the therapeutic efficacy of anti-CD40 treatments, they did not examine whether the use of targeted nanoparticles could reduce the severity or breadth of anti-CD40-induced systemic inflammation. Thus, identification of generalizable strategies to eliminate the common systemic side effects of immunotherapy agents remains an unmet need.
Here we tested the use of liposomes as nanoparticulate anchors for combinatorial immunotherapy in the setting of local therapy of established tumors, aiming to substantially alter the bio-distribution of anti-CD40 and CpG in vivo with the goal of blocking systemic toxicity while maintaining anti-tumor efficacy. Anti-CD40 and CpG were coupled to the surface of liposomes without loss of immunomodulatory function, via covalent conjugation or physical association to lipid anchors, and the controlled release of both ligands was demonstrated in vitro and in vivo (-). Using a poorly immunogenic aggressive melanoma model in which s.c. solid tumors were allowed to establish prior to treatment, we showed that an effective anti-tumor response could be stimulated by intratumorally-injected liposome-anchored anti-CD40/CpG without inducing the signatures of systemic inflammation observed following equivalent local doses of soluble anti-CD40/CpG (-). Mice that received combination liposome therapy also showed more consistent anti-tumor responses, with all mice (n=9) surviving past day 37. On the other hand, soluble immunotherapy induced a bimodal response in which 3 out of 9 mice showed minimal tumor growth for over 45 days, but the other 6 mice succumbed to tumor progression between days 28-37 (). Analysis of the tissue distributions of anti-CD40 and CpG by histology () and flow cytometry () confirmed that liposomal delivery sequestered both ligands at the tumor site and tumor-proximal lymph node, while agents injected in soluble form were detected at significantly higher levels in the systemic circulation, particularly for anti-CD40 (). The maleimide-thiol reaction used here for liposomal conjugation of anti-CD40 is generally applicable to any antibody, suggesting the potential use of this system for the delivery of additional immuno-agonists to tumors. Together, this data suggests that liposomal-anchored immunotherapies offer a promising strategy for more potent yet safe anti-tumor therapy, by providing a robust therapeutic regimen while simultaneously minimizing any indications of systemic inflammation. Further understanding of the change in therapeutic outcome obtained with liposomal delivery compared to soluble therapy (loss of a minor complete response population but gain in overall time to progression) represents a key area for further study and improvement of this approach.
A number of other studies have also described the use of biomaterial vehicles, ranging from liposomes and nanoparticles to larger microspheres and hydrogels, for the local delivery of anti-CD40, CpG, and immunomodulatory cytokines. The delivery of IL-2, IL-12, and/or GM-CSF by a variety of biomaterial carriers demonstrated significant anti-tumor effects in therapeutic challenge models, but the systemic inflammatory effects of such potent immunostimulatory treatments were not directly examined [56
]. On the other hand, in the setting of prophylactic vaccinations (or pre-tumor challenge), Hatzifoti et al [73
] found that liposomal entrapment reduced anti-CD40-induced toxicity (as measured by splenomegaly), while Bourquin et al [57
] showed that s.c. injection of cationic gelatin nanoparticles carrying CpG DNA and vaccine antigens reduced systemic cytokine induction relative to soluble injections of the agonist (via decreased systemic exposure to nanoparticle-bound CpG compared to unencapsulated CpG). Conversely, De Jong et al [60
] used liposomes to encapsulate CpG DNA, and surprisingly found that subcutaneous liposomal delivery dramatically increased inflammatory cytokine levels in plasma compared to subcutaneous free CpG. The disparity in systemic side effects reported in these studies might reflect differences in the stability of agonist entrapment in these various carriers, since soluble drugs or immuno-agonists released from locally-injected carriers are known to reach the systemic circulation as early as 6 hr post-injection [58
]. These results underline the benefits of physically anchoring immuno-modulatory compounds to locally retained particle carriers, as we have proposed in the current study, compared to more commonly used encapsulation/release strategies.
A variety of leukocytic cell populations have been implicated in mediating the anti-tumor effects of anti-CD40 and CpG therapies. TLR9 expression is found predominantly in APCs such as dendritic cells, macrophages, and B cells, and the activation of any of these cells by TLR9 stimulation is known to potentiate antigen cross-priming, the production of TH
1-skewed cytokines, and the induction of potent CTL and NK-cell responses [76
]. The mechanisms of anti-CD40 tumor inhibition are currently less well defined, as various studies [77
] have implicated DCs, macrophages, B cells, or combinations thereof as the primary cells responsible for priming potent CTL or NK-cell activity [15
], or T-cell independent [81
] immune responses. Nevertheless, the anti-tumor efficacy of anti-CD40 and CpG therapy likely depends on their ability to stimulate APCs present in the local tumor environment, as well as APCs in the tumor-draining lymph node, where the adaptive immune response is primed. Flow cytometry analysis () indicated that DCs and macrophages in both locales had taken up FAM-labeled CpG and rhodamine-labeled liposomes following the i.t. injection of combination liposomes, confirming that the coupling of immuno-stimulatory ligands to liposomal carriers had not prevented them from reaching target APCs. CpG-lipid delivered via combination liposomes actually reached the tumor-draining lymph node at a higher level than soluble free CpG (), although the diminished level of co-localizing rhodamine-lipid fluorescence suggests that this did not occur via the draining of intact liposomes, but rather by the draining of released CpG-lipid micelles. Whether the enhanced draining relative to free CpG was mediated by the micellar nature of the released CpG-lipid, and/or the presence of the PEG linker in the CpG-lipid conjugate, remains to be investigated. Nevertheless, this observation is consistent with the results reported by Bourquin et al [57
], in which subcutaneously injected nanoparticles increased the localization of a CpG oligonucleotide cargo to draining lymph nodes, relative to free CpG.