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Logo of hhmipaAbout Author manuscriptsSubmit a manuscriptHHMI Howard Hughes Medical Institute; Author Manuscript; Accepted for publication in peer reviewed journal
Biomaterials. Author manuscript; available in PMC 2012 February 1.
Published in final edited form as:
PMCID: PMC3140866

Induction of potent anti-tumor responses while eliminating systemic side effects via liposome-anchored combinatorial immunotherapy


Immunostimulatory therapies that activate immune response pathways are of great interest for overcoming the immunosuppression present in advanced tumors. Agonistic anti-CD40 antibodies and CpG oligonucleotides have previously demonstrated potent, synergistic anti-tumor effects, but their clinical use even as monotherapies is hampered by dose-limiting inflammatory toxicity provoked upon systemic exposure. We hypothesized that by anchoring immuno-agonist compounds to lipid nanoparticles we could retain the bio-activity of therapeutics in the local tumor tissue and tumor-draining lymph node, but limit systemic exposure to these potent molecules. We prepared PEGylated liposomes bearing surface-conjugated anti-CD40 and CpG and assessed their therapeutic efficacy and systemic toxicity compared to soluble versions of the same immuno-agonists, injected intratumorally in the B16F10 murine model of melanoma. Anti-CD40/CpG-liposomes significantly inhibited tumor growth and induced a survival benefit similar to locally injected soluble anti-CD40+CpG. Biodistribution analyses following local delivery showed that the liposomal carriers successfully sequestered anti-CD40 and CpG in vivo, reducing leakage into systemic circulation while allowing draining to the tumor-proximal lymph node. Contrary to locally administered soluble immunotherapy, anti-CD40/CpG liposomes did not elicit significant increases in serum levels of ALT enzyme, systemic inflammatory cytokines, or overall weight loss, confirming that off-target inflammatory effects had been minimized. The development of a delivery strategy capable of inducing robust anti-tumor responses concurrent with minimal systemic side effects is crucial for the continued progress of potent immunotherapies toward widespread clinical translation.

Keywords: tumor immunotherapy, agonistic anti-CD40, CpG oligonucleotides, localized biodistribution, liposome delivery, reduced toxicity

1. Introduction

Tumors elicit tolerized immune responses against tumor-associated self-antigens while simultaneously inducing local immune suppression as a mechanism to avoid detection and elimination by the host immune system [1,2]. A potential therapeutic strategy to counter this immunosuppression is the use of non-cell-based therapies such as cytokines, immune receptor-targeting monoclonal antibodies, or Toll-like Receptor (TLR) agonists to break tumor-associated tolerance, blocking tumor-induced suppressive factors, or directly providing the costimulatory signals necessary to prime an anti-tumor immune [3-9]. However, these potent agents are also prone to eliciting serious side effects following systemic infusion [10-13]. Thus, the clinical effectiveness of many immuno-agonists has remained limited by the lack of a strategy to achieve therapeutic efficacy while avoiding excessive systemic exposure.

Anti-CD40 antibodies and CpG oligonucleotides are two immunostimulatory agents that exemplify this two-edged nature of immunotherapy. Agonists against CD40, a co-stimulatory receptor expressed on the surface of antigen-presenting cells (APCs), have been studied extensively for their ability to promote anti-tumor immunity. Triggering of CD40 signaling via anti-CD40 ligation provides strong activating signals to APCs, endowing them with the capacity to prime strong anti-tumor cytotoxic T-cell responses [14-16]. Pre-clinical studies have shown the efficacy of anti-CD40 therapy against a wide variety of tumor models, either as a monotherapy [17-19], in combination with chemotherapy [20], or in combination with other immunostimulants such as interleukin (IL)-2 [21,22]. These promising results led to the use of anti-CD40 therapy in phase I clinical trials for human patients suffering from non-Hodgkins lymphoma, multiple myeloma, and other solid malignancies [23-26]. However, despite achieving moderate levels of therapeutic efficacy, the maximum tolerated dose for human therapy has been limited due to inflammatory effects in off-target organs [23-25]. Intravenous infusion of anti-CD40 results in widespread systemic exposure to the immunoagonist, leading to symptoms of cytokine release syndrome (fever, headaches, nausea, chills), noninfectious ocular inflammation, elevated hepatic enzymes (indicative of liver damage), and hematologic toxicities including T-cell depletion. Severe off-target inflammatory effects in the liver, lungs, and gut have previously been observed in mice as well, including similar evidence of systemic cytokine release [27-30]. Although the aforementioned side effects were mostly transient in nature, two recent studies in mice unexpectedly observed long-term immuno-suppression following anti-CD40 therapy as well, possibly relating to the activation-induced apoptosis of CD4+ or CD8+ T-cells [31-32].

CpG oligonucleotides, ligands for Toll-like receptor (TLR) 9 expressed by APCs, represent another class of potent immunostimulatory factors. Stimulation of the TLR9 receptor directs APCs towards priming potent, TH1-dominated T-cell responses, by increasing the production of pro-inflammatory cytokines and the presentation of co-stimulatory molecules to T cells [33]. Like anti-CD40, CpG therapy has been tested against a wide variety of tumor models in mice, and has consistently been shown to promote tumor inhibition or regression [6,34-36]. Furthermore, the combination of CD40 agonists and TLR ligands can synergize to stimulate highly potent anti-tumor responses in vivo [37-40]. However, recent studies have also suggested that systemic over-exposure to CpG can have potentially dangerous side effects, including lymphoid follicle destruction [41] and the suppression of adaptive T-cell immunity via indoleamine 2,3-dioxygenase (IDO) induction in the spleen [42,43]. Thus, both anti-CD40 and CpG show substantial anti-tumor potency concurrent with issues of systemic toxicity.

In light of the dangers of systemic immunostimulatory therapy, intratumoral or peritumoral treatments have been tested in an attempt to reduce the level of systemic exposure to potent immuno-agonists. In the clinical setting, local immunotherapy has so far been proposed primarily for the treatment of unresectable tumors or for post-surgical adjuvant therapy to prevent local recurrence [44-47]. However, pre-clinical studies in animal models have also shown that the generation of a local anti-tumor immune response can drive systemic/distal tumor inhibition, via the induction of tumor antigen-specific immune memory. The priming of an adaptive anti-tumor immune response is highly attractive since it could enable immunological targeting of unknown tumor metastases or disseminated malignancies, following locally delivered immunotherapy at a known tumor site. Local therapies applied at a single tumor site using anti-CD40 [18], CpG [36], target antibody-cytokine (IL-2) fusion proteins [48], or other immunostimulants [8,49-52] have successfully inhibited the growth of distal untreated tumors. Furthermore, the intratumoral injection of CpG has recently been tested in a phase I clinical trial against B-cell lymphoma in humans, and some patients exhibited anti-lymphoma clinical responses at distant, untreated tumor sites [53].

Despite such therapeutic benefits, pre-clinical and clinical studies have established that the local injection of soluble agonists [54-57] or controlled release of drugs from a local injection site [58-60] does not necessarily prevent such agonists from entering the systemic circulation and dispersing to distal organs. This could occur either by drainage through lymphatics to the thoracic duct or via direct entry into the bloodstream from leaky tumor vessels. In mice, subcutaneous or intratumoral administrations of the immunotherapeutic cytokines IL-2 [56] or IL-12/GM-CSF [59] resulted in rapid clearance from the local injection site and detection in other peripheral organs within minutes after injection. Similarly, in human patients, high circulating levels of IL-12 [61] or IL-2 [54] were observed within 30 minutes or 3 hours (respectively) after intratumoral/subcutaneous injection. Such observations have necessitated the use of isolated organ perfusion in order to withstand the systemic toxicity of some local recombinant cytokine therapies [62,63]. As a result, the maximum tolerated dose in local immunotherapy may still be restricted by the need to limit undesired widespread exposure and off-target inflammatory symptoms. With this motivation, we sought to develop a biomaterial-based delivery strategy for immunostimulatory factors that could physically retain injected therapeutics at a local tumor site and limit their tissue drainage, while retaining their potent therapeutic efficacy in activating an anti-tumor immune response.

In order to accomplish this, we developed a strategy to couple anti-CD40 and CpG to the surface of PEGylated unilamellar liposomes, for simultaneous co-delivery. We hypothesized that anchoring these molecules to liposomal carriers with a more confined bio-distribution following intratumoral injection would enhance the local retention of these ligands while maintaining their bioactivity. Intratumoral injections of anti-CD40/CpG combination liposomes were performed in established subcutaneous B16F10 tumors in order to investigate whether immunostimulatory effects could be limited to the treated tumor and the tumor-proximal lymph node, thereby driving tumor inhibition while avoiding the inflammatory side effects that result from systemic exposure to these agonists.

2. Materials and Methods

2.1. Materials

Monoclonal anti-CD40 (clone FGK4.5, rat IgG2a) was purchased from Bio X Cell (West Lebanon, NH). Cholesterol, dithiothreitol (DTT), and Tween 20 were obtained from Sigma-Aldrich (St. Louis, MO). Zeba desalting columns were from Pierce (Thermo Fisher Scientific, Rockford, IL). Phospholipids dioleoylphosphocholine (DOPC), polyethylene glycol (PEG)2000-distearoylphosphoethanolamine (DSPE), maleimide-PEG2000-DSPE, and rhodamine-dioleolyphosphoethanolamine (DOPE) were purchased from Avanti Polar Lipids (Alabaster, AL). Fluorescein amidite (FAM)-labeled CpG oligonucleotide (sequence 1826, with phosphorothioate backbone) and FAM-labeled CpG-PEG-lipid conjugate were synthesized in-house as previously described [64]. DNA synthesis reagents were purchased from Glen Research (Sterling, VA) or ChemGenes (Wilmington, MA). Anti-mouse CD45 (clone 30-F11), anti-mouse F4/80 (BM8), anti-mouse CD11c (N418), and polyclonal anti-rat IgG-HRP were obtained from eBioscience (San Diego, CA). Secondary anti-rat IgG was purchased from Jackson ImmunoResearch Labs (West Grove, PA). TNF-alpha and IL-6 ELISA kits were purchased from R&D Systems (Minneapolis, MN). Purified anti-human IgG and recombinant mouse CD40/human Fc fusion protein, for the sandwich ELISA of anti-CD40, were also purchased from R&D Systems.

2.2. Animals and cells

Animals were cared for in the USDA-inspected MIT Animal Facility under federal, state, local and NIH guidelines for animal care. C57BL/6 female mice were purchased from the Jackson Laboratory. For tumor experiments, all mice were inoculated between 6-8 weeks of age. B16F10 melanoma cells were purchased from American Type Culture Collection, and certified to be mycoplasma-free prior to inoculation into animals.

2.3. Synthesis of liposome-anchored anti-CD40 and CpG

Liposomes were synthesized with a composition of cholesterol/DOPC/maleimide-PEG-DSPE/PEG-DSPE 35/50/5/10 by mol%; 0.5 mol% of fluorescent rhodamine-labeled DOPE was also incorporated for biodistribution experiments (histology and flow cytometry). Lipids were vacuum-dried, rehydrated in PBS at 15 μmol/ml, and sonicated for 4 min (alternating 5-7 Watts) using a Misonix probe-tip sonicator (Qsonica, Newtown, CT) to form unilamellar liposomes. Anti-CD40 with exposed free hinge-region thiols was prepared by mixing anti-CD40 (12-15mg/ml) with a 25X molar excess of DTT for 20 min at 25°C in the presence of 10mM EDTA in PBS. The mildly reduced anti-CD40 was passed through a desalting column to remove DTT, and then immediately mixed with maleimide-functionalized liposomes at a ratio of 3 mg Ab:1 μmol liposomes for covalent coupling in the presence of 10 mM EDTA. The maleimide-thiol reaction was allowed to proceed for at least 10 hr at 25°C, followed by centrifugation of the resulting aggregated liposomes and multiple washes with PBS, to remove unbound antibody. Prior to use, anti-CD40-liposomes were syringe-extruded 25X through 100nm polycarbonate filter membranes (Avanti Polar Lipids).

Lipid-conjugated CpG was prepared as previously described [64]. Briefly, CpG oligonucleotides were synthesized using the ABI 394 DNA synthesizer (Applied Biosystems, Carlsbad, CA), and lipid phosphoramidite containing a 24-unit PEG spacer was synthesized as previously described [64]. Lipid phosphoramidite was then dissolved in dichloromethane and coupled to CpG DNA for 15min, using the DNA synthesizer. Following synthesis, CpG and CpG-PEG-lipid conjugates were purified by reverse phase HPLC. To create combination liposomes simultaneously bearing anti-CD40 + CpG, a post-insertion approach [65] was used to insert the purified CpG-lipid conjugate onto the surface of anti-CD40-conjugated liposomes: ~3nmol CpG-lipid was mixed with previously prepared anti-CD40-liposomes (1μmol) for 2hr at 25°C. The resulting combination liposomes were again centrifuged and washed multiple times with PBS, to remove unimer or micellar CpG-lipid, then syringe-extruded 25X through 200nm polycarbonate filter membranes (Avanti). Final liposome size distributions were characterized by dynamic light scattering (Brookhaven 90Plus Particle Size Analyzer, Worcestershire, UK). All liposomes were stored in PBS at 4°C until use.

2.4. Quantitation of anti-CD40 and CpG coupling to liposomes

Bioactive anti-CD40 conjugated to liposomes was quantified by a functional ELISA assay. Anti-CD40 liposomes were first mixed with PBS containing 0.5% Tween 20 surfactant to solubilize the lipids. Standard 96-well plates were coated with 2 μg/ml of goat anti-human IgG antibody, followed by the addition of 100 ng/ml recombinant mouse CD40/human Fc fusion protein. Use of recombinant CD40 as the capture agent for lipid-anchored anti-CD40 ensured that only bioactive anti-CD40 still capable of binding its target receptor would be quantitated. 200 ng/ml of HRP-conjugated goat anti-rat IgG was then added as the detection antibody, followed by an HRP-sensitive colorimetric substrate (R&D Systems). CpG and CpG-lipid were both tracked using a fluorescent FAM label on the 3’ end, and fluorescent measurements were taken at ex/em wavelengths of 480/520nm.

2.5. In vitro release of anti-CD40 and CpG from liposomes

To measure the in vitro release of anti-CD40 or CpG-lipid from combination liposomes, dialysis cassettes (1ml capacity) with a 300kD MWCO membrane were used (Float-a-lyzer G2, Spectrum Labs, Rancho Dominguez, CA). 400μl samples were dialyzed against 20ml PBS containing 10% fetal calf serum, with gentle agitation, at 37°C. Samples and dialysis buffers were collected at each indicated timepoint; anti-CD40 was measured by sandwich ELISA and CpG was measured by its fluorescent label FAM. All samples were mixed with 0.5% Tween 20 to disrupt intact liposomes prior to the anti-CD40 ELISA.

2.6. Tumor inoculation and tumor therapy experiments

For in vivo tumor experiments, C57BL/6 mice were anaesthetized and inoculated subcutaneously on the right hind flank with 5×104 B16F10 cells in Hank’s Balanced Salt Solution (HBSS). Tumors were allowed to establish for 8 days before the start of therapy, by which time they had reached an average area of ~12 mm2. Tumor sizes were measured every 1-2 days by electronic caliper and calculated as the product of 2 orthogonal diameters (D1 × D2). For anti-tumor therapy experiments, mice received intra-tumoral injections on days 8, 10, 12, and 14 of PBS, soluble anti-CD40 only, anti-CD40-liposomes, soluble anti-CD40 + CpG, or combination liposomes, diluted in 150 μl of PBS. Each dose, whether in soluble or liposomal form, consisted of 40 μg of anti-CD40, with or without 20 μg of CpG. Body mass of treated mice was measured daily, beginning on day 7 post-tumor inoculation, using an electronic scale. Serum was collected from mice at the indicated time points via retro-orbital bleeding, for the measurement of circulating inflammatory markers. Mice were euthanized when tumor areas exceeded 100 mm2, per institutional guidelines.

2.7. Serum measurements of systemic side effects

Blood was collected from mice at the indicated time points and centrifuged to remove the cellular fraction. Circulating serum levels of TNF-alpha and IL-6 were measured by ELISA detection kits (R&D Systems), according to the manufacturer’s instructions. The serum level of free anti-CD40 was measured by sandwich ELISA using recombinant CD40 receptor (described in section 2.4), and serum CpG was detected via its fluorescent FAM label (ex/em at 480/520nm). Serum levels of the hepatic enzyme ALT (alanine transaminase) were determined using a standard biochemical assay (Infinity ALT reagent kit, Thermo Fisher), according to the manufacturer’s instructions.

2.8. Histological and flow cytometric analysis

For biodistribution experiments, a single intratumoral injection of soluble or liposomal formulations was given at a dose of 40 μg anti-CD40 ± 20 μg CpG between days 8-10 post-inoculation when tumors were at a size of 12-15 mm2, and mice were euthanized 24hr or 48hr later. Tumors, tumor-proximal or distal lymph nodes, and spleens were harvested and either immediately snap-frozen using liquid nitrogen (for cryosectioning) or digested with 100μg/ml Liberase Blendzyme III (Roche Applied Sciences, Indianapolis, IN) for 15min at 37°C, followed by mechanical dissoctiation and rinsing through 40 μm nylon mesh cell strainers to obtain cell suspensions. Cryosections were sliced at a thickness of 7μm, and imaged directly without any further processing for the optimal detection of fluorescent FAM-labeled CpG, FAM-labeled CpG-lipid, and rhodamine-labeled liposomes. All brightfield and fluorescent images were taken using a Zeiss LSM 510 confocal microscope, and processed using Zeiss LSM 510 Image Browser. For secondary immunostaining against anti-CD40, cryosections were acetone-fixed and stained using a DyLight649-labeled anti-rat IgG, with minimal cross-reactivity against mouse IgG (Jackson Immuno). After washing, stained cryosections were mounted and covered using VectaShield HardSet mounting medium with DAPI (Vector Laboratories, Burlingame, CA). For flow cytometry analysis, recovered cells were re-suspended in PBS + 1% BSA and stained with fluorescent antibodies against CD45, CD11c, and F4/80, for 30-40min on ice. Flow cytometry was performed on an LSRII cytometer (BD Biosciences) and data was processed using FlowJo software (Tree Star, Ashland, OR). Cellular events were gated based on forward and side scatter, and tumor-infiltrating leukocytes were gated from other cell populations based on CD45 expression.

2.9. Statistical analysis

Data are shown as mean ± SEM, unless indicated otherwise. Comparisons of Kaplan-Meier survival curves were performed using a log-rank test. For all other data, comparisons of two experimental groups were performed using two-tailed unpaired t-tests unless indicated otherwise. Statistical analysis was performed using GraphPad Prism software.

3. Results

3.1. Synthesis and characterization of liposomes for the combined delivery of immunostimulatory anti-CD40 and CpG

Immunotherapy using soluble immuno-agonists can be effective but is known to have systemic toxicity even following local administration [54,55,62]. To test whether nanoparticle anchoring could restrict the dissemination of conjugated immunostimulatory ligands following local injection while maintaining their ability to effectively stimulate target cell populations, we began by preparing immunoliposomes for anti-CD40 delivery into tumors. Monoclonal agonistic anti-CD40 was mildly reduced to selectively cleave disulfides in the hinge region of the antibody [66], and then mixed with maleimide-functionalized small unilamellar vesicles (SUVs) (cholesterol:DOPC:PEG-DSPE:maleimide-PEG-DSPE in a 35:50:10:5 mol% ratio) with a mean diameter of 80 nm (± 15nm std. dev.) (Fig. 1A). Aggregation of liposomes occurred during the coupling reaction, consistent with the availability of multiple thiol groups per antibody, which could mediate crosslinking between liposomes. However, because the lipids themselves can readily reorganize during processing, we used this crosslinking phenomenon to quickly purify the reaction components: The aggregated liposomes were pelleted by centrifugation, washed to remove unbound antibody, then re-sized by membrane extrusion (Fig. 1A) to obtain homogeneous Ab-conjugated vesicles with a mean diameter of approximately 100 nm and a standard deviation of 14 nm (Fig. 1B). Quantitation of coupled anti-CD40 was performed by sandwich ELISA analysis of liposomes lysed by Tween 20 surfactant, using recombinant CD40 as the capture ligand to measure the amount of bioactive antibody linked to the vesicles. Anti-CD40 binding to the liposomes required the presence of maleimide-functionalized lipid, and the amount of bioactive Ab conjugated was only weakly influenced by the quantity of maleimide-PEG-lipid included in the vesicle composition (Supplementary Fig. 1). We thus chose to use liposomes with 5 mol% mal-PEG-DSPE for further studies, which consistently gave conjugations of ~55 μg bioactive conjugated anti-CD40 per mg lipid (Supplementary Fig. 1).

Figure 1
Synthesis of PEGylated liposomes bearing lipid-anchored anti-CD40 and CpG oligonucleotides. (A) Schematic illustration of the surface coupling of anti-CD40 to PEGylated liposomes via maleimide-thiol reaction, followed by the insertion of the CpG-lipid ...

To co-incorporate anchored CpG oligonucleotides into anti-CD40-bearing immunoliposomes, we employed a synthetic CpG-PEG-lipid conjugate recently developed in our lab (Fig. 1A) [64]. When suspended alone in aqueous buffer, the CpG-PEG-lipid conjugate self-assembled into stable micelles, but mixing of these oligo-micelles with anti-CD40-liposomes led to rapid spontaneous insertion of the lipophilic tails of the CpG conjugate into the liposomal membranes. Anti-CD40 was conjugated to liposomes as described above, and following washing to remove unbound antibody, the aggregated liposomes were mixed with lipid-CpG (Fig. 1A). After washing again to remove unbound CpG, the anti-CD40/CpG-coupled liposomes were membrane-extruded to a mean diameter of approximately 150 nm, with a std. dev. of 18 nm (Fig. 1B). Mixing fluorescently-tagged CpG-lipid (130 μg/ml) with anti-CD40-liposomes (14.4 mg lipid/mL) gave CpG insertion with ~90% efficiency, resulting in 20 ± 2.2 μg CpG oligonucleotides (~3 nmol) per total mg liposomes. The high efficiency of CpG-lipid association with liposomes and high level of CpG loading per lipid mass contrasted with the low efficiency of soluble non-lipidated CpG entrapment we could achieve by traditional liposomal encapsulation in PEGylated liposomes, where only 2 μg of CpG was encapsulated per mg lipid. The mean sizes of anti-CD40-liposomes or combination liposomes were stable for at least 7 days in storage at 4°C (data not shown).

To test the stability of anti-CD40 and CpG association with PEGylated liposomes, the release of the immunostimulatory ligands from vesicles was measured in vitro in the presence of serum: combination liposomes carrying anti-CD40 and CpG were dialyzed (300kD MWCO membrane) against PBS pH 7.4 containing 10% fetal calf serum at 37°C, and release of the ligands into the dialysis buffer was monitored over time. Soluble anti-CD40 and CpG both diffused freely through the membrane with substantial release from the dialysis cassette in <5 hr (Fig. 2A-B). By contrast, PEG-DSPE-anchored anti-CD40 effectively tethered the antibody to the liposome surfaces, with <10% of the initial loading released over 7 days (Fig. 2A). After 7 days, the liposomes were disrupted by adding 0.5% Tween 20 surfactant, and >90% of the coupled anti-CD40 was immediately recovered. CpG-lipid inserted into combination liposomes was retained by the vesicles less efficiently; nearly 80% of the oligo was released by 24 hr and release was complete by ~7 days (Fig. 2B). CpG release may reflect the ability of oligo-lipids to self-assemble favorably into highly stable micelles in aqueous buffers [64]. Gel electrophoresis of released samples confirmed that the fluorescent measurements of labeled CpG represented fully intact FAM-labeled CpG-lipid, with only a low level of degradation of these phosphorotioate backbone-stabilized oligos (or cleavage of the FAM label) by serum nucleases over 24 hr (data not shown). Thus, the vesicles efficiently retain anti-CD40 but release CpG over approximately one week under strong sink conditions. Combination liposomes stored in PBS at 4°C showed negligible loss of either anti-CD40 or CpG over at least 2 weeks (data not shown).

Figure 2
Controlled release of anti-CD40 and CpG from combination liposomes (filled symbols) by dialysis against serum-containing PBS in vitro, compared to the dialysis of freely soluble immuno-agonists (open symbols). (A) Release of conjugated anti-CD40 from ...

3.2. Anti-tumor efficacy and systemic toxicity following intratumoral injection of anti-CD40-displaying liposomes

Many previous preclinical studies have demonstrated that anti-CD40 monotherapy can lead to inhibition of tumor growth or even complete tumor regression in some animal models [11,15-18]. We thus initially tested the efficacy of anti-CD40-liposomes alone, without CpG, in the aggressive, poorly immunogenic B16F10 transplanted melanoma model [19]. We first confirmed that anti-CD40-liposomes did not directly exert cytotoxic effects on B16 tumor cells during in vitro culture, and that tumor cells exhibited minimal non-specific binding of immunoliposomes, consistent with the absence of CD40 surface expression on B16F10 cells (Supplementary Fig. 2 and data not shown). For in vivo experiments, C57BL/6 mice were inoculated s.c. in the flank with B16F10 tumor cells, which were allowed to establish into a solid tumor for 8 days, to a mean size of ~12 mm2. Starting on day 8, mice were given 4 intratumoral injections of anti-CD40-liposomes or soluble anti-CD40, every other day, at an equivalent dose of 40 μg antibody per injection. While locally administered soluble anti-CD40 strongly inhibited B16 tumor growth, anti-CD40-liposomes slowed tumor growth only ~50% as effectively as the soluble antibody (Fig. 3A-B).

Figure 3
B16 melanoma therapy using locally-injected soluble anti-CD40 or liposome-anchored anti-CD40. (A) Average tumor growth curves from a representative experiment (n = 5 mice/group). Arrows indicate 4 intra-tumoral injections (days 8,10,12,14 following tumor ...

In parallel with measuring the anti-tumor activity of these two different treatments, we also assessed systemic toxicity associated with each therapy. Even when administered locally, soluble anti-CD40 caused a substantial weight loss in treated mice for several days following the initiation of therapy, which was statistically significant (versus PBS-injected controls) on days 2-6 after the start of therapy. The weight loss peaked at ~10% on day 3, comparable to the loss experienced by mice in an inflammatory lipopolysaccharide (LPS)-induced acute phase response [67,68]. This result is consistent with the expectation that soluble anti-CD40 can drain rapidly from the injection site, enter the systemic circulation, and cause widespread inflammatory effects. The transient nature of the systemic response to soluble anti-CD40 has been previously observed and may reflect systemic tolerization to the antibody’s effects on repeated treatment [27,39]. In contrast, treatment with anti-CD40-liposomes did not cause significant weight loss relative to PBS-treated controls at any time point (Fig. 3C). Measurements of circulating serum levels of pro-inflammatory cytokines such as IL-6 provided a further indication of systemic inflammation in response to local soluble immunostimulant therapy (Fig. 3D). Intratumorally injected anti-CD40 caused a dramatic increase in serum IL-6 within 24hr, but anti-CD40-displaying liposomes induced a barely detectable (p=0.11, n.s.) increase in serum cytokine levels over control mice. Similar trends were observed with serum levels of the inflammatory cytokine tumor necrosis factor-alpha (TNF-α), and alanine transaminase (ALT) enzyme, a standard clinical measure of hepatic inflammatory damage (data not shown). While these results confirmed the potential benefits of liposomal anchoring for eliminating systemic side effects, liposomal anchoring clearly inhibited the anti-tumor efficacy of anti-CD40 monotherapy.

3.3. Therapeutic efficacy and systemic toxicity of combination anti-CD40/CpG liposomes

Combination therapies of anti-CD40 mixed with TLR agonists such as CpG oligonucleotides have previously been shown to provide substantially enhanced anti-tumor immune responses compared to anti-CD40 monotherapy [38-40]. We reasoned that the enhanced potency of anti-CD40 + CpG signaling combined with the controlled release of CpG-lipid from liposomes (as indicated by our in vitro data) might allow the liposome-delivered combination therapy to achieve potency comparable to soluble anti-CD40+CpG therapy, while still avoiding systemic inflammation that might be augmented by the addition of soluble CpG. We thus proceeded to test the anti-tumor efficacy of soluble or liposomal combination anti-CD40/CpG in the B16 model. As before, mice received 4 intratumoral injections (given every other day starting day 8 post tumor cell inoculation) of either soluble anti-CD40 alone, soluble anti-CD40 mixed with CpG, or anti-CD40/CpG liposomes, at equivalent doses of 40 μg anti-CD40 and 20 μg CpG per injection. As shown in Fig. 4A, 3/9 mice treated with soluble anti-CD40+CpG exhibited “complete responses”, defined as apparent cures of the animals and lack of tumor progression or new tumor nodule formation over 45 days. The remaining mice showed a delay in tumor growth compared to untreated animals but eventually all succumbed (Fig. 4B); we quantified this delay in tumor growth as a time to progression (time to tripling of initial tumor size from the start of therapy) (Fig. 4C). Such a mixture of complete and partial responses to immunotherapy has previously been reported and has been correlated with the tumor size at the start of treatment [8]. In the current study, however, the initial tumor burden of the three mice showing the strongest anti-tumor responses was only slightly smaller than that of the other six mice (mean 9.3 ± 0.7 mm2 vs. 12 ± 2 mm2, n.s.). The combination of soluble CpG with soluble anti-CD40 provided only a modest enhancement over soluble anti-CD40 alone, which induced 2/8 complete responses and showed a slightly longer time to progression for the remaining tumors (Fig. 4A-C). No significant difference in survival benefit was observed between either regimen of soluble therapy (Fig. 4B, p=0.33, n.s. by log-rank test).

Figure 4
Treatment of B16 melanoma via soluble anti-CD40 + CpG vs. liposome-anchored anti-CD40/CpG. (A) Individual tumor growth curves (n=8 or 9 mice/group), combined from two independent experiments. Mice were given 4 intra-tumoral injections (days 8,10,12,14) ...

In comparison, liposomal anti-CD40/CpG elicited no complete responses (0/9), but instead showed a significant increase in the time to progression for partial responders compared to the equivalent soluble therapy (p = 0.04), from a mean of 27.8 ± 1.4 days for soluble treatment to 33.4 ± 1.8 days for liposomal anti-CD40/CpG (Fig. 4A-C). Anti-CD40/CpG liposome therapy significantly prolonged the survival of tumor-bearing mice compared to PBS-treated controls (Fig. 4B, p<0.0001 by log-rank test), and induced a mean survival benefit comparable to both soluble immunotherapy regimens (p>0.05, not significant). Furthermore, combinatorial liposome therapy demonstrated a substantial increase in potency over liposomal anti-CD40 alone (Figs. 3B vs. vs.4A),4A), suggesting a synergistic effect in the particle-mediated co-delivery of both immuno-stimulants. Thus, though liposomal therapy showed the loss of a minority of complete responses seen with soluble immunostimulatory ligands, the majority of animals (partial responders in all groups) actually exhibited longer times to progression following liposomal therapy compared to the soluble treatment regimens.

To determine whether the therapeutic effects of anti-CD40/CpG liposomes was still achieved with minimized systemic side effects, overall weight loss and circulating serum inflammatory markers were monitored following locally-administered therapy. The addition of soluble CpG to soluble anti-CD40 intratumoral therapy greatly exacerbated the weight loss of animals through the entire course of treatment (Fig. 5A vs. Fig. 3C), showing statistically significant differences versus PBS-treated animals from day 9 through day 16 (p≤0.04). In contrast, anti-CD40/CpG-liposomes showed a mild, transient effect that reached statistical significance only on day 10 (mean 4% weight loss at this time point, p=0.04) (Fig. 5A). Serum levels of hepatic ALT enzyme were significantly elevated in mice at 24 hr after the first injection of soluble anti-CD40 + CpG, indicative of inflammatory hepatic damage (Fig. 5B), while mice treated with anti-CD40/CpG liposomes showed a barely detectable increase above background levels that did not reach significance (p=0.07, n.s.). Similarly, serum levels of the pro-inflammatory cytokines IL-6 and TNF-α were greatly increased in mice that received soluble anti-CD40/CpG, but not in mice that received liposome-anchored agonists (Figs. 5C-D). Taken together, these data demonstrate that dual-agonist immunotherapy employing liposomal delivery was capable of stimulating a potent local anti-tumor immune response, without inducing the systemic toxicity elicited by an equivalent dose of locally-administered soluble agonists.

Figure 5
Systemic toxicity following soluble vs. liposomal anti-CD40/CpG immunotherapy. (A) Weight loss following intratumoral injections of PBS, soluble anti-CD40+CpG, or anti-CD40/CpG liposomes, following an identical dosing regimen as described in Figure 4 ...

3.4. Biodistribution of liposome-anchored anti-CD40 and CpG

To determine whether the lack of systemic inflammation following liposome-anchored αCD40/CpG delivery was achieved as a result of lowered systemic exposure to these agents, we collected blood serum at various time points following intratumoral injections. Consistent with the in vitro release data, negligible levels of anti-CD40 could be detected in serum at 6 or 24 hr after local liposome-anchored αCD40/CpG therapy (Fig. 6A, or later times, not shown), indicating that lipid anchoring effectively prevented dissemination of the antibody to the systemic circulation. In contrast, locally-administered soluble anti-CD40+CpG or anti-CD40 alone resulted in high levels of circulating antibody within 6 hr post-injection (Fig. 6A and data not shown). The release of liposome-inserted CpG-lipid from combination liposomes occurred much more rapidly than the release of lipid-anchored anti-CD40 in vitro (Fig. 2), so a similar trend was expected in vivo. Fig. 6B shows that fluorescently-labeled CpG from combination liposome delivery could be detected in serum at 5 hr following intra-tumoral injection, and also remains present at a measurable level in serum up to 20 hr post-injection. However, the level of circulating CpG following liposomal delivery was approximately half that attained following intratumoral soluble anti-CD40+CpG therapy at both time points (p=0.0008 and p=0.002, respectively, Fig. 6B). Thus, liposome-anchored CpG delivery lowered the systemic exposure to this agent as well.

Figure 6
Systemic exposure to anti-CD40 and CpG following soluble or liposomal local immunotherapy. (A) Serum levels of anti-CD40 at 6 hr and 24 hr after an intratumoral injection of soluble or combination liposome therapy. P-values determined by unpaired t-test: ...

Low levels of anti-CD40 and CpG in the serum following liposomal delivery could either reflect preferential retention of the nanoparticle-anchored agonists in the tumor site/tumor draining lymph nodes or more rapid clearance/degradation of the particle-delivered agonists. We thus performed histological analysis on cryosections of tumors and tumor-draining lymph nodes, to directly visualize the local retention and proximal draining of intratumorally-injected immunostimulatory ligands. Established B16 tumors were given a single intratumoral injection of PBS, soluble unlabeled anti-CD40 + FAM-labeled CpG, or rhodamine-tagged liposomes conjugated with anti-CD40 and FAM-labeled CpG-lipid. Fig. 7A shows representative cryosections of tumors excised at 48hr following soluble or combination liposome therapy (refer to Supplementary Fig. 3A for PBS controls). Liposomes (red) were retained at a high level at the tumor for over 48 hr post-injection, while secondary anti-rat IgG staining (yellow) against anti-CD40 co-localized closely with the liposomes, confirming that anti-CD40 remained coupled to the liposome carrier in vivo. FAM-labeled CpG-lipid (green) was also highly retained at the tumor site for at least 48hr following liposomal delivery, but was more dispersed through the local dermal and subcutaneous tissue. This was consistent with the release of CpG-lipid from liposomes that had been observed in the presence of serum in vitro. Tumors that received soluble anti-CD40+CpG therapy also showed significant levels of CpG remaining in the tumor site at 24 and 48 hr, consistent with earlier reports of the nonspecific interactions of phosphorothioate-backbone CpG with tissue matrix proteins [69,70]. However, significantly lower amounts of anti-CD40 remained within the tumor mass following soluble therapy, (Fig. 7A), indicating rapid draining away from the injection site.

Figure 7
Histological analysis of anti-CD40 and CpG biodistributions following local immunotherapy. Immunohistochemical staining of frozen tissue sections showing fluorescent rhodamine-labeled liposomes (left panels, red); anti-CD40 (middle panels, yellow); and ...

The initiation of antigen-specific anti-tumor responses requires the interactions of activated APCs and T-cells in the tumor-proximal lymph node (LN). Since this is also the collection site for lymphatic draining from the tumor, we examined whether anti-CD40 and CpG reached the tumor-proximal LN following soluble or liposomal delivery. At 24 hr following treatment with anti-CD40/CpG liposomes, a low level of rhodamine-labeled lipid (Figure 7B, red) was observed, primarily at the subcapsular and intermediate sinus areas, co-localized with secondary staining for anti-CD40 (yellow). As with the tumor cryosections, FAM-labeled CpG-lipid (green) appeared to be distributed in a more dispersed pattern than the liposomes/anti-CD40. By 48hr, fluorescent signals from rhodamine-liposomes, FAM-CpG, or secondary anti-CD40 staining in the tumor-proximal LN of liposome-treated mice were greatly diminished, though still detectable above PBS-treated background levels (Supplementary Fig. 3B and data not shown). By comparison, mice that received soluble anti-CD40+CpG therapy showed surprisingly low levels of CpG in the tumor-draining LN, at both 24hr (Fig. 7B) and 48hr (not shown). Soluble anti-CD40, however, was readily detected in a diffuse pattern in the tumor-proximal LN at both timepoints, consistent with the tumor-site and serum observations that locally-administered soluble anti-CD40 drains rapidly from the injection site into lymphatic or systemic circulation.

Given the persistence of liposomal anti-CD40/CpG in the tumors and draining lymph node, and the observed colocalization of anti-CD40 with liposomes but dispersion of CpG-lipid, we used flow cytometry to separately trace the binding/uptake of CpG and liposomes by cells at these sites following local therapy. Established B16 tumors were injected once intratumorally with either PBS, soluble anti-CD40 + FAM-labeled CpG, or combination liposomes carrying anti-CD40/FAM-labeled CpG-lipid/fluorescent rhodamine-lipid. At 24 or 48 hr post-injection, the tumor tissues, distal and tumor-proximal lymph nodes, and spleens were harvested and processed into cell suspensions for analysis. Cells were gated on CD45 expression (common leukocyte marker), CD11c (marker for dendritic cells), and F4/80 (macrophages), since these antigen-presenting cells express both the CD40 receptor and the TLR9 receptor for CpG, and represent primary targets of this immunotherapy. Figure 8A shows representative flow cytometry histograms of excised tumor samples at 48 hr post-injection, illustrating that the majority of tumor-infiltrating DCs (CD45+CD11c+) and macrophages (CD45+F4/80+) were positive for rhodamine-labeled liposomes (with their coupled anti-CD40 cargo) and FAM-labeled CpG. As summarized in Figure 8B, this high level of uptake by tumor-infiltrating APCs was consistently observed at both 24 and 48 hr post-injection, indicating that liposomally delivered anti-CD40/CpG therapy had reached its intended targets for local immuno-modulation.

Figure 8
Flow cytometry analysis of excised tumors and proximal or distal lymphoid organs following local immunotherapy. (A) Representative histograms of excised tumor tissues at 48 hr following a single intratumoral injection of anti-CD40/CpG liposomes, showing ...

Consistent with the histological data, flow cytometry analysis of proximal and distal lymphoid organs following intratumoral injection confirmed that a small fraction of leukocytes in the tumor-proximal LN took up liposomes (Fig. 8C) and ~10% of these cells took up CpG-lipid (Fig. 8D). As expected, barely detectable levels of either component were observed in spleens or contralateral lymph nodes, consistent with the elimination of systemic toxicity via liposomal coupling and local retention. CD11c+ dendritic cells and F4/80+ macrophages in the tumor-draining LN both took up rhodamine-labeled liposomes and FAM-labeled CpG by 24hr post-injection (Fig. 8E), suggesting the successful delivery to APCs in the draining LN, and fluorescent CpG could still be detected at 48 hr post-injection. In addition, higher overall levels of FAM-labeled CpG were observed in the proximal LN following combination liposome delivery than following soluble delivery, as quantified by mean fluorescent intensity in cells at both time points (Fig. 8F). This suggests that the CpG-PEG-lipid conjugate achieved enhanced draining to the proximal lymph node compared to free soluble CpG.

4. Discussion

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 [12,13,23-26]. 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 [13,23-25]. 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 (Figs. 3, ,5),5), 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 (Figs. 1--2).2). 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 (Figs. 4--5).5). 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 (Fig. 4B). Analysis of the tissue distributions of anti-CD40 and CpG by histology (Fig. 7) and flow cytometry (Fig. 8) 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 (Fig. 6). 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 [56,59,71-72]. 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 [58,59]. 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 TH1-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 [77-79] have implicated DCs, macrophages, B cells, or combinations thereof as the primary cells responsible for priming potent CTL or NK-cell activity [15-17,80], 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 (Figs. 8B, E) 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 (Fig. 8F), 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.

5. Conclusions

Recent studies in immunomodulatory therapy have achieved potent levels of anti-tumor inhibition and regression in a wide variety of pre-clinical models, but the effectiveness of this strategy in clinical treatments remains hampered by dose-limiting toxicities caused by systemic over-exposure. We have reported here the formulation and characterization of a liposomal vehicle carrying a combined dose of anti-CD40 and CpG, two highly potent anti-tumor agents. Liposomal surface coupling of these ligands retains them at a high level in the tumor and the surrounding tissue following intratumoral injection, allowing them to be presented to APCs at the tumor and the tumor-draining lymph node while restricting them from entering systemic circulation or reaching distal lymphoid organs. Future studies will be needed to elucidate the underlying mechanisms governing differences in the therapeutic outcome of liposomal vs. soluble therapy, but the results described here suggest that nanoparticle anchoring can used to block systemic toxicity while maintaining anti-tumor effects for two distinct immunostimulatory agonists.

Supplementary Material


Supplementary fig. 1:

Efficiency of anti-CD40 conjugation to the surface of maleimide-functionalized liposomes. The molar percentage of maleimide-terminated lipid in the overall liposome composition was titrated from 0 to 10%, and 5% maleimide was chosen as the optimal composition for antibody binding and particle stability. Bioactive levels of conjugated antibody were measured by sandwich ELISA using recombinant CD40 as the capture ligand.


Supplementary fig. 2:

Two representative confocal images of B16F10 tumor cells after co-incubation with anti-CD40-conjugated liposomes in vitro. Cells were co-incubated with liposomes for 6 hours and then washed prior to imaging. Minimal non-specific adsorption or uptake of liposomes by tumor cells was detected, and there was no evidence of any direct cytotoxicity following co-culture with the liposomes. Scale bars = 20μm.


Supplementary fig. 3:

Representative cryosections of a subcutaneous B16 tumor and the surrounding tissue (A), and the tumor-proximal lymph node (B), from an untreated tumor-bearing mouse, illustrating background levels of fluorescence and non-specific staining. Left panels: red = fluorescent rhodamine-labeled liposomes; middle panels: yellow = secondary anti-isotype staining against anti-CD40; right panels: green = FAM-labeled CpG-lipid conjugate, overlaid with brightfield. Tissue sections are outlined in white; scale bars = 500μm.


This work was supported in part by the Dana-Farber/Harvard Cancer Center - MIT Bridge Project Fund. DJI is an investigator of the Howard Hughes Medical Institute. The authors would like to acknowledge the Swanson Biotechnology Center (SBC) of the Koch Institute at MIT for the use of flow cytometry and histology facilities. W. Zhang at the SBC performed the cryosectioning for histological analysis.


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