Journal of Controlled Release 313 (2019) 106–119

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Journal of Controlled Releasejournal homepage: www.elsevier.com/locate/jconrel

Lipid nanoparticles of Type-A CpG D35 suppress tumor growth by changing
tumor immune-microenvironment and activate CD8 T cells in miceT

Lisa Munakataa,1, Yoshihiko Tanimotob,1, Akio Osac,1, Jie Mengb, Yasunari Hasedab,Yujiro Naitoc, Hirotomo Machiyamac, Atsushi Kumanogohc, Daiki Omataa, Kazuo Maruyamad,⁎
Yasuo Yoshiokae,f,g, Yoshiaki Okadaf, Shohei Koyamac, Ryo Suzukia, ,2, Taiki Aoshib,gaLaboratory of Drug and Gene Delivery Research, Faculty of Pharma-Science, Teikyo University, Japan
Vaccine Dynamics Project, BIKEN Innovative Vaccine Research Alliance Laboratories, Research Institute for Microbial Diseases, Osaka University, Japanc
Department of Respiratory Medicine and Clinical Immunology, Graduate School of Medicine, Osaka University, JapandLaboratory of Ultrasound Theranostics, Faculty of Pharma-Science, Teikyo University, Japane
Vaccine Creation Project, BIKEN Innovative Vaccine Research Alliance Laboratories, Research Institute for Microbial Diseases, Osaka University, Japanf
Graduate School of Pharmaceutical Sciences, Osaka University, Japang
BIKEN Center for Innovative Vaccine Research and Development, The Research Foundation for Microbial Diseases of Osaka University, Suita, Osaka, Japanb

ARTICLE INFOABSTRACT

Keywords:
CpG
Lipid nanoparticle
Cancer immunotherapy
Oligodeoxynucleotide delivery
Microfluidics

Type-A CpG oligodeoxynucleotides (ODNs), which have a natural phosphodiester backbone, is one of the highest
IFN-α inducer from plasmacytoid dendritic cells (pDC) via Toll-like receptor 9 (TLR9)-dependent signaling.
However, the in vivo application of Type-A CpG has been limited because the rapid degradation in vivo results inrelatively weak biological effect compared to other Type-B, -C, and -P CpG ODNs, which have nuclease-resistantphosphorothioate backbones. To overcome this limitation, we developed lipid nanoparticles formulation containing a Type-A CpG ODN, D35 (D35LNP). When tested in a mouse tumor model, intratumoral and intravenous
D35LNP administration significantly suppressed tumor growth in a CD8 T cell-dependent manner, whereas
original D35 showed no efficacy. Tumor suppression was associated with Th1-related gene induction and activation of CD8 T cells in the tumor. The combination of D35LNP and an anti-PD-1 antibody increased thetherapeutic efficacy. Importantly, the therapeutic schedule and dose of intravenous D35LNP did not induce
apparent liver toxicity. These results suggested that D35LNP is a safe and effective immunostimulatory drug
formulation for cancer immunotherapy.

1. Introduction
Conventional cancer immunotherapies focusing on increasing
tumor-specific T cell responses using antigen/peptide- or dendritic cellbased vaccine have shown only limited clinical efficacies, potentially
due to tumor-mediated immune suppression [1]. Recent introduction of
immune checkpoint inhibitors, which release the tumor-mediated T cell
suppression, has been shown substantial therapeutic effect. In somepatients, this has significantly improved the overall survival [2,3]. Oneof the fundamental limitation of the immune checkpoint inhibitors is
the requirement for pre-existing antitumor T cell responses, which
many patients do not have [1,2].
To augment antitumor immunity, several immunomodulators including cytokines such as IFN-α and IL-12 [4,5], and toll-like receptor
agonists such as TLR4, TLR3, TLR7, and TLR9 [6–8] have been examined in clinical trials, and some were successfully approved forclinical use. However, these immunomodulators also have limitations
for cancer therapy due to the associated intolerable toxicities [9–11].

Abbreviations: DiR, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DOTAP, 1,2-dioleoyl-3trimethylammonium-propane; DOPE, 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine; D35LNP, D35-containing lipid nanoparticles; LPS, lipopolysaccharides;
DSPE-PEG(2k or 5k), N-(Carbonyl-methoxypolyethyleneglycol (2000 or 5000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine); TLR9, toll-like receptor 9; BM,
bone marrow; hPBMCs, human peripheral blood mononuclear cells; iMonocytes, inflammatory monocytes; ODNs, oligodeoxynucleotides; PFA, paraformaldehyde;
pDC, plasmacytoid dendritic cells; PMNs, polymorphonuclear cells; TANs, tumor-associated neutrophils⁎
Corresponding author at: 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.
E-mail addresses: r-suzuki@pharm.teikyo-u.ac.jp (R. Suzuki), aoshi@biken.osaka-u.ac.jp (T. Aoshi).
1
The authors contributed equally to this work.
2
Co-corresponding authors.
https://doi.org/10.1016/j.jconrel.2019.09.011
Received 6 June 2019; Received in revised form 10 September 2019; Accepted 18 September 2019
Available online 16 October 20190168-3659/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license(http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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CpG motif-containing oligodeoxynucleotides (ODNs) have been investigated as immunomodulatory and immunostimulatory drugs, which
are TLR9 agonists and induce innate immune responses such as interferon and inflammatory cytokine secretions. Four types of CpG ODNs
have been reported including Type-A, B, C, and P [12,13]. Type-A CpG
ODNs mostly consist of a natural phosphodiester backbone, and theystrongly induce IFN-α production from plasmacytoid dendritic cells(pDCs). Type-B CpG ODNs consist of an unnatural phosphorothioate
backbone, and they mainly activate B cells and induce IL-6 production.
Type-C CpG ODNs also consist of an unnatural phosphorothioate
backbone, and they mainly induce IL-6 production from B cells, but an
introduction of one palindromic sequence enables weak IFN-α induction. Type-P CpG ODNs similarly consist of an unnatural phosphorothioate backbone as Type-B and C, but they contain 2 palindromic
sequences, which contribute to induce strong IFN-α as well as IL-6
production. The most popular CpG ODNs examined in the clinicalstudies are Type-B CpG ODNs; however, repeated Type-B CpG ODNs
administration caused splenomegaly, lymphoid follicle destruction, andliver toxicity in mice [14–16], and hematologic adverse events, injection site reaction, and influenza-like symptoms in human cancer clinical
trials with no additional benefit [17,18]. This Type-B associated toxicity has been shown to be associated with TLR9 signaling (dependent
on the presence of CpG motif), but some are derived from the unnatural
phosphorothioate backbones. ODNs consisting of a phosphorothioate
backbone including Type-B, C, and P CpG ODNs are known to bindvarious proteins in nonspecific ways [19,20], causing various effects
such as platelet activation [21], complement activation [22], andclotting time prolongation [23,24], raising safety concerns. In contrast,
Type-A CpG ODNs is composed of mostly the natural phosphodiester
backbone, freeing from the above-mentioned phosphorothioate-associated side effects, and would be an attractive therapeutic candidate forcancer immunotherapy.
However, the use of Type-A CpG ODNs for cancer treatment has
been very limited [25,26]. One of the reasons for the unavailability ofType-A CpG ODNs-based drugs for cancer therapy is the requirement ofa natural phosphodiester backbone for high IFN-α-inducing activity[27], which is also associated with rapid in vivo degradation due to its
susceptibility to DNase. In addition, the handling of D35 is very difficult
because of the formation of uncontrolled large aggregates in salt-containing buffers such as saline and PBS [20,28,29]. Moreover, the negatively charged phosphodiester backbone of D35 does not bind to cell
membranes due to the electrostatic repulsion between nucleic acids andthe cell membrane, which results in poor cellular uptake [30]. To
overcome these limitations of D35 for immunotherapeutic drug application, development of practical drug delivery system for D35 is demanded.
In this study, we developed a lipid-based drug delivery system for
Type-A CpG ODN D35, and found that lipid nanoparticle formulation[31] is a promising pharmaceuticalization method. Evaluation of theanti-tumor effect and potential toxicity in mice demonstrated that ourdeveloped D35-containing lipid nanoparticles (D35LNP) formulation isa safe and promising immunostimulatory drug, especially for cancer
immunotherapy. D35LNP also showed a combinational effect with an
immune checkpoint inhibitor.

from FUJIFILM Wako Pure Chemical Corporation, Ltd., Osaka, Japan.
2.2. Preparation of D35-lipid nanoparticles (LNP)
D35LNP was prepared with NanoAssemblr Benchtop (Precision
NanoSystems Inc., BC, Canada) which can mediate bottom-up self-assembly for nanoparticle synthesis with microfluidic mixing technology.
Briefly, lipids were dissolved in ethanol. Lipid components werechanged in each experiment such as DOTAP: DPPC: Cholesterol: DSPEPEG(2k) = 50: 19.5-14: 30: 0.5–6, DOTAP: DPPC: Cholesterol: DSPEPEG(5k) = 50: 19.5: 30: 0.5 or DOTAP: DOPE: Cholesterol: DSPEPEG(2k) = 50: 19.5: 30: 0.5. D35 was prepared in 25 mM acetate
buffer at pH 4.0. The lipid solution (10 mg/mL) in ethanol and D35
solution were injected into the microfluidic mixer at a 1:3 vol respectively combined final flow rate of 15 mL/min (3.75 mL/min ethanol,
11.25 mL/min aqueous). The D35LNP mixtures were immediately
dialyzed (50 kD MWCO dialysis tubing, Repligen Corporation, MA)against 5% glucose solution to remove ethanol and unload D35.
D35LNP was then concentrated to approximately 0.7 mg/mL D35 using
Amicon Ultra centrifugal filters (100 kD MWCO, Merck KGaA,
Darmstadt, Germany) and filtered through a 0.22 μm PVDF filter(Merck KGaA). The theoretical D35-to-lipid ratios for all formulations
were maintained at N/P charge ratios (ratios of the charge on the cationic lipid, assuming it is in the positively charged protonated form, tothe negative charge on the D35 oligonucleotide) of 3. All LNP preparation work was carried out at room temperature.
2.3. Analysis of lipid nanoparticles
The size distribution of D35LNP was measured with dynamic lightscattering (Zetasizer Nano-ZS, Malvern Panalytical Ltd., UK). The
morphology of D35LNP was measured through negative staining (2%phosphotungstic acid) using transmission electron microscope (TEM
(JEM-1200EX at 80 kV), JEOL Led., Tokyo, Japan). The TEM images
were taken at the Hanaichi UltraStructure Research Institute (Aichi,
Japan). D35 concentration in D35LNP suspension was measured with
picogreen reagent. Briefly, D35LNP was incubated at 37 °C for 10 min
in the presence of 1% Triton X-100 (Wako Pure Chemical Industries),and picogreen reagent was added. The fluorescence intensity (Ex/Em:
485/528 nm) was measured.
2.4. Human PBMCs

2. Materials and methods

PBMCs were prepared from Japanese healthy adult volunteers withinformed consent. All experiments using human PBMCs were approvedby the Institutional Review Board of the Research Institute forMicrobial Diseases, Osaka University (Permit number: 26-5). After
preparing PBMCs using Ficoll-Paque PLUS (GE) and LeucoSep(Greiner), it was washed twice with RPMI 1640 medium, and resuspended in R-10 medium (RPMI 1640 medium supplemented with
10% fetal calf serum, 100 unit/mL penicillin, and 100 μg/mL streptomycin). PMN cells were isolated from human whole blood using
Polymorphprep™ according to the manufacturer’s instruction. For B
cells, pDCs, and monocytes isolation, each cell population was positively selected using CD19, CD304, and CD14 magnetic MicroBeads(Miltenyi) according to the manufacturer’s instruction.

2.1. Materials

2.5. Mice

D35 [28] was purchased from GeneDesign (Osaka, Japan). 1,2dioleoyl-3-trimethylammonium-propane (DOTAP) was purchased from
Lipoid GmbH. 1,2-Dipalmitoyl-sn-glycero-3-phosphocholine (DPPC),
1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE), and N-(Carbonyl-methoxypolyethyleneglycol (2000 or 5000)-1,2-distearoyl-snglycero-3-phosphoethanolamine (DSPE-PEG(2k or 5k)) were purchasedfrom NOF CORPORATION, Tokyo, Japan. Cholesterol was purchased

C57BL/6 mice were purchased from Sankyo Labo Service
Corporation Inc. or CLEA Japan. TLR7 and TLR9 KO mice were purchased from Oriental Bio Service Inc. Mice (6–10-week-old) were usedin all experiments. All animal experiments were approved according to
the Teikyo University guidelines for the welfare of animals in studies ofexperimental neoplasia, the Animal Care and Use Committee of Osaka
University. The experiment was carried out according to the107Journal of Controlled Release 313 (2019) 106–119

L. Munakata, et al.

Regulations on Animal Experiments at Teikyo University and Osaka
University.

cells were stained with anti-mouse CD8b-PE (BioLegend, Clone:
YTS156.7.7, 1:100).

2.6. Mouse BM cells

2.11. mRNA transcription measurement

The femurs and the tibiae of mice were removed and the bones werecleaned from the surrounding muscle tissues by scissors. The marrow
was flushed out with RPMI 1640 using a syringe with a 25 G needle.
The cells were treated with ACK lysis buffer for 5 min. After washing
the cells once in RPMI 1640 and counting the cell number, bonemarrow (BM) cells were resuspended in R-10 medium. To enrich or
deplete mouse pDCs in BM cells, Plasmacytoid Dendritic Cell Isolation
Kit (Miltenyi) was used. The flow-through cells were used as pDC-enriched cells and the column-trapped cells were used as pDC-depleted
cells.

The MC38 tumors were embedded in O.C.T. Compound (Sakura
Finetek), and sections were prepared using Cryostat. Total RNA wasextracted from the frozen section using NucleoSpin RNA (Takara Bio),and cDNA strands were generated with ReverTra Ace qPCR RT Kit(TOYOBO). Real-time PCR was performed with LightCycler 480 II(Roche) using KAPA SYBR FAST qPCR Master Mix Kit (NIPPON
Genetics). Expression of target mRNA was normalized to expression ofreference mRNA (GAPDH), and fold change was calculated based on the
ΔΔCt method [35]. The primers related to tumor marker were designed
by Universal ProbeLibrary Assay Design Center (Roche, ProbeFinder
Version: 2.53), and each primer sequence is described in Supplementary Table 1. The primers for examining gene expression of TCRVα andβ repertoires were designed based on a previous study [36], and eachprimer sequence is described in Supplementary Table 2.

2.7. In vitro stimulation
Human PBMC or mouse BM cells were plated on 96-well plates at
1 × 106 cells/well/200 μL. D35 (1 μM =6.3 μg/mL), D35LNP (1 μg/
mL as D35 amount), or R848 (1 μg/mL) (InvivoGen) was added to thecell cultures overnight at 37 ℃ in CO2 incubator. The centrifuged supernatant was collected and used for cytokine ELISA. Human IFN-α wasmeasured with Human IFN-α pan ELISA development kit (Mabtech).
Mouse IFN-α/β (type I IFN) production was measured using B16-Blue
IFN-α/β reporter cells (InvivoGen). Mouse IL-6 production was measured with Mouse IL-6 DuoSet ELISA (R&D Systems).

2.12. Immune cell isolation and Immune profiling using flow cytometry
The protocol of immune cell isolation has been described in previous study [37]. Briefly, MC38 tumor was resected and weighed, thenshredded into small pieces and incubated in dissociation buffer including 100 U/mL of collagenase type IV (Invitrogen), and 100 μg/mL
of DNase I (Roche) for 45 min. After incubation, tumor cells weretreated with red blood cell lysis buffer (Gibco), and cell suspensions
were made to pass through a 100-μm cell strainer (Falcon). The cellpellet was dissolved with 2% FBS in PBS and was used for flow cytometry analysis. Isolated cells were stained using the LIVE/DEAD Fixable
Dead Cell Stain Kit (Invitrogen). After Fc blocking (clone: 2.4G2) (BD
Biosciences), cells were subsequently stained with antibodies for surface antigens listed in Supplementary Table 3. Fixation/permeabilization buffers (Invitrogen by Thermo Fisher Scientific) were used for intracellular staining. Sample acquisition was performed on a BD FACS
Canto II cytometer equipped with Diva software and analyzed using
FlowJo. Gating strategy for identifying immune cell populations was
performed as described previously [38].

2.8. MC38 cells
The MC38 cell line was cultured with high glucose DMEM (Nacalai
Tesque) containing 10% fetal bovine serum (FBS, Gibco), L-glutamine,
MEM Nonessential Amino Acids Solution (Nacalai Tesque), penicillin/
streptomycin (Nacalai Tesque) and gentamicin (50 μg/mL, Nacalai
Tesque) in 10 cm polystyrene tissue culture dishes at 37 °C in a 5% CO2
incubator.
2.9. Tumor treatment
Mice were intradermally inoculated with MC38 (1 × 106 cells), due
to this inoculation amount was used for immunotherapeutic evaluationsof several immune checkpoint inhibitors and their combinations[32,33]. D35LNP was intratumorally (4.5 μg/mouse as D35 amount) or
intravenously (25 μg/mouse as D35 amount) injected three times aweek from day7-9 to day17-20. For CD8 T cell depletion experiments,
anti-CD8α (clone 53-6.7 (rat IgG)) (100 μg/mouse) (Bio X cell, NH) andisotype control antibody (normal rat IgG, Thermo Fisher Scientific, MA)(100 μg/mouse) were injected intraperitoneally at days 6 and 13 after
tumor inoculation. For D35LNP and immune checkpoint antibody
combination experiment, anti-PD-1 antibody (clone: 29 F.1A12)(200 μg/mouse) and isotype control antibody (clone: 2A3) (200 μg/
mouse) (both were purchased from Bio X cell, NH) were injected intraperitoneally at the same days as D35LNP administration. Tumor size
was measured using calipers, and tumor volume was calculated using
the following formula: (major axis × minor axis²) × 0.5 [34].

2.13. IVIS
D35LNP was labeled with lipophilic carbocyanine DiOC18 (7) (DiR)(1,1'-Dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine
iodide,Thermo Fischer Scientific Inc., MA). Briefly, DiR solution in ethanol wasadded into the D35LNP suspension (DiR/total lipid of D35LNP: 1.6 mg/
60 mg), and DiR-labeled D35LNP (DiR-D35LNP) was prepared. In the
prepared DiR-D35LNP, we confirmed that the remaining free DiR was
only less than 1% of initial concentration, and the particle size anddistribution was not changed compared with non-labeled D35LNP by
ultrafiltration and dynamic light scattering, respectively. Mice were
subcutaneously injected with MC38 cells (1 × 106). DiR-D35LNP was
intravenously (D35: 25 μg/mouse) injected. After 6 h, the mice weresacrificed, and the tissues were collected from the mice. The tissues
were observed with fluorescence imaging system (Ex/Em: 745/830 nm)(IVIS imaging system (Lumina XR), PerkinElma Inc., MA) and the signal
intensity was calculated using Living Image Software (PerkinElma Inc.).

2.10. Immunostaining
The MC38 tumors were embedded in O.C.T. Compound (Sakura
Finetek), and sections were prepared using Cryostat (Leica CM3050 s).
The sections were fixed in 4% paraformaldehyde (PFA)-PBS (Nacalai
Tesque) for 5 min, and washed 3 times and blocked with TBS (Thermo
fisher scientific). The samples were stained with the following fluorescent dye and antibodies. The nuclei were stained with DAPI (Dojindo,
0.25 μg/ml), the blood vessels were stained with anti-mouse CD31Alexa Fluor 647 (BioLegend, Clone: MEC13.3, 1:100) and the CD8 T

2.14. Liver toxicity
D35 (25 μg/mouse) or D35LNP (25 μg/mouse as D35 amount) was
intravenously injected into the mice. Lipopolysaccharides (LPS) (25 μg/
mouse) from Escherichia coli O111:B4 (Merck KGaA) was injected
through the tail vein of C57BL/6 J mice as positive control of liver
toxicity. After 1 day, the mice were sacrificed, and the blood was collected from tail vein by using heparin-treated glass capillary tubes. The
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liver was fixed in 10% Formalin Neutral Buffer Solution, Deodorized(Mildform 10 N (FUJIFILM Wako Pure Chemical Industries, Ltd.)), andembedded in paraffin. The slices were prepared with paraffin sectioning
method, stained with hematoxylin-eosin, and assessed in Kamakura
Techno-Science Inc. (Kamakura, Japan). ALT or AST level in the blood(plasma) samples was measured with Transaminase CII-test Wako
(FUJIFILM Wako Pure Chemical Industries, Inc.). We also assessed theliver toxicity after repeated D35LNP injection. In this experiment, D35(25 μg/mouse) or D35LNP (25 μg/mouse as D35 amount) was intravenously injected into mice 5 times every 2 days. After 2 days of the
final injection, the blood and the liver were collected, and the liver
toxicity was assessed the same way as described above.

were obtained with human PBMCs. IFN-α induction was only detectedin CD304+ pDC population, but not in polymorphonuclear cells(PMNs), CD19+ B cells, and CD14+ monocytes (Fig. 1G). These results
demonstrated that D35LNP induced IFN-α secretion via endosomal
TLR9 signaling and pDC dependent manner, as lipid free non-particulate original D35.
We next examined the physical property of these D35LNPs. D35LNP
showed narrow size distribution. The average sizes of D35LNP0.5% and
D35LNP3.0% were 54.0 nm (polydispersity index (PdI): 0.157) and
43.0 nm (PdI: 0.089), respectively (Fig. 1H), which were determinedthrough dynamic light scattering measurement. Their zeta potentials
were 1.12 ± 8.26 mV and 0.34 ± 11.2 mV, respectively, indicatingthat D35LNP had slightly positive or almost neutral surface charge
under physiological condition despite containing 50% cationic lipid
DOTAP. Transmission electron microscope revealed that both
D35LNP0.5% and D35LNP3.0% were spherical in shape with fully filled
packed core, indicating that our developed D35LNPs formed lipid nanoparticles (Fig. 1I).

2.15. Statistical analysis
The data were presented as mean ± SD, and One-way analysis ofvariance (ANOVA) with Tukey or Bonferroni test were used to analyze
the tumor growth and liver enzyme data when more than two groups
were compared. Two group comparison was analyzed by Student’s t-test
Gene expression data were presented as mean ± SEM and analyzed
with Mann-Whitney U test. All Statistics were calculated with BellCurvefor Excel (Ver. 3.00; Social Survey Research Information Co., Ltd.) or
Prism software (GraphPad Ver. 7.01).

3.2. D35LNP0.5% showed anti-tumor effect via intratumoral injection
We next examined D35LNPs in vivo using tumor model in mice.
MC38 tumor cells (murine colon adenocarcinoma) were inoculated
subcutaneously, and the mice were treated with D35LNPs through intratumoral (i.t.) injection at days 9, 11, 13, 15, and 17 after tumor
inoculation (Fig. 2A; upper). The tumor growth was significantly suppressed with D35LNP0.5% treatment at the time point of days 17 and21 compared with non-treated group, while D35LNP3.0% did not show
apparent therapeutic effect at all examined time points (Fig. 2B), suggesting “PEG dilemma” [39] that also affects in vivo bioactivity of
D35LNP. This difference in therapeutic effect was consistent with thedifference in IFN-α inducing ability in vitro between D35LNP0.5% and
D35LNP3.0% (Fig. 1B). Lipid free D35 showed no tumor suppressive
effect at all (Fig. 2B).
Next, we tested whether the anti-tumor effect of D35LNP0.5% wasdependent on CD8 T cells. CD8 T cell depletion with anti-CD8a antibody treatment exacerbated tumor progression without any othertreatment (Fig. 2C; CD8a-Ab), while isotype control antibody administration showed minimal effect on tumor growth compared to that in
the non-treated group (Fig. 2C and D; Isotype-Ab). With D35LNP0.5%
i.t. treatment, tumor growth was significantly suppressed compared tothat in the non-treated group at day 21, and CD8 T cell depletion with
anti-CD8a antibody completely canceled D35LNP0.5%-mediated antitumor effect (Fig. 2C and D; D35LNP0.5%+CD8a-Ab).
Histological examination of tumor at day 21 supported that antiCD8a antibody administration depleted CD8 T cells completely in thetumor tissue (Fig. 2E). Although MC38 tumor contains many CD8 T cell
infiltrations without any treatment, CD8 T cell infiltration in the tumor
was more aggressive with D35LNP0.5% i.t. treatment (Fig. 2E;
D35LNP0.5%). Collectively these results suggested that D35LNP0.5%
i.t. injection resulted in CD8 T cell activation at the tumor site, which
effectively suppressed tumor growth.

3. Results
3.1. Development of the drug delivery system for Type-A CpG D35
To develop an efficient drug delivery system for D35, we focused ona lipid-based drug delivery system. We prepared substantial numbers ofneutral, cationic, and anionic lipid based complexes with D35, andcarefully tested these lipid/D35 complexes. Through in vitro evaluations of size, stability, and cytokine production from human peripheral
blood mononuclear cells (hPBMCs), we practically chose DOTAP, acationic lipid as the main lipid component, and cholesterol and DPPC as
the other components (see Methods) to make the particulate D35/lipid
complex. As described later in this study, DOTAP based D35/lipid
complex formed the lipid nanoparticles (referred to as D35LNP)(Fig. 1H and I). We also tested various amounts (mol%) and lengths of
PEG-lipid incorporations. Successfully prepared samples were further
evaluated for their IFN-α inducing ability from both hPBMCs (Fig. 1A)
and mouse bone marrow (BM) cells (Fig. 1B). Among them, 0.5% of 2KPEG-lipid incorporated D35LNP showed the most effective and consistent IFN-α production from both human and mouse BM cells (Fig. 1A
and B). An increased amount of 2K-PEG mol% resulted in decreased
IFN-α production, suggesting the “PEG dilemma” [39] phenomenon.
When 5K-PEG-lipid was examined, 0.5% of 5K-PEG-lipid incorporated
D35LNP was consistently less effective than 0.5% of 2K-PEG-lipid incorporated D35LNP (Fig. 1A and B). Based on these results, we chose
0.5% of 2K-PEG-lipid incorporated D35LNP (D35LNP0.5%), and occasionally 3.0% of 2K-PEG-lipid incorporated D35LNP (D35LNP3.0%) for
further evaluation.
Next, we examined the molecular and cellular mechanisms of the
D35LNP-mediated IFN-α induction. IFN-α induction with D35LNP wascompletely dependent on the presence of CpG-motif in the D35 sequence (Fig. 1C). When CpG-motif was replaced with GpC-motif (notstimulatory for TLR9), IFN-α induction ability was diminished in bothhuman PBMCs (Fig. 1C; left panel) and mouse BM cells (Fig. 1C; rightpanel). In addition, incorporation of pH-sensitive-endosome-disrupting
lipid DOPE [40] reduced IFN-α induction to about 50% (Fig. 1D). In
accordance with these results, IFN-α induction with D35LNP wascompletely dependent on TLR9 but not TLR7, using TLR9 and TLR7 (ascontrol)-deficient BM cells from mice (Fig. 1E). We also enriched ordepleted pDC from wild type mouse BM cells and stimulated them with
D35LNP0.5%. IFN-α induction remained higher in pDC enriched BM
cells, but diminished in pDC depleted BM cells (Fig. 1F). Similar results

3.3. D35LNP0.5% changes gene expression in the tumor microenvironment
To examine immune related gene expression change at the tumor
site after D35LNP0.5% i.t. injection, we purified the mRNA of tumors
from groups shown in the Fig. 2C at day 21. The gene expression induction of interferon, chemokine, tumor microenvironment, and T celldifferentiation type such as Th1-, Th2-, Th17-, and Treg- related genes
were determined by real-time PCR. Among these, Ifna6, Ifnb1, Ifng,
Cxcl9, Cxcl10, Cxcr3, Cd274 (PD-L1), Stat4, Stat6, Il6, Il1b, and Tgfb1
significantly increased in the D35LNP0.5%-treated group (n = 5)
(Fig. 3A) compared to non-treated group (n = 5). Notably, overall gene
expression levels of Ifng- and Th1-related genes were substantiallyhigher than those of Th2-, Th17-, and Treg-related genes (Fig. 3A). A
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Fig. 1. Development of lipid nanoparticle for Type-A CpG D35.
(A–B) IFN-α production from human PBMCs (A) or mouse bone marrow (BM) cells (B) with various amount of 2K- or 5K-PEG-lipid incorporated D35LNP stimulationin vitro. (C) CpG-motif dependency of D35LNP mediated IFN-α production in both human PBMC (left panel) and mouse BM cells (right panel). (D) Better IFN-α
induction of human PBMCs without pH sensitive endosome escapable lipid DOPE, suggesting the importance of D35 delivery to the endosome. Each bar indicates
IFN-α production with D35LNP stimulation (1, 0.5, and 0.25 μg/mL as D35 amount). (E) TLR9 but not TLR7 dependent IFN-α induction from mouse BM cells. R848
(1 μg/mL), D35 (1 μM =6.3 μg/mL), and D35LNP (1 μg/mL as D35 amount). (F) pDC is a source of IFN-α in mouse BM cells stimulated with D35LNP. D35 (1 μM),
and D35LNP (1 μg/mL). (G) pDC is a source of IFN-α in human PBMCs stimulated with D35LNP(1 μg/mL). (H) The size distribution measurement by dynamic lightscattering of D35LNP 2K-PEG 0.5% (D35LNP0.5%) and D35LNP 2K-PEG 3.0% (D35LNP3.0%). (I) TEM image of D35LNP0.5% and D35LNP3.0%. Black bars indicate
400 nm. Bar graph indicates a representative data of at least two independent experiments with similar results.
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Fig. 2. Anti-tumor effect with D35LNP intratumoral treatment.
(A) Treatment procedure. C57BL/6 mice were intradermally inoculated with MC38 tumor (1 × 106 cells). D35LNP was injected intratumorally at days 9, 11, 13, 15
and 17 (A; upper panel). For CD8 T cell depletion experiment, mice were injected anti-CD8a antibody or isotype control antibody intraperitoneally (A; lower panel) in
addition to D35LNP intratumoral injection at days 9, 11, 13, 15 and 17, and at days 6 and 13. (B–C) Tumor growth curves. Tumor size was measured at days 9, 11, 13,
15, 17, and 21. Values are expressed as the mean ± S.D., and asterisk (*) means P < 0.05. (D) Individual tumor growth curves of Fig. 2C. (E) Immunofluorescenthistology staining of MC38 tumor with nuclei (DAPI, blue), CD31 (green), and CD8b (red). Scale bars indicate 200 μm. Note that CD8a-antibody treatment completely depleted CD8 T cells in tumor tissue. All statistical significances were calculated by one-way analysis of variance (ANOVA) with Bonferroni test.

strong increase in the expression of Ifng, Cxcl9, and Cxcl10 suggests that
D35LNP0.5% i.t. treatment provoked “T cell-inflamed” status [41,42].
Ccl22 and Cd274 (PD-L1) induction also suggested that the activatedCD8 T cell induced regulatory mechanisms, which is affecting tumor
microenvironment [43,44]. Interestingly, most of the elevated gene
expressions induced by D35LNP0.5% i.t. treatment were canceled with
anti-CD8a antibody treatment, whereas Ifna6 gene expression remained
high after anti-CD8a antibody treatment (Fig. 3A; Ifna6, D35LNP+ CD8Ab vs. D35LNP). These results indicated that D35LNP0.5% i.t.
injection caused initial IFN-α expression in the tumor, and then induced
Th1-skewed immune-environment and CD8 T cell activation, which
finally resulted in tumor growth suppression.
We also examined TCR repertoire gene expression in the tumors ofboth non-treated (n = 5) and D35LNP0.5%-treated groups (n = 5). The
expression of TCR Vα6-5/6-7 and Vα12 genes significantly increased intumors treated with D35LNP0.5% i.t. compared to that in the nontreated group, while TCR repertoire gene expression in the spleen did
not change in both groups (Fig. 3B). These data supported that
D35LNP0.5% i.t. injection induced tumor-specific T cell responses intumors.
3.4. D35LNP0.5% intravenous injection showed anti-tumor effectWe also tested D35LNP intravenous (i.v.) treatment against subcutaneous the MC38 tumor. Tumor growth was significantly suppressedwith D35LNP0.5% i.v. treatment, while neither D35 nor D35LNP3.0%
treatment was effective (Fig. 4A). Histological examinations showed noapparent differences in CD8 T cells infiltration among these groups(Fig. 4B). Although the level of gene expression was not as strong asthat with i.t. treatment (Fig. 3), the expression of Ifng, Cxcl9, and Stat4
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Fig. 3. Gene expression change at the tumor site with D35LNP0.5% intratumoral injection.
Tumor tissues (n = 4 or 5) of each group in Fig. 2C were evaluated for mRNA expression for several panels of genes by qRT-PCR method. (A) The panels includes
interferons (Ifna6, Ifnb1, Ifng), chemokines (Cxcl9, Cxcl10, Cxcl11, Cxcr3), tumor environment (Ccl22, Ctnnb1, Cd274(PD-L1))(C), Th1(Stat4, Tbx21 (Tbet))(D), Th2(Stat6, Gata3), Th17 (Il6, Il1b), and Treg (Stat5a, Tgfb1). Values are expressed as the mean ± SEM of gene expression relative to GAPDH. *P < 0.05, **P < 0.01.
(B) TCR repertoire gene expression changes in the tumor and the spleen at day 21. Black bar indicates non-treated samples, and red bar indicates D35LNP treatedsamples. *P < 0.05, ***P < 0.001. Statistical significance was calculated by Mann-Whitney U test.

(all of them were Th1-related genes) significantly increased in the
D35LNP0.5% i.v. treated group (n = 4) compared to non-treated group(n = 5) (Fig. 4C), which is consistent with the tumor-suppressive effect(Fig. 4A). These results suggested that D35LNP0.5% i.v. treatment also
induced weak but substantial Th1 skewed immune environment intumor site, as seen in i.t. treatment (Fig. 3). We also performed
D35LNP0.5% i.v. treatment with or without anti CD8a depletion antibody injection. The tumor suppressive effect by D35LNP0.5% i.v.
treatment was completely canceled by CD8 T cells depletion (Fig. 4D),
suggesting that D35LNP0.5% i.v. also effectively changed tumor immune-environment and activated CD8 T cells to suppress tumor growth.

3.5. Combination treatment with D35LNP0.5% and a PD-1-blocking
antibody
To investigate whether D35LNP0.5% exerts a combinational effectwith immune checkpoint inhibitors against MC38 tumors, MC38 subcutaneous tumor bearing mice were treated with D35LNP0.5% i.t. and/or PD-1 blocking antibody intraperitoneally (i.p.) 3 times a week between 8–20 days after tumor inoculation (Fig. 5A). The groups treatedwith the PD-1 antibody (anti-PD-1 Ab) and the combination (Combination; D35LNP0.5% and anti-PD-1 antibody) showed significant
tumor-suppressive effect in both tumor volume (Fig. 5B; left panel) andtumor weight (Fig. 5B; right panel) compared to the isotype antibody
control group (Isotype).
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Fig. 4. MC38 tumor growth was also suppressed with D35LNP0.5% intravenous treatment.
(A) Tumor growth curve. C57BL/6 mice were injected intredermally with MC38 tumor cells (1 × 106). D35LNP was injected intravenously at days 9, 11, 13, 15 and17. *P < 0.05. Statistical significance was calculated by one-way ANOVA with Bonferroni test. (B) Immuno-fluorescence staining of MC38 tumor for nuclei (DAPI,
blue), CD31 (green), and CD8b (red). (C) mRNA expression of the same gene panels as Fig. 3A measured by qRT-PCR method. **P < 0.01. Statistical significancewas calculated by Mann-Whitney U test. (D) Tumor growth curve with or without CD8a antibody treatment. *P < 0.05. Statistical significance was calculated by
one-way ANOVA with Bonferroni test.

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Fig. 5. Intratumoral D35LNP0.5% andanti PD-1 antibody combination treatment.
(A) Treatment procedure. C57BL/6
mice were injected subcutaneouslywith MC38 tumor cells (1 × 106). BothD35LNP0.5% and anti-PD-1 antibody
were simultaneously injected at days 8,
11, 13, 15, 18, and 20. (B) Tumor volume curves (left) and tumor weight at
22 days after tumor inoculation (right)(Isotype Ab, n = 7; D35LNP0.5%,
n = 8;
Anti
PD-1
Ab,
n = 9;
Combination, n = 9). Statistical significances were calculated by one-way
ANOVA with Tukey multiple comparison test. (C) Immune cell population
analysis of tumors by FACS from
Isotype Ab (n = 6) vs. D35LNP0.5%
treated mice (n = 5). * CD8/4:
p = 0.0102, CD8/Foxp3: p = 0.0133.
Statistical significances were calculatedby Student’s t-test

We also investigated immune cell populations in tumors by flow
cytometry. The ratio of CD8/CD4 to CD8/Foxp3 in tumor-infiltrating T
cells significantly increased in the D35LNP0.5% treated group compared to that in the Isotype group (Fig. 5C). Note that we could not
perform immune cell population analysis for the other two groups (antiPD-1 Ab and Combination), because the tumor samples were too small
to recover sufficient numbers of the immune cells.
To further investigate whether this combinational therapeutic effectis affected by the administration route of D35LNP0.5%, we also treated
MC38 tumor bearing mice with D35LNP0.5% i.v. and/or PD-1 blocking
antibody i.p. using similar protocol as shown in Fig. 5A (3 times a week
between 7–19 days after tumor inoculation). The Combination showedsignificant inhibitory effect on tumor growth compared to that in the
Isotype group (Fig. 6A). Although the difference in anti-tumor efficacy
between the anti-PD-1 Ab group and the Combination group was notstatistically significant (Fig. 6A), the Combination group showed moreconsistent tumor-suppressive effect in individual mice (Fig. 6B). We
also analyzed tumor infiltrating immune cell populations in each groupby flow cytometry. The absolute count of CD8 T cells, and both CD8/

CD4 and CD8/Foxp3 ratio significantly increased in the Combinationgroup compared to Isotype and D35LNP groups (Fig. 6C). Regarding thefunction of CD8 T cells, the Ki-67 positive population significantly increased in the anti-PD-1 Ab group compared to that in the Isotype group(p = 0.0126), and the IFNγ-positive population significantly increasedin the Combination group compared to that in the Isotype group(p = 0.0420). (Fig. 6C). While tumor-associated neutrophils (TANs)
significantly reduced in the Anti-PD-1 Ab and Combination groups(both contain Anti-PD-1 Ab treatment), inflammatory monocytes (iMonocytes) significantly reduced in the D35LNP0.5% and Combinationgroups (both contain D35LNP0.5% treatment). These results suggestthat the combination of D35LNP0.5% and PD-1 blockade improved
anti-tumor effect irrespective of the D35LNP0.5% administration route,and either D35LNP0.5% or PD-1 blockade might have preferentiallyinhibited different inhibitory myeloid cell populations such as iMonocytes or TANs, respectively (Fig. 6C).

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Fig. 6. Intravenous D35LNP0.5% and anti PD-1 antibody combination treatment.
(A) Tumor volume curves (left) and tumor weight 20 days after tumor cell inoculation (right) (Isotype Ab, n = 6; D35LNP0.5%, n = 7; Anti PD-1 Ab, n = 7;
Combination, n = 9). (Left: * Isotype vs. anti PD-1 p < 0.05; ** Isotype vs. Comb p < 0.01; Right: * Isotype vs. PD-1 only p = 0.0111, ** Isotype vs. Combp = 0.0014). (B) Tumor volume curves from each mouse in 4 different groups. (C) Immune cell populations were evaluated in each treatment group (*p < 0.05**p < 0.01). Isotype vs. Comb (CD8 T: p = 0.0192, CD8 T/CD4 T ratio: p = 0.0431, CD8/Foxp3 ratio: p = 0.0129). D35LNP vs Comb (CD8 T: p = 0.0181, CD8 T/
CD4 T ratio: p = 0.0207, CD8/Foxp3 ratio: p = 0.0033). All statistical significances were calculated by one-way ANOVA with Tukey multiple comparison test.

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Fig. 7. Tissue distribution and liver toxicity after intravenous D35LNP administration.
(A) DiR fluorescence dye labeled D35LNP0.5% and D35LNP3.0% were administered intravenously. In 6 h later, biodistribution was evaluated by IVIS for theindicated organs. (B) Quantified DiR signals in the indicated organs. (C) ALT and AST in the plasma were evaluated after single D35LNP intravenous administration.
**p < 0.01. Statistical significance was calculated by one-way ANOVA with Bonferroni test. (D) The liver histology after single D35LNP intravenous administration.
Scale bars; 50 μm. (E) ALT and AST in the plasma after 5 times repeated D35LNP intravenous administrations. (F) The liver histology after 5 times repeated D35LNP
intravenous administrations. Scale bars; 50 μm.

3.6. Biodistribution and liver toxicity evaluation after D35LNP intravenous
administration

Because the liver was the organ with highest D35LNP distribution,
we next examined liver toxicity after single or repeated (5 times every 2days similar to the tumor treatment schedule) i.v. injection of
D35LNP0.5% and D35LNP3.0%. After single injection, no elevation in
AST and ALT was observed in the plasma compared to LPS, as the liver
damage-inducing positive control (Fig. 7C). Consistent with this result,histological examination also showed no apparent liver damage with
D35LNP0.5% while the LPS group showed decreased glycogen deposition as shown by the white space reduction in hepatocytes and theappearance of increased small round vacuoles in the cytosol (Fig. 7D).
Moreover, no liver damage was observed after repeated D35LNP i.v.
injection 5 times within 2 weeks (Fig. 7E and F). These results indicatedthat the therapeutic schedule and dose of D35LNP i.v. injection did not

To assess the biodistribution, D35LNP, D35LNP0.5% and
D35LNP3.0% were labeled with DiR, a near infrared fluorescence dye.
At 6 h after intravenous injection, most DiR-labeled D35LNP0.5% and
D35LNP3.0% were observed in the reticuloendothelial system and organs such as the liver, the lung, and the spleen (Fig. 7A and B). We also
observed DiR signal in tumor tissues, but it was weaker and lesser inintensity than that of the kidney (Fig. 7B). These results suggested thatthe overall organ distribution of D35LNP0.5% and D35LNP3.0% was
almost the same, and ∼1.0% of D35LNP reached the tumor after i.v.
injection.
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induce apparent liver damage in mice.

would be the main reason for the difference of the anti-tumor activity
between D35LNP0.5% and D35LNP3.0%.
Although our results clearly showed that D35LNP injection activated anti-tumor immunity in the host, the detailed cellular mechanism
in vivo is still not fully elucidated. The key role of pDC in IFN-α induction by D35LNP stimulation in vitro (Fig. 1F and G) strongly suggested that pDC activation by D35LNP should be important in vivo. It
has been reported that CpG-activated pDC administration in tumor induced antitumor effect through pDC-NK-cDC-CD8T cell activation
where pDC produced CCL3/4/5 and NK cell expressed CCR5 [50].
Another report showed that activated NK cells produced CCL5 and
XCL1, and both chemokines were involved in cDC1 recruitment [51]. In
another report, activated CD8 T cells have been shown to secrete XCL1
and CCL3/4 to recruit XCR1+ cDC1 and CCR5+ pDCs, respectively, andto make clusters of CD8 T/pDC/cDC1 for efficient CD8 T cell priming[52]. Tumor residing cDC1 cells also produced CXCL9/CXCL10 to recruit CXCR3+ cells [53]. Summarily, these reports suggested that
D35LNP-mediated pDC activation provoked these redundant chemokine networks to form NK/pDC/cDC1 clusters for initiating efficientanti-tumor cell responses.
Our created D35LNP showed anti-tumor effect both in i.t. and in i.v.
administration routes. This is different from other reported CpG based
immune stimulators for cancer immunotherapy, in which only i.t. or s.c.
was evaluated [25,54–57]. Although a very small amount of i.v.-administered D35LNP reached the tumor site (Fig. 7A and B), tumor
growth suppression with D35LNP i.v. treatment was dependent on CD8
T cells, which was the same with i.t. administration. Based on thesefacts, we speculated that i.t. and i.v. treatment of D35LNP shared partially similar mechanism of actions. However, expression of Treg-related genes such as Stat5 and Tgfb1 increased after i.t. administration(Fig. 3G), but decreased after i.v. administration (Fig. 4C), suggestingthat i.v. administration of D35LNP may invoke additional or different
systemic actions, and this possibility requires examination in future
experiments.
After i.v. treatment, majority of D35LNP accumulated in the reticuloendothelial system (liver, lung, and spleen), which has been
known to capture other particulate drugs such as liposomes. Even withhigh accumulation in the liver, we could not detect any abnormal ALT
and AST elevation after D35LNP i.v. administration (Fig. 7C and E).
Although the exact reason for this low liver toxicity by D35LNP requires
examination in future experiments, natural phosphodiester backbone of
D35 may contribute to high safety profile of D35LNP.
Through these developmental processes in this study, we consider
that D35LNP0.5% is one of the optimized formulations of D35 forcancer therapy without liver toxicity. Notably, original lipid free D35
did not show any therapeutic effect on cancer in vivo (Fig. 2A and A).
Currently, no other CpG-based immunostimulator that can be administered systemically has been reported. Our developed D35LNP
formulation suppressed tumor growth after administration through not
only i.t. but also with i.v. routes. Combination with anti-PD-1 antibody
induced more consistent tumor suppression. Summarily, D35LNP is apromising immunostimulator for future cancer therapy, which can be
combined with a variety of other approaches such as chemotherapy andradiation to improve its therapeutic efficacy [58,59].

4. Discussion
Among the 4 different types of CpG ODNs (including A, B, C and P),
Type-A CpG is one of the strongest IFN-α inducer, which has been
considered a good cytokine for cancer therapy [45,46], and one of theweakest inflammatory IL-6 inducer which potentially promotes tumor
progression [47,48]. However, the use of Type-A CpG ODNs for cancer
treatment has been limited, because it is prone to degradation anduncontrolled aggregation formation in vivo. To overcome this limitation, we developed D35LNP. D35LNP induced high amount of IFN-α
secretion from human PBMC and mouse bone marrow cells (Fig. 1).
This IFN-α production was dependent on the presence of CpG motif in
D35 ODN sequence, and TLR9 mediated signaling in pDCs (Fig. 1C, E, F,
G). Optimal in vitro IFN-α production was achieved by 0.5% of 2k-PEGlipid incorporation, but 3% of 2k-PEG-lipid incorporation resulted invery weak IFN-α production, suggesting that surface PEG modification
greatly influenced D35LNP’s activity (Fig. 1A and B).
According to these in vitro results, D35LNP0.5% but not
D35LNP3.0% showed anti-tumor effect in vivo. In the MC38 subcutaneous tumor model, tumor growth suppression was dependent on
CD8 T cells, irrespective of the administration route of D35LNP0.5%
such as i.t. or i.v., suggesting that CD8+ T cells work as a final antitumor effector cells. T cell repertoire examination also supported that
D35LNP treatment induced tumor-specific T cells, which has a significantly skewed TCR repertoire gene expression (such as Vα6-5/6-7
and Vα12) in the tumor, but not in the spleen (Fig. 3B). The otherimmune-related tumor mRNA also changed with D35LNP0.5% treatments. As expected, IFN-α response was induced by D35LNP0.5%
treatments. mRNA expression levels of interferons (Ifna6, Ifnb1, Ifng),
CD8 T cell recruitment chemokines and chemokine receptors (Cxcl9,
Cxcl10, Cxcl11, and Cxcr3), and Th1 related genes (Stat4 and Tbx21)
were significantly increased, and their strong induction was associatedwith tumor suppression after i.t. treatment (Fig. 3A). Similar results
were also seen after i.v. treatment (Fig. 4C). Although the expression ofthese marker genes was higher with i.t. treatment, significant inductionof Ifng and Cxcl9 expression after i.v. treatment was associated withtumor-suppressive D35LNP0.5% i.v. treatment but not with tumor nonsuppressive D35LNP3.0% i.v. treatment (Fig. 4C). These results suggested that D35LNP treatment changed the tumor microenvironment
toward a Th1 and CD8 T cell-activated environment, irrespective of i.t.
or i.v. treatment. This Th1/CD8 T cell skewed tumor microenvironment
induced by D35LNP administration also enhanced anti-PD-1 antibody’s
therapeutic effect. D35LNP0.5% and anti-PD-1 antibody combination
treatment increased the number of CD8 T cell and IFN-γ+ CD8 T cells,
and the ratios of CD8/CD4 and CD8/Foxp3, and elicited more consistent tumor growth suppression (Fig. 6). This suggests that
D35LNP0.5% accelerated anti-tumor immunity by inducing Th1/CD8
skewed tumor microenvironment, while anti-PD-1 antibody released
the brake on these T cells.
The exact mechanisms of the anti-tumor activity difference between
D35LNP0.5% and D35LNP3.0% need further examination. It has been
known that PEGylated lipid particles showed two different types ofphenomena; “PEG dilemma” [39] and “accelerated blood clearance(ABC) phenomenon” [49]. Theoretically, it is possible that both phenomena influence the difference between D35LNP0.5% and
D35LNP3.0%. “PEG dilemma” suggested that PEGylation amount dependent inhibition of cellular uptake, and ABC phenomenon suggested
anti-PEG antibody induction dependent rapid clearance of PEGylated
lipid particles. Both would result in the biological activity reduction of
PEGylated lipid particles in vivo via intravenous administration route.
However, when D35LNP was directly injected into tumor, and
D35LNP0.5% was more effective than D35LNP3.0% (Fig. 2B). In thiscase, we may exclude the possibility of ABC phenomenon involvementin the anti-tumor effect reduction of D35LNP3.0%, and PEG dilemma

Author contributions
S.K., R.S., and T.A. designed research, L.M., Y.T., A.O., J.M., andY.H. conducted the research, Y.N., H.M., A.K., D.O., K.M., Y.Y., andY.O. contributed critical reagents/tools used for the study, L.M., Y.T.,
A.O., S.K., R.S., and T.A. analyzed data,; and L.M., Y.T., S.K., R.S., andT.A. wrote the paper.
Declaration of Competing Interest
These authors disclose the following: Yasuo Yoshioka and Taiki
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Aoshi are employed by the Research Foundation for Microbial Diseasesof Osaka University. Shohei Koyama, Ryo Suzuki, and Taiki Aoshi have
filed a patent application related to the content of this manuscript. The
remaining authors declare no conflicts.

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Significance statement
Lipid nanoparticle formulation of Type-A CpG oligodeoxynucleotide
D35 enabled the conversion of otherwise in vivo ineffective D35 totumor suppressive immunomodulatory D35 in vivo. Our formulation
could be administered intratumorally or intravenously to induce Th1
and CD8 T cells -skewed immune environment in the host without
apparent liver toxicity. Our D35-containing lipid nanoparticles(D35LNP) formulation can be used an immunomodulator for cancer
immunotherapy and potentially in the treatment of other diseases that
require Th1 and CD8 T cells -skewed immune activation.
Acknowledgments
We thank Yutaka Kono, Toshihiro Nukiwa, and Koichi Yamanishi
for useful advices, Miki Tsutsui and Megumi Yamashita for technicalassistance, and Saiko Ito for secretary assistance. We acknowledge thefinancial support from the Japan Agency for Medical Research andDevelopment (AMED) with grant number (16cm0106310h0001,
17cm0106310h0002, 18cm0106310h0003).
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.jconrel.2019.09.011.
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