Intratumoral delivery of IL-12 and IL-27 mRNA using lipid nanoparticles for cancer immunotherapy

Reagents

All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise noted. DOPE was purchased from Avanti Polar Lipids, Inc (Alabaster, AL, USA) and DMG-PEG₂₀₀₀ was purchased from NOF America Corporation (White Plains, NY, USA).

Synthesis of DALs

Compounds b, c and DAL1 were synthesized according to methods reported previously³⁷.

Synthesis of DAL2: DAL1 (250 mg, 0.35 mmol) was first hydrolyzed by NaOH aqueous (1 M) in a mixture of THF and MeOH at 70 °C for 3 hrs. Next, 100 mL CH₂Cl₂ was added, which was dried with MgSO₄. Then, the solvent was evaporated and 10 mL anhydrous THF was added to the residue. NHS (125 mg, 1.05 mmole) and DCC (230 mg, 1.05 mmole) were added to the solution. The resulting mixture was stirred at room temperature overnight. Tert-butylamine (80 mg, 1.05 mmole) and TEA (150 μL, 1.2 mmole) were added to the above reaction mixture. The resulting mixture was stirred at room temperature overnight. After the solvent was removed under reduced temperature, the residue was purified by column chromatography using a CombiFlash Rf system with a RediSep Gold Resolution silica column (Teledyne Isco) with gradient elution (CH₂Cl₂ and ultra) from 100% CH₂Cl₂ to 80% CH₂Cl₂ (ultra, CH₂Cl₂/MeOH/NH₄OH =75/22/3 by volume) to generate 96 mg of oil like DAL2. Using different head groups, DAL3-7 were synthesized following the same procedure as used for the synthesis of DAL2. The amine used for synthesizing DAL4 was 5-Fluoro-2-aminomethylphenylboronic acid, pinacol ester. The pinacol ester was hydrolyzed during purification through column chromatography to yield pure DAL4. The ¹H NMR spectra of DAL2–7 are provided in Supplementary Fig. 1.

mRNA synthesis

IL-12, IL-27, and GM-CSF plasmids were purchased from InvivoGen (San Diego, CA, USA) and were amplified to generate templates for in vitro transcription. DNA sequences of the cytokines used in this study are listed in supplementary data. mRNA transcripts were synthesized as reported previously^{37, 38}. mRNAs were synthesized with full substitution of UTP by pseudouridine-5’-triphosphate (TriLink, USA) using AmpliScribe T7-Flash Transcription Kit (Lucigen, USA) following the manufacturer’s instruction. The resulting mRNAs were then purified by RNA Clean & Concentrator (Zymo, USA) and capped using Vaccinia Capping System (NEB, USA) and Cap 2´-O-Methyltransferase (NEB, USA). After one final round of purification, mRNA concentrations were measured using a NanoDrop 2000 Spectrophotometer (ThermoFisher, USA) and stored at −80°C for future use.

Preparation and characterization of LNPs-mRNA

LNPs-mRNA were prepared by mixing lipid materials dissolved in ethanol and mRNA diluted in 10 mM citrate buffer, pH=3³⁹. The molar ratio of MC3 LNPs was MC3: DSPC: Cholesterol: DMG-PEG=50:10:38.5:1.5. For DAL-LNPs, the molar ratio was DALs: DOPE: Cholesterol: DMG-PEG=20:30:40:0.75. LNPs-mRNA were prepared by rapidly blending the aqueous and ethanol phases with a pipette for in vitro studies. LNPs-mRNA were prepared by a microfluidic device for in vivo studies (Precision NanoSystems, Vancouver, BC, Canada) and dialyzed in PBS (Slide-A-Lyzer^™ Dialysis Cassettes, 3.5K MWCO, ThermoFisher, USA). The parameters of microfluidic device: flow rate: total 12mL/min, aqueous phase/ethanol phase = 3/1, room temperature. The size and zeta potential of DAL-LNPs were measured by Zetasizer (Malvern, USA). The mRNA encapsulation efficiency (EE%) was determined by the RiboGreen assay (Invitrogen, USA). Cryo-EM image of DAL4-LNP-IL-12+IL-27 was obtained by Glacios Cryo Transmission Electron Microscope (ThermoFisher, USA) using a similar method reported before³⁷.

In vitro delivery of cytokine mRNAs LNP to B16F10 melanoma cells

B16F10 cells were originally purchased from ATCC and were maintained in RPMI 1640 Medium (Corning) with fetal bovine serum 10% (v/v). To quantify the delivery efficacy and production of mRNA encoded cytokines in MC3-LNP or DAL4-LNP, we treated the cultured B16F10 cells in a 96 well plate with MC3-IL-12/IL-27/GM-CSF, or DAL4-LNP-IL-12/IL-27/GM-CSF with a dose of 50 ng mRNA of each cytokine mRNA. After 18 hrs incubation, the supernatants of cell cultures were collected, and respective cytokines were quantified by ELISA following standard procedures.

In vivo anti-cancer efficacy of cytokine mRNA LNPs

All animal experiments were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of The Ohio State University and were approved by the Animal Ethics Committee of Institutional Animal Care and Use Committee (IACUC). To establish the mice tumor model, 1 × 10⁵ B16F10 cells in 100 μL PBS were subcutaneously injected in female C57BL/6 mice. Mice were randomly grouped (n = 5–7 in each group) when the tumor reached about 50 mm³. Tumor volume is calculated by length × (width)²/2. For the biodistribution study, intratumoral injection of GFP mRNA in DAL4-LNP (10 μg mRNA/injection) was performed one-time. GFP expression in the tumor microenvironment was analyzed at 24 hrs. For treatment efficacy studies, intratumoral injections of cytokine mRNA LNPs (2 μg mRNA/injection in Fig. 4; 2 μg IL-12 mRNA/injection or 6 μg IL-27 mRNA/injection in Supplementary Fig. 2) were performed every other day for six doses in total. For toxicity studies, intratumoral treatment with cytokine mRNA LNPs (2 μg mRNA/injection) was performed every other day for three times in total. The tumor size was checked every other day. The body weight was checked on days 8, 12, and 14 after tumor inoculation. Paraffin-embedded tissues (heart, lung, liver and kidney) were prepared from DAL4-LNP-IL-12 + IL-27 mRNA treated mice (n=5) or control-treated mice (n=5). Hematoxylin and eosin (H.E.) stained slides were evaluated under a microscope. Representative images were photographed and are provided in Supplementary Fig 4.

Antibodies and flow cytometry

Fluorescence labeled monoclonal antibodies to mouse CD45 (30-F11), CD11b (M1/70), F4/80 (745–2342), NK1.1 (Pk136), CD3 (145–2c11), CD4 (GK1.5), CD8α (53–6.7), CD19 (1D3), IFN-γ (XMG1.2), TNF-α (XT22), and isotype control antibodies were purchased from BD Biosciences or Biolegend. Mononuclear cells from tumors were prepared as described^{40, 41}. For staining cell surface markers, cells were incubated with antibodies in staining buffer (0.1 M PBS, 1% FCS and 0.1% sodium azide) on ice for 30 minutes. Cells were washed three times after staining and fixed in 1% paraformaldehyde. For intracellular staining of IFN-γ and TNF-α, cells were first stimulated with cell stimulation cocktail (Invitrogen, USA) for 4 hrs. The cells were then stained for the cell surface markers (CD3/4/8/NK1.1) followed by a standard intracellular cytokine staining procedure. Stained cells were harvested using a Celesta flow cytometer (BD) and data was analyzed using FlowJo software (Tree Star, Inc., OR).

Statistical analysis

Mann-Whitney test and one-way ANOVA with Dunnett’s multiple comparisons were used to analyze in vitro data (GraphPad Prism, CA, USA); Two-way ANOVA with repeated measurements was used to analyze tumor volume and body weight data (R3.4.3, The R Foundation); the survival of tumor-bearing mice was analyzed using log-rank tests (GraphPad Prism). All tests were two-tailed and P < 0.05 was considered statistically significant.

Synthesis of diamino lipid derivatives

We first designed and synthesized a library of seven di-amino lipid materials (DALs) with the same diamino core and carbon chains with different head groups (Fig. 1A). These DAL compounds provide beneficial characteristics. For example, various functional groups can be installed on the phenyl ring such as boronic acid, hydroxyl group, and halogen. These groups enable us to fine-tune several physicochemical properties such as size, hydrophobicity, and charge of the lipids and their corresponding nanoparticles. Fig. 1B displays a representative synthetic route. Compound a underwent a substitution reaction with b to yield intermediate c. Subsequently, a reductive amination reaction between c and d produced DAL1³⁷. Removal of ethyl group on DAL1 formed e, which was further reacted with 5-Fluoro-2-aminomethylphenylboronic acid, pinacol ester and hydrolyzed to synthesize DAL4. We confirmed the structures of all these DALs using both ¹H NMR and mass spectrum (MS) (Supplementary Data and Supplementary Fig. 1).

Formulations of DAL lipid nanoparticles (DAL-LNPs) for in vitro mRNA delivery and cytokine expressions

Next, we studied the mRNA delivery efficiency of DAL lipid nanoparticles (DAL-LNPs) using luciferase mRNA in B16F10 melanoma tumor cells in vitro. We formulated DALs with 1,2-dioleoylsnglycero-3-phosphoethanolamine (DOPE), cholesterol (Chol), 1,2-dimyristoyl-racglycero-3-methylpolyoxyethylene (DMG-PEG₂₀₀₀, PEG) (molar ratio: DAL/DOPE/Chol/PEG = 20/30/40/0.75), and luciferase mRNA to prepare the nanoparticles (DAL-LNP-Luc) as described previously³⁷. The mRNA encapsulation efficiency (Fig. 2A), formulation size distribution, (Fig. 2B) and polydispersity index (PDI) (Fig. 2B) were also determined. The mRNA encapsulation efficiency of DAL-LNPs ranged from 2.6% in DAL1-LNP-Luc to 90.3% in DAL4-LNP-Luc (Fig. 2A). These nanoparticles had a size of 150–200 nm and PDI less than 0.2 as measured by dynamic light scattering (Fig. 2B). After treating the B16F10 cells with DAL-LNPs for 18 hours, the luciferase mRNA delivery efficiency was quantified by a bioluminescence reporter assay. As shown in Fig. 2C, DAL4-LNP induced the highest luminescence signal in cultured cells when compared with other DAL-LNPs and Lipofectamine^™ 3000. DAL4-LNP displayed a spherical morphology when visualized by cryo-EM microscopy (Fig. 2D). Thus, we chose DAL4-LNP as a lead candidate to encapsulate cytokine mRNAs for further testing.

We investigated the cytokine mRNA delivery and protein expression in vitro by an ELISA assay. We treated B16F10 cells with DAL4-LNP encapsulating either IL-27 mRNA (DAL4-LNP-IL-27), IL-12 mRNA (DAL4-LNP-IL-12), or GM-CSF mRNA (DAL4-LNP-GM-CSF) for 18 hours and collected culture supernatants. The results (Fig. 2E–G) showed that each of the cytokine proteins can be expressed and secreted in the supernatants of cell culture, which was more efficient than MC3 formulated LNPs, an FDA approved LNP formulation. These results indicated that the DAL4-LNP can effectively deliver cytokine mRNA in cells.

DAL4-LNP can effectively deliver mRNA into tumors for protein expression

After selecting DAL4-LNP from in vitro assays, we tested if DAL4-LNP could be used to deliver mRNA to established tumors for protein expression in vivo. For this purpose, we generated DAL4-LNP loaded with mRNA encoding GFP and i.t. injected DAL4-LNP-GFP to established B16F10 tumors. 12 hours after injection of LNP-GFP mRNA, GFP⁺ cells were readily detected in tumors, ranging from 3–15% of total tumor cells by flow cytometry (Fig. 3A). Among GFP⁺ cells, approximately 66.7% were CD45^‒ non-immune cells which are primarily tumor cells and fibroblasts, and 31.9% of cells were CD45⁺ immune cells (Fig. 3B). Among GFP⁺ immune cells, about 98% were CD19⁺ B cells, and 2% were monocyte or macrophages, while GFP⁺ T cells were barely detected (Fig. 3B). Thus, DAL4-LNP can serve as an efficient vehicle for mRNA delivery to tumors for protein expression.

Intratumoral delivery of LNP-IL-12/IL-27 inhibits tumor growth

Cytokines such as IL-12⁷, IL-27^22–24 and GM-CSF^{26, 27} have demonstrated anti-tumor activity when delivered systemically or locally. To compare the therapeutic potential of each cytokine mRNA in DAL4-LNP, we first probed the treatment efficacy in a subcutaneous B16F10 mouse tumor model by delivering a single cytokine mRNA with DAL4-LNP via intratumoral injection every other day for six doses. DAL4-LNP loaded with IL-12 mRNA (DAL4-LNP-IL-12) exhibited the strongest tumor inhibitive effect when compared to DAL4-LNP encapsulated with IL-27 mRNA (DAL4-LNP-IL-27) or GM-CSF mRNA (DAL4-LNP-GM-CSF) with slower tumor growth and prolonged survival (Fig. 4A–B).

Previous gene therapy based on the systemic application of IL-27 and IL-12 has shown significant synergy of these two cytokines in tumor inhibition⁴². To determine if intratumoral injections of cytokine mRNA nanoparticles could induce stronger tumor growth inhibition, we tested IL-12 mRNA in combination with IL-27 and/or GM-CSF mRNA in DAL4-LNP. DAL4-LNP-IL-12+IL-27 outperformed other combinations by retarding tumor growth (Fig. 4C) and extending the survival of tumor-bearing mice (Fig. 4D). Interestingly, the combination of all three cytokine mRNAs did not induce better tumor inhibition or prolong survival compared to the IL-12+IL-27 combination. To further calibrate the dosage of DAL4-LNP-IL-12+IL-27, we changed the IL-27 mRNA dose from 2 μg to 6 μg per mouse for six doses while the IL-12 mRNA dose remained at 2 μg (Supplementary Fig. 2). We observed that the increased dosage of DAL4-LNP-IL-27 also showed significant tumor inhibition compared to the control group, and DAL4-LNP-IL-12+IL-27 resulted in 100% survival on day 35. To assess potential systemic toxicity, we comprehensively monitored body weight changes in addition to overall survival during the treatment process and analyzed histology of major organs after DAL4-LNP treatments. No significant body weight changes were observed as compared with control mice (Supplementary Fig. 3). Histology analysis of major organs (heart, lung, liver and kidney) from five treated mice demonstrated no observed inflammatory changes (Supplementary Fig. 4) as compared to control mice. Consequently, intratumoral delivery of DAL4-LNP-IL-12+IL-27 mRNA did not cause noticeable systematic toxicity within the dosages tested.

Intratumoral delivery of DAL4-LNP-IL-12 + IL-27 mRNA induces robust infiltration of immune effector cells into tumors

Both IL-12 and IL-27 have been shown to be potent cytokines capable of inducing T and NK cell responses in tumor models^{7, 22–24}. We hypothesized that intratumoral injection of DAL4-LNP-IL-12 + IL-27 mRNA can induce potent immune responses that lead to the infiltration of immune effector cells into tumors. To test this hypothesis, we analyzed tumors from mice receiving the treatment regimen and examined the density of immune cells in tumors by flow cytometry. DAL4-LNP-IL-12 + IL-27 mRNA-treated tumors resulted in a nearly 10-fold increase of total CD45⁺ leukocytes compared to control-treated tumors (Fig. 5A). The percentages of almost every subtype of leukocytes among the total tumor mononuclear cells, including CD19⁺ B cells, F4/80⁺ macrophages, CD4⁺ T cells, CD8⁺ T cells, and NK cells, increased when compared to the control group. Additionally, tumor-infiltrating T and NK cells from DAL4-LNP-IL-12 + IL-27 mRNA treated tumors significantly increased the number of IFN-γ (Fig. 5B) and TNF-α (Fig. 5C) producing cells. Thus, intratumoral delivery of DAL4-LNP-IL-12+IL-27 mRNA induced robust infiltration of immune effector cells into tumors.