Intratumoral Interleukin-12 Mrna Therapy Promotes TH1 Transformation of the Tumor Microenvironment

Author Manuscript Published OnlineFirst on August 17, 2020; DOI: 10.1158/1078-0432.CCR-20-0472 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Intratumoral interleukin-12 mRNA therapy promotes TH1 transformation of the tumor microenvironment

Susannah L. Hewitt1, Dyane Bailey1, John Zielinski1, Ameya Apte1, Faith Musenge1, Russell Karp1, Shannon Burke2, Fabien Garcon2, Ankita Mishra1, Sushma Gurumurthy1, Amanda Watkins2, Kristen Arnold1, James Moynihan3, Eleanor Clancy-Thompson3, Kathy Mulgrew3, Grace Adjei2, Katharina Deschler2, Darren Potz1, Gordon Moody3, David A. Leinster2, Steve Novick2, Michal Sulikowski2, Chris Bagnall2, Philip Martin3, Jean-Martin Lapointe2, Han Si3, Chris Morehouse3, Maja Sedic1, Robert W. Wilkinson2, Ronald Herbst3, Joshua P. Frederick1*, Nadia Luheshi2*

1Moderna Inc., 200 Technology Square, Cambridge, MA 02139, USA;

2AstraZeneca, Oncology R&D Unit, Aaron Klug Building, Granta Park, Cambridge, CB21 6GH, UK;

3AstraZeneca, Oncology R&D Unit, One MedImmune Way, Gaithersburg, MD 20878, USA.

*Joint Corresponding Authors: Nadia Luheshi, AstraZeneca, Aaron Klug Building, Granta Park, Cambridge, CB21 6GH, UK, +44 (0)203 749 6373, [email protected], Joshua Frederick, Moderna Inc., 200 Technology Square, Cambridge, MA 02139, USA, 617- 599-2205, [email protected]

Running title: IT IL-12 mRNA drives TH1 transformation of TME

Disclosure of Potential Conflicts of Interest AstraZeneca is developing MEDI1191 as a potential anticancer therapy for patients with solid tumors suitable for direct intratumoral dosing. J.P.F. and S.L.H. are inventors on a patent application for “Polynucleotides encoding Interleukin-12 (IL12) and Uses Thereof.” (WO2017/201350). S.L.H., D.B., J.Z., A.A., F.M., R.K., A.M., S.G., K.A., D.P., M.S. and J.P.F. are either current or previous employees of Moderna Inc. and received salary and stock options as compensation for their employment. S.B., F.G., A.W., G.A, K.D., G.D., A.L., S.N., M.S., C.B., P.M., J-M. L., E.C-T., J.M., H.S., C.M., R.W.W., R.H. and N.L. are either current or previous employees of AstraZeneca.

Downloaded from clincancerres.aacrjournals.org on September 27, 2021. © 2020 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 17, 2020; DOI: 10.1158/1078-0432.CCR-20-0472 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Statement of translational relevance

Preclinical data reported here in syngeneic mouse tumor models and patient tumor slice cultures demonstrate that IT IL-12 mRNA drives TH1 transformation of the tumor microenvironment, leading to IFNγ and cytotoxic T cell-dependent anti-tumor immunity that is further enhanced by PD-L1 blockade. This preclinical activity of IL-12 mRNA indicates the potential of MEDI1191 (human IL-12 mRNA) as a novel treatment for patients with solid tumors otherwise unresponsive to immune checkpoint blockade, alone and in combination with inhibitors of the PD-L1 / PD-1 checkpoint. MEDI1191 is currently being evaluated in a phase I trial in patients with solid tumors (NCT03946800).

Abstract

Purpose: Whilst immune checkpoint inhibitors such as anti–PD-L1 are rapidly becoming the standard of care in the treatment of many cancers, only a subset of treated patients have long- term responses. Interleukin 12 (IL-12) promotes anti-tumor immunity in mouse models, however systemic recombinant IL-12 had significant toxicity and limited efficacy in early clinical trials.

Experimental design: We therefore designed a novel intratumoral (IT) IL-12 mRNA therapy to promote local IL-12 tumor production whilst mitigating systemic effects.

Results: A single IT dose of mouse (m)IL-12 mRNA induced IFNγ and CD8+ T cell- dependent tumor regression in multiple syngeneic mouse models, and animals with a complete response demonstrated immunity to re-challenge. Anti-tumor activity of mIL-12 mRNA did not require NK and NKT cells. mIL-12 mRNA anti-tumor activity correlated with TH1 tumor microenvironment (TME) transformation. In a PD-L1 blockade monotherapy- resistant model, anti-tumor immunity induced by mIL-12 mRNA was enhanced by anti–PD- L1. mIL-12 mRNA also drove regression of un-injected distal lesions, and anti–PD-L1 potentiated this response. Importantly, IT delivery of mRNA encoding membrane-tethered mIL-12 also drove rejection of un-injected lesions with very limited circulating IL-12p70, supporting the hypothesis that local IL-12 could induce a systemic anti-tumor immune response against distal lesions. Furthermore, in ex vivo patient tumor slice cultures, human IL-12 mRNA (MEDI1191) induced dose-dependent IL-12 production, downstream IFNγ expression and TH1 gene expression.

Conclusions: These data demonstrate the potential for intratumorally delivered IL-12 mRNA to promote TH1 TME transformation and robust anti-tumor immunity.

Introduction

Patients who respond to programmed death-1 / programmed cell death 1 ligand (PD-1 / PD-L1) immune checkpoint blockade (ICB) tend to have an inflamed, TH1-polarized tumor microenvironment (TME), characterized by expression of interferon-γ (IFNγ) and PD-L1 (1). Novel therapies that induce TH1 transformation of the patient TME therefore have the potential to enhance anti-tumor responses in patients that are currently non-responders to ICB. As a central mediator of TH1 immune responses, IL-12 is known to play a key role in driving anti-tumor immunity. IL-12 guides the differentiation of TH1 T cells and enhances the activation and cytotoxic activity of natural killer cell (NK), natural killer T cells (NKT), and cytotoxic T cells (CTL) (2). Many of the downstream effects of IL-12 are mediated via induction of the key TH1 cytokine, interferon-γ (IFNγ), by innate and adaptive immune cells. IL-12 acts both directly and via IFNγ to promote antigen presentation, reduce myeloid immunosuppression, and to enhance T cell recruitment through production of other TH1 chemokines including IFNγ-inducible protein 10 (IP-10) (3,4). IL-12 also exerts anti- angiogenic effects through IP-10 (5). Systemic recombinant IL-12 (recIL-12) promotes IFNγ- and CTL-dependent anti- tumor immunity in a wide variety of mouse syngeneic tumor models (6,7). NK and NKT cells also play a key role in driving antitumor activity of IL-12 in some mouse models (8-10). However, systemic recIL-12 was poorly tolerated in early clinical trials, and efficacy of systemic recIL-12 has been limited at tolerated doses (11,12). The narrow therapeutic margin of systemic recIL-12 has led to the development of alternative local strategies for direct delivery of IL-12 to the TME. IL-12 plasmid intratumoral (IT) injection with electroporation drives local IL-12 production and cytotoxic T cell dependent anti-tumor immunity in mice bearing various syngeneic tumors (13,14). IT administration of IL-12-encoding adenovirus and oncolytic viruses promotes anti-tumor immune responses in preclinical models, and this activity is enhanced by combination with ICB therapy (15-17). IT delivery of dendritic cells or of antitumor CD8+ T cells engineered to express IL-12 also drives tumor regression in preclinical models (18,19). IL-12 plasmid (tavokinogene telseplasmid) IT delivery by electroporation demonstrated clinical activity in metastatic melanoma patients, inducing regression of both treated and untreated lesions (20). However, the requirement for an electroporation device limits tavokinogene telseplasmid utility to patients with superficial lesions.

Here we report on the development of a novel lipid nanoparticle (LNP)-formulated, IL-12 mRNA-based therapy designed for intratumoral (IT) injection in patients with superficial and deep-seated lesions. We found that a single IT dose of mouse IL-12 mRNA induced tumor regression in multiple tumor models. This was both CD8+ T cell and IFNγ dependent and correlated with upregulation of signature TH1 immune response genes, thus TH1 TME transformation. In a PD-L1-resistant model, anti-tumor immunity induced by mouse IL-12 mRNA was enhanced by PD-L1 blockade. Mouse IL-12 mRNA also drove regression of un-injected distal lesions, and anti–PD-L1 potentiated this response. IT delivery of mRNA encoding membrane-tethered mouse IL-12 induced regression of untreated lesions in the absence of increases in circulating IL-12, supporting the hypothesis that local IL-12 could induce a systemic anti-tumor immune response against distal lesions. Finally, we report that in ex vivo patient tumor slice cultures human IL-12 mRNA (MEDI1191) induced dose- dependent IL-12 production, IFNγ expression and TH1 TME transformation.

Materials and Methods

mRNA design, synthesis and formulation mIL-12 mRNA and MEDI1191 mRNA incorporate wild-type mouse IL12B and IL12A sequences (NM_001303244.1 and NM_001159424.2) and optimized human IL12A and IL12B mRNA sequences (human NM_002187.2 and NM_000882, in-house algorithm applied to optimize translation). For both MEDI1191 and mIL-12 mRNA, a linker was added between the IL12B and IL12A sequences as published (21) to generate a linked monomeric IL-12p70 (IL12B-IL12A). The IL12B signal peptide was retained for secretion, and the IL12A signal peptide was removed [first 22 amino acids for both mouse and human IL12A, according to Uniprot (mouse P43431 and human P29459)]. For tethered mIL-12, the C terminus of the single chain IL12B-IL12A construct was linked via a peptide linker

(SG3SG4SG4SG4SG3SLQ) to a PDGFRβ transmembrane domain, followed by a G4S linker and V5 peptide tag at the intracellular end. mRNA constructs incorporated a miR122 binding site in the 3′ UTR (22), except where indicated in Fig. 1B. Control mRNA is non-translating where the initiating AUG codon and any other potential initiating codons were modified. mRNA was synthesized and LNP formulated as described previously (23-26) and briefly detailed in the Supplementary Methods.

In vitro IL-12p70 expression and bioactivity assays In vitro IL-12p70 expression was quantified by DuoSet ELISA (R&D Systems) or Mesoscale Discovery assay (Mesoscale Discovery) in the supernatants and lysates of IL-12 mRNA-treated cells. Human and mouse CD8+ T cells were cultured with species-specific CD3/CD28 DynabeadsTM and either HeLa supernatant containing mIL-12 mRNA- or MEDI1191-derived IL-12p70 or HeLa cells transfected with tethered mIL-12 mRNA. IFNγ levels were measured by DuoSet ELISA (R&D Systems).

Syngeneic and PDX tumor models All in vivo experiments were carried out either in accordance with the Institutional Animal Care and Use Committee (IACUC) at Moderna Inc., MI Bioresearch and Crown Bioscience San Diego, Medimmune (Gaithersburg, MD), or the UK Animal (Scientific Procedures) Act for studies at MedImmune. For syngeneic models, female C57BL/6 mice (The Jackson Laboratory or Charles River UK) or Balb/c mice (Charles River Laboratories) were implanted SC with MC38-S, MC38-R, B16F10-AP3 or A20 tumor cells. For PDX models non-obese diabetic, severe combined immunodeficient (NOD-SCID) mice were implanted with SC melanoma (ME12057, ME12058) or head and neck (HN5111, HN5116) tumor cell slurries (Crown Bioscience). Tumors were measured with calipers, and volumes were calculated using the formula Volume = (Length x Width2) / 2. Tumor volumes at randomization (24 hours prior to IT mRNA treatment or on the day of treatment) are listed in Supplementary Table 2. mRNAs were administered IT in a fixed 25µL volume to mice bearing established tumors. Antibodies were administered IP as detailed in Supplementary Table 3. Survival events were either recorded on the day when total tumor volume exceeded 1500 or 2000mm3, or on the day when animals were removed from study due to specific negative clinical signs (e.g. tumor ulceration, weight loss > 20%), whichever event occurred first. CRs were defined as animals with no measurable tumor at all implant sites at study completion. CR rates and Kaplan-Meier survival plots are based on all animals enrolled in each group, except for the A20 tumor model where any animals that grew secondary masses are removed from the displayed data. IL-12p70 and IFN were quantified with the ProcartaPlex Mix&Match Mouse 5-plex, Cytokine and Chemokine 36-Plex Mouse ProcartaPlex Panel 1A (Thermo Fisher), electrochemiluminescent assay for mouse IL-12p70

or mouse IFNγ (V-plex, Meso Scale Discovery), or DuoSet ELISA (R&D Systems) as indicated.

Flow cytometry analysis Syngeneic tumors were dissociated to single cells, counted, normalized and stained for viability (Fixable Viability Dye or Live/Dead Aqua (Thermo Fisher), Supplementary Table 4). Peripheral blood and spleen cells were stained with Zombie NIR viability dye or Live/Dead Aqua (Thermo Fisher). Tumor and spleen cells were blocked with TruStain FcXTM (anti-mouse CD16/32, BioLegend) prior to fluorescent antibody staining (antibodies detailed in Supplementary Table 4). Tumor samples were run on an LSRFortessa or FACSymphony (BD Biosciences) with 123count eBeadsTM (Thermo Fisher). Peripheral blood and spleen samples were run on an Attune NXT flow cytometer or FACSymphony (BD Biosciences). All analyses were carried out using FlowJo V10 (TreeStar).

Splenocyte restimulation with tumor antigenic peptides 1 × 105 splenocytes per well were seeded into pre-coated 96-well mouse IFNy ELISpot plates (MABTECH) in RPMI with 10% FBS, 1% penicillin and streptomycin, 50nM 2-Mercaptoethanol, 10ng/ml IL-2 (Roche). Splenocytes were stimulated with 1 or 10ug/ml tumour antigenic peptides (H-2Kb MuLV P15E peptide KSPWFTTL and H-2K TRP-2 peptide SVYDFFVWL both from MBL). Plates were incubated at 37°C, 5% CO2 for 48 hours before completing the IFNγ detection assay. Plates were counted on a CTL ImmunoSpot 6 Ultra-V analyzer.

Patient tumor slice culture All samples were obtained with written informed consent from patients. Studies were conducted in accordance with recognized ethical guidelines (Declaration of Helsinki) and were approved by the East of England—Cambridge East Research Ethics Committee (MedImmune Research Tissue Bank, RTB 16/EE/0334, HTA license number 12283). Fresh endometrial, colorectal adenocarcinoma and colorectal liver metastasis tumor samples from surgical resections (Nottingham Health Service Biobank, Tissue Solutions and Scievita) were transferred to MedImmune Ltd in ice-cold Aqix RS-I solution (Life Science Group). Upon receipt (< 24h post-resection), samples were embedded in 4% low-melting point agarose (VWR) prepared in PBS, and vibratome sectioned (300 μm slices, VT-1200S, Leica). Fresh tumor slices were transferred to an organotypic insert (Millipore) in a 6-well plate containing

RPMI1640 with 10% FBS and 1% Penicillin/Streptomycin. MEDI1191 or control RNA was added directly to the media at the desired concentration. Every 24 hrs, an aliquot of culture media was collected for analysis, and replaced with the same volume of fresh complete media. Culture supernatants and tissue slices were harvested at endpoint after 3 – 5 days. IL- 12p70, CXCL-10 and IFNγ were quantified in endpoint culture supernatants by MesoScale Discovery assay (MesoScale Discovery) and ELISA (R&D Systems).

Transcriptomic analysis mRNA was isolated from snap-frozen syngeneic tumors using an RNeasy mini kit (Qiagen) and quantified on the Affymetrix mouse 430 2.0 microarray. Interchip normalisation was performed using the fRMA method (27). Technical outlier samples were identified by principle component analysis and removed from the analysis. Significantly differentially expressed genes (DEG) in mIL-12 mRNA versus control mRNA-treated tumors were identified as those with fold change ≥ 2 with an adjusted p-value < 0.05. ToppFun pathway analysis was performed on DEG lists (https://toppgene.cchmc.org/) with a false discovery-corrected p-value cut-off of 0.05. Full DEG lists and pathway analysis results (Supplementary Table 1) underwent further manual curation to identify relevant groups of transcripts prior to the generation of heatmaps (TIBCO Spotfire). Microarray data have been deposited in the ArrayExpress database at EMBL-EBI (www.ebi.ac.uk/arrayexpress) under accession number E-MTAB-9316. Patient tumor slices were placed in Trizol (Invitrogen), disrupted in a Tissue Lyser (Quiagen) and total RNA was extracted with a Direct-zol RNA miniprep kit (Zymo Research). 50 ng total RNA was evaluated using the 770-gene PanCancer IO 360TM panel (nanoString Technologies). Gene counts were normalized to internal reference standards using the nSolver TM software. A further inter-chip normalization was performed with the ComBat algorithm (28) as implemented in R (R Foundation for Statistical Computing), and genes were identified which were upregulated >1.5 fold versus matched tumor slices treated with control mRNA with an unadjusted p value of < 0.05. All significantly upregulated genes are displayed in Fig. 6H.

Immunohistochemistry Syngeneic tumors collected at study endpoint and representative patient tumor slices collected immediately after vibratome sectioning were formalin fixed and paraffin embedded. Sectioning and immunostaining of all samples was performed as detailed in supplementary

methods. Slides were digitally scanned (Aperio AT2 system, Leica Biosystems) and analysed with Tissue Studio 4.4.2 (Definiens) or HALO 2.1 software (Indica Labs).

Statistical analyses MC38-R and B16F10-AP3 tumor flow cytometry data statistical analysis was performed in R (R Foundation for Statistical Computing). Cell count and CD8 / Treg ratio

data were log10 transformed, and percentage data were arc-sine square-root transformed prior to ANOVA analysis with p value correction according to the method of Edwards and Berry (29). All other statistical analysis was performed in GraphPad Prism. Comparisons of survival curves between groups were conducted using the log-rank test without any p value adjustment for multiple testing. Comparisons of plasma mIL-12p70 or mIFNγ concentrations between groups were conducted by first performing a Log10 transformation, then using 1- way analysis of variance (ANOVA) statistical analysis with Tukey’s honest significance difference post-hoc test. Statistics were not carried out on IL-10 levels due to zero values in controls. Comparison of tumor slice culture supernatant cytokine levels was performed using a Friedman test with Dunn’s multiple comparison post-test. For all other datasets, comparisons of two groups were made using a Student’s t-test, and comparisons between multiple groups were made by ANOVA with post-hoc Tukey’s multiple comparison test. Data are presented as mean ± standard deviation (s.d.) unless otherwise indicated. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Results

Intratumoral administration of a single dose of mIL-12 mRNA induces dose-dependent tumor regression in multiple models We first designed mouse (m) IL-12 mRNA to enable us to investigate responses in immunocompetent syngeneic mouse models of cancer, as human IL-12p70 protein is not bioactive in mice (30). mIL-12 mRNA (Fig. 1A) encodes a subunit-linked, monomeric mIL- 12p70 protein as previously described (21). Lipid nanoparticle (LNP)-encapsulated mIL-12 mRNA (Fig. 1A) induced dose-dependent release of mIL-12p70 from colorectal MC38 tumor cells in vitro (Supplementary Fig. S1A), and mIL-12 mRNA-derived mIL-12p70 protein had comparable activity to heterodimeric recombinant mIL-12p70 on mouse CD8+ T cells (Supplementary Fig. S1B). A miR122-3p (miR122) binding site was included in the mIL-12

mRNA 3ʹUTR to inhibit IL-12 protein production in hepatocytes without impacting levels in cancer cells (Fig. 1B, (22)). The pharmacodynamic effects and anti-tumor activity of mIL-12 mRNA were investigated in mice bearing established subcutaneous (SC) syngeneic tumors with differing sensitivities to ICB. The SC A20 lymphoma model is a well-established ICB sensitive system (31). We recently reported that the MC38-resistant (MC38-R) variant of the MC38 colorectal cell line is less responsive to inhibition of PD-1 and PD-L1 versus MC38-sensitive (MC38-S) tumors after SC implantation in mice (23). mIL-12p70 expression was investigated in MC38- R and A20 tumor-bearing mice following a single intratumoral (IT) dose of LNP- encapsulated mIL-12 mRNA. In both models, mIL-12 mRNA induced dose-dependent tumor expression of mIL-12p70, which was also associated with increased circulating plasma mIL- 12p70 concentrations (Fig. 1C and D, Supplementary Fig. S1C and S1D). IL-12 levels remained elevated out to seven days post dose (Fig. 1C and D, Supplementary Fig. S1C and S1D). This contrasts with the 5 – 10 hour half-life of recombinant IL-12 (32) and is consistent with previous reports that mRNA administration leads to continued protein production and so prolonged bioavailability versus recombinant protein administration (23,33). A single dose of mIL-12 mRNA induced dose-dependent tumor growth delay, tumor regression and in some cases complete responses (CRs) in mice bearing established A20, MC38-S and MC38-R tumors (Fig. 1E–G, Supplementary Fig. S1F). In contrast to mIL-12 mRNA, non-translating control mRNA had no effect on tumor growth or overall survival versus untreated animals. A single dose of as little as 0.05 µg mIL-12 mRNA significantly extended overall survival versus control mRNA-treated mice in all three models (p ≤ 0.0032, Fig. 1E–G). Animals treated with 0.5 µg and 5 µg mIL-12 mRNA survived significantly longer than those treated with the lowest 0.05 µg dose of mIL-12 mRNA (p ≤ 0.0472, Fig. 1E–G). Complete regression of these established tumors was also observed in a larger proportion of animals in the 0.5 µg and 5 µg mIL-12 mRNA groups versus in the 0.05 µg mIL-12 mRNA group in all three models (Supplementary Fig. S1F). mIL-12 mRNA had greater anti-tumor activity in the A20 and MC38-S models versus the MC38-R model (Fig. 1E–G), consistent with the previously observed higher sensitivity of the A20 and MC38-S models to PD-1 / PD-L1 blockade compared to MC38-R (23,31). Whilst weekly repeated dosing continued to induce IL-12 as well as downstream cytokines (Supplementary Fig. S2), repeated dosing did not significantly enhance mIL-12 mRNA anti-tumor activity versus a single dose in the MC38-S, A20 or MC38-R models (Supplementary Fig. S1E and S1G).

mIL-12 mRNA was well tolerated, particularly at the 0.05 and 0.5 µg dose levels, with less than 10% body weight loss detected (Supplementary Fig. S3A–S3D). The 5-µg mIL-12 mRNA dose level was similarly well tolerated in three out of four studies and the one outlier study is shown in Supplementary Fig. S3D, where an average of 6.98% body weight loss was observed at day 17 post-implantation. Importantly, in nearly all animals with a CR to mIL-12 mRNA, tumors did not grow at the re-challenge implantation site in all three models (Fig. 1H, Supplementary Fig. S1H). This infers that an immune memory response was established to A20, MC38-S or MC38-R cells in most animals with a CR to mIL-12 mRNA treatment.

CD8+ T cells are required for optimal mIL-12 mRNA anti-tumor activity Since mice with a CR to mIL-12 mRNA showed evidence of the establishment of an immune memory response to tumor cells, we investigated the role of the adaptive immune system and specifically CD8+ T cells in mIL-12 mRNA anti-tumor activity. In MC38-R tumor-bearing animals, mIL-12 mRNA induced a dose-dependent increase in tumor- infiltrating CD8+ T cells 7 days post-dose (Fig. 2A). mIL-12 mRNA did not alter tumor FoxP3– effector CD4+ cells or FoxP3+ Treg numbers (Fig. 2B and C). A dose-dependent increase in the ratio of CD8+ T cells to FoxP3+ Treg cells was therefore observed 7 days after a single dose of 0.05 µg or 0.5 µg mIL-12 mRNA (Fig. 2D). To further investigate the impact of mIL-12 mRNA on the MC38-R TME, we performed tumor transcriptomic evaluation followed by pathway analysis and manual review of differentially expressed genes. This analysis highlighted a significant increase in transcripts relating to T cell lineage abundance 7 days post-dose (> 2 fold change, false discovery rate < 0.05, Fig. 2E, Supplementary Table 1). CD8+ T cell depletion significantly reduced mIL-12 mRNA anti-tumor activity in the MC38-R model compared to mice with an intact immune system (p < 0.0001, Fig. 2F). mIL- 12 mRNA still delayed tumor growth in CD8+ T cell-depleted mice (p < 0.0001 versus CD8+ T cell-depleted mice treated with control mRNA), but mIL-12 mRNA no longer induced any CRs in CD8+ T cell-depleted animals. In contrast, CD4+ T cell depletion had no impact on mIL-12 mRNA anti-tumor activity in this model. Tumor and peripheral T cell depletion was confirmed by flow cytometry (Supplementary Fig. S4). These data suggest that CD8+ T-cell- dependent adaptive immunity is required for optimal anti-tumor activity of mIL-12 mRNA in the MC38-R tumor model.

mIL-12 mRNA-induced anti-tumor immunity is IFNγ-dependent and associated with TH1 transformation of the MC38-R TME Many of the known effects of IL-12 are dependent on IFNγ release from activated NK and T cells (12). Therefore, we next investigated the role of T and NK cell activation and IFNγ release in mIL-12 mRNA-induced anti-tumor immunity. Within 24 hours, mIL-12 mRNA induced dose-dependent activation of tumor NK and NKT cells (increase in the activation marker CD69, by % positive cells; Fig. 3A and Supplementary Fig. S5A). In contrast, no early changes were observed in the activation of CD4+ or CD8+ T cells within MC38-R tumors (Supplementary Fig. S5B–S5D). NK cell numbers in tumors were unchanged 7 days post-dose (Supplementary Fig. S5E) and NKT cell numbers in tumors were mildly but significantly increased 7 days post-dose (Supplementary Fig. S5F). Tumor transcriptomic analysis revealed the upregulation of additional NK cell activation markers (Supplementary Fig. S5G). Tumor NK activation within the first 24 hours post-dose was followed by a dose dependent increase in tumor and plasma IFNγ levels in MC38-R tumor- bearing mice (Fig. 3B and C). A similar dose-dependent increase in IFNγ levels was also observed in A20 tumor bearing mice (Supplementary Fig. S5H and S5I). In order to determine the role of IFNγ in the anti-tumor immune response to mIL-12 mRNA more directly, we treated MC38-R tumor bearing mice with an IFNγ-blocking antibody prior to administration of a single IT dose of mIL-12 mRNA. Blockade of IFNγ completely abrogated the anti-tumor activity of mIL-12 mRNA in this model (Fig. 3D and E; p < 0.0001 mIL-12 mRNA plus isotype versus mIL-12 mRNA plus anti-IFNγ; p = 0.5878 mIL-12 mRNA + anti-IFNγ versus control mRNA + anti-IFNγ). Since NK cell activation has the potential to lead to both IFNγ release and tumor cell cytotoxicity, we investigated the impact of NK and NKT cell depletion on mIL-12 mRNA- induced IFNγ expression and anti-tumor activity in the MC38-R model. NK and NKT cell- depletion with anti-NK1.1 led to a small but significant reduction in the peripheral IFNγ response to mIL-12 mRNA in mice (Supplementary Fig S6A and S6B). However, NK and NKT cell depletion had no statistically significant impact on mIL-12 mRNA anti-tumor activity in this model (Fig. 3F; Supplementary Fig. S6C and S6D). NK and NKT cell depletion was confirmed by flow cytometry (Supplementary Fig. S6E). By 7 days post-dose in MC38-R tumor-bearing mice, mIL-12 mRNA induced TH1 TME transformation (upregulation of TH1 immune response genes, Fig. 3G). mIL-12 mRNA-driven TH1 TME transformation also included an early increase in the maturation of

dendritic cells (DCs) including CD103+ and CD8+ cross-presenting DC subsets (increased % cells positive for the maturation marker CD86; Fig. 3H; Supplementary Fig S7A and S7B), followed by a more general increase in the expression of genes relating to DC abundance and antigen presentation 7 days post-dose (Fig. 3G). Importantly, non-translating control mRNA did not induce non-specific DC activation (Fig. 3H; Supplementary Fig S7A and S7B), consistent with the modifications incorporated into the synthetic mRNA to reduce non- specific immune activation, and with our previous results (23,24). Additional pathway analysis of the transcriptomic landscape of MC38-R tumors revealed the broad extent of TME transformation 7 days post-dose with mIL-12 mRNA. This included increases in expression of T and NK cell activation genes, cytokines and chemokines, complement factors, as well as genes relating to extra cellular matrix (ECM) modulation and lipid metabolism (Supplementary Fig S7C–S7F; Supplementary Table 1). The broad extent of MC38-R tumor transcriptomic changes 7 days-post dose correlated with significant inhibition of tumor growth induced by mIL-12 mRNA at this timepoint (Supplementary Fig S7G).

In the PD-L1 resistant MC38-R model, anti-tumor immunity induced by mIL-12 mRNA is enhanced by PD-L1 blockade IFNγ is a potent inducer of PD-L1 expression on both tumor and myeloid cells (1,34). We therefore sought to determine whether IT mIL-12 mRNA induces PD-L1 expression in the tumor microenvironment of syngeneic mouse tumor models. Indeed, IT mIL-12 mRNA increased PD-L1 expression on immune infiltrating cells in MC38-R tumors 7 days post-dose (Fig. 4A). Flow cytometry analysis revealed PD-L1 expression increased on tumor infiltrating CD11b+ myeloid cells and on CD11b+ Ly6Chi monocytes (Fig. 4B, Supplementary Fig. S8A). No significant change in PD-L1 expression on MC38-R tumor cells was detected at any timepoint (Supplementary Fig. S8B). On tumoral total CD11b+ myeloid cells, PD-L1 was only induced 24 hours post-dose by the low dose and 72 hours post-dose by the higher dose of mIL-12 mRNA, and PD-L1 levels on these cells had returned to baseline 7 days post-dose (Supplementary Fig. S8A). In contrast, both 0.05 and 0.5 µg hi mIL-12 mRNA increased PD-L1 expression on Ly6C monocytes in the tumor, and this increase was sustained from 24 hours through to 7 days post-dose (Fig. 4B). - The CD11b+ myeloid compartment includes neutrophils, monocytes, macrophages, MDSC and some DC. The more sustained expression of PD-L1 on the Ly6Chi monocyte / mMDSC subset versus other CD11b+ myeloid populations might for example reflect greater suppressive functions of mMDSC. In the blood of MC38-R tumor bearing mice, mIL-12

hi mRNA also induced PD-L1 expression on Ly6C monocytes 24h post-dose, and this PD-L1 induction was significantly inhibited in the presence of an IFNγ-blocking antibody (Supplementary Fig. S8C). Since PD-L1 interaction with PD-1 can suppress anti-tumor immunity, the effect of PD-L1 blockade (anti–PD-L1 antibody) on mIL-12 mRNA anti-tumor activity was investigated in animals bearing established MC38-R tumors. As expected, anti-PD-L1 plus control mRNA had no impact on tumor growth or overall survival in the ICB-resistant MC38-R model (p = 0.1793 for anti-PD-L1 plus control mRNA compared to isotype antibody plus control mRNA, Fig. 4C). In contrast, the combination of anti–PD-L1 with a single dose of 0.5 or 5 µg mIL-12 mRNA significantly improved overall survival and increased the number of animals with CRs, compared to the same doses of mIL-12 mRNA alone (p ≤ 0.0250, Fig. 4C). However, the combination of anti-PD-L1 with the lowest dose of 0.05 µg mIL-12 mRNA did not significantly improve overall survival versus 0.05 µg mIL-12 mRNA alone (p = 0.9689; Fig. 4C). In addition, there was a nonsignificant trend toward improved survival with three weekly doses of mIL-12 mRNA in combination with anti–PD- L1 versus a single dose of mIL-12 mRNA in combination with anti–PD-L1 (p = 0.1225, Supplementary Fig. S8D). The combination of anti–PD-L1 with three doses of mIL-12 mRNA also led to more CRs (12/15 CRs, 80%), versus anti–PD-L1 in combination with a single dose of mIL-12 mRNA (8/15 CRs, 53%). We next investigated the mechanism by which anti–PD-L1 enhanced the anti-tumor activity of mIL-12 mRNA in MC38-R tumor-bearing mice. We quantified the frequency of splenic tumor peptide-reactive T cells by re-challenging splenocytes with the MC38 immunodominant retroviral antigenic peptide p15E (35). mIL-12 mRNA alone expanded tumor-reactive T cells in the spleen 7 days post-dose, but anti–PD-L1 combined with mIL-12 mRNA did not further expand these cells versus mIL-12 mRNA monotherapy-treated animals (Fig. 4D). However, the combination of mIL-12 mRNA with anti–PD-L1 did significantly increase the number of CD8+ T cells infiltrating into tumors compared to treatment with either anti–PD-L1 alone (p < 0.0001) or mIL-12 mRNA alone (p < 0.05, Fig. 4E and F). In particular, a dense CD8+ immune infiltrate was detected surrounding a completely necrotic tumor core 7 days post-dose in 4 of 8 animals treated with 0.5 µg mIL-12 mRNA plus anti–PD-L1 (Fig. 4G, these 4 animals indicated by star symbol in Fig. 4F). In most animals with a CR to mIL-12 mRNA plus anti–PD-L1, no tumor growth was observed following re-challenge implantation of MC38-R cells on the opposite flank of the animal (Supplementary Fig. S8E and S8F). Together, these data infer that PD-L1 blockade enhances

CD8+ T cell recruitment to MC38-R tumors and CD8+ T cell-mediated tumor lysis in response to mIL-12 mRNA. We also investigated mIL-12 mRNA anti-tumor activity alone and in combination with anti–PD-L1 treatment in the B16F10-AP3 model, a PD-L1 blockade-resistant melanoma tumor model with minimal immune infiltrate (36). In this model, a single dose of mIL-12 mRNA transformed the TME, activating NK cells and DCs (Supplementary Fig. S9A and S9B), increasing CD8+ T cell infiltration and the CD8+ T cell to Treg ratio (Supplementary Fig. S9C and S9D), inducing IFNγ release (Supplementary Fig. S9E) and additionally increasing PD-L1 expression on both tumor and myeloid and cells (Supplementary Fig. S9F– S9H). A single dose of 0.5 µg mIL-12 mRNA significantly increased survival compared to control mRNA (p < 0.0001, Supplementary Fig. S9I and S9J). There was a non-significant trend toward improved survival and an increased number of CRs in animals treated with mIL- 12 mRNA plus anti–PD-L1 versus mIL-12 mRNA (Supplementary Fig. S9I and S9J). However, in the B16F10-AP3 model neither mIL-12 mRNA alone nor mIL-12 mRNA combination with αPD-L1 expanded splenic tumor peptide-reactive T cells (Trp2 or p15E peptides; Supplementary Fig. S9K), suggesting that mIL-12 mRNA is less efficient at inducing anti-tumor immunity in B16F10-AP3 versus MC38-R tumor-bearing mice.

Locally administered mIL-12 mRNA drives regression of un-injected distal lesions, and anti–PD-L1 potentiates this response We next tested the hypothesis that IT mIL-12 mRNA alone and in combination with anti–PD-L1 would drive regression of distal, untreated tumors in mice bearing bilateral MC38-S tumors (Fig. 5A). A single injection of the lower 0.5 µg dose of mIL-12 mRNA induced complete regression of both treated and untreated tumors in 3/20 animals (bilateral CRs, Fig. 5A) and significantly increased overall survival versus animals treated with isotype antibody plus 5 μg control mRNA (p < 0.0001; Fig. 5B). The higher 5 μg mIL-12 mRNA dose induced significantly greater overall survival and a larger number CRs compared to 0.5 μg mIL-12 mRNA (p < 0.0001, 16/20 CRs, Fig. 5A and B). Anti–PD-L1 antibody only had marginal anti-tumor activity in this model, providing a modest but statistically significant survival benefit versus animals treated with isotype antibody plus control mRNA without extending median overall survival or inducing any CRs (p = 0.0214, Fig. 5B). In contrast, the combination of αPD-L1 Ab with 0.5 or 5 µg mIL-12 mRNA significantly improved overall survival compared to the same doses of mIL-12 mRNA alone (p ≤ 0.0373) and compared to anti–PD-L1 alone (p < 0.0001, Fig. 5A and B). The anti-tumor activity of the combination of

anti–PD-L1 and mIL-12 mRNA was also dose-dependent in this model, with anti–PD-L1 plus 5 µg mIL-12 mRNA providing a significantly greater survival benefit and larger number of CRs versus anti–PDL1 + 0.5 µg mIL-12 mRNA (p < 0.0001, 20/20 CRs and 8/20 CRs respectively, Fig. 5B). IT injection of mIL-12 mRNA induces local tumoral mIL-12p70 expression, with some increases in peripheral mIL-12p70 exposure in syngeneic tumor-bearing mice (Fig. 1C and D). To investigate the ability of local IL-12p70 alone to drive regression of un-injected lesions, we used mRNA designed to encode a membrane-tethered mIL-12p70 to limit peripheral mIL-12p70 exposure (Fig. 5C). Whilst cell-associated mIL-12p70 was detected in HeLa cells transfected with tethered mIL-12 mRNA, mIL-12p70 release into the cell culture supernatant was very low relative to the secreted IL-12 variant (Fig. 5D). HeLa cells expressing tethered mIL-12 mRNA enhanced IFNγ release from co-cultured mouse CD8+ T cells, confirming the bioactivity of this membrane–associated mIL-12p70 protein (Fig. 5E). IT delivery of tethered mIL-12 mRNA in mice bearing bilateral MC38-S tumors led to complete regression of both treated and distal tumors in 4/20 mice (bilateral CRs, Fig. 5F). Minimal increases in circulating mIL-12p70 were detected above controls in tethered mIL-12 mRNA-treated animals, in contrast to animals treated with secreted mIL-12 mRNA (Fig. 5G). Furthermore, plasma IFNγ induction was also greatly reduced in animals treated with tethered versus secreted mIL-12 mRNA (Fig. 5H). These data with tethered mIL-12 mRNA indicate that local tumor mIL-12p70 expression can induce regression of untreated lesion in the absence of increases in circulating mIL-12p70 levels.

MEDI1191 (mRNA encoding human IL-12 formulated in LNPs) induces IL-12 production in PDX models Having observed promising anti-tumor activity of mIL-12 mRNA in mouse syngeneic tumor models, we developed MEDI1191 as a potential new treatment for patients with solid tumors. MEDI1191 is an LNP-formulated mRNA encoding a linked monomeric bioactive human (h) IL-12p70 protein. MEDI1191 induced dose-dependent release of hIL-12p70 from human monocyte-derived macrophages and from a panel of 16 human tumor cell lines. hIL- 12p70 production varied significantly between tumor cell lines in vitro, whereas it was very consistent between monocyte derived macrophages from different donors (Supplementary Fig. S10A and B). MEDI1191-derived linked monomeric hIL-12p70 activity was comparable to that of recombinant heterodimeric hIL-12p70 in a human CD8+ T cell IFNγ release assay (Supplementary Fig. S10C).

We investigated the ability of MEDI1191 to drive IL-12p70 production following IT administration in 2 melanoma and 2 head and neck squamous cell carcinoma (HNSCC) PDX models. IT MEDI1191 induced dose-dependent increases in tumor and plasma hIL-12p70 concentrations in the ME12057 melanoma and HN5111 HNSCC PDX models, the two in which multiple dose levels were assessed (Supplementary Fig. S10D). No significant difference in tumor or plasma hIL-12p70 levels was observed between all four PDX models 6 hours post-dose with 0.5 µg MEDI1191 (Supplementary Fig. S10E). 24 hours post-dose, hIL- 12p70 levels in tumor and plasma were significantly lower in the HN5116 HNSCC tumor- bearing mice versus the other models.

MEDI1191 induces dose-dependent IL-12p70 protein release, IFNγ production and increased TH1 gene expression in ex vivo patient tumor slice cultures Finally, we investigated the ability of MEDI1191 to transform the microenvironment of patient colorectal and endometrial tumors in an ex vivo tumor slice culture system. Fresh samples of patient tumors collected following surgical resection were vibratome sectioned (37) and cultured in vitro at the air / media interface in the presence of MEDI1191 or control mRNA. Details on patient tumor samples are in Supplementary Fig. S11. MEDI1191 induced significant, dose-dependent hIL-12p70 release from all patient tumor slice cultures tested (Fig. 6A). MEDI1191 also induced dose-dependent IFNγ and CXCL10 release, whereas control mRNA had minimal effect on expression of these mediators (Fig. 6B and C). Whilst MEDI1191 induced hIL-12p70 release from every patient tumor tested, IFNγ and CXCL10 release was more variable between patient tumor slices (Fig. 6A–C). We therefore investigated whether MEDI1191-dependent induction of an early IFNγ response in tumor slice cultures might depend on the density of tumor infiltrating NK and T cells at baseline. NK and T cell densities were quantified by immunohistochemistry in patient tumor slices fixed immediately after vibratome sectioning (Fig. 6D and E). No correlation was observed between patient tumor slice IFNγ release and baseline T cell infiltration in this acute model (Fig. 6F, IFNγ release measured after 3 – 5 days in culture). However, a positive correlation was observed between patient tumor slice acute IFNγ release and baseline NK cell infiltration (Fig. 6G). Finally, since mIL-12 mRNA induced TH1 TME transformation in the murine MC38-R model, we investigated whether MEDI1191 also induced similar TME transformation in patient tumor slices. Indeed, nanoString transcriptomic analysis of patient tumor slices treated with MEDI1191 revealed significant increases in expression of TH1 response genes compared to tumor slices treated with control mRNA (Fig. 6H). Notably,

IFNG, STAT1 and GBP2 expression was upregulated in both MEDI1191-treated patient tumor slices and mIL-12 mRNA treated MC38-R tumors (Fig. 6H, Fig 3D). The remaining TH1 genes induced by MEDI1191 in patient tumor slices all also came from the same gene families as TH1 genes upregulated in mIL-12 mRNA-treated tumors (e.g. human CXCL10 vs mouse Cxcl9).

Discussion

We report here that in syngeneic mouse tumor models and patient tumor slice cultures IL-12 mRNA drives TH1 transformation of the tumor microenvironment. In mouse models this leads to IFNγ and cytotoxic T cell-dependent anti-tumor immunity that is further enhanced by PD-L1 blockade. A single IT dose of mIL-12 mRNA induced dose-dependent mIL-12p70 expression in syngeneic tumor-bearing mice. Similarly, MEDI1191 induced robust, dose-dependent hIL- 12p70 protein production in all patient tumor-derived models tested, including 2 melanoma and 2 HNSCC PDX models and 7 colorectal and endometrial cancer patient tumor slice cultures. Our data indicate that IL-12p70 expressed in mouse syngeneic models and PDX models is likely derived from both cancer cells and tumor-infiltrating myeloid cells transfected with mRNA. Both human macrophages and human tumor cell lines produced hIL-12p70 in response to LNP-formulated MEDI1191 in vitro. It is difficult to directly determine which cells in the TME are responsible for IL-12 mRNA-induced IL-12p70 production in vivo, since IL-12p70 is a secreted protein. However, we recently reported that IT delivery of LNP-formulated mRNA encoding transmembrane OX40L in syngeneic tumors led to protein translation in cancer cells, tumor-infiltrating myeloid cells and tumor draining lymph node myeloid cells (23). Repeated doses of IL-12 mRNA continued to induce IL-12 expression and IFNγ release, but did not significantly enhance the anti-tumor activity of mIL-12 mRNA. This may reflect the limitations in using rapidly growing syngeneic models to address questions of dose and schedule. Alternatively, this may suggest that repeated IT injection of IL-12 mRNA as a monotherapy in the same tumor has limited benefit. Indeed, whilst we found that the anti- tumor response to a single dose of IL-12 mRNA requires IFNγ, others have reported that chronic IFNγ can lead to upregulation of immune checkpoints, T cell dysfunction and impaired anti-tumor immunity (38).

mRNA-derived IL-12p70 protein drove TH1 TME transformation, consistent with the expected activity of IL-12p70 as a key driver of type 1 immune responses (2). In syngeneic mouse tumors, early tumor NK activation after a single dose of mIL-12 mRNA was followed by dose-dependent increases in IFNγ expression, DC activation, and upregulation of a broad range of TH1 response genes. TH1 TME transformation in syngeneic mouse tumors led to dose-dependent tumor regression, complete responses and induction of anti-tumor immunity via a mechanism requiring both IFNγ and CD8+ T cells. The activity of mIL-12 mRNA observed in multiple tumor models suggests IL-12 mRNA therapy has the potential to benefit patients with varying TME composition. MEDI1191 also drove TH1 gene expression in patient tumor slices, building confidence in the potential for translation of this therapeutic approach to a clinical setting. IFNγ expression in MEDI1191-treated slice cultures correlated positively with baseline NK infiltration, but not with baseline CD3+ T cell infiltration. Detailed T cell subset analysis might reveal the contribution of T cells to IFNγ release in this model (e.g. separating Treg, CD4+ effector and CD8+ cytotoxic T cells). Analysis of a larger number of patient tumor samples might reveal a contribution of T cells to IFNγ production in a subset of tumors. In addition, this ex vivo system likely underestimates the contribution of T cells due to the short experimental time-course and the lack of modelling of immune cell migration between tumor, lymph node and blood. Importantly, a single dose of mIL-12 mRNA also promoted regression of untreated lesions in a dual flank MC38-S model, consistent with the ability of mIL-12 mRNA to promote systemic anti-tumor immunity. The hypothesis that IT mIL-12 mRNA drives true abscopal activity was supported by the discovery that mRNA encoding membrane-tethered IL-12 induced complete regressions in un-injected distal tumors, albeit with reduced potency versus mRNA encoding secreted mIL-12. It is therefore likely that the abscopal effects are due to the IL-12 mRNA-induced expansion of tumor-specific T cells observed in the periphery of tumor-bearing mice, rather than due to induction of circulating IL-12p70. IT delivery of an adenovirus-encoded, membrane-tethered IL-12 also inhibited growth of distal CT26 mouse colorectal cancer lesions in a dual flank model (39). Abscopal effects of IT IL- 12 plasmid plus electroporation have been observed in un-injected lesions in metastatic melanoma patients in the absence of detectable elevations of circulating IL-12 (20,40). Whilst a dual flank syngeneic model does not fully recapitulate the complexities of disseminated cancer in patients, these data highlight the potential for locally delivered MEDI1191 to drive immune-mediated regression of distal lesions in patients with metastatic disease.

Our preclinical data support the hypothesis that MEDI1191 might expand the utility of PD-1/PD-L1 blockade to some patients currently resistant to ICB (1). mIL-12 mRNA monotherapy drove regression of ICB-resistant MC38-R mouse tumors, and combination with PD-L1 blockade enhanced anti-tumor immunity in this model. These results are in agreement with improved anti-tumor activity of both an IL-12 immunocytokine and an IL-12- expressing oncolytic virus in combination with anti-PD-1 or anti-PD-L1 in an otherwise PD-1 / PD-L1 blockade-resistant MC38 model (15,41). Our data suggest that mIL-12 mRNA may overcome PD-L1 resistance by bypassing the need for tumor DC-derived IL-12 in MC38-R tumors, priming an anti-tumor immune response that can be enhanced through blockade of PD-1 signaling by de novo expressed PD- L1 in the TME. IL-12 release from tumoral DC was reported recently to be required for effective anti-PD-1 immunotherapy in a PD-1 blockade-sensitive MC38 model (42). We reported previously that the PD-L1 blockade-resistant MC38-R model has a smaller DC infiltrate compared to the PD-L1 blockade-sensitive MC38-S model (23). Here we demonstrate that IT mIL-12 mRNA alone increased tumoral cytotoxic T cell infiltration in the MC38-R model, expanded peripheral tumor-specific T cells and increased PD-L1 expression in the TME. Combination with PD-L1 blockade further enhanced cytotoxic T cell recruitment to MC38-R tumors and increased the incidence of tumor regression. In the ICB-resistant B16F10-AP3 model which has a particularly small immune infiltrate (23,36), mIL-12 mRNA had significant monotherapy activity, but mIL-12 mRNA plus PD-L1 blockade combination therapy only provided a modest, non-significant survival benefit versus mIL-12 mRNA alone. Notably, mIL-12 mRNA also did not drive peripheral expansion of tumor-peptide reactive T cells in B16F10-AP3 tumor-bearing mice. Thus, different, unknown mechanisms may be driving ICB resistance in the B16F10-AP3 model versus in MC38-R. In patients, a wide variety of mechanisms are emerging as drivers for primary and adaptive resistance to ICB (43), and combinatorial therapeutic approaches will likely be required to overcome resistance in these diverse patient populations. The development of strategies for tumor-targeted IL-12 delivery has been driven by extensive preclinical evidence that IL-12 can drive anti-tumor immunity, combined with the poor tolerability and limited efficacy of systemic recIL-12 in early clinical trials (11,12). Tumor-targeted IL-12 delivery may improve this therapeutic margin by driving local IL-12 exposure (required for anti-tumor immunity) whilst limiting circulating IL-12 exposure (likely reducing undesirable toxicity).

Various strategies have therefore promoted tumor-targeted IL-12 delivery and they have all demonstrated promising activity in mouse tumor models (14-17,41,44,45). Systemically administration of tumor-targeted IL-12 would avoid the complexity of IT injection in patients. However, systemically delivered IL-12 immunocytokines lead to high peripheral versus tumor IL-12 protein exposures, and a potentially still narrow narrow therapeutic window in patients (46). Delivery of IT IL-12 plasmid plus electroporation (tavokinogene telseplasmid) in metastatic melanoma patients has reported clinical activity (20,40). However, a separate electroporation device is required, limiting this therapeutic approach to patients with cutaneous or subcutaneous lesions (47). An LNP-formulated mRNA therapy can be delivered electroporation which expands the potential of IT administration to patients with deep-seated lesions. Following IT viral IL-12 delivery, the challenge of maintaining tight control over IL-12 expression has lead to complex genetic switch technology whereby a second orally dosed small molecule controls IL-12 expression (17). In contrast, the limited half-life of LNP-formulated mRNA used here (25) leads to controlled and transient protein production, as demonstrated in multiple syngeneic and PDX models. MEDI1191 was therefore developed as an optimized IL-12 mRNA for IT delivery. A research version of LNP-formulated IL-12 mRNA, delivered systemically, inhibited tumor growth in a mouse model of hepatocellular carcinoma (44). However, higher protein expression was observed in normal liver tissue and spleen than in tumors, indicating that this approach was insufficient to target IL-12 protein expression to tumors. Improving on this early research version, a miR122-binding site was incorporated in the MEDI1191 mRNA 3′UTR to minimize peripheral IL-12 expression in miR122-positive normal hepatocytes (22). In addition, MEDI1191 incorporates aspects of a novel LNP formulation that demonstrates improved pharmacokinetics and better local mRNA endosomal escape and translation (25). These advances, in conjunction with direct intratumoral injection, aim to provide a clinically viable therapeutic window between efficacy and toxicity, and have enabled testing of MEDI1191 in patients. In conclusion, the robust preclinical activity reported here of MEDI1191 (IL-12 mRNA) and its mouse surrogate provide support for the development of MEDI1191 as a potential novel treatment for patients with both superficial and deep-seated solid tumors, alone and in combination with inhibitors of the PD-L1 / PD-1 checkpoint. IT MEDI1191 is currently being evaluated in a phase I trial in patients with solid tumors in combination with durvalumab (anti–PD-L1, NCT03946800).

Authors’ Contributions

Conceptualization, A.L., J.P.F., N.L., R.H., R.W.W., S.G., S.L.H.; Methodology, A.L., A.M., A.W., C.B., D.B., F.G., J.Z., K.A., M. Sulikowski, N.L., R.K., S.B., S.L.H.; Investigation, A.A., A.M., A.W., C.B., D.B., D.P., E.C-T., F.G., F.M., G.A., G.M., J.M., J.M.L., J.Z., K.D., K.M., M. Sulikowski, M. Sedic, P.M., R.K., S.B., S.L.H.; Formal Analysis, A.M., A.W., C.B., C.M., D.B., E.C-T., F.G., H.S., J.M., J.M.L., J.Z., K.A., K.D., K.M., M. Sulikowski, N.L., P.M., S.B., S.L.H., S.N.; Writing – Original Draft, N.L., S.L.H.; Writing – Review & Editing, J.P.F., N.L., R.H., R.W.W., S.L.H.; Visualization, A.A., A.M., A.W., D.B., E.C.T., F.G., F.M., J.M.L., J.Z., K.A., K.D., N.L., P.M., R.K., S.B., S.L.H.; Supervision and Project Administration, J.P.F., K.A., N.L., R.H., R.W.W., S.G., S.L.H.

Acknowledgments

Support for mRNA manufacture and LNP formulation was provided by Moderna Inc, in particular Sarah Peterson, Mengfei Sun and Weijia Wang. We acknowledge support from teams within Moderna including Toxicology and Pathology and Non-clinical Sciences, and from teams within AstraZeneca including IVS and Core Tissue Culture. We thank Jill Grenier, Philippe Garnier and Lisa Johansen for program management.

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Figure Legends

Figure 1. A single intratumoral injection of mIL-12 mRNA induces dose-dependent tumor regression in multiple syngeneic models. A, Design of IL-12 mRNA constructs for

IT injection. B, IL-12 protein secretion by ELISA from cells transfected with mIL-12 mRNA by lipofectamine, with inclusion of a miR122 binding site. Asterisks indicate statistical significance by ANOVA. C and D, Time-course of mIL-12p70 protein in MC38-R tumors. (C) or circulating plasma (D) by 5-multiplex panel following IT injection of LNP-formulated mIL-12 mRNA, or LNP-formulated control nontranslating mRNA (mean ± SD). E–G, Survival curves and complete regressions of established tumors in three subcutaneous syngeneic models following IT mIL-12 mRNA, with individual tumor volumes shown for MC38-S (F, bottom panel). A20 dosed day 18, MC38-S day 12 and MC38-R day 11 post- implantation. Asterisks indicate statistical significance between dose levels, log-rank test: control mRNA to 0.05 µg, 0.05 µg to 0.5 µg, 0.5 µg to 5 µg; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001. H, Protective immunity after CRs rechallenged with the same tumor type; numbers indicate animals with no tumor growth after second implantation (out of total rechallenged; A20 and MC38-S one experiment, MC38-R five experiments combined). Data representative of three (G), two (B–D and F), one (E) or up to five (H) independent experiments. IT = intratumoral, CR = complete responder, out of group size.

Figure 2. CD8+ T cells are required for optimal mIL-12 mRNA anti-tumor activity. Response of MC38-R tumor-bearing mice to IT treatment with a single dose of 0.05 or 0.5 µg mIL-12 mRNA, or control mRNA. A, Number of tumoral CD8+ T cells. B, Number of tumoral CD4+ FoxP3– effector T cells. C, Number of tumoral CD4+ FoxP3+ regulatory T cells (Treg). D, Ratio of CD8+ T cell / Tregs in tumors. Analyses by flow cytometry, points represent individual samples and bars are mean ± SD of n ≥ 6 animals. Asterisks indicate statistical significance between groups: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 by ANOVA (A–D). E, Microarray analysis of T cell mRNA levels upregulated > 2 fold following 0.5 µg mIL-12 mRNA versus 5 µg control mRNA treatment 24h and /or 7d post-dose. Columns represent individual mice, rows represent genes. False discovery rate < 0.05, n ≥ 7 per treatment per time point. F, Effect of CD8α+ or CD4+ cell–depleting antibodies combined with mIL-12 mRNA treatment dosed d13 post-implantation on survival (top panel) or individual tumor volumes (bottom panel). Antibody treatment regime detailed in Fig. S2B. Control mRNA dosed at 0.5 µg (A–D and F) or 5 µg (E). Data representative of two (A–D) or one (E and F) independent experiments. H = hours, d = days post treatment.

Figure 3. mIL-12 mRNA-induced anti-tumor immunity is IFNγ-dependent and associated with TH1 transformation of the tumor microenvironment. Response of

MC38-R tumor-bearing mice to IT treatment with a single dose of 0.05 or 0.5 µg mIL-12 mRNA, or control mRNA. A, Percent of tumoral NK cells positive for CD69. B and C, Time- course of IFNγ protein in tumors (B) or circulating plasma (C), 5-multiplex panel (mean ± SD). D and E, Effect of IP IgG1 isotype or IFNγ–blocking antibody (d11, 12, 13 (0.5 mg) and d17 (0.2 mg) post-implantation) combined with 0.5 µg mIL-12 mRNA treatment (d13 post- implantation) on individual MC38-R tumor volumes (D) or survival (E). Statistical significance by log-rank test, ns = not significant. F, Effect of NK cell depletion by anti- NK1.1 with mIL-12 mRNA treatment on individual tumor volumes. Vertical dashed line = IT mIL-12 or control mRNA dosed day 11, diagonal dash line = slope of tumor growth for untreated tumors. G, Microarray analysis of significant changes ≥ 2 fold in mRNA levels following 0.5 µg mIL-12 mRNA versus 5 µg control mRNA treatment 24h and/or 7d post- dose. Columns represent individual mice; rows represent genes. False discovery rate < 0.05, n ≥ 7. H, Percent of tumoral CD103+ cDC1 cells positive for CD86. Analyses by flow cytometry, statistical significance by ANOVA (A and H). Control mRNA dosed at 0.5 µg (A, D–E, F and H) or 5 µg (B–C, G). Data representative of two (A–C and H) or one (D–G) independent experiments.

Figure 4. Anti-tumor immunity induced by mIL-12 mRNA is enhanced by PD-L1 blockade in the ICB-resistant MC38-R model. A, PD-L1 expression in MC38-R tumors by IHC, 7d post 0.5 µg mIL-12 or control mRNA treatment. PD-L1 expression in endothelial cells in blood vessels (arrow head), and in immune infiltrates (arrows). N = 8/group. B, Percent of Ly6Chi monocytes positive for PD-L1 in MC38-R tumors by flow cytometry after IT delivery of mIL-12 or control mRNA treated on day 11. C, Survival of MC38-R tumor- bearing animals after IT administration of mIL-12 or control mRNA, alone or in combination with isotype antibody control or anti–PD-L1 (20mg/kg IP, days 11, 14, 18, and 21; mRNA day 11 post-implantation). N = 15/group, asterisks indicate statistical significance by log-rank test. D, IFNγ ELISPOT quantification of p15E peptide reactive T cells in splenocytes isolated from MC38-R tumor-bearing mice 14d post-implantation, after a single IT dose of 0.5 µg control or IL-12 mRNA on day 7, alone or with αPD-L1 (10 mg/kg IP, days 7 and 10). N = 8/group. E and F, CD8α staining in MC38-R tumors 14d post-implantation with representative images (E) and quantification (F), after a single IT dose of 0.5 µg control or IL-12 mRNA on day 7, alone or with αPD-L1 (10 mg/kg IP, days 7 and 10). N = 8/group. G, Dense CD8+ infiltrate (arrows) surrounding a necrotic tumor core [Nec] in mIL-12 mRNA + αPD-L1 combination treated samples with no viable tumor remaining: detected in 4/8

of one (A, E–G) or two (B–D) independent experiments. IP = intraperitoneal.

Figure 5. Locally administered mIL-12 mRNA drives regression of uninjected distal lesions and anti–PD-L1 potentiates this response. A and B, Dual tumor model with scheme of mIL-12 mRNA and anti–PD-L1 treatments (lower panel, a), treated and distal MC38-S tumor volumes (upper panel, A), or survival (B) after a single IT administration of mIL-12 or control mRNA alone or in combination with isotype control or anti–PD-L1 antibody (20 mg/kg IP, days 13, 17, 20, and 24; mRNA d13). CRs refer to combined tumor volume (A and B), n = 20, asterisks indicate statistical significance by log-rank test. C, Design of tethered mIL-12 mRNA construct. D, IL-12 protein expression in HeLa cell lysate and supernatant, after mRNA transfection. nd = no data, below limit of detection. E, IFNγ production from activated CD8+ T cells following co-culture with either recombinant mIL-12 or tethered mIL-12 mRNA- transfected HeLa cells. F, Volumes of MC38-S tumors treated IT with 5 µg secreted or tethered mIL-12 mRNA on day 13, or controls as in (G). Asterisks indicate statistical significance of survival between control mRNA and mIL-12 mRNA treated groups, log-rank test. G and H, Circulating mIL-12p70 (G) or IFNγ (H) protein by 36-multiplex panel in plasma following IT delivery of mIL-12 or control mRNA. Data representative of one (A and B) or two (D to H) independent experiments.

Figure 6. MEDI1191 induces dose-dependent IL-12p70 protein release, IFNγ production and increased TH1 gene expression in ex vivo patient tumor slice cultures. A–C, Quantification of IL-12p70 (A), IFNγ (B) and CXCL10 (C) in supernatants of ex vivo cultured tumor slices from patients with colorectal adenocarcinoma (CRC), colorectal liver metastases (CRLM) and endometrial carcinoma (UCEC) treated with control mRNA or MEDI1191. Each point represents the average of two to three slices from a single patient tumor. LLOD is lower limit of detection. Statistical analysis on log-transformed data using a Friedman test with Dunn’s multiple comparison post-hoc test. D and E, Representative images of immunohistochemical staining for CD3 (D) and NKp46 (E) in patient tumor slices collected prior to ex vivo culture. F and G, Quantification of total number of CD3+ (F) and NKp46+ (G) cells in whole tissue slices compared to IFNγ production from slices of the same tumors for CRC, CRLM and UCEC tumors. Data are mean ± SD. Best fit line is shown. H, mRNA levels of indicated transcripts from the tumor slices treated with MEDI1191. Data are

mean of 4 tumor samples, showing all measured genes that were upregulated >1.5 fold (with P < 0.05) versus control mRNA (770-gene PanCancer IO 360TM panel, Nanostring).

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Treated 0 25 50 75 100

) 0

3 Days post implant

2000 m

m Secreted mIL-12 mRNA (

1500

* e

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tumor

* *

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* o 5 µg + PD-L1 *

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PD-L1 * T 20 40 60 80 100 20 40 60 80 100 20 40 60 80 100 20 40 60 80 100 5 µg Control mRNA + isotype Days post implant

HeLa cells + IT mIL-12 IP αPD-L1 D Supernatant Lysate E Recombinant IL-12 Tethered IL-12

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Figure 6

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F G Colorectal 2500 2500

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n * *

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Intratumoral interleukin-12 mRNA therapy promotes TH1 transformation of the tumor microenvironment

Susannah L Hewitt, Dyane Bailey, John Zielinski, et al.

Clin Cancer Res Published OnlineFirst August 17, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/1078-0432.CCR-20-0472

Supplementary Access the most recent supplemental material at: Material http://clincancerres.aacrjournals.org/content/suppl/2020/08/15/1078-0432.CCR-20-0472.DC1

Author Author manuscripts have been peer reviewed and accepted for publication but have not yet Manuscript been edited.

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