NUVEC. Non-viral adjuvant delivery system for vaccines and cancer treatments. Allan Hey, Head of CMC, N4 Pharma Ltd

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1 NUVEC Non-viral adjuvant delivery system for vaccines and cancer treatments Allan Hey, Head of CMC, N4 Pharma Ltd 1

2 N4 Pharma plc o Established in 2014 o Listed on Alternative Investment Market (AIM) in 2017 o Focussed on developing Nuvec as an enabling technology for immuno-oncology 2

3 Introduction: Immuno-Oncology o Cancers survive by remaining hidden from the immune system o May be recognised, but without an effective Th1 immune response o Immunotherapy works by waking up the immune system to the threat o Checkpoint inhibitors (antibodies to PD-1/PD-L1) stop inhibition of immune response o Adoptive cell transfer (e.g. CAR-T) boosts numbers or response of patient s own T-cells o Cancer vaccines work by presenting cancer antigens in a more immunogenic setting o Need to induce a Th1 immune response o Often DNA or mrna-based o Bias towards RNA in recent times (safety concerns for DNA-based vaccines) 3

4 Delivery of nucleic acid vaccines o Three basic problems o Nucleases in extracellular space will digest unprotected nucleic acids o Getting a negatively charged, hydrophilic molecule past the negatively charged, hydrophobic cell membrane o Ensuring/enhancing immunogenicity of products o Lipid-based nanoparticles are current standard: o Protect nucleic acid from nucleases o Can be produced with cationic outer membrane to allow direct cell entry o Some disadvantages: o Chemically and physically unstable difficult to make and store o Aqueous core not ideal to ensure stability of nucleic acids, particularly mrna o Often still have to get nucleic acids past a membrane, in order to load them o Often require additional adjuvant/immunomodulator to ensure immunogenicity of product o Cationic lipids have been shown to cause toxic effects From: Islam, M et al (2015). Biomaterials science / c5bm00198f. 4

5 What is Nuvec? o Non-viral delivery particle for cancer immunotherapy and viral vaccines o Silica nanoparticles (180 nm) with unique (hairy, spiky) structure coupled with a suitable polycation such as polyethyleneimine (PEI) to allow binding of nucleic acids o Economic, scalable manufacturing process o High capacity loading o Simple / efficient formulation o Well tolerated even at high doses o Inherent adjuvant activity opotential use of similar particles for peptides, proteins and other drugs 5

6 Nuvec Manufacture 6

7 Manufacturing of Nuvec 1. Synthesis of initial resorcinol formaldehyde (RF) core 2. Addition of Tetraethyl Orthosilicate (TEOS) and further RF to form structures 3. Calcination at 550 C to produce hollow sphere with spikes 7

8 Manufacturing of Nuvec o Successfully scaled up from 500mg to 17g batch production, further scale-up relatively simple o Excellent particle characterisation o Uniform shape and structure by Scanning Electron Microscopy (SEM) o Transmission Electron Microscopy (TEM) analysis shows hollow core with silica spikes o Coupled with PEI to give surface positive charge at neutral ph o DNA or RNA loading achieved by simple co-incubation in PBS 8

9 How does Nuvec work? 9

10 Nuvec mechanism of action to deliver DNA/RNA 1. Topography & PEI protect pdna or mrna from nuclease digestion 5. Protein antigen expression (peptides loaded onto MHC Class I & II molecules on antigen presenting cells) 2. Cellular uptake (general and dynamic dependent endocytosis) --Nuvec --pdna or mrna --Protein antigen 3. Endosome escape (PEI loaded on Nuvec enables rupturing of endosome) 4. mrna released in cytoplasm, pdna delivered to the nuclei 10

11 In vivo Pharmacology 11

12 Transfection of luciferase plasmid in vivo o Female mice injected subcutaneously with Nuvec containing Luciferase-pcDNA3 plasmid DNA (1:10 DNA:Nuvec ratio) o Nanoparticle-associated bioluminescence (NABL) detected at two time-points (24 hr and 48 hr) after injection. o Signal for at least 48 hours, remains local to injection site 12

13 pdna-ova loaded Nuvec administered s.c. to induce OVAspecific immune responses 1 st Injection 2 nd Injection 3 rd Injection Spleen and Blood collection (for OVA re-call assay and serum antibody assay) Day 0 Day 7 Day 14 Day 28 Compound ID PBS pdna-ova pdna-ova/in vivojetpei pdna-ova/nuvec (25 mg/kg Nuvec ) pdna-ova/nuvec (50 mg/kg Nuvec ) pdna-ova/nuvec (75 mg/kg Nuvec ) Nuvec (75 mg/kg Nuvec ) pdna:nuvec ratio N/A N/A N/A 1:10 1:20 1:30 N/A 50 µg pdna-ova s.c. injection in 1:10, 1:20, 1:30 ratio of Nuvec (Nuvec-DNA 25 mg/kg, 50 mg/kg, 75 mg/kg) Dose 100 µl Serum samples diluted in certain ranges and analysed by ELISA for OVA specific total IgG/IgG 1 /IgG 2a determination 13

14 Normalised to PBS Vehicle only OVA-specific IgG 1 and IgG 2a OVA-specific IgG 1 and IgG 2a antibodies (@ 1/200 serum dilution) Increase in OVA-specific IgG 1 and IgG 2a observed with increasing dose of Nuvec, even above in vivo-jetpei control note that Nuvec promotes IgG 2a more than IgG 1, indicating a good Th1 immune response IgG1 IgG2a Group (n=6/group) 14

15 Cytokine pg/ml (Mean ± SEM) Th1 vs Th2 response: Cytokine profile IL-5 and IFNɣ response to OVA challenge in isolated spleen cells Cytokine analysis shows dose dependent secretion of IFN gamma, but no increase in Il-5, indicating robust dose-dependent Th1 response IL-5 IFNɣ 0 15

16 Nuvec Toxicology 16

17 Si (ppb w/v, Mean±SD) Si measured at injection site after 50 mg/kg s.c. injection 7000 Silicon in Rat Injection Site by ICP-MS 6000 Increased Si detected at Injection Site versus Vehicle Control at Day 3 but almost completely gone by Day Group (n=3/group) 17

18 Si measured in plasma after 50 mg/kg s.c. injection Si is detected in Plasma but levels remained similar to Vehicle Control throughout the duration of the study 18

19 Nuvec detected by TEM in plasma 1hr 2hr 4hr Nuvec detected in plasma up to Day 3 following s.c. administration at 50 mg/kg, beyond which no Nuvec could be positively identified D2 D3 D7 D15 D29 D57 Note: Dark surface-like 1hr Control structures on some of the plasma TEM images are the edges of the squares on TEM grids, where the particles seemed to be congregating in many cases. 8hr 19

20 Summary of toxicology findings to date o No major toxicology findings across rat/mouse in vivo and PBMC/spleen in vitro studies. o Single high-dose (up to 900 mg/kg) s.c. administration of SiNPs in rats well tolerated, with no dose-limiting toxicity o The level of SiNP and PEI in Nuvec seems well tolerated when locally administered even at doses much higher than used for transfection in vivo o Localised dose dependent granuloma formation at injection site. o Histopathology confirms immune cell infiltrate is localised mainly to injection site and the draining lymph node (adjuvant-like effect). o No evidence for systemic inflammation in liver or kidney. o Transient spill over of pro-inflammatory cytokines (IL-6, TNF-a) and monocyte and T cell chemokines detected in plasma at high doses. o Dose dependent increase in platelet aggregation observed in human blood but not at all efficacious doses. 20

21 Conclusions 21

22 Nuvec is a novel nucleic acid delivery system for vaccines and therapeutic applications o Shows great promise as a non viral delivery vehicle for pdna/mrna o Key advantages compared to lipid and viral systems o High capacity loading and protection of nucleic acid o Potential to be dose-sparing o Excellent localised in vivo transfection efficiency o Subcutaneous/Intratumoural routes o Well tolerated even at high doses in rats o No induction of high levels of inflammatory cytokines o Immune response observed for both pdna and mrna o Th1 response suggests good potential for cancer immunotherapy 22

23 Thank you for your attention! Website: 23