de Gunst, T., Yahyanejad, S., den Boer, H., Vos, R., Alemdehy, F., Telford, B., van den Bosch, M., Stegink, M., van Pinxteren, L., Schaapveld, R. & Janicot, M.

InteRNA Technologies B.V., Life Science Incubator. Yalelaan 62. Utrecht. The Netherlands

A novel lipid-based nanoparticle formulation mediating safe and robust functional delivery of siRNA/miRNA

abstract poster #27

OTS 2019Munich, Germany

Do not miss poster #138

Correspondence to: degunst@interna-technologies.com

Mounting evidence suggests that small interfering RNA- (siRNA) and microRNA- (miRNA) basedtherapies hold great promise for development of therapeutic modalities. However, despite the earlypromise and exciting potential, several specific technical barriers (e.g., extracellular and intracellularstability as well as effective and safe delivery) still need to be overcome. While unmodified RNAs arerapidly degraded in circulation, chemically modified versions hardly penetrate cells and require anadditional delivery system to allow intracellular molecular effects. New strategies overcome the coreobstacles to cellular delivery of siRNAs/miRNAs which include: (i) ribonuclease- (RNase) mediateddegradation; (ii) short biological half-life; (iii) lack of endosomal escape; (iv) lack of tissue targeting; (v)inefficient biodistribution; and (vi) side effects. InteRNA Technologies has pursued new approaches toidentify a robust drug delivery modality for its drug candidate, INT-1B3 (formulated miR-193a-3pmimic) which is expected to enter soon into first-in-human clinical evaluation. During the last decade,InteRNA has tested various delivery technologies using in vivo screening in which formulated siRNAtargeting reference gene HPRT-1 was administered (bolus i.v.) to human melanoma A2058 tumor-bearing immune compromised mice. Tissue samples from spleen, liver, and tumor were evaluated fortissue distribution and pharmacodynamic (target engagement) effects. In addition, safety was assessedby cytokine expression in blood, and blood chemistry.

LNP SCREENING SAFETY OF LNP IN VIVO

✓ InteRNA Technologies has identified a novel lipid-based nanoparticle (LNP) which safely and effectively delivers the si-/miRNA to tumor tissues and tissue beyond liver

✓ This formulation (4.2) was selected for further development and became a key component of INT-1B3 (formulated 1B3)

✓ An in vitro experimental approach to test various chemical modifications led to improved metabolic stability and target recognition

✓ Efficacy of INT-1B3 has been evaluated upon systemic administration in experimental tumor-bearing mouse models

✓ Significant tumor growth inhibition has been demonstrated at well-tolerated doses in a large panel of human and syngeneic tumor models

✓ A full PK/PD and safety profile has been assessed in mice

✓Optimization of large-scale production of drug product has allowed IND-enabling GLP-Tox studies in rats and non-human primates to support further clinical development

100nm

Effect of repeated (QDx2) i.v. bolus administrations of INT-1B3(10 mg/kg/admin.) on cytokine expression in the plasma ofmurine TNBC 4T1 orthotopic tumor-bearing immune-competent mice.

LIPID NANOPARTICLE

1B3 SCREENING

INTRODUCTION

1B3 : chemically-modified miR-193a-3p mimic

(miR-193a-Iv11)

SCREENING FORMULATION COMPOSITION

Sequence and chemical modification patterns as compared to

1B3 (−) indicates same nucleotide as 1B3 (Iv11); (◼) indicates

2’-O-methyl nucleotide; (◼) indicates 2’-fluoro nucleotide; ()

indicates phosphorothioate bond; and (◼) indicates

nucleotides in the seed sequence.

✓ Preferred delivery and superior target

engagement of formulation # 4.2 in

tumor samples (in comparison to

clinically tested formulation # 1.0)

PBS 1.1 3.2 4.2 6.01.0 3.1 4.1 5.0

Target engagement

CONCLUSIONS

A2058 tumor cells were implanted at day 0, animals wererandomized at mean TV of 100mm3 and treatment wasinitiated with a QDx2 for two weeks. Animals weresacrificed at day 14.

PBS

INT-1

B3

(788

)

INT-1

B3

(799

)

INT-1

B3

(808

)

INT-1

B3

(810

)

INT-1

B3(

812)

1000

2000

Tum

or

wei

ght

( m

g )

( M

edia

n w

ith

i.q

ra

ng

e )

PBS 4.2.1 4.2.2 4.2.3 4.2.4 4.2.5

10 ( mg/kg/admin. )

TGI in Melanoma (A2058)

✓ Confirmation of LNP-subtype

Cytokine expression

Anti-tumor efficacy in syngeneic tumor models

siRNA delivery

In the last decades, development in the chemistry ofdouble-stranded RNAi formulations enabled newstrategies to overcome the core obstacles to cellulardelivery of siRNA/miRNA molecules, which include: (i)poor ribonuclease (RNase) resistance; (ii) shortbiological half-life; (iii) lack of tissue targeting; (iv)inefficient cellular uptake; and (v) undesirable toxicity.

Electron Microscopy

✓ No significant immune activation (cytokine storm)

✓ Transient mild upregulation of IL-6 (and CCL2, data not shown) and mirror image transient decrease IL-13

▪ Well tolerated dosing for 8 weeks (data not shown)

▪ IND-enabling GLP-Tox ongoing

Hep3b tumor fragments were implanted at day 0, animals wererandomized at day 21 based on plasma AFP when treatmentwas initiated with a QDx3 in the first week and Q3D (mon/thu)for 3 more weeks. Animals were sacrificed at day 49.

✓ Confirmation of Lipid Nanoparticle

PBS 1.0 4.2.1 4.2.1 4.2.1 SoC 6.0

1 3 10 ( mg/kg/admin. )

TGI in HCC (Hep3B)

PBS

NO

V340

3mg/k

g

INT1B

3 1m

g/kg

INT1B

3 3m

g/kg

INT1B

3 10

mg/k

g

Sorafe

nib D

22

SOS 3

mg/k

g

0

2000

4000

6000

Tum

or w

eigh

t(

mg

)( M

edia

n w

ith

i.q r

ange

)

INT-1B3 IN VIVO VALIDATION

Representative electron microscopy image of INT-1B3 presented on the left side is associated to a schematic view of the various elementsof a Lipid Nanoparticle-formulated oligonucleotide on the right side. Composition of the LNP formulation is detailed in the table below.This example image is taken from http://www.arbutusbio.com/LNP%20Illustration.jpg

Murine tumor cells were injected in the flank ofcorresponding immuno-competent syngeneic mice.When subcutaneous tumors reached a volume of ~100-mm3 (established tumor models), treatment witheither PBS () or 10 mg/kg/admin. INT-1B3 () via i.v.bolus dosing on a twice weekly schedule, and up to 8administrations (CrownBio’s MuScreenTM format) wasinitiated. Animals were euthanized according todefined humane criteria. (A) pancreatic Pan02 tumormodel. (B) HCC H22 tumor model. (C) Lewis Lung LL/2tumor model. (D) colon MC-38 tumor model. Resultsare presented as median with interquartile range.

✓ INT-1B3 treatment resulted in asignificant and a potent tumor growthinhibition (TGI) on the primarysubcutaneous tumors in the varioussyngeneic tumor models presented.

API : lipid ratio ~ 1 : 10.3

% molar ratio % mg lipids/mL

XL-10 40 % 48 %

DSPC 10 % 14 %

Cholesterol 48 % 30 %

MPEG 2KDa-DSG 2 % 8 %

% are based on a molar ratio

siH

PR

T-1

siH

PR

T-1