Spherical (SNA) TLR9 Agonists Induce Long-Term Tumor- Specific Immune Responses In Synergy With PD-1 Checkpoint Inhibition Bart R. Anderson, SubbaRao Nallagatla, Richard S. Kang, and Ekambar R. Kandimalla Exicure, Inc., 8045 Lamon Avenue, Skokie, IL 60077

INTRODUCTION RESULTS Toll-like receptor 9 (TLR9) belongs to the family of pattern recognition SNA Is Taken Up More Than Linear

receptors in the innate immune system and is predominately expressed in )

human B cells and plasmacytoid dendritic cells (pDCs). CpG dinucleotides A Linear B . U

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l 2 5 F compared to conventional “linear” oligonucleotides that are not in SNA n 0 configuration. SNAs enter cells and localize to endosomes, which is where SNA structure a e 1 1 0 TLR9 are localized, making SNAs ideal TLR9 agonists. M [F lu o re s c e in -la b e le d o lig o n u c le o tid e ] (µ M ) Immune checkpoints are inhibitory pathways crucial for maintaining self-tolerance and modulating Figure 1. Cellular uptake of SNA and linear oligonucleotides. (A) Primary human foreskin keratinocyte (HFK) cells were treated with 100 nM Cy5-labeled oligonucleotide in linear or SNA format. At the duration and amplitude of physiological immune responses. However, tumors use immune- 24 hours the uptake was assessed by fluorescence microscopy. checkpoint pathways, particularly the PD-1 / PD-L1 pathway, as a major mechanism of immune (B) Healthy human volunteer peripheral blood mononuclear cells (PBMCs) were isolated from whole blood. PBMCs were treated for resistance. 24 hours with fluorescein-labeled oligonucleotide in linear or SNA format. Flow cytometry was used to assess the uptake of oligonucleotides, and was performed in the presence of 2 mg/mL trypan blue to quench extracellular fluorescein. Mean ± SEM of Here, we investigated the ability of TLR9-agonist SNAs to synergize with an anti-PD-1 checkpoint N=4 PBMC donors is shown. 2-way RM ANOVA with Holm-Sidak's multiple comparisons correction -values: ** < 0.01, *** < 0.001, inhibitor to produce long-term, specific anti-tumor immunity. **** < 0.0001. TLR9-Agonist SNA Induces TLR9-Dependent TLR-Agonist SNA Induces Immunostimulation in Non-Human Primates TLR9-Agonist SNA Treatment Upregulates Immunostimulation In Mice PD-1 and PD-L1 Expression in EMT-6 Tumors A B 1 0 0 IFN  4 0 IP -1 0 4 0 0

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p P 5 0 0 0 0 H o u r s p o s t-a d m in is tr a tio n 5 0 0 k k k k k k P B S S N A a n ti-P D -1 S N A + p p p p p p m m m m m m a n ti-P D -1 .3 1 3 .5 6 3 0 4 0 0 Figure 4. PD-1 axis expression in EMT-6 tumors S N A , 3 m g /k g C o n tro l S N A , 3 m g /k g T L R 9 -K O W T T L R 9 -K O W T S N A C o n to l On study day 0, BALB/C mice were subcutaneously inoculated S N A with EMT-6 cells to establish flank tumors. On study day 10 Figure 2. Cytokine induction by SNAs in WT and TLR9-KO Figure 3. Immunostimulation by SNAs in non-human primates (when tumor volume reached ~ 100 mm3) and study day 18, mice Cynomolgus macaques were injected subcutaneously with SNA and blood was subsequently drawn for assessment of serum cytokine mice were treated with 1.2 mg/kg SNA or PBS vehicle Male C57BL/6 mice aged 17-18 weeks, either wild-type (WT) or levels and activation of immune cell subsets. Mean + SEM of N=4 (2 male and 2 female) macaques per group. administered intratumorally, with 10 mg/kg anti-PD-1 antibody lacking TLR9 (TLR-KO), were injected subcutaneously with 3 (A) Macaques were treated with TLR9-agonist SNA or a non-CpG negative control SNA at 3 mg/kg. Serum cytokine levels were administered intraperitoneally, or both. At 9 days after initiating mg/kg SNA or vehicle (PBS). At 10 hours post-administration, quantified using a Luminex panel. treatment (study day 19), the tumors were removed and the blood was drawn and processed to serum. Serum cytokine (B) Macaques were treated with TLR9-agonist SNA or a non-CpG negative control SNA at the indicated dose levels. Flow cytometry was expression of PD-1 and PD-L1 mRNA was assessed using qRT- levels were quantified using a multiplex ELISA (Quansys). Mean used to quantify changes in immune cell activation at 24 hours post-administration. Activated plasmacytoid dendritic cells (pDCs) were PCR. mRNA levels were normalized to GAPDH using the 2-ΔΔCt + SEM of N=4 mice is shown. defined as CD3/8/14/20- HLADR+ CD11c- CD123+ CD86+. Activated B cells were defined as CD3- CD20+ CD86+. method.

TLR9-Agonist SNA Treatment Synergizes With Checkpoint Inhibition CONCLUSIONS

2 0 0 0

) • TLR9-agonist SNAs induced potent, 3 V e h ic le ( P B S )

m a n ti- P D - 1 TLR9-dependent TH1-type immune

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( L in e a r O lig o n u c le o tid e + a n ti- P D - 1 responses in mice and monkeys and

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V • SNA plus anti-PD-1 combination therapy

r N a iv e M ic e , E M T - 6 c h a lle n g e induced tumor-specific immunity and o 5 0 0 m memory.

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• These data support the clinical

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a 0 investigation of SNAs in immuno-

e S N A , 0 .8 m g /k g , IT S N A + a n ti- P D - 1 tr e a te d m ic e , C T - 2 6 c h a lle n g e E M T - 6 4 T 1 o r C T -2 6 oncology. One such SNA, AST-008, is M A n ti-P D -1 , 1 0 m g /k g , IP N a iv e M ic e , C T - 2 6 c h a lle n g e re c h a lle n g e c h a lle n g e undergoing a Phase 1a clinical trial and 0 1 0 2 0 3 0 4 0 5 0 6 0 7 0 8 0 9 0 1 0 0 1 1 0 1 2 0 1 3 0 is planned for testing in cancer patients S tu d y d a y combined with an anti-PD-1 antibody. Figure 5. Anti-tumor effects of SNA and anti-PD-1 On study day 0, BALB/C mice were subcutaneously inoculated with EMT-6 cells to establish flank tumors. Beginning on study day 3, SNA treatment began, dosing 0.8 mg/kg subcutaneous (SC) peritumorraly every 3 days for a total of 5 doses. Beginning on study day 5, anti-PD-1 antibody treatment began, dosing 10 mg/kg intraperitoneally (IP) every 5 days for a total of 3 doses. Starting N=8 animals per group. For the SNA plus anti-PD-1 group, complete tumor remission was observed in 7/8 animals. Those 7 animals were re-challenged with EMT-6 tumor cells again on day 44. On day 115, those animals were challenged with either 4T1 or CT-26 tumor cells. www.exicuretx.com