TTI-10001, A Next Generation Small Molecule STING Agonist, Demonstrates

Potent Anti-Tumor Activity In Mice Following Intravenous Or Oral Administration

Trillium Therapeutics Inc., Mississauga, ON, Canada

SITC2019

P668

TTI-10001 Displays Favorable PK Properties after IV and

Oral AdministrationOral administration of TTI-10001 Induces Complete Tumor

Regression and Generates a Memory Response

Key In Vitro Properties of TTI-10001

Conclusions

Natasja Nielsen Viller, Peter Dove, David Rosa, Bolette Bossen, Tran Truong, Tapfuma Mutukura, Debbie Jin, Marilyse Charbonneau, Laura Brinen, Karen Dodge, Gloria H. Y. Lin, Jeff Winston, Robert A. Uger, Malik Slassi, Zezhou Wang

TTI-10001 has desirable physical-chemical properties

TTI-10001 is a novel, potent, non-CDN, small molecule pan-STING agonist

TTI-10001 is well tolerated in mice by systemic administration (IV and oral)

TTI-10001 induces durable complete regressions of tumors and induces a memory

response

TTI-10001 demonstrates an excellent PK/PD profile that results in potent anti-tumor

activity by both intratumoral and systemic administration

These data highlight the potential of TTI-10001 to achieve best-in-class status among

next generation STING agonists

IV administration of TTI-10001 Induces Complete Tumor

Regression and Generates a Memory Response

TTI-10001 displays pan STING allele activity

TTI-10001 has a favorable in vitro safety profile

STING Bridges Innate and Adaptive Anti-Tumor Immunity

STING has emerged as an attractive cancer immunotherapy target due to its key role in induction of

type I interferons (IFNs) and other pro-inflammatory cytokines that promote tumor-specific antigen

cross-presentation and effective T cell priming

We have previously demonstrated that TTI-10001, a novel non-cyclic dinucleotide (CDN) small

molecule STING agonist with pan-allele STING activity and ideal drug properties, potently induces the

STING pathway in vitro and results in robust anti-tumor activity in vivo following intratumoral (IT)

administration.

Here we report that TTI-10001 demonstrates favorable pharmacokinetic (PK) and pharmacodynamics

(PD) properties, is well tolerated in vivo, and induces potent anti-tumor monotherapy activity in mice

after intravenous (IV) or oral dosing

A. STING agonist binds to and activates STING

B. STING activation leads to expression of type I

interferons

C. Interferons stimulate dendritic cell (DC)

maturation

D. Dendritic cells displaying tumor antigen

migrate to regional lymph nodes

E. Dendritic cells cross-present tumor-derived

antigens and activate tumor-specific CD8+

cytotoxic T cells

F. CD8+ cytotoxic T cells infiltrate the tumor and

destroy tumor cells

STING

Allele

Human

Frequency

(%)

EC50 (uM)*

TTI-10001 ADU-S100

WT 57.9 0.55 1.60

HAQ 20.4 0.61 3.04

REF 13.7 4.41 >20

AQ 5.2 0.63 2.31

Q 1.5 0.69 9.49

Mouse - 0.13 0.35

*STING reporter assay (n>3)

TTI-10001 does not induce cytokine production from STING KO bone marrow derived dendritic cells

Property TTI-10001

Ideal

(for systemic

dosing)

Molecular Weight <350 Da <500 Da

tPSA (oral/CNS) 85.2/85.2 <140

Ligand Efficiency (LE) 0.42 >0.29

LE Dependent

Lipophilicity (LELP)5 <10

cLogP 2.17 1 – 4.5

Solubility (in water) >20 mg/ml -

Rotatable Bonds 6 ≤10

N + O (Hydrogen Bond

Acceptor)6 ≤10

NH + OH (Hydrogen

Bond Donor)1 ≤5

CYP3A/2D6 hERG SafetyScreen 44TM 1 KinaseExpress Screen45 TM 2

IC50 >30 µM IC 50 >100 µM No inhibition at 3 µM No inhibition at 3 µM

1) 44 enzymes/ion channels/transporters/GPCRs screen (Eurofins)

2) 45 kinase screen (Eurofins)

Key pharmacokinetic parameter features:

High oral bioavailability: F = 85%

Longer half-life in tumors (6.6 hours, IV; 4.4 hours, oral) than in plasma (3-4

hours, IV or oral)

Longer retention time in tumors than in plasma following IV administration

Tissue

2.5 mg/kg, IV 2.5 mg/kg, Oral

Apparent t1/2

(h)

Cmax

(ng/ml)

AUC0-tlast

(h*ng/ml)

Apparent t1/2

(h)

Cmax

(ng/ml)

AUC0-tlast

(h*ng/ml)

Tumor 6.56 1100 3162 4.43 622 1982

Plasma 4.07 3510 5677 3.07 2177 4795

C57BL/6 mice (n = 3) bearing MC38 tumors were dosed with 2.5 mg/kg TTI-10001 through IV or oral route. Plasma and

tumors were harvested at 0.5, 2, 4, 6 and 24h post dosing. TTI-10001 concentrations were measured by tandem liquid

chromatography-mass spectrometry at InterVivo Solutions.

TTI-10001 is Well Tolerated after Repeated IV or Oral

Administration

No deaths, body weight loss, or overt morbidity observed with doses are equal to or below

100 mg (2.5mg/kg) IV or 200 mg (10 mg/kg) oral 3 times every other day

Different doses of TTI-10001 were administered via IV or oral routes to naïve C57BL/6 mice (n = 3-4 per dose group), at

day 0, 2 and 4. Clinical observations for morbidity and mortality were made daily while body weights were measured on the

day of the first dose and 3 times per week for 11 days.

Oral AdministrationIV Administration

A) MC38 tumor model, mice dosed 1x IV with 10, 50 or 100 µg of TTI-10001

B) MC38 tumor model, mice dosed 4 x IV with 10 or 50 mg of TTI-10001

D) Cured mice are resistant to rechallengeC) IV dosing of TTI-10001 is well tolerated

A-B) MC38 cells were implanted subcutaneously in C57BL/6 (n=10 mice per group) on day 0. Mice were randomized when the

tumor size was approximately 65-70mm3 and received IV injections of TTI-10001 as indicated. Tumor growth was measured 3 times

per week for the duration of the study. Signs of morbidity and mortality were measured daily for the duration of the study. C) Body

weight of mice was measured on the first day of dosing and was measured 3 times a week during the duration of the study. D) TTI-

10001-treated mice free from tumors for 30 days were re-challenged with MC38 cells in the right flank. Naïve C57BL/6 mice were

inoculated with MC38 cells as control. Tumor re-growth was monitored for 35 days. Growth curves were analyzed by one way

ANOVA with Tukey’s multiple comparison test on day 21 (panel A) and day 23 (panel B). Survival curves were analyzed by Logrank

(Mantel-Cox) test (corrected for multiple comparisons). CR: complete tumor regression; * p<0.05, ** p<0.01, *** p<0.001, ****

p<0.0001

TTI-10001 Correlation Between PK, PD, and Efficacy

B) Oral dosing of TTI-10001 is well tolerated C) Cured mice are resistant to rechallenge

A) MC38 tumor model, mice dosed 4 x oral with 50 µg of TTI-10001

TTI-10001 exhibits dose- dependent systemic drug exposure, PD marker activation, and

efficacy in vivo across different administration routes

A) TTI-10001 was administered either through, IT, IV or oral routes as

indicated into C57BL/6 mice bearing MC38 tumors. Plasma and

tumors were harvested at several time points and IFNb levels were

measured by cytometric bead array to determine the maximum levels

of induction. B) MC38 tumor bearing mice were randomized when the

tumor size was approximately 65-70mm3 and were dosed with TTI-

10001 as indicated. Tumor growth was measured 3 times per week

for the duration of the study. Signs of morbidity and mortality were

measured daily for the duration of the study. Survival curves were

analyzed by Logrank (Mantel-Cox) test (corrected for multiple

comparisons). * p<0.05, *** p<0.001, **** p<0.0001

A) MC38 cells were implanted subcutaneously in C57BL/6 (n = 8 mice per group) and were randomized when the tumor size was

approximately 65-70mm3. Mice then received 50 mg of TTI-10001 via oral administration as indicated. B) Body weight of mice was

measured on the first day of dosing and was measured 3 times a week during the duration of the studies. C) TTI-10001-treated

mice free from tumors for 30 days were re-challenged with MC38 cells in the right flank. Naïve C57BL/6 mice were inoculated with

MC38 cells as control. Tumor re-growth was monitored for 35 days. Growth curves were analyzed by unpaired t-test on day

29. Survival curves were analyzed by Logrank (Mantel-Cox) test (corrected for multiple comparisons). CR: complete tumor

regression; *** p<0.001

A)

B)2.5 mg/kg TTI-10001 was administered via IV or oral routes into C57BL/6 mice

bearing MC38 tumors. Plasma and tumors were harvested at several time

points as indicated and IFNb levels were measured by cytometric bead array.

TTI-10001 Displays a Favorable PD Profile with IV or Oral

Administration

Peak IFNβ induction is at least 30 fold higher

in tumors than in plasma (IV and oral).

More sustained IFNβ levels in the tumor vs

the plasma (IV and oral).

More rapid reduction of IFNβ in the plasma

than the tumor.