A Systemically Administered, Conditionally Active TLR8 for the Treatment of HER2-Expressing Tumors Kara Moyes, Ty Brender, Sean W. Smith, Hengyu Xu, Ben Setter, Li-Qun Fan, Rebecca Brunette, Justin Killebrew, Phil Tan, Craig Coburn, Robert DuBose, Peter Baum, Valerie Odegard Silverback Therapeutics, Seattle, WA

Introduction Figure 1: SBT6050 Induces a HER2-Dependent Figure 3: HER2-TLR7 is a Mouse Surrogate for SBT6050 Figure 6: HER2-TLR7 Treatment Activates Intratumoral Pro-Inflammatory, Th1-Polarizing Myeloid Cell Response Clinical development of systemically administered innate immune cell has been hindered by acute Innate and Adaptive Immune Responses toxicities due to peripheral activation of the targeted cell types. Intratumoral administration, the route of Macrophages 1000 pos A B C delivery typically used for innate immune/myeloid cell agonists, is limited by tumor accessibility and a A HER2 (BT474) HER2-TLR7 Mouse Macrophages A Tumor Antigen Specific T cells B Polyfunctional Tumor Specific dependence on abscopal responses. SBT6050 Mouse HER2 binding domain 1500 T cell Response Cell Type HER2 pos

) TLR7 TLR8 L L HER2 mAb

(SK-BR-3) I ** 35

)

+ m

T 60

/

L TLR8 small molecule Dendritic Cells +++ +++ 8 *

HER2 neg +

Agonism of TLR8 (toll-like 8) has been shown to drive anti-tumor immune responses. Here, we g m

D 30

8 / p 1000

( 500 50

(MDA-MB-468) C

g

D

describe a TLR8 agonist conjugate, SBT6050 designed for systemic administration, that utilizes cell surface Macrophages +++ +++

f

p

 C

TLR7 ( 25

o

neg

F f

HER2 (MDA-MB-468) 40

+ N

expression of HER2 to localize activation of TLR8 for the treatment of HER2-expressing tumors. Monocytes +++ +++ agonist o

F

T 

SBT6050 + 20

r N

500 F e NK Cells - - T 30

HER2 mAb N 15

m

m

T

• SBT6050 activates human monocytes and macrophages only in the presence of HER2-expressing a r

T Cells - - +

0 TLR8 small molecule t 20

 10

e t

tumor cells with moderate or high expression levels. 0 0.01 0.1 1 10 100 1000 N

Tumor Cells - - 0 -

F 1

10 I 5

nM 0 0.01 0.1 1 10 100 H

mIgG2a Fc A % PBMCs HER2-TLR7 (nM) 0 0 • Systemic delivery of a SBT6050 surrogate in mice shows durable, single agent efficacy in a % HER2 mAb HER2-TLR7 Her2 mAb HER2-TLR7 15000 4000 checkpoint refractory tumor model without the induction of peripheral production or B HER2pos (NCI-N87) associated CRS-like toxicity. Mice do not express a functional homolog of human TLR8, but mouse TLR7 phenotypically matches the myeloid expression of human ) SBT6050 (TNF) TLR8 (A). HER2-TLR7 surrogate (B) mediates HER2-dependent macrophage activation similar to that of SBT6050 (C). M1/M2 Ratio

L Neutrophils ) CC

3000 L

m HER2 mAb (TNF)

s

/ l

m 10000

l

g /

e 50

p **

• SBT6050 is currently in preclinical development for patients with moderate or high HER2-expressing g SBT6050 (IL-12p70) ( 10 c

**

p

s (

2000 0

+

HER2 mAb (IL-12p70) M 7 tumors and is projected to enter the clinic in 2020. 5

 Figure 4: HER2-TLR7 Monotherapy Results in Tumor Clearance Without A

4 40

p

F

T

2 D

5000

N

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+ C

- neg

T 1000 6 Significant Systemic Cytokine Release f

L HER2 (MDA-MD-468)

0 30

I

o

2 +

SBT6050 (TNF) D 5

b

C

SBT6050 is Designed for Systemic Administration with TME-Localized Activity B HER2 Expression Levels 1 20 0 /

0 SBT6050 (IL-12p70)

1

+

D I

0.01 0.1 1 10 100 1000 80000 SKBR3 I

A C

3+ BT474 s 10

pos +

nM NCI-N87 s 1

HER2 CT26 a l

60000 r

C

N

G

pos neg A MDA-MB-453 0 0

In vitro differentiated macrophages (A) or PBMCs (B) were co-cultured with HER2 or HER2 tumor cell 100 E PBS p<0.0001 ZR-75-1 2+ % M HER2 mAb HER2-TLR7 HER2 mAb HER2-TLR7 lines in the presence of SBT6050, unconjugated anti-HER2 mAb or TLR8 small molecule agonist, as - 40000

HER2-TLR7 O CT26-HER2

E l

indicated. In contrast to SBT6050, TLR8 small molecule activity was not dependent on HER2 (A). SBT6050 p<0.0001 G

a 75 HER2 mAb 20000 MDA-MB-175 1+ v displays enhanced potency compared to the TLR8 small molecule alone (A). TNFα and IL-12 are shown as i IFN v MCP-1 r D representative indicators of pro-inflammatory and Th1-polarizing myeloid cell responses, respectively (B). 0 MDA-MB-468 neg u 0.0001 0.001 0.01 0.1 1 10 100 s 2000 ** 50000

t 50 **

e

n

e u

e -HER2 (g/mL) u

CR=3/9 s s

c 40000

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r s

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t

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t

25 g

Figure 2: Delivery of TLR8 Agonist to Myeloid Cells P g

C Cytokine/ HER2-TLR7 PBS

/

/ 30000

e

Chemokine (pg/mL) (pg/mL) e 1000

n n

Requires Moderate to High HER2 Expression and i

i k 0 k 20000

IL-6 ND ND o

o t 0 10 20 30 40 t Fc:Fc R Interactions y

γ y 500

c c

IL-10 ND ND

10000 g

Days post-treatment g p TNFα ND ND p A Tx IL-1β ND ND 0 0 HER2 Expression HER2 Molecules/Cell HER2 mAb HER2-TLR7 HER2 mAb HER2-TLR7 Tumor Cell Line Activation MCP-1 60 ND by IHC (x106) HER2pos CT26 tumor-bearing mice were treated intravenously with 5 mg/kg HER2-TLR7 or unconjugated Mice bearing subcutaneous HER2pos CT26 tumors were treated intravenously with HER2-TLR7 at 5 mg/kg, unconjugated HER2 mAb at 5 HER2 mAb. On days 2 (A) and 7 (B, C) post-treatment, intratumoral immune cell populations were SKBR3 3+ 1.1 +++ mg/kg, or PBS; CR=Complete Response (A). Relative HER2 expression of cell lines determined by flow cytometry (B). analyzed by flow cytometry for anti-tumor T cells (A, B) and innate immune cells (C). Intratumoral Cytokine/chemokine expression as assessed in blood drawn 24 hours after dosing; ND=Not Detected (C). BT474 3+ 0.65 +++ were assayed on day 2 post-treatment (D). Statistical significance was determined by unpaired T-test. NCI-N87 3+ 0.47 +++ *p<0.05, **p<0.01, p<0.001. MDA-MB-453 2+ 0.08 +++ Figure 5: HER2-TLR7 Induces Durable Anti-Tumor Immunity That Protects ZR-75-1 2+ 0.04 ++ Conclusions Human TLR8 Expression Profile Supports Development of a TLR8-Selective Payload MDA-MB-175-VII 1+ 0.02 - Against Tumor Re-challenge MDA-MB-468 0 <0.01 - SBT6050 activates human myeloid cells only in the presence of HER2-expressing TLR4 TLR7 TLR8 TLR9 STING RIG-I • A HER2pos CT26 B HER2neg CT26 cells, enabling systemic administration with tumor-localized activity. Dendritic Cells +++ + ++++ - ++ ++ B 2500 Wildtype IgG1 1500 Re-challenge 2500 Re-challenge

) • Systemic administration of a SBT6050 mouse surrogate results in durable anti-

) 3 IgG1 Null 3 N=6 N=3 2000 m m tumor efficacy in the absence of peripheral cytokine production, consistent with

Macrophages ++++ ++ +++ - ++ ++ ) 2000

m L

m Naive N=20 Naive N=5

(

(

m tumor-localized activity. / 1000 e

1500 e

g m

Myeloid Cells Cells Myeloid 1500

m p

MDSC +++ +++ +++ - ++ ++ (

u

u

l

l

 o

1000 o • In mouse HER2-expressing tumors, a surrogate molecule of SBT6050 drives

F V

V 1000

N r

r 500 T

Fibroblasts ++ ++ - - +++ +++ o activation of both innate and adaptive immune response characterized by -

500 o m m 500

u activation of tumor-associated myeloid cells, infiltration of neutrophils, persistent

u

Non

T T Myeloid Endothelial Cells +++ ++ - - + ++ 0 increases in local cytokine and chemokine production, and the generation of a 0 0.01 0.1 1 10 100 0 0 10 15 20 10 15 20 25 robust, neo-Ag specific anti-tumor CTL response. HER2+ Tumor nM - - - - ++ ++ Cell Days post-inoculation Days post-inoculation Tumor Activation determined by PBMC co-culture assay (A). Purified monocytes co-cultured with BT-474 tumor • SBT6050 is currently in preclinical development for patients with moderate or high cells together with TLR8 conjugates bearing either WT IgG1 or IgG1Null, a mutant IgG1 Fc rendered Mice cleared of HER2pos CT26 tumor with HER2-TLR7 treatment were re-challenged with HER2pos (A) or HER2neg (B) CT26 cells. HER2-expressing tumors and is projected to enter the clinic in 2020. Expression levels were determined using publicly available RNA-Seq datasets incapable of binding to FcγRs by targeted mutations in Fc: L234A, L235A, G237A and K322A (B). Re-challenge was performed 60 days after initial tumor clearance. Naïve mice were included as a control for tumor cell growth.

Abstract 3830, AACR Annual Meeting, March 29-April 3, 2019