Table 1. Expression levels were determined using publicly available RNA-Seq datasets.

Introduction

Despite advances in treatment options for patients with HER2-expressing tumors, significant unmet medical need remains. Checkpoint inhibition (CPI) therapy has been largely ineffective in HER2-expressing tumors (2+ and 3+ by IHC), likely due to the absence of T cell infiltrate. In contrast, it is well established that HER2-expressing tumors are replete with myeloid cells offering the opportunity for intratumoral immune activation. Local administration, the typical delivery route used for innate immune/myeloid cell agonists, is limited by tumor accessibility and a dependence on abscopal responses. SBT6050 is a novel ImmunoTAC™ therapeutic comprised of a HER2-directed monoclonal antibody conjugated to a potent and highly specific TLR8 agonist, allowing for systemic delivery of a myeloid cell agonist with activity localized to HER2-expressing tumor sites.

Conclusions

• SBT6050, a HER2-directed monoclonal antibody conjugated to a potent and highly specific TLR8 agonist, activates multiple anti-tumor immune mechanisms in a HER2-dependent manner, allowing for systemic delivery and tumor-localized activity.

• SBT6050-S demonstrates robust, durable single agent activity in tumor models with low tumor infiltrating lymphocytes, highlighting the potential for clinical activity with SBT6050 in HER2-expressing malignancies.

• SBT6050-S is curative as a single agent in a HER2-expressing xenograft model lacking T, B, and NK cells, demonstrating the potential of myeloid cells to mediate robust efficacy.

• The combination of a low dose of SBT6050-S together with trastuzumab in a HER2-positive mouse tumor model enhanced the single agent activity observed with either agent alone.

• Collectively, these data support clinical evaluation of SBT6050 as a single agent and in combination with trastuzumab in relevant HER2-expressing tumor types.

SBT6050 is currently in late preclinical development for patients with moderate or high HER2-expressing tumors and is projected to enter the clinic later this year.

Figure 5: SBT6050 Mouse Surrogate (SBT6050-S) Matches the Functional Profile of SBT6050 on Human Myeloid Cells

Figure 7: SBT6050-S Induces Robust Single Agent Tumor Clearance in T, B, and NK Cell Deficient Mice

SBT6050, a HER2-Directed TLR8 Therapeutic, is a Systemically Administered, Tumor-Targeted Human Myeloid Cell Agonist Heather Metz, Monica Childs, Jamie Brevik, Damion Winship, Ty Brender, Michael Comeau, Jenny R Chang, Jeffrey Adamo, Ben Setter, Hengyu Xu, Li-Qun Fan, Sean W Smith, Phil Tan, Robert DuBose, Yvette Latchman, Peter Baum and Valerie OdegardSilverback Therapeutics™, Seattle, WA | Contact information: info@silverbacktx.com

Figure 8: Combination of SBT6050-S and Trastuzumab EnhancesIn Vivo Efficacy

The data presented here demonstrate the following:

• SBT6050 potently activates human myeloid cells in the presence of HER2-expressing tumor cells with 2+ and 3+ levels of expression.

• Activation of myeloid cells by SBT6050 drives an innate immune response for direct tumor killing and can also nucleate a T cell response.

• SBT6050 mouse surrogate (SBT6050-S) is efficacious as a single agent in HER2-expressing, tumor-infiltratinglymphocyte (TIL) deficient tumor models.

• SBT6050 and trastuzumab recognize distinct epitopes on HER2.

• Combination of SBT6050-S and trastuzumab in a mouse tumor model leads to enhanced efficacy compared toeither agent alone.

Collectively, these data support clinical evaluation of SBT6050 as a single agent and in combination with trastuzumab in relevant HER2-expressing tumor types.

Figure 1: SBT6050 is an ImmunoTAC™ Therapeutic Designed for Systemic Administration with TME-Localized Activity

Cell Type TLR4 TLR7 TLR8 TLR9 STING RIG-I

Mye

loid

Cel

ls Dendritic Cells +++ +/- ++++ - ++ ++

Macrophages ++++ + +++ - ++ ++

Non

-Mye

loid Fibroblasts ++ ++ - - +++ +++

Endothelial Cells +++ ++ - - + ++

Tum

or

HER2+ Tumor Cells - - - - ++ ++

Figure 5. (A) TLR7 RNA expression in mouse myeloid cells is comparable to that of TLR8 in human. RNA expression data obtained from public databases. (B) SBT6050-S mediates HER2-dependent macrophage activation in vitro with a potency similar to SBT6050.

A B

Figure 6: SBT6050-S Induces Durable Single Agent Efficacy in a T Cell Excluded Syngeneic Model

HER2-expressing EMT6 Tumors are Infiltrated by Myeloid Cells, not T Cells A

B

Tumor (H&E) T Cells Macrophages

Robust Single-Agent Anti-Tumor Efficacy

0/8 CR 0/8 CR 4/8 CR

Figure 6. (A) IHC of untreated EMT6 tumors. (B) Monotherapy efficacy of established tumors. Unconjugated HER2 mAb denotes SBT6050-S without payload. Mice (n=10) bearing HER2-expressing EMT6 tumors were treated with PBS, 10mg/kg unconjugated HER2 mAb, or 10mg/kg SBT6050-S. Arrows indicate doses administered. (C) Mice cleared of tumors were re-challenged with HER2-expressing or HER2 negative EMT6 tumor cells 60 days after initial tumor clearance. Naïve mice were included as a control for tumor growth.

0/10 CR 0/10 CR 10/10 CR

Figure 7. Unconjugated HER2 mAb denotes SBT6050-S without an agonist payload. Isotype control matched to unconjugated HER2 mAb. SCID-Beige mice (n=10) bearing NCI-N87 tumors were treated with 10 mg/kg isotype control, 10mg/kg unconjugated HER2 mAb, or 10mg/kg SBT6050-S. Arrows indicate doses administered.

Figure 8. SBT6050-S was administered at 1mg/kg to allow for combinatorial benefit to be observed. SCID mice (n=10) bearing NCI-N87 tumors were treated with the indicated combinations. Unconjugated HER2 mAb denotes SBT6050-S without payload. (A) Isotype controls matched to unconjugated HER2 mAb (1mg/kg) and trastuzumab (5mg/kg). (B) Isotype control for trastuzumab (5mg/kg) + unconjugated HER2 mAb (1mg/kg). (C) Isotype control for trastuzumab (5mg/kg) + SBT6050-S (1mg/kg). (D) trastuzumab (5mg/kg) + isotype control for unconjugated HER2 mAb (1mg/kg). (E) trastuzumab (5mg/kg) + unconjugated HER2 mAb (1mg/kg). (F) trastuzumab (5mg/kg) + SBT6050-S (1mg/kg). Arrows indicate doses administered.

Table 1: Human TLR8 Myeloid-Restricted Expression Profile Supports Development of a TLR8-Selective Payload

Tumor Cell Line

HER2 #/Cell (x106)

IHC Score

SKBR3 1.10 3+

NCI-N87 0.65 3+

BT-474 0.47 3+

MDA-453 0.08 2+

ZR-75-1 0.04 1+/2+

MDA-468 <0.02 0

Figure 2. IHC for HER2 and TLR8 performed on a ductal breast carcinoma sample.

Figure 2: HER2 And TLR8 Are Adjacent In Human Tumor

HER2 TLR8

0 . 0 1 0 . 1 1 1 0 1 0 0 1 0 0 0

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

C o n c ( n M )

TN

Fa

(p

g/m

L)

S K - B R - 3

N C I - N 8 7

B T - 4 7 4

M D A - 4 5 3

Z R - 7 5 - 1

0

0.220.250.220.19

~ 0.21

E C 5 0 ( n M )

M D A - 4 6 8

0 . 0 1 0 . 1 1 1 0 1 0 0 1 0 0 0

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

C o n c ( n M )

TN

Fa

(p

g/m

L)

S K - B R - 3

N C I - N 8 7

B T - 4 7 4

M D A - 4 5 3

Z R - 7 5 - 1

0

0.220.250.220.19

~ 0.21

E C 5 0 ( n M )

M D A - 4 6 8

0 . 0 1 0 . 1 1 1 0 1 0 0 1 0 0 0

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

C o n c ( n M )

TN

Fa

(p

g/m

L)

S K - B R - 3

N C I - N 8 7

B T - 4 7 4

M D A - 4 5 3

Z R - 7 5 - 1

0

0.220.250.220.19

~ 0.21

E C 5 0 ( n M )

M D A - 4 6 8

0 . 0 1 0 . 1 1 1 0 1 0 0 1 0 0 0

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

C o n c ( n M )

TN

Fa

(p

g/m

L)

S K - B R - 3

N C I - N 8 7

B T - 4 7 4

M D A - 4 5 3

Z R - 7 5 - 1

0

0.220.250.220.19

~ 0.21

E C 5 0 ( n M )

M D A - 4 6 80 . 0 1 0 . 1 1 1 0 1 0 0 1 0 0 0

0

2 0 0 0

4 0 0 0

6 0 0 0

8 0 0 0

1 0 0 0 0

C o n c ( n M )

TN

Fa

(p

g/m

L)

S K - B R - 3

N C I - N 8 7

B T - 4 7 4

M D A - 4 5 3

Z R - 7 5 - 1

0

0.220.250.220.19

~ 0.21

E C 5 0 ( n M )

M D A - 4 6 8

Figure 3: SBT6050 Designed to Activate Myeloid Cells in the Presence of 2+ and 3+ Levels of HER2 Expression

Figure 3. Tumor cell lines were co-cultured with human PBMC and the indicated concentrations of SBT6050. Activation was determined by TNFα production.

ADCC

Figure 4: SBT6050 Drives a Broad Spectrum of T Cell Dependent and Independent Anti-Tumor Immune Mechanisms

Figure 4. (A) Human PBMC were co-cultured with HER2-expressing (BT-474, 3+; MDA-MB-453, 2+) or HER2-negative (MDA-MB-468) tumor cell lines in the presence of SBT6050. Data representative of multiple donors. No activation of PBMC was observed when co-cultured with tumor cells and unconjugated HER2 mAb (data not shown). (B) ADCC was assessed using human PBMC co-cultured with labelled BT-474, MDA-MB-453, or MDA-MB-468 tumor cell lines in the presence of SBT6050, unconjugated HER2 mAb or a negative control conjugate (isotype control mAb conjugated with TLR8 agonist payload) as indicated and 24 hours later analyzed by flow cytometry. Data representative of multiple donors.

A

BProtection From Tumor Re-challenge is HER2 IndependentC

ASCO 2020, Abstract #3110

Dendritic c

ells

Macrophage

s

Monocytes

0

2

4

6

8

log2

(TPM

+1)

Human TLR8

Human TLR7

Mouse TLR7