Figure 6: Mouse surrogate (ASGR1-S) matches the functional profile of ASGR1-TLR8

Table 1: Expression based upon RNA levels in public databases.

Introduction

Conclusions

• ASGR1-TLR8 ImmunoTAC therapeutic, designed to activate an anti-HBV response by liver-localized activation, potently activates myeloid cells in an ASGR1-positive cell dependent manner

• ASGR1-TLR8 myeloid activation promotes a strong IFNγ response and B cell activation in co-culture assays

• ASGR1-S drives HBsAg seroconversion with lowering of HBsAg and viral DNA titers, without evidence of liver damage in an AAV-HBV mouse model

• ASGR1-S significantly induces IFNγ+ anti-HBcAg and HBsAg T cell responses as well as an anti-HBsAg B cell response

• Together, these data support the continued preclinical development of ASGR1-TLR8 as a potential future treatment for patients with cHBV

Figure 5: ASGR1-TLR8 activation of myeloid cells leads to increases in B cell activation

Figure 8: ASGR1-S induces anti-viral antigen T and B cell responses, reflective of those required for cHBV functional cure

ASGR1-TLR8, an ASGR1-Directed TLR8 ImmunoTAC Therapeutic, is a Potent Myeloid Cell Agonist with Liver-Localized Activity for the Treatment of Chronic HBVPeter Baum, Ty Brender, Jeff Adamo, Li-Qun Fan, Jenny R. Chang, Hengyu Xu, Ben Setter, Brenda Stevens, Mike Comeau, Monica Childs, Ray Carrillo, David Purdy, Yvette Latchman, Robert Dubose, Graham Jang, Phil Tan, Sean Smith, Valerie OdegardSilverback Therapeutics, Seattle, WA | Contact information: info@silverbacktx.com

Figure 9: ASGR1-S treatment was well-tolerated in mice includingno signs of liver damage

• ASGR1-TLR8 conjugate potently activates human myeloid cells in an ASGR1-dependent manner • ASGR1-TLR8 myeloid activation promotes a strong IFNγ response and activates B cells• ASGR1-S drives seroconversion with lowering of HBsAg and viral DNA plasma titers in the mouse AAV-HBV

model• ASGR1-S significantly increases anti-viral antigen IFNγ+ T cell and B cell anti-HBsAg responses while being

well tolerated with no evidence of liver damage

Figure 1: ASGR1-TLR8 is an ImmunoTAC Therapeutic designed for systemic treatment and liver-localized immune activation

Cell Type TLR4 TLR7 TLR8 STING RIG-I

Mye

loid

Cel

ls

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

Macrophages ++++ + +++ ++ ++

Non

-Mye

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

Endothelial Cells +++ ++ - + ++

Figure 5: CD86 levels on CD19pos B cells were determined by flow cytometry after co-culture of PBMC with ASGR1pos or ASGR1neg cells in presence of test articles at 10nM.

Figure 6: A) RNA expression levels determined from public databases. B) Bone marrow derived macrophages (BMDM) were co-cultured with HEK293 cells transfected with mouse ASGR1 and test articles. Negative control conjugate is the TLR7 agonist conjugated to a non-ASGR1 IgG2a antibody. TNF-α in supernatants determined by ELISA after 24 hr.

Figure 8: Splenocytes were isolated from animals on d35 post-dose initiation and 2.5e5 cells/plate seeded on indicated ELISPOT plates. A) IFNγ+ T cells were enumerated after culture in the presence or absence of viral HBV core (HBc) or HBV surface (HBs) peptide pools. B) Anti-HBsAg B cells were enumerated using anti-mouse IgG coated plates with an HBsAg capture ELISA. P values were determined by Mann-Whitney U test.

Table 1: Human TLR8 myeloid-restricted expression profile supports developmentof a TLR8-selective payload

Figure 2: ASGR1-TLR8 is designed for liver-localized TLR8 activation

Figure 3: ASGR1-TLR8 activation of myeloid cells is ASGR1 dependentand more potent than the free payload

Figure 3: PBMC were co-cultured with ASGR1pos (HepG2 cells transfected to express more physiological ASGR1) or ASGR1neg (HEK293 cells) with titration of test articles. Negative control-TLR8 conjugate binds an irrelevant antigen and unconjugated TLR8 agonist was the unconjugated small molecule. TNF-α was measured by ELISA in the culture supernatants after 24 hr.

Figure 4: ASGR1-TLR8 potently activates human myeloid cells, resulting in a robust IFNγ response

Figure 4: PBMCs were co-cultured with ASGR1pos cells in the presence of indicated conjugates or control antibody for 24 hr. Supernatants were tested for cytokine levels by MSD assay or ELISA.

ASGR1-TLR8, an ImmunoTAC™ therapeutic comprised of a TLR8 agonist conjugated to an antibody directed to the liver-restricted ASGR1, is designed to promote functional cure of chronic HBV (cHBV). cHBV infection remains a global unmet medical need contributing to an estimated 887,000 deaths per year. Clinical and preclinical evidence demonstrate that effective IFNγ+ T cell and IgG B cell anti-viral immune responses can lead to functional cure, and a major goal for therapy is increasing these immune-mediated responses. Prior studies have shown that TLR8 is particularly effective in generating IFNγ-skewed adaptive immunity through myeloid cell activation. Consistent with this, oral administration of a TLR8 agonist small molecule, GS-9688, attained seroconversion in animal models of chronic HBV and has shown signs of clinical activity. However, toxicities due to systemic activation of TLR8 may limit dose and liver exposure in patients. We believe that a systemically delivered TLR8 agonist with liver-localized activity could better realize the potential for effective therapy and functional cures. There is significant unmet need for cHBV therapies that can elicit a functional cure, which is defined as sustained loss of hepatitis B surface antigen (HBsAg) in the blood. Many of the approved therapies for cHBV have low functional cure rates or lack durability over time. Here we describe the activities of our ASGR1-TLR8 conjugate and an ASGR1-TLR8 mouse surrogate (ASGR1-S) that demonstrate the potential of liver localized myeloid cell activation for promoting functional cure in cHBV. The presented data show:

High affinity; nM binding to human and cyno ASGR1 High specificity for ASGR1; no binding to closely

related proteins Initial developability assessment favorable Non-ligand blocking, allowing for combination with

GalNac-based therapies

Anti-ASGR1 mAbASGR1-TLR8 Conjugate

ASGR1 mAb Does Not Block ASGR Ligand Binding

Antigen Binding Domain: ASGR1

Payload: Proprietary TLR8 Agonist

Fc: IgG1

Figure 2: Ligand blocking was determined by a FACS-based competition assay using detection of binding of a triantennary GalNacASGR ligand binding to ASGR1pos cells at indicated concentrations in the presence of excess ASGR1 antibodies.

ASGR1pos Cells ASGR1neg Cells

TNF-α: Myeloid activation, increases viral Ag presentation

Potent IFNγ Activation

IL-12: DC Activation

Figure 7: ASGR1-S induced myeloid cell activation leads to HBsAg reduction and seroconversion in the AAV-HBV mouse model

Figure 7: C57BL/6 mice were transduced with AAV8-HBV1.2 vector and sorted into treatment groups (n=10). Animals received 0.5mg of ASGR1-S, 0.5mg of unconjugated mASGR1 mAb, or vehicle, subcutaneously QWx5 beginning d0 (black arrows). In A) dotted line is LLOQ for HBsAg ELISA. In B), average plasma anti-HBsAg IgG was determined by ELISA. p-values determined by T-test by comparison to vehicle group.

ASGR1-TLR8 increases B cell expression of CD86, an activation marker indicative of increased antigen presentation and IgG secretion

TLR8 is highly expressed in human myeloid cells; as is TLR7 in mouse myeloid cells

Note: Mouse TLR8 is not a functional homologue of human TLR8

ASGR1-S

A B

HBsAg plasma levels SeroconversionB

p= <.0001

• ASGR1-S led to seroconversion in 9 of 10 animals• ASGR1-S also lowered average plasma viral DNA titers 2.5 logs p=<.001 t test

p= .02

A

A ASGR1-S increases IFNγ+ anti-viral T cells B ASGR1-S increases B cells producing anti-HBs antigen antibodies

No elevation in ALTNormal body weight gain

• No histopathology findings or elevated liver enzymes observed after ASGR1-S treatment.

Antigen Binding Domain: mouse ASGR1

Payload: Proprietary TLR7 Agonist Fc: mIgG2a

Functional Profile

AASLD TLMdX November 2020

IL-18: Synergizes with IL-12 for Th1 response