Table 1. Expression levels were determined using publicly available RNA-Seq datasets.
Introduction
Many solid tumors, including those expressing HER2, are resistant to immunotherapy due to immune-suppressive mechanisms, loss of human leukocyte antigen (HLA), low neoantigen availability, and/or minimal T cell infiltrates. These tumors frequently contain abundant populations of tumor-associated myeloid cells. Activation of these cells through TLR agonism has emerged as a promising approach in overcoming resistance mechanisms to current cancer immunotherapies. TLR8 is highly expressed in human myeloid cells known to be prevalent in human tumors such as conventional DCs and macrophages. Agonism of TLR8 in human myeloid cells activates a broad spectrum of anti-tumor immune mechanisms, including proinflammatory cytokine production, repolarization of suppressive myeloid cells and the priming of cytotoxic T-lymphocyte (CTL) responses. SBT6050 is a novel ImmunoTAC™ therapeutic comprised of a HER2-directed monoclonal antibody conjugated to a potent and specific TLR8 agonist, allowing for systemic delivery of a myeloid cell agonist with activity localized to HER2-expressing tumor sites. Here we present data demonstrating the superiority of SBT6050 at activating human myeloid cells compared to HER2 antibody conjugates that use either a selective TLR7 agonist or resiquimod.
Conclusions
• SBT6050, an ImmunoTAC™ therapeutic, is a HER2-directed monoclonal antibody conjugated to a TLR8-specific agonist which potently activates multiple human anti-tumor immune mechanisms in a HER2-dependent manner, enabling tumor-localized activity via systemic delivery.
• The activity of SBT6050 on human myeloid cells cannot be achieved with TLR7-specific or resiquimod antibody conjugates, despite these agents activating mouse myeloid cells.
• SBT6050 surrogate demonstrates potent single agent activity and durable anti-tumor responses upon re-challenge in tumor models with low tumor infiltrating lymphocytes, highlighting the potential for clinical activity in tumors with low tumor-infiltrating lymphocytes (TIL).
• SBT6050 mouse surrogate (SBT6050-S) is curative as a single agent in a xenograft model lacking T, B, and NK cells, demonstrating the potential of myeloid cells to mediate robust efficacy.
• 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-Induced Myeloid Cell Production of IL-18, IL-12p70 Leads to T, NK Cell Effector Responses
Figure 8: SBT6050-S Induces Durable Single Agent Efficacy in a T Cell Excluded Syngeneic Model
SBT6050, a HER2-Directed TLR8 ImmunoTAC™ Therapeutic, is a Potent Human Myeloid Cell Agonist that Provides Opportunity for Single Agent Clinical ActivityMichael R Comeau, Ty Brender, Monica Childs, Jamie Brevik, Damion Winship, Heather Metz, Jenny R Chang, Jeffrey Adamo, Ben Setter, Hengyu Xu, Li-Qun Fan, Brenda Stevens, Sean W Smith, Phil Tan, Robert DuBose, Yvette Latchman, Peter Baum and Valerie OdegardSilverback TherapeuticsTM, Seattle, WA | Contact information: [email protected]
Figure 9: SBT6050-S Induces Robust Single Agent Tumor Clearance in T, B, and NK Cell Deficient Mice
The data presented here demonstrate the following:
• SBT6050, but not TLR7 agonist or resiquimod HER2 conjugates, drives a broad spectrum of anti-tumor immune mechanisms in vitro.
• SBT6050, but not TLR7 or resiquimod HER2 conjugates, activate pro-inflammatory cytokine production from polarized human macrophages.
• SBT6050 potently activates human myeloid cells only in the presence of HER2-expressing tumor cells.
• SBT6050 induction of IL-12 and IL-18 from human myeloid cells results in the secondary activation of Granzyme-B and IFN-γ production by cells such as T cells and NK cells.
• SBT6050 mouse surrogate (SBT6050-S) is efficacious as a single agent in a HER2-expressing syngeneic model with low T cell infiltrate.
• SBT6050-S is curative as a single agent in a HER2-expressing tumor model deficient in T, B, and NK cells.
Figure 1: SBT6050 is an ImmunoTAC™ Therapeutic Designed for Systemic Administration with TME-Localized Activity
Figure 6: SBT6050 is Equipotent on Differentially Skewed Human Macrophages
Table 1: Human Myeloid-Restricted Expression Profile Supports Development of a TLR8-Selective Payload
Figure 2: (A) IHC of human breast carcinoma. (B) PBMCs were co-cultured with HER2-expressing (BT-474, MDA-MB-453) or HER2-negative (MDA-MB-468) tumor cell lines in the presence of SBT6050. No activation of PBMCs was observed when co-cultured with tumor cells and unconjugated HER2 mAb (SBT6050 antibody without the TLR8 agonist); data not shown. Data representative of multiple donors.
Figure 2: HER2 and TLR8 are Adjacent in Tumor, Consistent with Activation Requirements of SBT6050
Figure 3: SBT6050, a TLR8 Agonist Conjugate, Potently Activates Human Myeloid Cells
Figure 4: SBT6050, But Not TLR7 Agonist or Resiquimod Conjugates, Drives a Broad Spectrum of Human Anti-Tumor Immune Mechanisms
AACR 2020, Poster Number: 4537
HER2 and FcγR Engagement Required for SBT6050 Activity
B) SBT6050 Potently Activates Human Myeloid Cells in a HER2-Dependent Manner
A) IHC of Human Breast Carcinoma Shows HER2 and TLR8 in Close Proximity
HER2 TLR8
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1As reported by InvivoGen using cell-based reporter assays2Patent App. Publication US20150299194A1
Test Article hTLR8 (EC50)
hTLR7 (EC50)
SBT6050 Agonist 163 nM Inactive
TLR7 Agonist(SBT6050 Surrogate
Agonist)Inactive 9 nM
Resiquimod1 2900 nM 288 nM
Resiquimod Variant2 580 nM 60 nM
B) Unconjugated Small Molecule Agonist Activityon TLR8, TLR7 Cell-based Reporter Lines
Figure 3: (A) PBMC were co-cultured with HER2pos BT-474 tumor cells and TNF-α measured in the culture supernatants after 24 hours. HER2-TLR7 agonist, HER2-Resiquimod Variant and HER2-Resiquimod are conjugates incorporating the same anti-HER2 monoclonal antibody utilized in SBT6050 conjugated to their respective agonists. HER2 mAb is the unconjugated monoclonal antibody utilized in SBT6050. Control Conjugate is a conjugate incorporating an irrelevant monoclonal antibody conjugated to the TLR8 agonist utilized in SBT6050. All conjugates displayed similar HER2 and FcγR binding (data not shown). (B) Unconjugated small molecule agonist potencies determined in TLR specific cell-based reporter assays.
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Figure 4: PBMCs were co-cultured with HER2pos (BT-474) tumor cells in the presence of indicated conjugates or control anti-bodies. 24 hours later, supernatants were tested by MSD assay or ELISA. No significant IFN-α was detected with test reagents. No activity was seen in PBMC + HER2neg (MDA-MB-468) tumor cell co-cultures (data not shown).
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Figure 5: PBMCs were co-cultured with HER2pos (BT-474) tumor cells and 1 nM SBT6050 in presence or absence of 10 nM IL-18 and IL-12p70 neutralizing antibodies. 24 hours later, supernatants tested by ELISA. No activity was seen in PBMC + HER2neg
(MDA-MB-468) tumor cell co-cultures (data not shown). IL-18 synergizes with IL-12p70 for IFN-γ and granzyme-B production and both factors are induced by TLR8 agonism in PBMC cultures (Gorski et al Int Immunol 2006; Dietsch et al PLOS One 2016).
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Figure 6: Macrophages differentiated from peripheral blood human monocytes were co-cultured with HER2pos (BT-474) or HER2neg (MDA-MB-468) tumor cell lines in the presence of indicated conjugates or control antibodies. 24 hours later, super-natants were tested for TNF-α production levels by ELISA. No TNF-α production was observed when macrophages were co-cultured with HER2neg (MDA-MB-468) tumor cells (data not shown).
Figure 7: SBT6050 Mouse Surrogate (SBT6050-S) Matches the Functional Profile of SBT6050 on Myeloid Cells
Baseline Activity with Unconjugatedanti-HER2 mAb
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Figure 7: (A) RNA expression data was obtained from public databases. (B) Mouse bone marrow-derived macrophages were co-cultured with the human HER2pos (SK-BR-3) or HER2neg (MDA-MB-468) tumor cell lines in the presence of indicated conjugates or control antibodies. 24 hours later, supernatants were tested for mouse TNF-α production levels by ELISA.
B) TLR7 Conjugate (SBT6050-S) on Mouse Myeloid Cells and SBT6050 on Human Myeloid Cells are Equipotent
A) TLR7 Expression in Mouse Myeloid Cells is Comparable to that of TLR8 in Human
ImmunoTAC™ Conjugate EC50 (nM)
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Figure 8: (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, 10 mg/kg unconjugated HER2 mAb, or 10 mg/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.
A) HER2-expressing EMT6 Tumors are Infiltrated by Myeloid Cells, not T Cells Tumor (H&E) T Cells Macrophages
0/8 CR
B) Robust Single-Agent Anti-Tumor Responses
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C) Protection From Tumor Re-challenge is HER2-Independent
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Figure 9: 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, 10 mg/kg unconjugated HER2 mAb, or 10 mg/kg SBT6050-S. Arrows indicate doses administered.
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loid Fibroblasts ++ ++ - - +++ +++
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Concentration (nM) Concentration (nM)