Generation and structure-activity relationships of novel imidazo-thienopyridine based TLR7 agonists: application as payloads for immunostimulatory antibody drug-conjugates.

Pairing immunostimulatory small molecules with the targeting capability of an antibody has emerged as a novel therapeutic modality with the potential to treat a variety of solid tumors. A series of compounds based on an imidazo-thienopyridine scaffold were synthesized and tested for their ability to agonize the innate immune sensors toll-like receptor 7 and 8 (TLR7/8). Structure-activity relationship (SAR) studies revealed that certain simple amino-substituents could enable TLR7 agonism at low nanomolar concentrations. Drug-linkers containing either payload 1 or 20h were conjugated to the HER2-targeting antibody trastuzumab at the interchain disulfide cysteine residues using a cleavable valine-citrulline dipeptide linker and stochastic thiol-maleimide chemistry. In vitro, these immune-stimulating antibody drug-conjugates (ADCs) were found to induce cytokine release in a murine splenocyte assay when co-cultured with the HER2-high NCI-N87 cancer cell line. In vivo, tumor regression was observed with a single dose in an NCI-N87 gastric carcinoma xenograft model in BALB/c nude mice.

An anti-HER2 biparatopic antibody that induces unique HER2 clustering and complement-dependent cytotoxicity.

Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that plays an oncogenic role in breast, gastric and other solid tumors. However, anti-HER2 therapies are only currently approved for the treatment of breast and gastric/gastric esophageal junction cancers and treatment resistance remains a problem. Here, we engineer an anti-HER2 IgG1 bispecific, biparatopic antibody (Ab), zanidatamab, with unique and enhanced functionalities compared to both trastuzumab and the combination of trastuzumab plus pertuzumab (tras + pert). Zanidatamab binds adjacent HER2 molecules in trans and initiates distinct HER2 reorganization, as shown by polarized cell surface HER2 caps and large HER2 clusters, not observed with trastuzumab or tras + pert. Moreover, zanidatamab, but not trastuzumab nor tras + pert, elicit potent complement-dependent cytotoxicity (CDC) against high HER2-expressing tumor cells in vitro. Zanidatamab also mediates HER2 internalization and downregulation, inhibition of both cell signaling and tumor growth, antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), and also shows superior in vivo antitumor activity compared to tras + pert in a HER2-expressing xenograft model. Collectively, we show that zanidatamab has multiple and distinct mechanisms of action derived from the structural effects of biparatopic HER2 engagement.

HIV-1 Tat exacerbates lipopolysaccharide-induced cytokine release via TLR4 signaling in the enteric nervous system.

The loss of gut epithelium integrity leads to translocation of microbes and microbial products resulting in immune activation and drives systemic inflammation in acquired immunodeficiency syndrome (AIDS) patients. Although viral loads in HIV patients are significantly reduced in the post-cART era, inflammation and immune activation persist and can lead to morbidity. Here, we determined the interactive effects of the viral protein HIV-1 Tat and lipopolysaccharide (LPS) on enteric neurons and glia. Bacterial translocation was significantly enhanced in Tat-expressing (Tat+) mice. Exposure to HIV-1 Tat in combination with LPS enhanced the expression and release of the pro-inflammatory cytokines IL-6, IL-1ß and TNF-α in the ilea of Tat+ mice and by enteric glia. This coincided with enhanced NF-κB activation in enteric glia that was abrogated in glia from TLR4 knockout mice and by knockdown (siRNA) of MyD88 siRNA in wild type glia. The synergistic effects of Tat and LPS resulted in a reduced rate of colonic propulsion in Tat+ mice treated with LPS. These results show that HIV-1 Tat interacts with the TLR4 receptor to enhance the pro-inflammatory effects of LPS leading to gastrointestinal dysmotility and enhanced immune activation.