Targeted Delivery of TLR7 Agonists to the Tumor Microenvironment Enhances Tumor Immunity via Activation of Tumor-Resident Myeloid Cells

Abstract

Toll-like receptors (TLR) are phylogenetically conserved mediators of innate immunity that are essential for establishing adaptive immune responses against invading pathogens. TLR7 is an endosomal receptor expressed predominantly in myeloid and B cells. Activation of TLR7 induces Type I interferon and proinflammatory responses; therefore, targeting TLR7 is a promising strategy for antitumor therapy. Although the use of bacterial components to trigger innate immune responses in cancer patients started a century ago, the effectiveness of systemic TLR agonists has been rather underwhelming in clinical trials, partly due to nonspecific immune activation leading to safety and tolerability issues. Antibody-drug conjugates (ADCs) constitute a proven therapeutic modality amenable to systemic administration with limited toxicity concerns via a targeted delivery platform. We generated TLR7 agonist-antibody conjugates that recognize tumor antigens expressed on the surface of tumor cells. Generated ADCs demonstrated robust activity in in vitro tumor antigen-presenting cell (APC) coculture systems as indicated by dose-dependent upregulation of PD-L1 and CD86 on macrophages. TLR7 agonist-ADC provided superior tumor growth control compared to intravenously (IV) administrated free TLR7 agonist. Treatment with TLR7 agonist-ADC led to prolonged activation of myeloid cells in the tumor microenvironment (TME) with minimum immune activation in the periphery. Systemic and tissue exposure studies demonstrated tumor-specific free drug release by targeted ADC treatment. In summary, the TLR7 agonist-ADC can potentially activate immune cells in the TME to generate tumor antigen-specific T-cell responses, making it an attractive approach for precision cancer therapy.

Figure 1

Generation and characterization of site-specific TLR7 agonist ADC. (a) Chemical structures of the TLR7 agonist (1) used as a payload and the linker payload (2). (b) Bacterial transglutaminase (bTGase)-mediated bioconjugation for the introduction of the payload to the C-termini of the light chains of a monoclonal antibody. (c, d) Liquid chromatography–mass spectrometry (LC-MS) analysis of the naked antibody (c) and the TLR7 agonist ADC (d) after partial reduction showed light chain and heavy chain signals. The mass shift observed for the light chain signal indicates the site-specific attachment of drugs to the light chain. The unchanged heavy chain signal demonstrates conjugation site specificity.

Figure 2

TA99 ADC delivers the TLR7 agonist to the TME. (a) Tissue exposure of TA99-ADC and nontargeted control ADC after a single iv administration of ADCs at 10 mg/kg (N = 3). (b) Direct detection and quantification of TLR7 agonist in the complex mixtures by HPLC-MRM. (c) Tumor exposure of free drug after a single iv dosing with 10 mg/kg of ADCs. (d) Tumor exposure of unconjugated TLR7 agonist after a single iv dosing of 2.5 mg/kg.

Figure 3

TA99-TLR7 agonist activates macrophages in vitro. (a) BMDMs were cocultured with GP75 expressing tumor cells, treated with TA99-TLR7 agonist conjugate (top concentration 30 μg/mL, 1:3 serial dilutions) or an equimolar small-molecule TLR7 agonist. 24 h later, activation markers on macrophage were assessed by flow cytometry. (b, c) Treatment of TA99-TLR7 agonist conjugate resulted in upregulation of PD-L1 (b) and CD86 (c) expression on macrophages. Figure 3a was created with BioRender.com.

Figure 4

Systemic administration of the anti-mGP75 antibody (TA99)-TLR7 agonist ADC led to local myeloid activation within the tumor microenvironment (TME). C57Bl6 × Balb/c F1 hybrid mice bearing CT26-mGP75 tumors were treated intravenously with either 10 mg/kg TA99 ADC or 2.5 mg/kg TLR7 agonist. Tissues were collected at 1, 4, and 24 h post-treatment, mechanically dissociated into single-cell suspensions, and stained for flow cytometry analysis. The Y-axis represents the percentages of PD-L1 positive cell subpopulations within the entire CD11c+MHCII+ myeloid cell (cDC) population. The percentage of PD-L1+ cDCs within the entire cDC population was displayed for (a) tumor and (b) spleen. The percentage of IFN-a+ pDC within the entire pDC population was displayed for (c) tumor and (d) spleen. The percentage of TNF-a+ pDCs within the entire pDC population was displayed for (e) tumor and (f) spleen.

Figure 5

Anti-mGP75 antibody (TA99)-TLR7 agonist ADC provides superior tumor growth control. C57Bl6 × Balb/c F1 hybrid mice were implanted subcutaneously with CT26 tumor cells expressing the mouse GP75 antigen. When tumors reached a volume of 100 mm³, the mice were treated IV with 10 mg/kg of TA99 ADC, nontargeted control ADC, naked TA99 or control antibody (single dose), or 2.5 mg/kg of TLR7 agonist (QWx3). PD-1 mIgG1 D265A was dosed at 10 mg/kg (IV Q4Dx3). Tumors were measured twice per week, and the volume was calculated using the formula 1/2 (length × [width]²). Tumor volumes for each group at Day 27 post-treatment are shown in (a). Tumor growth curves for animals treated with TA99-ADC and TLR7 agonist 1 are displayed in (b).