Introduction: Toll like receptor 7 (TLR7) activators are effective cancer immunotherapeutic drugs. However, the only clinically approved TLR7 ligand, imiquimod (IMQ), is absorbed into the systemic circulation inducing off-target adverse effects. To overcome this problem, we synthesized phospholipid-conjugated TLR7-specific activators. The objective of this project was to evaluate the safety and the anti-cancer activities of two TLR7 ligand-phospholipid conjugates, designated TMX-201 and TMX-202.

Procedures: In vitro immune activities of TLR7 agonists were compared in human and mouse leukocytes. Anti-malignant effects were evaluated using Inv-MycERTAM transgenic model of pre-malignant keratinocyte proliferation and murine models of 4T1 breast cancer and B16 melanoma.

Results: TMX-201 and TMX-202 were more potent cytokine inducers than IMQ in both mouse and human mononuclear cells. TMX-201 and TMX-202 showed less off-target binding compared to IMQ. The maximum tolerated doses of IMQ, TMX-201, and TMX-202 by i.v. administration were 15, 75 and 150 mg/kg, respectively. Topical administration of TMX-202 gel significantly reduced acanthosis in the Inv-mycERTAM model without inducing systemic cytokine production. Systemic administration of TMX-201 (4mg/kg) suppressed the growth of established subcutaneous B16 melanoma by 46%, without discernible adverse effects. Systemic or intra-tumor administration of TMX-201, in combination with anti-CTLA4 antibody, significantly suppressed lung metastasis formation in the 4T1 breast cancer model.

Conclusion: TMX-201 and TMX-202 displayed more potent immune stimulatory activity than IMQ with fewer off-target effects. These phospholipid-conjugated TLR7 agonists showed anti-cancer efficacy in three different mouse models. The new compounds may be useful topical and systemic therapeutic agents for cancer therapy, alone or in combination with checkpoint inhibitors.

Table 1.

TMX-201 effect on lung metastases in the 4T1 breast cancer model

Treatment Metastatic nodules 
Vehicle + isotype control 33.5 ± 5.2 
TMX-201 23.6 ± 4.9 
Anti-CTLA4 32.4 ± 13.6 
TMX-201 i.p. + anti-CTLA4 16 ± 2.7* 
TMX-201 i.t. + anti-CTLA4 13 ± 2.6* 
Treatment Metastatic nodules 
Vehicle + isotype control 33.5 ± 5.2 
TMX-201 23.6 ± 4.9 
Anti-CTLA4 32.4 ± 13.6 
TMX-201 i.p. + anti-CTLA4 16 ± 2.7* 
TMX-201 i.t. + anti-CTLA4 13 ± 2.6* 

*p<0.05 vs vehicle+isotype control group

Citation Format: Dennis A. Carson, Tomoko Hayashi, Brian Crain, Shiyin Yao, Jeffrey Cheng, Nadia Passini, Roberto Maj, Emanuela Mura, Howard Cottam, Johanna Holldack, Alcide Barberis. Application of novel phospholipid conjugated Toll like receptor 7 ligands for cancer therapy by topical and systemic administration. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2568. doi:10.1158/1538-7445.AM2014-2568