Weichuan Luo's Dissertation

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REPOLARIZATION OF TUMOR-INFILTRATING MYELOID CELLS TO AUGMENT CAR T CELL THERAPIES

thesis
posted on 29.04.2021, 18:08 by Weichuan Luo

Although CAR T therapies have demonstrated efficacy in treating hematopoietic cancers, related CAR T cell therapies have been ineffective in treating solid tumors, in part due to the tumor’s immunosuppressive microenvironment. While this immunosuppressive tumor microenvironment (TME) is created by multiple cell types, critical to the suppressive properties of the TME are tumor-associated macrophages (TAMs) and myeloid-derived suppressor cells (MDSCs) that express checkpoint co-receptors (e.g. PD-L1), release immunosuppressive cytokines (e.g. TGFβ and IL-10), secrete growth factors (e.g. FGF, VEGF, and PDGF), and inactivate T cells by nitrosylating T cell receptors. Motivated by the fact that TAMs and MDSCs can change when exposed to an opposing immune stimulus, we investigated strategies to reprogram TAMs and MDSCs from their protumorigenic to an anti-tumorigenic state. Here, we demonstrate that targeting of a strongly proinflammatory TLR7 agonist specifically to TAMs and MDSCs can reverse an immunosuppressive TME by converting anti-inflammatory (M2-like) TAMs to pro-inflammtory (M1-like) macrophages and by decreasing the intratumoral content of MDSCs.

Our approach to the targeting of TLR7 agonists to TAMs and MDSCs is based on the fact that tumor-infiltrating myeloid cells over-express folate receptor β (FRβ) that is essentially absent from quiescent myeloid cells and all other cell types. By covalently linking a TLR7 agonist to folate, we demonstrate that the resulting conjugate (FA-TLR7a) accumulates specifically in TAMs and MDSCs, while avoiding uptake in other immune cells and healthy tissues. Because FRβ resides in the same endosome as TLR7, internalization of FA-TLR7a by the TAMs and MDSCs leads to activation of TLR7 and the consequent repolarization of the TAMs and MDSCs to their pro-inflammatory states. Moreover, because other immune cells express no FRβ and since the murine tumor models employed (4T1, CT26, EMT6 cells) also express neither FR nor TLR7, we conclude that the effects of FA-TLR7a described below derive primarily from repolarization of TAMs and MDSCs.

To test the ability of FA-TLR7a to improve the efficacy of a classic anti-CD19 CAR T cell therapy, we transduced the above 3 cancer cell lines with CD19 and tested the ability of anti-CD19 CAR T cells to eradicate the tumors in the presence and absence of FA-TLR7a. Infusion of CAR T cells alone exerted only moderate efficacy, while co-administration of FA-TLR7a greatly enhanced CAR T cell tumoricidal activity. Flow cytometric analysis of cell surface markers on TAMs and MDSCs demonstrated that the targeted TLR7 agonists shifted TAM markers from M2-like to M1-like while significantly decreasing the numbers of tumor infiltrating MDSCs. Similar analyses of other immune cells revealed increased numbers of T cells and anti-CD19 CAR T cells and enhanced expression of activation markers on the T cells. We conclude that the FA-TLR7 agonist greatly augments CAR T cell efficacy in solid tumors via stimulation of FR+ TAMs and MDSCs.

History

Degree Type

Doctor of Philosophy

Department

Chemistry

Campus location

West Lafayette

Advisor/Supervisor/Committee Chair

Dr. Philip S. Low

Additional Committee Member 2

Dr. Timothy L. Ratliff

Additional Committee Member 3

Dr. Qing Deng

Additional Committee Member 4

Dr. Tony R. Hazbun