TLR7/8 Agonist and SHP2 Inhibitor Loaded Nanoparticle Enhances Macrophage Immunotherapy Efficacy

Abstract

Tumor-associated macrophages (TAMs) are among the most abundant immune cells in the solid tumor microenvironment, making them an attractive target for cancer immunotherapy. However, there are two important challenges. First, tumors repolarize the TAMs predominantly to M2 tumor-aiding phenotype by secreting various immunosuppressive cytokines. Second, CD47 on cancer cells interacts with signal-regulating protein a (SIRPa) expressed on macrophages. This crosstalk provides a downregulatory signal in the form of activation of SHP1/2 that inhibits cancer cell phagocytosis. These challenges can be overcome by engineering a nanoparticle that can deliver a rationale combination of immunomodulatory agents to the TAMs that can repolarize the M2 macrophages to M1 phenotype efficiently and concurrently block CD47-SIRPa interactions by inhibiting SHP2 signaling. A lipid nanoparticle (LNP) system loaded with amphiphilic R848-cholesterol (TLR7/8 agonist) and SHP099 (SHP2 inhibitor) in a predefined ratio has been designed. In vitro studies show that the LNPs system repolarized to M2 macrophages to M1 phenotype and expressed co-stimulatory molecules while enhancing phagocytic potential. In vivo efficacy studies in 4T1 tumor-bearing mice show that LNPs exhibit superior anti-tumor efficacy than other treatments. Thus, the lipid nanoparticle-mediated co-delivery of a rational combination of TLR7/8 agonist and SHP2 inhibitor in the TAMs can enhance macrophage immunotherapy.

Supramolecular nanotherapeutics enable metabolic reprogramming of tumor‐associated macrophages to inhibit tumor growth

“…The concentration of the TLR agonist that was to be injected was 5 mg/kg. This value was determined from past studies that have evaluated the toxicity profiles of systemic injection of TLR agonists 25,26,35 . Treatment groups consisted of either MSNPs, R848‐SNP, ETO‐SNP, R848 + ETO FD, or untreated control.…”

“…Previous studies involving R848 do show that R848 does not have any effects on the expression of CD206. We attribute that the low dosing of Etomoxir could serve as a reason why there are no significant changes in CD206 levels 26 . Flow cytometric analysis were further validated through cytokine quantification of whole tumor and TUNEL experiments evaluating the cytotoxicity.…”

“…The TLR agonist amphiphile was first synthesized by an EDC, DMAP facilitated esterification reaction, where the active drug Resiquimod (R848) is conjugated to a cholesterol linker using an enzymatically active ester bond, which upon entering the cytosol of macrophages that have upregulated amounts of esterase, break the bond, hence releasing the active TLR agonist. 26 For synthesizing the Etomoxir amphiphile, cholesterol hemisuccinate was first modified by conjugation to the reactive amine group present on N-Boc-ethylenediamine using EDC-Sulfo-NHS chemistry. The Boc group was then deprotected, and the resulting Cholesterol-amine intermediate was conjugated to the carboxylic acid group present in Etomoxir to form the FAO inhibiting amphiphile (Figure 2A).…”

“…This value was determined from past studies that have evaluated the toxicity profiles of systemic injection of TLR agonists. 25,26,35 Treatment groups consisted of either MSNPs, R848-SNP, ETO-SNP, R848 + ETO FD, or untreated control. A total of three doses were administered every alternate day, and the body weight of the mice was monitored to determine the toxicity of treatments (Figure 4A).…”

“…We identified that the TL7/8 agonist R848 is reported in the literature to promote glycolysis and Etomoxir to inhibit FAO and OXPHOS. [24][25][26] Co-delivery of these drugs to TAMs could metabolically reprogram macrophages from an M2 phenotype to an M1 phenotype.…”