Abstract C024: CD206 small molecule activation recruits TLR1/2 to enhance cancer cell phagocytosis in reprogrammed tumor-associated macrophages and improves tumor control
Authors: Singh Sitanshu Sekhar , Sable Rushikesh V. , Kumari Anju , Hu Xin , Kapoor Abhijeet , Calvo Raul , Major Emily , Odzorig Taivan , Bassel Laura , Kozolov Serguei , Henderson Mark , Marugan Juan , Rudloff Udo 
Published: November 2022

Abstract
Introduction: Targeting innate immune checkpoints on tumor-associated macrophages (TAMs) like CD40 activation or CD47 blockade is currently tested as a promising new therapeutic strategy in pancreas cancer. The mannose receptor CD206 is expressed on M2-like TAM populations and activation of CD206 can recalibrate these immune suppressive cells towards an anti-tumor M1-like phenotype to elicit effective innate immune responses and tumor control. The lack of a detailed understanding of the molecular mechanisms of CD206-mediated macrophage reprogramming, however, is an impediment for the design of novel, effective combination studies or the development of accurate biomarkers. Methods: Single-cell RNA-Seq analysis of TAM populations of autochthonous KP16 and KPC tumors treated with a CD206 checkpoint modulator was used to classify CD206 positive M2-like TAMs into responsive vs non-responsive to CD206 modulation. Candidates of differentially expressed innate immune regulators were evaluated for their role in CD206 activation in macrophages derived from transgenic knockout mouse models deficient for different innate immune regulators. The induction of CD206 protein-protein interactions with other innate immune regulators was examined via proximal ligation assays (PLA) or co-immunoprecipitation, as well as a series of CD206 mutants expressed in different reporter assays. CCR2-RFP transgenic mice were used to study cancer cell phagocytosis in vitro and in vivo. Results: CD206 activation via small molecule candidate NCGC00413972 induces a conformational change in CD206 which induces the formation of a TLR1/2-CD206 trimeric complex, activates SYK kinase, recruits MyD88, and activates NF-κB and type I interferon signaling. Carbohydrate recognition domain 3 (CRD3) of the non-activated CD206 receptor negatively regulates CD206-TLR1/2 interaction. CD206 small molecule ligation, or the deletion of the N-terminal CRDs of CD206, leads to the formation of active TLR1/2-CD206 complexes, NF-κB activation, and TAM reprogramming. Loss of SYK kinase binding sites in the cytoplasmatic domain of CD206 or inhibition of SYK abrogates NF-κB activation and reduces cell death of M2-like macrophages but does not affect interferon signaling or cancer cell phagocytosis. The combination of an FDA-approved SYK inhibitor with a CD206 small molecule activator increased the fraction of M2-to-M1 reprogrammed TAMs and cancer cell phagocytosis in the tumoral stroma of KPC mice. CD206 and TLR2 activators also cooperated in the reprogramming of TAMs, resulting in a recalibration of the immune landscape towards an anti-tumor phenotype and enhanced tumor control. Conclusions: Deconvolution of the mechanism of action of CD206 small molecule activation identifies toll-like receptor signaling as an essential co-regulator for the induction of innate immune responses. Co-stimulation of CD206 and TLR2 demonstrates therapeutic potential as an effective immunotherapy in a recalcitrant disease, as it cooperatively increases NF-κB signaling, cancer cell phagocytosis, and recalibrates myeloid cells.
Citation Format: Sitanshu Sekhar Singh, Rushikesh V. Sable, Anju Kumari, Xin Hu, Abhijeet Kapoor, Raul Calvo, Emily Major, Taivan Odzorig, Laura Bassel, Serguei Kozolov, Mark Henderson, Juan Marugan, Udo Rudloff. CD206 small molecule activation recruits TLR1/2 to enhance cancer cell phagocytosis in reprogrammed tumor-associated macrophages and improves tumor control [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C024.