Boosting Natural Killer Cell-Mediated Targeting of Sarcoma Through DNAM-1 and NKG2D.

Ece Canan Sayitoglu,Renee Potens,Tolga Sutlu,Robin Krueger,Evren Alici,Kim Kusser,Didem Ozkazanc,Adil Doganay Duru,Harry Thomas Temple,Benjamin J Josey,Adil Mardinoglu,Cheng Zhang,Michael Chrobok,Tamara M Chinn,Cevriye Pamukcu,Anna-Maria Georgoudaki,Muhammad Arif,Michelle Hartman

doi:10.3389/fimmu.2020.00040

Abstract

Sarcomas are malignancies of mesenchymal origin that occur in bone and soft tissues. Many are chemo- and radiotherapy resistant, thus conventional treatments fail to increase overall survival. Natural Killer (NK) cells exert anti-tumor activity upon detection of a complex array of tumor ligands, but this has not been thoroughly explored in the context of sarcoma immunotherapy. In this study, we investigated the NK cell receptor/ligand immune profile of primary human sarcoma explants. Analysis of tumors from 32 sarcoma patients identified the proliferative marker PCNA and DNAM-1 ligands CD112 and/or CD155 as commonly expressed antigens that could be efficiently targeted by genetically modified (GM) NK cells. Despite the strong expression of CD112 and CD155 on sarcoma cells, characterization of freshly dissociated sarcomas revealed a general decrease in tumor-infiltrating NK cells compared to the periphery, suggesting a defect in the endogenous NK cell response. We also applied a functional screening approach to identify relevant NK cell receptor/ligand interactions that induce efficient anti-tumor responses using a panel NK-92 cell lines GM to over-express 12 different activating receptors. Using GM NK-92 cells against primary sarcoma explants (n = 12) revealed that DNAM-1 over-expression on NK-92 cells led to efficient degranulation against all tested explants (n = 12). Additionally, NKG2D over-expression showed enhanced responses against 10 out of 12 explants. These results show that DNAM-1+ or NKG2D+ GM NK-92 cells may be an efficient approach in targeting sarcomas. The degranulation capacity of GM NK-92 cell lines was also tested against various established tumor cell lines, including neuroblastoma, Schwannoma, melanoma, myeloma, leukemia, prostate, pancreatic, colon, and lung cancer. Enhanced degranulation of DNAM-1+ or NKG2D+ GM NK-92 cells was observed against the majority of tumor cell lines tested. In conclusion, DNAM-1 or NKG2D over-expression elicited a dynamic increase in NK cell degranulation against all sarcoma explants and cancer cell lines tested, including those that failed to induce a notable response in WT NK-92 cells. These results support the broad therapeutic potential of DNAM-1+ or NKG2D+ GM NK-92 cells and GM human NK cells for the treatment of sarcomas and other malignancies.

Highlights

Sarcomas are a group of rare, heterogeneous, and aggressive tumors of mesenchymal origin that may arise in a range of different tissues, including bone, cartilage, connective tissue, muscle, fat, vasculature, and peripheral nerves
In order to understand the immunological landscape of sarcomas and to assess possible immune defects that may contribute to the poor clinical responses observed, we characterized the major tumor-infiltrating leukocyte (TIL) populations and compared their frequencies to matched PBMCs from the same patients (Figure 1 and Table 1)
When assessing CD16 (FcγRIII) and collectively KIR2DL1 (CD158a), KIR2DL2/L3 (CD158b), and KIR3DL1(CD158e) in seven sarcoma patients, we observed a generalized decrease in the CD16+KIR+ and CD16+KIR− NK cell population in the TILs compared to NK cells in matched PBMC (n = 14, p = 0.0002 and 0.0107, respectively) (Figure 1C; Figure S1A)

Summary

Introduction

Sarcomas are a group of rare, heterogeneous, and aggressive tumors of mesenchymal origin that may arise in a range of different tissues, including bone, cartilage, connective tissue, muscle, fat, vasculature, and peripheral nerves. Several clinical trials have included sarcoma patients in the testing of various immunotherapy strategies, including: (i) immune checkpoint blockade [8,9,10]; (ii) tumorspecific or tumor-associated peptide vaccines [11,12,13,14]; and (iii) adoptive immune cell therapies with allogeneic NK cells (ongoing clinical trials), autologous T cells [15], CAR-T cells (ongoing clinical trials) and NY-ESO-1-specific T cells [16] to name a few [17] These studies aim to target general tumor markers found in a variety of solid tumors and hematological malignancies, not targeting sarcomas. This highlights the need for more precise tumor characterization, targeted immunomodulation of the individual tumor microenvironment and targeting of sarcoma-specific molecules

Methods

Results

Conclusion