Bispecific NKG2D-CD3 and NKG2D-CD16 Fusion Proteins as Novel Treatment Option in Advanced Soft Tissue Sarcomas.

Ilona Hagelstein,Kim L Clar,Martina S Lutz,Jonas S Heitmann,Elke Malenke,Clemens Hinterleitner,Gundram Jung,Yanjun Zhou,Moritz Schmidt,Melanie Märklin,Hans-Georg Kopp,Helmut R Salih

doi:10.3389/fimmu.2021.653081

Abstract

Soft tissue sarcoma (STS) constitutes a rare group of heterogeneous malignancies. Effective treatment options for most subtypes of STS are still limited. As a result, especially in metastatic disease, prognosis is still dismal. The ligands for the activating immunoreceptor NKG2D (NKG2DL) are commonly expressed in STS, but generally absent in healthy tissues. This provides the rationale for utilization of NKG2DL as targets for immunotherapeutic approaches. We here report on the preclinical characterization of bispecific fusion proteins (BFP) consisting of the extracellular domain of the NKG2D receptor fused to Fab-fragments directed against CD3 (NKG2D-CD3) or CD16 (NKG2D-CD16) for treatment of STS. After characterization of NKG2DL expression patterns on various STS cell lines, we demonstrated that both NKG2D-CD16 and NKG2D-CD3 induce profound T and NK cell reactivity as revealed by analysis of activation, degranulation and secretion of IFNγ as well as granule associated proteins, resulting in potent target cell lysis. In addition, the stimulatory capacity of the constructs to induce T and NK cell activation was analyzed in heavily pretreated STS patients and found to be comparable to healthy donors. Our results emphasize the potential of NKG2D-CD3 and NKG2D-CD16 BFP to target STS even in an advanced disease.

Highlights

Soft tissue sarcomas (STS) comprise a rare, heterogenic group of malignancies derived from tissues of mesenchymal lineage [1]
We developed NKG2D-CD16 and NKG2D-CD3 bispecific fusion proteins (BFP) consisting of the extracellular domain of NKG2D fused to anti-CD16 or anti-CD3 Fab-fragments instead of an optimized Fc part, which allow for recruitment of NK cells and T cells, respectively (Figure 1)
Whereas MICA, ULBP2 and 3 mRNA levels were found to be broadly expressed in all STS cell lines, MICB and ULBP4 mRNA signatures were only detected to a very low amount

Summary

Introduction

Soft tissue sarcomas (STS) comprise a rare, heterogenic group of malignancies derived from tissues of mesenchymal lineage [1]. With more than 100 different entities, biological and clinical characteristics in STS vary from low-grade tumors to highly aggressive cancers with an enormous metastatic potential [1, 2]. Reflecting its heterogeneity, genetic alterations in STS are NKG2D BFP in Sarcoma Treatment highly variable. Some STS histotypes are characterized by distinct genetic alterations including EWS-ATF1 in Ewing sarcomas, cKIT mutations in GISTs or PAX3-FKHR in alveolar rhabdomyosarcomas [3]. Some STS subtypes have recently been associated with epigenetic dysregulations triggered by a single or small number of genetic alterations [3, 7]

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Results

Conclusion