The bispecific immunoligand ULBP2‐aCEA redirects natural killer cells to tumor cells and reveals potent anti‐tumor activity against colon carcinoma

Abstract

NKG2D, an activating receptor expressed on NK cells and T cells, is critically involved in tumor immunosurveillance. In this study, we explored the potential therapeutic utility of the NKG2D ligand ULBP2 for the treatment of colon carcinoma. To this end we designed a fusion protein consisting of human ULBP2 and an antibody-derived single chain targeting the tumor carcinoembryonic antigen (CEA). The bispecific recombinant fusion protein re-directed NK cells towards malignant cells by binding to both, tumor cells and NK cells, and triggered NK cell-mediated target cell killing in vitro. Moreover, tumor growth was significantly delayed in a syngeneic colon carcinoma mouse model in response to immunoligand treatment. The anti-tumor activity could be attributed to the stimulation of immune cells with an elevated expression of the activation marker CD69 on NK, T and NKT cells and the infiltration of CD451 immune cells into the solid tumor. In summary, it was demonstrated that immunoligands provide specific tumor targeting by NK cells and exert anti-tumor activity in vitro and in vivo. This technology represents a novel immunotherapeutic strategy for solid tumors with the potential to be further developed for clinical applications.Colorectal cancer is fatal in advanced stages of the disease. Curative treatment options are commonly reserved for tumors not yet metastasized or with resectable metastasis. Colorectal cancer is susceptible to chemotherapy from the onset, but due to therapeutic pressure cancer cells usually become resistant to the standard therapies. In addition, the standard therapies are frequently associated with severe side effects. Thus, there is a high demand for targeted therapies. Most recently developed antibody-based immunotherapies including cetuximab, bevacizumab and the fully human antibody panitumumab have not only added new impulses to colorectal cancer therapy, but essentially improved clinical outcome. 1 They have shown to overcome resistance to conventional chemotherapy and have clearly demonstrated that colorectal carcinoma is accessible to antibody therapy.The potential of immune modulation as well as the role of antibody therapy to enhance a cell-based immunotherapy is currently evaluated. 2-4 Increasing attention has been drawn onto NK cells, which are part of the innate immune system and can attack malignant cells without prior antigen stimulation. 5,6 Numerous ways of tumor cell recognition by NK cells have been described. NK cell cytotoxicity against tumors can be triggered by cells lacking expression of self-recognized MHC class I molecules. This mechanism has been referred to as the "missing-self"-hypothesis and is controlled through a group of inhibitory receptors on the NK cell surface. 7 In addition, an anti-tumor immune response can be induced by the upregulation of ligands for triggering NK cell receptors on the surface of tumor cells. 8 This mechanism has been referred to as "induced-self" and provokes an effective immune response even in the presence of inhibitory signalling....

NK cells and cancer: you can teach innate cells new tricks

“…The first of this kind was a RAET1H-anti-CD138 fusion protein that bound to CD138, which is overexpressed in multiple myeloma 186 , and crosslinked NKG2D on NK cells. Similarly, a RAET1H-anti-carcinoembryonic antigen (CEA) bispecific protein showed significant in vitro and in vivo efficacy against colon carcinoma 187 . Another study used recombinant MICA conjugated to mono clonal antibodies specific for CEA, HER2 or CD20, and their use resulted in NKG2D-dependent tumour cell lysis by NK cells 188 .…”

Mono- and dual-targeting triplebodies activate natural killer cells and have anti-tumor activityin vitroandin vivoagainst chronic lymphocytic leukemia

“…19 We reported the first such immunoligand, ULBP2-BB4 (scFv against CD138), which successfully activated and retargeted NK cells through ULBP2 against CD138-positive multiple myeloma cells both in vitro and in vivo. 20 Subsequently, two additional immunoligands in similar formats, ULBP2-aCEA (scFv against CEA) 21 and ULBP2-aPSMA (scFv against PSMA) 22 and immunoligands fused to other ligands, [23][24][25] validated our approach. Several trispecific immunoconstructs targeting the FcgRIIIa receptor on NK cells have been developed and compared with their bispecific counterparts.…”

“…19,30 So far, these novel approaches mainly involved bispecific recombinant proteins by our group and others to exploit NKG2D-dependent NK cell activation against tumor. [19][20][21] In this study, our strategy was to design NKG2D-triggering triplebodies that involved targeting of CD19 antigen or CD19/CD33 in combination. ULBP2-aCD19-aCD19 targets CD19 on CLL cells by two subsequent antiCD19 scFvs separated by 20 amino acid long Gly/Ser linker.…”

“…We previously reported the activation of NKT and T cells from spleen and peripheral blood of a syngeneic mouse model by ULBP2-aCEA immunoligand. 21 Although, g/d T cells represent a small subset of T cells, NKG2D co-stimulation on Vg9Vd2 T cells promotes secretion of IFNg, TNFa and cytotoxicity. 39 Recently, recombinant antibodies could successfully retarget g/d T cell effector functions against HER2-positive tumor cells.…”

Cancer Immunotherapy: New Technology to Target Cancer Cells

“…Many researchers have already harness the potential of NK cells by developing immuno ligands that target NKG2D via its cognate ligand (ULBP2) and bind to the target cells via an antibody derived single chain, which is specific for a tumour-associated antigen [15,16]. These immuno ligands have demonstrated antitumour activity both in vitro and in vivo.…”