Proteolytische Freisetzung von NKG2D Liganden durch Tumorzellen

The immunoreceptor NKG2D promotes immunosurveillance of malignant cells and protects the host from tumour initiation by activating NK cells and costimulating CD8 T cells. MICA and other ligands of NKG2D are frequently expressed by tumour cells. Human tumour cells are thought to avert NKG2D-mediated-immunosurveillance by shedding MICA and other NKG2D ligands (NKG2DL). This thesis shows that the GPI-anchored NKG2DL ULBP2 is released from the cell surface of tumour cells by the action of metalloproteases similarly to the type I transmembrane proteins MICA and MICB. In addition, soluble ULBP2 was detected in the serum of patients with hematopoetic malignancies. Shedding of MICA and ULBP2 from tumours was induced by activation of protein kinase C (PKC) and was inhibited by the same compounds suggesting that ULBP2 and MIC molecules are released by the same or closely related proteases. Further, the molecular mechanisms of MICA shedding were defined to characterise the proteases involved. Amino acid deletions in the membrane-proximal stalk region of the MICA ectodomain greatly impaired MICA shedding, whereas amino acid substitutions had no significant effect. Further, MICA shedding was blocked by specific inhibitors of “a disintegrin and metalloprotease” (ADAM) proteases and was markedly reduced when ADAM10 and/or ADAM17 were down-regulated by RNA-interference. Altogether, these data demonstrate that ADAM10 and ADAM17 are critically involved in the proteolytic release of soluble MICA by tumours and thereby likely contribute to tumour immune evasion. Therefore, therapeutic blockade of ADAM10 and ADAM17 activities may represent a novel attractive approach to improve the efficacy of immunotherapeutic cancer treatment.NKG2D ist ein aktivierender NK-Zell Rezeptor und ein kostimulierender Rezeptor auf CD8 T Zellen. Die Expression der NKG2D Liganden (NKG2DL) wird durch Zellstress, virale Infektion und im Zuge maligner Transformation induziert und ermöglicht so dem Immunsystem die Erkennung und Elimination von veränderten und potentiell „gefährlichen“ Körperzellen (z. B. Tumoren). Die Freisetzung von löslichen NKG2D Liganden durch Metalloproteasen wird als ein wichtiger Mechanismus der Tumorzellen zur Vermeidung einer Immunantwort erachtet. In dieser Arbeit konnte gezeigt werden, dass auch der NKG2DL ULBP2, wie z. B. die MIC-Moleküle MICA und MICB, in löslicher Form von der Zelloberfläche von Tumorzellen freigesetzt wird. Lösliches ULBP2 konnte im Serum von Leukämie-Patienten nachgewiesen werden. Die Tumor-assoziierte Freisetzung sowohl von ULBP2 als auch von MICA ließ sich durch Aktivierung der Protein Kinase C verstärken und durch die gleichen Inhibitoren blockieren. Dies führte zu der Schlussfolgerung, dass sowohl die MIC Moleküle als auch ULBP2, als Vertreter der GPI-verankerten ULBPs, durch die gleichen oder nah verwandte Proteasen freigesetzt werden. Zur Identifizierung dieser Proteasen wurde beispielhaft der NKG2D Ligand MICA gewählt. Es konnte gezeigt werden, dass MICA in der Stielregion, die sich zwischen der Transmembranregion und der Ektodomäne befindet, gespalten wird. Wichtig für die Freisetzung ist nicht die Aminosäuresequenz, sondern die Länge dieser Stielregion. Des Weiteren konnte die Bildung von löslichem MICA durch Inhibitoren, die spezifisch gegen Mitglieder der ADAM (eine Disintegrin und Metalloprotease) Proteasen gerichtet sind, verringert werden. Die aufgrund dieser Experimente getätigte Annahme, dass ADAM10 und ADAM17 an der Freisetzung von löslichem MICA beteiligt sind, konnte in zwei unabhängigen Versuchsansätzen durch transiente Expressionssuppression dieser ADAMs in zwei Tumorzelllinien bestätigt werden. Zusammenfassend konnte gezeigt werden, dass die Metalloproteasen ADAM10 und ADAM17 an der Freisetzung von löslichem MICA von Tumorzellen beteiligt sind und somit einen interessanten Ansatzpunkt zur Verbesserung der Immuntherapien von Tumoren bieten

Sustained in vivo signaling by long-lived IL-2 induces prolonged increases of regulatory T cells.

Regulatory T cells (Tregs) expressing FOXP3 are essential for the maintenance of self-tolerance and are deficient in many common autoimmune diseases. Immune tolerance is maintained in part by IL-2 and deficiencies in the IL-2 pathway cause reduced Treg function and an increased risk of autoimmunity. Recent studies expanding Tregs in vivo with low-dose IL-2 achieved major clinical successes highlighting the potential to optimize this pleiotropic cytokine for inflammatory and autoimmune disease indications. Here we compare the clinically approved IL-2 molecule, Proleukin, with two engineered IL-2 molecules with long half-lives owing to their fusion in monovalent and bivalent stoichiometry to a non-FcRγ binding human IgG1. Using nonhuman primates, we demonstrate that single ultra-low doses of IL-2 fusion proteins induce a prolonged state of in vivo activation that increases Tregs for an extended period of time similar to multiple-dose Proleukin. One of the common pleiotropic effects of high dose IL-2 treatment, eosinophilia, is eliminated at doses of the IL-2 fusion proteins that greatly expand Tregs. The long half-lives of the IL-2 fusion proteins facilitated a detailed characterization of an IL-2 dose response driving Treg expansion that correlates with increasingly sustained, suprathreshold pSTAT5a induction and subsequent sustained increases in the expression of CD25, FOXP3 and Ki-67 with retention of Treg-specific epigenetic signatures at FOXP3 and CTLA4.This work was supported by Wellcome Trust Grant 091157, JDRF International Grant 9-2011-253, the National Institute for Health Research Cambridge Biomedical Research Centre, and the Medical Research Council Cusrow Wadia Fund. The Cambridge Institute for Medical Research (CIMR) is in receipt of a Wellcome Trust Strategic Award (100140). U.M.N. was the recipient of a Hoffmann-La Roche postdoctoral fellowship.This is thefinal version. It was first published by Elsevier at http://www.sciencedirect.com/science/article/pii/S089684111400146

A novel three-dimensional heterotypic spheroid model for the assessment of the activity of cancer immunotherapy agents

The complexity of the tumor microenvironment is difficult to mimic in vitro, particularly regarding tumor–host interactions. To enable better assessment of cancer immunotherapy agents in vitro, we developed a three-dimensional (3D) heterotypic spheroid model composed of tumor cells, fibroblasts, and immune cells. Drug targeting, efficient stimulation of immune cell infiltration, and specific elimination of tumor or fibroblast spheroid areas were demonstrated following treatment with a novel immunocytokine (interleukin-2 variant; IgG-IL2v) and tumor- or fibroblast-targeted T cell bispecific antibody (TCB). Following treatment with IgG-IL2v, activation of T cells, NK cells, and NKT cells was demonstrated by increased expression of the activation marker CD69 and enhanced cytokine secretion. The combination of TCBs with IgG-IL2v molecules was more effective than monotherapy, as shown by enhanced effects on immune cell infiltration; activation; increased cytokine secretion; and faster, more efficient elimination of targeted cells. This study demonstrates that the 3D heterotypic spheroid model provides a novel and versatile tool for in vitro evaluation of cancer immunotherapy agents and allows for assessment of additional aspects of the activity of cancer immunotherapy agents, including analysis of immune cell infiltration and drug targeting.ISSN:0340-7004ISSN:1432-085

Cergutuzumab amunaleukin (CEA-IL2v), a CEA-targeted IL-2 variant-based immunocytokine for combination cancer immunotherapy: Overcoming limitations of aldesleukin and conventional IL-2-based immunocytokines

We developed cergutuzumab amunaleukin (CEA-IL2v, RG7813), a novel monomeric CEA-targeted immunocytokine, that comprises a single IL-2 variant (IL2v) moiety with abolished CD25 binding, fused to the C-terminus of a high affinity, bivalent carcinoembryonic antigen (CEA)-specific antibody devoid of Fc-mediated effector functions. Its molecular design aims to (i) avoid preferential activation of regulatory T-cells vs. immune effector cells by removing CD25 binding; (ii) increase the therapeutic index of IL-2 therapy by (a) preferential retention at the tumor by having a lower dissociation rate from CEA-expressing cancer cells vs. IL-2R-expressing cells, (b) avoiding any FcγR-binding and Fc effector functions and (c) reduced binding to endothelial cells expressing CD25; and (iii) improve the pharmacokinetics, and thus convenience of administration, of IL-2. The crystal structure of the IL2v-IL-2Rβγ complex was determined and CEA-IL2v activity was assessed using human immune effector cells. Tumor targeting was investigated in tumor-bearing mice using 89Zr-labeled CEA-IL2v. Efficacy studies were performed in (a) syngeneic mouse models as monotherapy and combined with anti-PD-L1, and in (b) xenograft mouse models in combination with ADCC-mediating antibodies. CEA-IL2v binds to CEA with pM avidity but not to CD25, and consequently did not preferentially activate Tregs. In vivo, CEA-IL2v demonstrated superior pharmacokinetics and tumor targeting compared with a wild-type IL-2-based CEA immunocytokine (CEA-IL2wt). CEA-IL2v strongly expanded NK and CD8+ T cells, skewing the CD8+:CD4+ ratio toward CD8+ T cells both in the periphery and in the tumor, and mediated single agent efficacy in syngeneic MC38-CEA and PancO2-CEA models. Combination with trastuzumab, cetuximab and imgatuzumab, all of human IgG1 isotype, resulted in superior efficacy compared with the monotherapies alone. Combined with anti-PD-L1, CEA-IL2v mediated superior efficacy over the respective monotherapies, and over the combination with an untargeted control immunocytokine. These preclinical data support the ongoing clinical investigation of the cergutuzumab amunaleukin immunocytokine with abolished CD25 binding for the treatment of CEA-positive solid tumors in combination with PD-L1 checkpoint blockade and ADCC competent antibodies

A novel three-dimensional heterotypic spheroid model for the assessment of the activity of cancer immunotherapy agents

The complexity of the tumor microenvironment is difficult to mimic in vitro, particularly regarding tumor–host interactions. To enable better assessment of cancer immunotherapy agents in vitro, we developed a three-dimensional (3D) heterotypic spheroid model composed of tumor cells, fibroblasts, and immune cells. Drug targeting, efficient stimulation of immune cell infiltration, and specific elimination of tumor or fibroblast spheroid areas were demonstrated following treatment with a novel immunocytokine (interleukin-2 variant; IgG-IL2v) and tumor- or fibroblast-targeted T cell bispecific antibody (TCB). Following treatment with IgG-IL2v, activation of T cells, NK cells, and NKT cells was demonstrated by increased expression of the activation marker CD69 and enhanced cytokine secretion. The combination of TCBs with IgG-IL2v molecules was more effective than monotherapy, as shown by enhanced effects on immune cell infiltration; activation; increased cytokine secretion; and faster, more efficient elimination of targeted cells. This study demonstrates that the 3D heterotypic spheroid model provides a novel and versatile tool for in vitro evaluation of cancer immunotherapy agents and allows for assessment of additional aspects of the activity of cancer immunotherapy agents, including analysis of immune cell infiltration and drug targeting. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00262-016-1927-1) contains supplementary material, which is available to authorized users