Investigation of a NKG2D-dependent tumor immune escape mechanism of head and neck squamous cell carcinoma and proof of concept of a clinical intervention strategy

Natural killer cells are large granular lymphocytes of the innate immune system, which play an important role in the recognition and elimination of malignantly transformed and infected cells. The major activating NK cell receptor NKG2D, which is also expressed as co-stimulatory receptor on T cell subsets, is thereby a key modulator for tumor recognition and immune cell cytotoxicity. NKG2D recognizes several structurally related cellular ligands (MICA, MICB and ULBP1-6) commonly overexpressed on tumor cells. However, soluble NKG2D (sNKG2D) ligands released from malignant cells by proteolytical shedding, or on exosomes, can inhibit NKG2D-dependent NK and T cell cytotoxicity. Consequently, previous studies showed a correlation of elevated individual sNKG2D ligand plasma levels with impaired NK cell cytotoxicity in cancer patients. Moreover, in a phase I/II trial with high-risk neuroblastoma patients, sMICA was shown to inhibit not only autologous NK cells, but also adoptively transferred allogeneic NK cells after haploidentical stem cell transplantation. Head and neck squamous cell carcinoma (HNSCC) is a highly heterogeneous and aggressive tumor of the upper aero-digestive tract accounting for 300,000 annual deaths worldwide due to failure of current therapies. In a previous study, sMICA in HNSCC patients’ plasma was correlated with impaired NK cell function, indicating that NKG2D-dependent tumor immune escape might play a role for evasion of HNSCC from immunosurveillance. However, little is known about the cumulative role of all sNKG2D ligands and their impact during course of disease. Therefore, the aim of this thesis was the characterization of the NKG2D-dependent tumor immune escape in HNSCC to identify a patient cohort, which might benefit from restoration of NKG2D-dependent tumor immunosurveillance. In this respect, a proof of concept for the depletion of sNKG2D ligands from patients’ plasma as future therapeutic intervention strategy to restore NKG2D-dependent antitumor immune cell function was established. In this thesis, profiling of the plasma from 44 HNSCC patients in comparison to age-matched healthy donors revealed significantly elevated levels of sMICA, sMICB and sULBP1-3. The cumulative levels of sNKG2D ligands were patient specific and correlated with disease progression and tumor load. Moreover, these data suggested a prominent role of sULBP1 and sULBP3 in NKG2D-dependent tumor immune escape in HNSCC. In addition, the immunosuppressive state of HNSCC patients augments NKG2D-dependent escape especially through high plasma levels of TGF-β1. In in vitro cytotoxicity assays, high levels of sNKG2D ligands and TGF-β1 in patients’ plasma correlated with NK cell inhibition by blocking the NKG2D receptor. Importantly, the specific depletion of sNKG2D ligands by antibody-coupled magnetic beads successfully restored NK cell cytotoxicity of primary human NK cells against single tumor cell suspensions and in a 3D tumor spheroid model. Consequently, all sNKG2D ligands are indicative for NKG2D-dependent tumor immune escape in HNSCC and determine a time point at which malignant cells critically benefit from bypassing immunosurveillance. Based on tumor spheroids, it could be shown that inhibition of NKG2D-dependent cytotoxicity correlated with drastically reduced NK cell infiltration. These data were in accordance with low infiltration frequencies found in primary tumors of HNSCC patients. Therefore, interference with NKG2D-dependent tumor immune escape might boost both, NK cell cytotoxicity and redirect NK cells to infiltrate tumors. As proof of concept, a biofunctionalized matrix for adsorption apheresis based on a monoclonal antibody for the representative ligand MICA was generated and showed high depletion capacity of soluble MICA allelic variants from human plasma in vitro. The data from HNSCC patients indicate that the simultaneous depletion of all sNKG2D ligands might be crucial for a clinical intervention strategy. Therefore, a pan-specific bioreactive surface using recombinant human IgG1-Fc fusion proteins of the NKG2D ectodomain was generated. In vitro depletion experiments demonstrated quantitative depletion of sMICA/B and sULBPs from human plasma. To validate adsorption apheresis of sNKG2D ligands, the anti-MICA antibody adsorber was used in a proof of concept study in rhesus monkeys (Macaca mulatta). The pre-clinical study in rhesus monkeys infused with soluble human MICA demonstrated that adsorption apheresis of sNKG2D ligands is technically feasible and that quantitative removal of sMICA can be achieved after three plasma volume exchanges. In conclusion, the cumulative level of sNKG2D ligands and TGF-β1 might provide a novel diagnostic and/or prognostic marker combination to decipher a patient cohort, which benefits from restoration of NKG2D-dependent tumor immunosurveillance. Adsorption apheresis of sNKG2D ligands is proposed as therapeutic intervention strategy to improve the efficacy of autologous immune cells by restoration of tumor infiltration and cytotoxicity. Furthermore, ad-sorption apheresis as preconditioning strategy combined with cellular immunotherapies might help to improve the efficacy of therapeutically administered wildtype or engineered cytotoxic lymphocytes.