OA-02: α-NKG2A blockade overcomes multiple myeloma resistance to BCMA and NKG2D CAR-NKAE cells

Introduction: CAR NK cells have emerged as a promising, safer and off-the-self therapy for cancer patients. However, inhibitory immune checkpoints (IC) can constrain their anti-tumor efficacy, as occurs in CAR-T. HLA-E is overexpressed in several tumors, including metastatic cells, and correlates with a poor prognosis. Here, we have studied the impact of HLA-E and NKG2A expression in a high cohort of MM patients at different stages and we propose the combination of α-NKG2A blocking antibodies with BCMA and NKG2D-CAR NK cell immunotherapies to enhance their anti-MM efficacy.

Methods: HLA-E and NKG2A expression was analysed in bone marrow (BM) samples from healthy donors (HD) (n=18) and MM patients (n=71) and IFNγ from these sera was quantified. HLA-E modulation by IFNγ and bortezomib (BTZ) as well as the efficacy of α-NKG2A blocking monotherapy against plasma cells (PC), within BM mononuclear cells (BMMCs) cultures, were also studied by multiparametric flow cytometry (MFC).
Activated and expanded NK (NKAE) cells were obtained from HD’ peripheral blood MCs (PBMCs) after stimulation with irradiated K562-mb21-41BBL cells and IL-2. NKAE cells were then purified and lentivirally transduced with either BCMA or NKG2D CAR. The combination activity of these CAR therapies with mouse and human α-NKG2A antibodies was tested in vitro against primary MM cells, PBMCs and CD34+ cells, by MFC and calcein-release assays, and in vivo using NSG-Tg hIL-15 mice bearing U-266 ffLucGFP MM cells.

Results: MM patients at relapse/progression exhibit higher HLA-E expression in malignant PC compared to healthy populations and elevated NKG2A expression in NK cells. IFNγ, highly increased in MM patients’ BM sera, augments HLA E expression. BTZ can diminish this effect in sensitive MM cells but not in BTZ resistant, suggesting HLA-E/NKG2A as an important IC in this relapse/refractory setting. However, mouse or human α-NKG2A monotherapy treatment does not restore MM patients’ NK cells anti tumor efficacy.
BCMA-CAR and NKG2D-CAR NKAE cells also have an elevated NKG2A expression and release high levels of IFNγ that augment HLA-E levels in MM cells. In this instance, the combination of CAR NKAE therapies with mouse or human α NKG2A antibodies significantly increases their cytotoxicity, degranulation and IFNγ release against MM cells along with a lack of toxicity against HD’ PBMCs and CD34+ from MM patients. Differential immunophenotype after α NKG2A blockade in CAR NKAE cells is being studied by Cytof.
Notably, human α-NKG2A pretreatment in NKG2D-CAR NKAE cells have significantly increased mice survival, controlling the tumor in treated mice. α-NKG2A combination with BCMA-CAR therapy is being tested.

Conclusions: HLA-E and NKG2A are overexpressed in malignant PC and NK cells, respectively, from relapse/progression MM patients. Despite the lack of efficacy of α-NKG2A monotherapy, the combination of α NKG2A antibodies with BCMA and NKG2D-CAR therapies is able to overcome MM CAR target independent resistance in vitro and in vivo.