S256: hla-e/nkg2a checkpoint drives multiple myeloma resistance to car-nk therapy

Background: HLA-E is highly expressed in several tumor cells, including metastatic cells, and correlates with a poor prognosis. Its interaction with the inhibitory receptor NKG2A, expressed in T lymphocytes and NK cells, enables tumor cell escape. Although α-NKG2A antibodies have not shown positive preclinical and clinical results as monotherapy so far, their combination with other blocking antibodies has shown promise. Allogeneic CAR-NK cells have recently emerged as a safer immunotherapy than CAR-T cells, but their efficacy could be limited due to their high NKG2A expression. Aims: The aim of our study is to combine an allogeneic CAR-NK cell therapy with an α-NKG2A blocking antibody to overcome HLA-E/NKG2A resistance in MM. Methods: Bone marrow (BM) samples were extracted from healthy donors (HD) (n=11) and MM patients (n=47) to analyse HLA-E and NKG2A expression, and IFNγ from serum was quantified by flow cytometry. Mouse or human anti-human NKG2A antibodies or their corresponding isotypes were added to BM mononuclear cells (BMMCs) from MM patients to test their efficacy against plasma cells (PC). Peripheral blood mononuclear cells (PBMCs) were obtained from HD’ samples and co-cultured with the irradiated K562-mb21-41BBL cell line and IL-2. Then, activated and expanded NK (NKAE) cells were isolated and lentivirally transduced with second generation NKG2D-CAR or BCMA-CAR. NKG2D-CAR or BCMA-CAR NKAE cells pretreated with α-NKG2A blocking antibodies or their corresponding isotypes were co-cultured with MM cell lines or primary cells. Functional assays were carried out to study their anti-MM effect and hematotoxicity, in BMMCs co-cultures. In vivo activity was also tested in an orthotopic xenograft model of disseminated U-266 ffLucGFP MM in NSG-Tg hIL-15 mice. Results: HLA-E is significantly overexpressed in malignant PC from MM patients compared to healthy populations (non-malignant PC and CD34+ cells), especially at progression (Fig.1). IFNγ concentration is significantly increased in BM serum from MM patients (p<0.05) compared to HD, and rises HLA-E expression in MM cells (p<0.01). Although NKG2A is expressed in MM patients´ NK cells, α-NKG2A in vitro treatment of MM patients´ BMMCs does not decrease tumor PC number. Due to NK cell exhaustion in this setting, we have generated two allogeneic CAR-NK therapies, NKG2D-CAR and BCMA-CAR NKAE cells, whose ligands are overexpressed in MM cells. These immune effectors have a high percentage of NKG2A+ cells and release high levels of IFNγ that increase HLA-E expression in MM cells, suggesting that the HLA-E/NKG2A axis may limit CAR-NKAE cell activity. Combination of CAR-NKAE cells with either mouse or human α-NKG2A blocking antibodies significantly and equally increases CAR-NKAE cell cytotoxicity of MM cell lines (Fig.2A) and primary MM cells without showing toxicity against PBMCs and CD34+ cells. CAR-NK cell degranulation levels and intracellular IFNγ percentage also significantly augment with both antibodies in co-culture with tumor cell lines. Remarkably, the preclinical efficacy of the combination of CAR-NK and α-NKG2A is corroborated in the NSG-Tg hIL-15 mice bearing MM (Fig.2B), where cancer elimination is observed. Summary/Conclusion: Overexpression of HLA-E in MM cells and NKG2A in NK cells from MM patients at progression suggest a role of the HLA-E/NKG2A axis in tumor progression. Although α-NKG2A monotherapy does not restore NK cell activity in this cancer, the combination of allogeneic CAR-NKAE therapies with α-NKG2A antibodies overcomes MM resistance. These data support the efficacy and non-hematotoxicity of this proposal for MM treatment.Keywords: Resistance, Multiple myeloma, Cancer immunotherapy, NK cell