Multiple myeloma (MM) is a hematologic cancer characterized by clonal proliferation of plasma cells in the bone marrow (BM). The progression, from the early stages of the disease as monoclonal gammopathy of undetermined significance (MGUS) and smoldering multiple myeloma (SMM) to MM and occasionally extramedullary disease, is drastically affected by the tumor microenvironment (TME). Soluble factors and direct cell–cell interactions regulate MM plasma cell trafficking and homing to the BM niche. Mesenchymal stromal cells, osteoclasts, osteoblasts, myeloid and lymphoid cells present in the BM create a unique milieu that favors MM plasma cell immune evasion and promotes disease progression. Moreover, TME is implicated in malignant cell protection against anti-tumor therapy. This review describes the main cellular and non-cellular components located in the BM, which condition the immunosuppressive environment and lead the MM establishment and progression.

Despite the impressive results of autologous CAR-T cell therapy in refractory B lymphoproliferative diseases, CAR-NK immunotherapy emerges as a safer, faster, and cost-effective approach with no signs of severe toxicities as described for CAR-T cells. Permanently scrutinized for its efficacy, recent promising data in CAR-NK clinical trials point out the achievement of deep, high-quality responses, thus confirming its potential clinical use. Although CAR-NK cell therapy is not significantly affected by the loss or downregulation of its CAR tumor target, as in the case of CAR-T cell, a plethora of common additional tumor intrinsic or extrinsic mechanisms that could also disable NK cell function have been described. Therefore, considering lessons learned from CAR-T cell therapy, the emergence of CAR-NK cell therapy resistance can also be envisioned. In this review we highlight the processes that could be involved in its development, focusing on cytokine addiction and potential fratricide during manufacturing, poor tumor trafficking, exhaustion within the tumor microenvironment (TME), and NK cell short in vivo persistence on account of the limited expansion, replicative senescence, and rejection by patient’s immune system after lymphodepletion recovery. Finally, we outline new actively explored alternatives to overcome these resistance mechanisms, with a special emphasis on CRISPR/Cas9 mediated genetic engineering approaches, a promising platform to optimize CAR-NK cell function to eradicate refractory cancers.

Background:Chimeric antigen receptors (CARs) have been used in the past several years in cancer therapy to redirect immune effector cells. Despite impressive preliminary efficacy of CAR‐T cells in multiple myeloma (MM), NK cell engineering has emerged as a competitive and safer approach. NK‐92 is a universal, cheap and fast “off‐the‐shelf” cellular therapeutic previously used in clinical trials. Although modest responses with these cells have been reported in MM, their oncolytic potential can be enhanced by genetic modification. So far, two preclinical studies have been performed with CAR NK‐92 against MM, targeting CD138 or CS1 (SLAMF7). However, there are still reasonable doubts about its clinical outcomes due to on‐target off‐tumor effect or fratricide, respectively.Aims:Thus, the aim of our study is to generate and compare two novel CAR NK‐92 products for MM treatment.Methods:NK‐92MI cells were lentivirally transduced with the full‐length ectodomain sequence of the human native NKG2D receptor or with an anti‐BCMA scFv, both containing identical 4–1BB costimulatory and CD3‐ζ signaling domains (Figure 1A). To compare the efficacy between these 2nd generation NKG2D‐CAR NK‐92MI and BCMA‐CAR NK‐92MI cells, the same MOI 10 was used to transduce them; both populations were then purified by FACS sorting to obtain stable modified cell lines and vector copy number was measured by qPCR to ensure similar CAR expression. Cytotoxicity assays were performed by 3‐hours Calcein‐AM analysis. We used MM cell lines with different expression of target ligands: U266, BCMAhigh and NKG2DLhigh; XG‐1, a NK resistant cell line, BCMAhigh and NKG2DLlow; NCI H929 R20, a bortezomib resistant cell line with NKG2DLlow. K562, a leukemia cell line with BCMAnegative and NKG2DLhigh expression, was used as negative control.Results:NKG2D‐CAR NK‐92MI cells consistently showed much higher in vitro antitumor activity than the parental line NK‐92MI against U266 (84 ± 2% vs 40.7 ± 4% at a 1:1 E:T ratio), XG‐1 (67.9 ± 9% vs 18.5 ± 4% at a 16:1 E:T ratio) and NCI H929 #R20 (50.9 ± 6% vs 23.7 ± 2% at a 16:1 E:T ratio) cell lines (Figure 1B). NKG2D‐CAR NK‐92MI proved specificity in a blocking assay with an anti‐NKG2D antibody (82% decrease compares to baseline lysis). Furthermore, the oncolytic potential of NKG2D‐CAR NK‐92MI was not altered by physiological levels of soluble MICA, described in MM patients, or mandatory 10Gy irradiation prior to clinical use. Next, we compared cytotoxicity between NKG2D‐CAR NK‐92MI and BCMA‐CAR NK‐92MI against U‐266 (84 ± 2% vs 91.9 ± 3% at a 1:1 E:T ratio), XG‐1 (67.9 ± 9% vs 89.9 ± 2% at a 16:1 E:T ratio) and K562 (94 ± 3% vs 25.74 ± 4% at a 1:1 E:T ratio) cell lines (Figure 1C). Strikingly, there were no significant differences between NKG2D‐CAR NK and the gold standard BCMA‐CAR against MM cell lines with high and similar BCMA and NKG2DL expression. In addition, correlation between target ligands expression on the tumor and efficacy and specificity of both CARs was also shown. None of the CAR NK‐92MI studied populations showed toxicity against PBMCs from healthy donors and in vivo MM orthotopic xenograft mouse model experiments are ongoing.Summary/Conclusion:We have generated two novel and stable CAR NK‐92 immunoproducts that improve the oncolytic efficacy of the parental cell line. Indeed, NKG2D‐CAR NK‐92MI cells are as equally efficient as BCMA‐CAR NK‐92MI cells to eradicate diverse MM cells. To summarize, all these data show the feasibility to use this NK ‘off‐the‐shelf’ approach as immunotherapy for MM.image

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.