Edited by: Ali A. Zarrin, TRex Bio, United States
Reviewed by: Camille Guillerey, University of Queensland, Australia; Rafael Solana, University of Cordoba, Spain
*Correspondence: Tony Reiman,
This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology
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Innate immune surveillance of cancer involves multiple types of immune cells including the innate lymphoid cells (ILCs). Natural killer (NK) cells are considered the most active ILC subset for tumor elimination because of their ability to target infected and malignant cells without prior sensitization. NK cells are equipped with an array of activating and inhibitory receptors (IRs); hence NK cell activity is controlled by balanced signals between the activating and IRs. Multiple myeloma (MM) is a hematological malignancy that is known for its altered immune landscape. Despite improvements in therapeutic options for MM, this disease remains incurable. An emerging trend to improve clinical outcomes in MM involves harnessing the inherent ability of NK cells to kill malignant cells by recruiting NK cells and enhancing their cytotoxicity toward the malignant MM cells. Following the clinical success of blocking T cell IRs in multiple cancers, targeting NK cell IRs is drawing increasing attention. Relevant NK cell IRs that are attractive candidates for checkpoint blockades include KIRs, NKG2A, LAG-3, TIGIT, PD-1, and TIM-3 receptors. Investigating these NK cell IRs as pathogenic agents and therapeutic targets could lead to promising applications in MM therapy. This review describes the critical role of enhancing NK cell activity in MM and discusses the potential of blocking NK cell IRs as a future MM therapy.
Multiple myeloma (MM) is characterized by the accumulation of malignant plasma cells (PCs), resulting in increased monoclonal protein in the blood and urine (
In recent years, there have been several notable therapeutic advancements for MM. Hematopoietic stem cell transplantation (HSCT) (
Natural killer (NK) cells are an intriguing immune cell type in MM given the recent development of monoclonal antibodies (mAbs), elotuzumab (anti-SLAMF7), and daratumumab (anti-CD38) that enhance NK cell-mediated tumour cell toxicity by activating the antibody dependent cellular cytotoxicity (ADCC) mechanism (
Given the success of blocking T cell IRs in multiple cancer types, blocking the IRs on NK cells offers another possibility to enhance anti-myeloma cell immunity. This review discusses NK cell IRs (
Restoring NK Cells by Targeting Their IRs.
NK cells are a cytotoxic subset of innate lymphoid cells (ILCs). They are the first responders against malignant and infected cells and are functionally classified by their innate capacity to eliminate cells without prior sensitization or recognition of presented antigens (
NK cells comprise 5% to 15% of peripheral blood lymphocytes (
When an NK cell encounters a cell, it does not necessarily induce cell lysis. Instead, cytotoxicity is dependent on expression of AR and IRs on the NK cells that are engaged by specific ligands expressed on target cells (
NK cell Surveillance of Cancer Cell
While the “missing self” mechanism of cell death works primarily through the lack of inhibitory signals, NK cells can also kill cancer cells with adequate activation signals (
When an NK cell comes in contact with a stressed cell, different patterns of inhibitory and activating ligand expression are detected through the NK cell’s IRs and ARs and the balance of these ligands and receptors dictates NK cell function. Activated NK cells can send suicide or self-destruction signals to the target cell and induce cell lysis through direct exocytosis of granzyme and perforin (
NK cells are integral members of anti-cancer immunity. While the cytotoxic mechanisms presented above represent ideal scenarios, the complex cancer immune microenvironment is marked by NK cell dysfunction and impairment. Deciphering how NK cell dysfunction contributes to tumorigenesis is essential to improve patient outcomes.
Several studies have reported the importance of NK cells in the immunosurveillance of tumor growth. An epidemiological study described that low activity of NK cells increased the risk of cancer specifically stomach, lung, and intestine (
NK cell numbers and functions have been linked to the prognosis of different blood cancers (
Likewise, the correlation between NK cells and the progression of MM is controversial (
Due to the complexity, heterogeneity, and plasticity of NK cells in cancer patients, the discrepancies are difficult to distill into a single explanation (
Finally, heterogeneity in ligand and receptor expressions within patient subsets may account for variability in research studies. For example, some patients may have reduced levels of activating ligands such as MICA/B that normally send signals to the activating receptor of the NK cell, NKG2D (
NK Cell Restoration Approaches for Multiple Myeloma Immunotherapy.
These studies not only provide a possible explanation for a discrepancy regarding the role of NK cells in MM, but also point to how an imbalance in ARs and IRs could lead to NK cell dysfunction in MM (
NK cells play an integral role in tumor surveillance, but are thought to be dysfunctional in MM patients. Immunosuppressive cells and cytokines, low NK cell numbers, IR and AR imbalance, and AR downregulation all lead to NK cell impairment and their inability to kill MM cells (
The mAb-ADCC approach recruits NK cells to myeloma cells that may otherwise be unrecognizable as stressed cells due to low activating ligand expression and IR/AR imbalance. As CD56bright NK cells mature to CD56dim cells, they express the Fcγ receptor III (also called CD16) that is important for ADCC against mAb-coated cancer cells (
Currently approved mAbs targeting MM cells include elotuzumab and daratumumab, targeting SLAMF7 and CD38 respectively. Both mAbs enhance NK cell cytotoxicity
Adoptive NK cell therapy aims to restore patient innate immune surveillance and control cancer progression by supplementing with new NK cells. This approach has shown promise against MM and other hematological malignancies including leukemia (
Early clinical trials using anti-CD33 CAR-NK-92 cells showed no major adverse effects in relapsed/refractory acute myeloid leukemia (AML) patients, supporting the notion that CAR-NK cells could be a safe alternative to CAR-T cells in hematological malignancies (
IMiDs have become a staple of MM treatment in the last two decades. Although their canonical mechanism of action is not often thought to include NK cells, IMiDs can act to restore NK cell activity. IMiDs reduce the NK cell activation threshold (
Proteosome inhibitors, such as bortezomib, also enhance anti-MM NK cell killing by downregulating HLA-I (
Expectedly, certain cytokines have been shown to augment NK cell function in MM and other hematological cancers. These cytokines include IL-2 and TNF-α (
Targeting NK IRs may unleash the breaks preventing NK cells from detecting and killing myeloma cells. Common IRs include KIRs, NK group 2 member A (NKG2A), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), T-cell Ig and ITIM domain (TIGIT), V-domain Ig-containing suppressor of T cell activation (VISTA), programmed death-1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and lymphocytic-activation gene 3 (LAG-3) (
The significant ligands of the KIR family include the HLA-I molecules. The most well-characterized inhibitory ligand is HLA-C where KIR2DL1 binds the C2 allele of HLA-C, and KIR2DL2 binds the C1 allele (
In non-transplantation settings, blocking the KIR-ligand axis may improve tumor immunity, similar to other checkpoint inhibitors (
Despite this pre-clinical success, heterogeneity in KIR expression can make mAbs targeting of these KIRs difficult in a clinical setting (
A phase II clinical trial treating MM patients with anti-KIR2D mAb (IPH2101) showed a surprising decrease in NK cell activity and KIR2D expression (
Selected clinical trials evaluating the safety, tolerability and efficacy of potential NK IRs for Multiple Myeloma NK cell-based immunotherapy (access date: August 10, 2020).
Receptor | Trial | Disease | Drugs | Phase | Participants | Results | Last Update Posted |
---|---|---|---|---|---|---|---|
KIR | NCT01217203 | Relapsed multiple myeloma | IPH2101, Lenalidomide | I | 15 | Complete; Objective response in 5 patients; Severe adverse events in 5 patients; No autoimmunity | February 28, 2014 |
NCT01222286 | Smoldering multiple myeloma | IPH2101 | II | 30 | Complete; No objective response; Adverse events in all patients | May 9, 2014 | |
NCT00999830 | Multiple myeloma | IPH2101 | II | 27 | Completed; Primary response in one patient (based on M-protein); Adverse events in 25 of 27 patients | March 24, 2016 | |
NCT00552396 | Multiple myeloma | Anti-KIR (1-7F9) | I | 32 | Complete; No dose-limiting toxicity; Severe adverse event in 1 patient; Increased patient NK cell cytotoxicity against MM |
March 31, 2016 | |
NCT02252263 | Multiple myeloma | Elotuzumab, Lirilumab, Urelumab | I | 44 | Complete; No results | November 1, 2017 | |
NCT01592370 | Non-Hodgkin’s lymphoma, Hodgkin lymphoma, multiple myeloma | Nivolumab, Ipilimumab, Lirilumab, Daratumumab, Pomalidomide, Dexamethasone | I/II | 375 | Ongoing | May 18, 2020 | |
NCT01248455 | Multiple myeloma, smoldering multiple myeloma | Anti-KIR | II | 9 | Terminated; Lack of patients meeting primary objective (50% decline in M-protein) | November 19, 2019 | |
NKG2A | NCT02921685 | Hematologic malignancies | Monalizumab | I | 18 | Ongoing | September 19, 2018 |
LAG-3/TIGIT | NCT04150965 | Relapsed refractory multiple myeloma | Elotuzumab, Pomalidomide, Dexamethasone, Anti-LAG-3, Anti-TIGIT | I/II | 104 | Ongoing | July 7, 2020 |
PD1 | NCT02903381 | Smoldering multiple myeloma | Nivolumab, Lenalidomide, Dexamethasone | II | 41 | Suspended; Safety concerns | July 21, 2020 |
NCT02636010 | Multiple myeloma | Pembrolizumab | II | 20 | Complete; No results | April 29, 2020 | |
NCT02331368 | Multiple myeloma | Autologous Stem Cell Transplant, Melphalan, Lenalidomide, MK-3475 | II | 32 | Terminated; Complete response in 7 of 23 evaluable patients; Severe adverse events in 14 of 32 total patients | July 27, 2018 | |
NCT03848845 | Multiple myeloma | GSK2857916, Pembrolizumab | II | 40 | Ongoing | August 5, 2020 | |
NCT03605719 | Recurrent plasma cell myeloma | Carfilzomib, Dexamethasone, Nivolumab, Pelareorep | I | 62 | Ongoing | November 25, 2019 | |
NCT03530683 | Lymphoma, multiple myeloma | TTI-622, Rituximab, PD-1 Inhibitor, Proteasome-inhibitors | I | 156 | Ongoing | September 12, 2019 | |
NCT03111992 | Multiple myeloma | PDR001, CJM112, LCL161 | I | 26 | Complete; No results | April 21, 2020 | |
NCT03357952 | Multiple myeloma | Daratumumab, JNJ-63723283 | II/III | 10 | Ongoing; All patients with treatment emergent adverse events; No dose limiting toxicity so far | January 3, 2020 | |
NCT03221634 | Multiple myeloma | Pembrolizumab, Daratumumab | II | 0 | Withdrawn; Business reasons | March 25, 2019 | |
NCT03292263 | Multiple myeloma | Melphalan, Nivolumab, Autologous Stem Cell Transplantation | I/II | 30 | Ongoing | March 17, 2020 | |
NCT02906332 | Multiple myeloma | Pembrolizumab, Lenalidomide, Dexamethasone | II | 16 | Ongoing; Combination is well tolerated; Preliminary data show potential efficacy | January 31, 2020 | |
NCT02807454 | Multiple myeloma | Daratumumab, Durvalumab, Pomalidomide, Dexamethasone | II | 37 | Ongoing | July 2, 2020 | |
NCT02685826 | Multiple myeloma | Durvalumab, Lenalidomide, Dexamethasone | I/II | 56 | Ongoing; Majority of patients with adverse events; Dose-limiting toxicity in 2 patients | April 27, 2020 | |
NCT02616640 | Multiple myeloma | Durvalumab, Pomalidomide, Dexamethasone | I | 114 | Ongoing | April 17, 2020 | |
NCT02576977 | Multiple myeloma | Pembrolizumab, Pomalidomide, Dexamethasone | III | 251 | Terminated; Anti-PD1 treatment combination had unfavourable benefit-risk profile in relapsed refractory multiple myeloma | July 31, 2020 | |
NCT02579863 | Multiple myeloma | Pembrolizumab, Lenalidomide, Dexamethasone | III | 310 | Terminated; Anti-PD1 treatment combination had unfavourable benefit-risk profile in newly diagnosed multiple myeloma | August 3, 2020 | |
NCT02289222 | Multiple myeloma | MK-3475, Pomalidomide, Dexamethasone | I/II | 48 | Terminated; Due to inclusion of an IMiD in combination with pembrolizumab | November 5, 2019 | |
NCT02077959 | Multiple myeloma | Lenalidomide, Pidilizumab | I/II | 20 | Complete; No results | May 30, 2019 | |
NCT02036502 | Multiple myeloma | Pembrolizumab, Lenalidomide, Dexamethasone, Carfilzomib | I | 77 | Complete; Tolerable safety profile; Notable anti-tumor activity | July 13, 2020 | |
NCT02726581 | Multiple myeloma | Nivolumab, Elotuzumab, Pomalidomide, Dexamethasone | III | 348 | Ongoing | August 10, 2020 |
Both ligand and receptor are highly expressed in patient samples across tumor types (
As proof-of-principle that NKG2A is important for NK cell activity and that it might serve as a relevant therapeutic target, NKG2A protein expression was knocked out in a human retroviral NK cell model to generate NKG2A-null NK cells. When NKG2A expression was lost, these cells showed higher cytotoxicity toward HLA-E positive cancer cells (
Accordingly, monalizumab, a novel IgG4 humanized antibody developed to block CD94/NKG2A, was shown to cause cancer cell death (
Although there is limited knowledge regarding the role of NKG2A in MM,
Like other IRs, expression of TIM-3 was also observed in circulating NK cells from cancers including lung adenocarcinoma (
Preclinical studies harvesting NK cells from patients with solid tumors have shown that blocking TIM-3 with anti-TIM-3 antibodies unleashed NK cell activity and induced IFN-γ production in NK cells (
It is important to note that there is still debate in the literature with contradictory assumptions about TIM-3 interaction with its ligands (
TIM-3 blocking mAbs are under clinical investigation either alone or in combination with anti-PD-1/PD-L1 mAbs. Initial results of TIM-3 blocking in solid tumors reported a manageable safety profile and revealed early signs of activity even in patients previously treated with PD-1 or PD-L1 mAb (
Overall, preliminary data suggests TIM-3 is a promising therapeutic target in several cancer types and supports the further clinical development of anti-TIM-3 inhibitors. No studies have specifically explored the role of TIM-3 in MM.
The majority of studies evaluating the role of TIGIT on tumor progression have focused on T cells and shown that TIGIT suppresses activity of T cells. In MM, refractory MM patients treated with DARA-pomalidomide combined therapy showed an increase in the exhausted T cells expressing CD28–veLAG3+veTIGIT+ve (
When focusing on NK cells, TIGIT has been shown to both contribute to and inhibit tumor progression. In one study, Jia et al. (
Given the dynamic nature of TIGIT expression on both NK cells and T cells as well as the dual role of TIGIT ligand PVR, particularly in MM, understanding how TIGIT affects NK cell function is critical. The complexity of this immune environment highlights the necessity to profile patients for expression of key IRs and ARs in order to better understand how to specifically harness NK cells to mediate an anti-tumor response in MM. CD8+veT cells in the BM of newly diagnosed and relapsed MM patients expressed higher levels of TIGIT compared with those in the healthy group. In this cohort, the investigators observed moderate levels of TIGIT on the NK cells from newly diagnosed or relapsed patients (
The high expression of TIGIT on NK cells suggests that a blockade of TIGIT as a monotherapy or in combination with other therapies may reverse NK cell exhaustion and enhance their activation (
Blocking TIGIT in combination with other therapies has also shown pre-clinical success. Combined treatments with anti-TIGIT and anti-PD-1 antibodies in a mouse models showed significant growth reductions in lymphoma (
Anti-TIGIT mAbs are now in phase I/II clinical trials as monotherapy or in combination with anti-PD-1 in solid tumors. Preliminary data from one trial (NCT02964013) showed a manageable safety profile and positive clinical response. Functional studies assessing how anti-TIGIT mAbs affect NK cells activity through cytokine production and NK cell degranulation in preclinical and clinical MM models may lead to an improved understanding of how to utilize NK cells in MM therapy.
Recent work using mouse models argues PD-1 also plays a robust inhibitory role in NK cells, elucidating the responsiveness of anti-PD-1 treated patients with low tumor HLA expression who would not be expected to show high T cell activity (
While some studies show that PD-1 is only lowly expressed on activated NK cells (
Despite success in solid tumors, anti PD-1/PD-L1 mAbs failed as monotherapy in MM (
Other combination regimens, such as anti-PD-1-radiation therapy, anti-PD-1-tumor vaccination, or combinations with other IR blockades may enhance the prognosis of refractory MM patients. Currently, blocking PD-1 with CARs has attracted the interest of investigators, with a new phase II trial (NCT04162119) recruiting patients to explore the safety and efficacy of BCMA-PD1-CAR-T cells in RRMM. BCMA-PD-1-CAR-T cell therapy works by administering T cells modified to target BCMA and secrete a PD-1Fc fusion protein capable of blocking the PD-L1/PD-1 inhibitory axis.
Given that low numbers of anti-MM T cells are a commonality amongst relapsed patients while NK cell-mediated MM cytotoxicity can be enhanced by anti-PD-1 therapy (
While PD-1 and CTLA-4 were the focus of initial immune checkpoint therapies, LAG-3 is part of the next wave of IRs being clinically investigated (
In MM, one study looked at immune checkpoint expression in the pathological shift from smoldering MM to symptomatic MM and demonstrated that LAG-3 expression on T cells increased with disease progression, suggesting LAG-3 as a potential target for immunotherapy (
Although LAG-3 is expressed on NK cells, it should not be considered a canonical immune checkpoint because of its low or absent expression in healthy patients (
A more recent analysis of the status of LAG-3 on the NK cell surface following exposure to IFN-α demonstrated an increased expression of LAG3 (
Further characterization showed that LAG3 was expressed in the NK cells populations that show high expression of activation and maturation markers. Additionally,
An anti-LAG-3 mAb is currently under clinical investigation in hematological malignancies (
Targeting IRs with mAbs has shown preliminary success in early clinical trials with positive response rates for some cancers. Emerging evidence also suggests that targeting IRs expressed on NK cells in MM remains a viable option and requires further exploration with particular attention paid to understanding the heterogeneity in ligand expression both within and across MM patients, the interplay between NK and T cells in response to IR blockade therapy, and how NK-targeted therapy can be combined with existing therapeutic options in MM patients.
In this review, we have highlighted the preclinical evidence that IRs on the NK cell such as KIRs, NKG2A, TIGIT, TIM-3, PD-1, and LAG-3 may impact MM biology and response to treatments. KIRs remain the most promising target. Not only were anti-KIR antibodies shown to be well tolerated, but they were also shown to enhance NK cell function (
Many of the IRs such as TIGIT, LAG-3, PD-1, and VISTA are expressed not only on NK cells, but also on T cells. Theoretically, blocking an IR expressed on both NK cells and T cells should enhance the anti-cancer effects of both immune cell types. However, the bulk of research on these IRs, particularly in the case of PD-1 and VISTA, has only elucidated their role on T cells, while neglecting to explore the role of NK cells. This is the case within the MM field as well as within the broader cancer community, highlighting the need for a more comprehensive understanding of how each immune cell type independently and collectively contributes to an anti-cancer effect. Specifically, the importance of NK cells has been shown in a study where the presence of NK cells affects the efficacy of a TIGIT-blocking mAb (
Tailoring treatment to patient-specific expression of receptors is widely adopted within the solid tumor community, but its use in MM is still relatively new. To ensure success of IR blockade therapy in MM, it is essential to estimate the patient’s expression of NK specific inhibitory ligands on malignant MM cells as well as the expression and functionality of targetable IRs on NK cells or T cells. Similarly, assessing the NK cells’ percentage, viability and functionality prior to the initiation of therapy may predict response to therapy. Previous trials proposed that the intra-tumoral level of IRs such as PD-1 on TILs were significant determinants of success for IR mAb therapies (
With this knowledge, prescription of specific IRs mAb relevant to individual expression patterns will more likely augment the immune cells to eradicate the cancer cells by hampering their evasion strategies in a precision-focused manner.
Early failure in clinical trials blocking these IRs is likely a complicated story reflecting not only intra- and inter-patient heterogeneity discussed above, but may also reflect the impaired immune landscape that is also temporally dynamic. A better understanding of how NK cell proliferation, function, and expression of receptors or their ligands changes during disease progression as well as in response to specific chemotherapeutics will improve our ability to effectively target NK cells to enhance their anti-tumor response. Specifically, studies have shown that expression of ligands such as PVR, PD-L1 can be enhanced by chemotherapy and/or IFN-γ (
Additionally, given the complex immune landscape in which NK cells reside, blocking a single NK cell IR may be insufficient in overcoming NK cell impairment in patients with severely compromised immune systems. Combination therapies further restoring immune and NK cell function may enhance NK cell IR therapies and elicit better patient outcomes. For example, a trial assessing both TIGIT and LAG-3 targeting in combination with anti-PD-1 is ongoing (NCT04150965). Similarly, IR-targeted therapy not only has implications for the intrinsic cytotoxic capabilities of NK cells, but can also be used within an ADCC and CAR-NK cell context. Considering the unique characteristic of the NK cells, which mediating ADCC, combining mAb against specific antigens expressed on myeloma cells with mAb targeting specific NK IRs according to their functional level and their cognate ligands on myeloma cells could enhance the NK cells killing. The combination of PD-1 blockade with mAbs daratumumab or elotuzumab are intriguing possibilities currently under investigation (
To conclude, we can say that there is a body of knowledge supporting the role of NK cells, IRs and cancer progression, although the evidence characterizing NK cells and their subpopulations in myeloma patients or the myeloma-NK cell interaction is still lacking. Therefore, we envision some key steps and factors to be considered in order to build on the foundation of myeloma-NK cell biology:
Profile NK cell receptors and subpopulations, NK cell activity and abundance, and NK cell function in myeloma.
Profile the expression of NK cell receptor cognate ligands in myeloma.
The immunosuppressive nature of myeloma poses a general challenge to immunotherapies in myeloma including those involving NK cells, and understanding and overcoming this challenge is critical to success.
Investigate the mechanisms that control specific ligands on the surface of myeloma.
Study ligand expression at the transcriptional and protein levels.
Evaluate the role of chemokines and soluble factors released in the microenvironment and if they positively or negatively mediate NK receptors and/or their ligands.
Explore the integration of NK cell-based therapies with traditional myeloma therapies pre-clinically to optimize clinical trial design.
Pursue the promise of CAR-NK cells in clinical trials.
-Myeloma–NK cell interaction
-Impact of NK cell receptor targeting on the effectiveness of mAb therapy for myeloma
-Personalizing approaches to NK cell-based therapies using knowledge of NK cell function and myeloma-NK cell ligand expression heterogeneity.
-Approaches to building CAR-NK cells as myeloma therapeutics (autologous, allogeneic, “off the shelf”)
Mobilizing NKs in MM is particularly attractive due to their natural capacity to distinguish damaged cells from healthy cells, allowing them to specifically eliminate only the damaged cells. Utilizing NK-based immunotherapy in MM remains an interesting and understudied area of research. This review highlights the important role that NK IRs may play in MM. With more research, we propose the development of a patient-specific strategy that incorporates precise IR blocking that can be adjusted according to patient-specific responses and changes due to different treatments regiments. This will involve more investigation into NK cell characteristics, their related ligands and NK cells subpopulations in MM patients as well as the MM microenvironment throughout disease stages. With this understanding comes the potential for novel IR-blockade immunotherapies regimen that could improve disease control and thus increase survival outcomes.
All authors contributed to the article and approved the submitted version. HA and TR conceptualized and designed the manuscript. JW and HA designed and created the figures and generated the clinical trial table. SG critically edited the manuscript.
This work is supported by a grant from The Canadian Institutes of Health Research (CIHR)-The New Brunswick Health Research Foundation (NBHRF) iCT SPOR grant SMC-151513, Canadian Cancer Society (CCS) 706194 and The Terry Fox Research Institute (TFRI) 1067.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.