Exploring the potential of combining IL-2-activated NK cells with an anti-PDL1 monoclonal antibody to target multiple myeloma-associated macrophages.

Multiple myeloma (MM) is an incurable disease, characterized by malignant plasma cells in the bone marrow. MM growth is largely dependent on the tumor microenvironment (TME), consisting of complex cellular networks that shape a tumor-permissive environment. Within the TME, tumor-associated cells (TAC) comprise heterogeneous cell populations that collectively support immunosuppression. Reshaping the TME toward an immunostimulatory environment may enhance effectiveness of immunotherapies. Here, we investigated interactions between donor-derived natural killer (NK) cells and TAC, like tumor-associated macrophages (TAM) and M1 macrophages, and assessed whether anti-tumor effector functions of NK cells could be enhanced by an ADCC-triggering antibody targeting macrophages. Monocytes were polarized in vitro toward either M1 or TAM before co-culture with high-dose IL-2-activated NK cells. NK cell responses were assessed by measuring degranulation (CD107a) and IFN-γ production. We found that NK cells degranulated and produced IFN-γ upon interaction with both macrophage types. NK cell responses against PD-L1+ M1 macrophages could be further enhanced by Avelumab, an anti-PD-L1- and ADCC-inducing antibody. Additionally, NK cell responses were influenced by HLA class I, shown by stronger degranulation in NK cell subsets for which the corresponding HLA ligand was absent on the macrophage target cells (KIR-ligand mismatch) compared to degranulation in the presence of the HLA ligand (KIR-ligand match). Our results suggest that NK cells could, next to killing tumor cells, get activated upon interaction with TAC, like M1 macrophages and TAMs, and that NK cells combined with PD-L1 blocking antibodies with ADCC potential could, through IFN-γ secretion, promote a more immune-favorable TME.

An in vitro model to monitor natural killer cell effector functions against breast cancer cells derived from human tumor tissue.

Adoptive natural killer (NK) cell-based immunotherapy poses a promising treatment approach in cancer. Despite minimal toxicities associated with NK cell infusion, the potential of NK cell therapy is inhibited by the immunosuppressive tumor microenvironment (TME). Multiple approaches to improve anti-cancer NK cell effector functions are being investigated. While much of this preclinical research is currently performed with commercially available tumor cell lines, this approach lacks the influence of the TME and heterogeneity of the primary tumor in patients. Here, we describe a comprehensive protocol for NK cell cytotoxicity- and degranulation assays against tumor cells derived from primary breast cancer tissue. Treatments to boost NK cell anti-tumor effector functions can be implemented in this model. Moreover, by using culture supernatants in follow up assays or by including additional cell types in the co-culture system, other NK cell effector mechanisms that further orchestrate innate and adaptive immunity could be studied.

Inhibitory receptors for HLA class I as immune checkpoints for natural killer cell-mediated antibody-dependent cellular cytotoxicity in cancer immunotherapy.

Natural killer (NK) cells mediate potent anti-tumor responses, which makes them attractive targets for immunotherapy. The anti-tumor response of endogenous- or allogeneic NK cells can be enhanced through clinically available monoclonal antibodies that mediate antibody-dependent cellular cytotoxicity (ADCC). NK cell activation is regulated by interaction of inhibitory receptors with classical- and non-classical human leukocyte antigens (HLA) class I molecules. Inhibitory receptors of the killer immunoglobulin-like receptor (KIR) family interact with HLA-A, -B or -C epitopes, while NKG2A interacts with the non-classical HLA-E molecule. Both types of inhibitory interactions may influence the strength of the ADCC response. In the present review, we provide an overview of the effect of inhibitory KIRs and NKG2A on NK cell-mediated ADCC, which highlights the rationale for combination strategies with ADCC triggering antibodies and interference with the NK cell relevant inhibitory immune checkpoints, such as KIR and NKG2A.

Polymorphic differences within HLA-C alleles contribute to alternatively spliced transcripts lacking exon 5.

The HLA genes are amongst the most polymorphic in the human genome. Alternative splicing could add an extra layer of complexity, but has not been studied extensively. Here, we applied an RNA based approach to study the influence of allele polymorphism on alternative splicing of HLA-C in peripheral blood. RNA was isolated from these peripheral cells, converted into cDNA and amplified specifically for 12 common HLA-C allele groups. Through subsequent sequencing of HLA-C, we observed alternative splicing variants of HLA-C*04 and *16 that resulted in exon 5 skipping and were co-expressed with the mature transcript. Investigation of intron 4 sequences of HLA-C*04 and *16 compared with other HLA-C alleles demonstrated no effect on predicted splice sites and branch point. To further investigate if the unique polymorphic positions in exon 5 of HLA-C*04 or *16 may facilitate alternative splicing by acting on splicing regulatory elements (SRE), in-silico splicing analysis was performed. While the HLA-C*04 specific SNP in exon 5 had no effect on predicted exonic SRE, the HLA-C*16 specific exon 5 SNP did alter exonic SRE. Our findings provide experimental and theoretical support for the concept that polymorphisms within the HLA-C alleles influence the alternative splicing of HLA-C.

ADCC-Inducing Antibody Trastuzumab and Selection of KIR-HLA Ligand Mismatched Donors Enhance the NK Cell Anti-Breast Cancer Response.

Natural killer (NK)-cell-based immunotherapies are an attractive treatment option for cancer. We previously showed that alloreactive mouse NK cells cured mice of 4T1 breast cancer. However, the tumor microenvironment can inhibit immune responses, and these suppressive factors must be overcome to unfold the NK cells' full anti-tumor potential. Here, we investigated the combination of antibody-dependent cellular cytotoxicity (ADDC) and the selection of KIR-HLA-ligand mismatched NK cells to enhance NK cell anti-breast cancer responses in clinically relevant settings. Donor-derived and IL-2-activated NK cells were co-cultured with patient-derived breast cancer cells or cell lines MCF7 or SKBR3 together with the anti-HER2 antibody trastuzumab. NK cells mediated anti-breast cancer cytotoxicity under normoxic and hypoxic conditions. Under both conditions, trastuzumab vigorously enhanced NK cell degranulation (CD107a) against HER2-overexpressing SKBR3 cells, but we observed a discrepancy between highly degranulating NK cells and a rather modest increase in cytotoxicity of SKBR3. Against patient-derived breast cancer cells, the anti-tumor efficacy was rather limited, and HLA class I expression seemed to contribute to inhibited NK cell functionality. KIR-ligand-mismatched NK cells degranulated stronger compared to the matched NK cells, further highlighting the role of HLA. In summary, trastuzumab and KIR-ligand-mismatched NK cells could be two strategies to potently enhance NK cell responses to breast cancer.

Activated Natural Killer Cells Withstand the Relatively Low Glucose Concentrations Found in the Bone Marrow of Multiple Myeloma Patients.

Infusion of ex vivo expanded and cytokine-activated natural killer (NK) cells is a promising alternative way to treat multiple myeloma (MM). However, the tumor microenvironment (TME) may suppress their function. While reduced glucose availability is a TME hallmark of many solid tumors, glucose levels within the TME of hematological malignancies residing in the bone marrow (BM) remain unknown. Here, we measured glucose levels in the BM of MM patients and tested the effect of different glucose levels on NK cells. BM glucose levels were measured using a biochemical analyzer. Compared to the normal range of blood glucose, BM glucose levels were lower in 6 of 9 patients (479-1231 mg/L; mean=731.8 mg/L). The effect of different glucose levels on NK cell cytotoxicity was tested in 4-hour cytotoxicity assays with tumor cells. 500 mg/L glucose (representing low range of MM BM) during the 4-hour cytotoxicity assay did not negatively affect cytotoxicity of activated NK cells, while higher glucose concentrations (4000 mg/L) diminished NK cell cytotoxicity. Since clinical application of NK cell therapy might require ex vivo expansion, expanded NK cells were exposed to a range of glucose concentrations from 500-4000 mg/L for a longer period (4 days). This did not reduce cytotoxicity or IFN-γ secretion nor affected their phenotypic profile. In summary, low glucose concentrations, as found in BM of MM patients, by itself did not compromise the anti-tumor potential of IL-2 activated NK cells in vitro. Although follow up studies in models with a more complex TME would be relevant, our data suggest that highly activated NK cells could be used to target tumors with a reduced glucose environment.

Defucosylation of Tumor-Specific Humanized Anti-MUC1 Monoclonal Antibody Enhances NK Cell-Mediated Anti-Tumor Cell Cytotoxicity.

Antibodies are commonly used in cancer immunotherapy because of their high specificity for tumor-associated antigens. The binding of antibodies can have direct effects on tumor cells but also engages natural killer (NK) cells via their Fc receptor. Mucin 1 (MUC1) is a highly glycosylated protein expressed in normal epithelial cells, while the under-glycosylated MUC1 epitope (MUC1-Tn/STn) is only expressed on malignant cells, making it an interesting diagnostic and therapeutic target. Several anti-MUC1 antibodies have been tested for therapeutic applications in solid tumors thus far without clinical success. Herein, we describe the generation of fully humanized antibodies based on the murine 5E5 antibody, targeting the tumor-specific MUC1-Tn/STn epitope. We confirmed that these antibodies specifically recognize tumor-associated MUC1 epitopes and can activate human NK cells in vitro. Defucosylation of these newly developed anti-MUC1 antibodies further enhanced antigen-dependent cellular cytotoxicity (ADCC) mediated by NK cells. We show that endocytosis inhibitors augment the availability of MUC1-Tn/STn epitopes on tumor cells but do not further enhance ADCC in NK cells. Collectively, this study describes novel fully humanized anti-MUC1 antibodies that, especially after defucosylation, are promising therapeutic candidates for cellular immunotherapy.

Tuning Natural Killer Cell Anti-multiple Myeloma Reactivity by Targeting Inhibitory Signaling via KIR and NKG2A.

Natural killer (NK) cells are attractive candidates for allogeneic cell-based immunotherapy due to their potent antitumor effector function and good safety profile. NK cells express killer immunoglobulin-like receptors (KIRs) and the NKG2A receptor important for NK cells education as well as providing inhibitory signals upon encountering HLA-expressing target cells. Multiple myeloma (MM) is an example of a tumor expressing relatively high levels of HLA molecules. In this review, we discuss the functional relevance of inhibitory KIRs and NKG2A for NK cells anti-MM response and strategies to lower these inhibitory signaling to enhance clinical efficacy of allogeneic NK cells in MM.

NKG2A Expression Is Not per se Detrimental for the Anti-Multiple Myeloma Activity of Activated Natural Killer Cells in an In Vitro System Mimicking the Tumor Microenvironment.

Natural killer (NK) cell-based immunotherapy is a promising therapy for cancer patients. Inhibitory killer immunoglobulin-like receptors (KIRs) and NKG2A are required for NK cell licensing, but can also inhibit NK cell effector function. Upon reconstitution in a stem cell transplantation setting or after ex vivo NK expansion with IL-2, NKG2A is expressed on a large percentage of NK cells. Since the functional consequences of NKG2A co-expression for activated NK cells are not well known, we compared NKG2A+ vs NKG2A- NK cell subsets in response to K562 cells, multiple myeloma (MM) cell lines and primary MM cells. NK cells were isolated from healthy donors (HLA-C1+C2+Bw4+) and activated overnight with 1,000 U/ml IL-2. NK cell degranulation in subsets expressing KIRs and/or NKG2A was assessed at 21 or 0.6% O2. Activated NKG2A+ NK cell subsets degranulated more vigorously than NKG2A- subsets both at 21 and 0.6% O2. This was irrespective of the presence of KIR and occurred in response to HLA-deficient K562 cells as well as HLA competent, lowly expressing HLA-E MM cell lines. In response to primary MM cells, no inhibitory effects of NKG2A were observed, and NKG2A blockade did not enhance degranulation of NKG2A+ subsets. KIR- NK cells expressing NKG2A degranulated less than their NKG2A- counterparts in response to MM cells having high levels of peptide-induced membrane HLA-E, suggesting that high surface HLA-E levels are required for NKG2A to inhibit activated NK cells. Addition of daratumumab, an anti-CD38 to trigger antibody-dependent cell-mediated cytotoxicity, improved the anti-MM response for all subsets and degranulation of the KIR-NKG2A- "unlicensed" subset was comparable to KIR+ or NKG2A+ licensed subsets. This demonstrates that with potent activation, all subsets can contribute to tumor clearance. Additionally, subsets expressing KIRs mismatched with the HLA ligands on the target cell had the highest level of activation in response to MM cell lines as well as against primary MM. Our current study demonstrated that if NK cells are sufficiently activated, e.g., via cytokine or antibody activation, the (co-)expression of NKG2A receptor may not necessarily be a disadvantage for NK cell-based therapy.