Low Dose Cyclophosphamide in Combination with Elotuzumab - a Novel Immunotherapeutic Strategy for Multiple Myeloma

Abstract Introduction Cyclophosphamide (CTX) is a widely used anti-neoplastic, performing as an alkylating agent at high doses and immunomodulatory agent at low doses 1.. Combining CTX with monoclonal antibody (mAb) therapy has proven beneficial in potentiating relapsed and/or refractory multiple myeloma (RRMM) therapies, with daratumumab-directed MM cell death enhanced in the presence of CTX 2,3.. Elotuzumab (ELO), the second mAb approved for treating RRMM, promotes MM cell clearance by enhancing macrophage-mediated phagocytosis and CD16- and SLAMF7-directed NK cell cytotoxicity. ELO has been approved for use alongside dexamethasone and lenalidomide 4 or pomalidomide (POM) 5.. However, potential therapeutic benefits of ELO in combination with immunomodulatory drugs such as CTX and POM have yet to be examined. Our research investigates, the efficacy of combining low-dose CTX, alone or in combination with POM, and ELO in enhancing macrophage and NK cell infiltration and function in the MM tumour microenvironment. Materials and Methods Multiple myeloma cells (MM1S and H929) were treated with low-dose CTX and/or POM for 24hrs, washed to remove residual drug and resuspended in fresh media for tumour cell secretome (TCS) generation. Direct effects of CTX and/or POM on surface expression of checkpoint proteins (PD-1 and CD47) on MM cells was assessed by mean fluorescent intensity (MFI) flow cytometry. CD32/CD64 receptor expression on THP-1 macrophages, NKG2D, CD2, DNAM-1, CD96 and KIR2DL1 receptors on KHYG1 and primary NK cells, were measured using flow cytometry as a measure of activation. Migration of serum-starved, CFSE-labelled macrophages and NK cells towards CTX and/or POM TCS was assessed after 4hrs, with total number of migrated cells quantified using the Accuri flow cytometer. Immune cell function following indirect conditioning of macrophages/NK cells with MM cell TCS was measured by quantifying antibody-directed cellular phagocytosis (ADCP) or antibody-directed cellular cytotoxicity (ADCC), respectively. Conditioned immune cells were co-cultured with MM cells in a 2:1 effector to target ratio for 4hrs in the absence/presence of mAbs (ELO, nivolumab and anti-CD47), after which MM cell clearance was quantified by flow cytometry and presented as relative uptake (ADCP) and cytotoxicity (ADCC). One-way ANOVA statistical analysis was performed, followed by Tukey post hoc tests, with significance recognized at p<0.05. Results Direct treatment of MM cells with CTX increased surface expression of immune evading checkpoint proteins PD-1 and CD47 (p<0.05,n=3). POM monotherapy did not alter PD-1/CD47 expression, however dual therapy of CTX and POM supported the CTX-driven effect (p<0.001,n=3). Expression of CD32/CD64 macrophage activation markers was significantly increased on THP-1 cells following CTX-TCS conditioning (p<0.001,n=3). POM altered CD32, but not CD64, however dual treatment with CTX and POM significantly increased expression of both CD32 and CD64 (p<0.001, n=3). Migration of macrophages towards CTX-TCS was enhanced in a dose-dependent manner (p<0.01,n=3). CTX and POM dual therapy supported this CTX driven effect (p<0.001,n=3). Migration trends of both primary and KHYG1 NK cells were also increased towards the secretome from CTX treated MM cells. ADCP and ADCC were increased by CTX alone or in combination with POM (p<0.05, n=3). Effects of CTX on ADCP were not significantly enhanced by ELO, however ELO did significantly augment ADCC by CTX-conditioned primary NK cells (p<0.05,n=3). Given the increased expression of PD-1 and CD47, we investigated if the inclusion of nivolumab and anti-CD47 mAbs potentiated ADCC. Although ADCC was increased in all combinations, there was no significant difference between ELO alone versus ELO in combination with either nivolumab or anti-CD47. Conclusions Low-dose CTX and POM potentiated the immunomodulatory effects of ELO, with NK-directed cytotoxicity of MM cells enhanced in the presence of this mAb. Our data therefore indicates that the inclusion of low-dose CTX and or POM in combination with ELO could be a novel immunotherapeutic strategy for treating RRMM. References 1. Swan et al., Hemasphere. 2020;4(2). 2. Pallasch et al., Cell. 2014; 156(3):590-602. 3. Naicker et al., Oncoimmunology. 2021; 10(1):1859263 4. Dimopoulos et al., Blood Cancer Journal. 2020 10:91 5. Hose et al., Journal of Cancer Research and Clinical Oncology. 2021; 147:205-212 Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Cytokine Activation Induces CD25 Expression and a Signaling-Competent High-Affinity IL-2 Receptor On CD56dim Human NK Cells.

Blood ◽

10.1182/blood.v120.21.2159.2159 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2159-2159

Author(s):

Julie M Chase ◽

Jeffrey W Leong ◽

Rizwan Romee ◽

Todd A. Fehniger

Keyword(s):

Flow Cytometry ◽

Nk Cells ◽

Low Dose ◽

Nk Cell ◽

Surface Expression ◽

Receptor Expression ◽

Published Data ◽

High Affinity ◽

Cd25 Expression ◽

Cytokine Activation

Abstract Abstract 2159 Introduction. NK cells are innate immune lymphocytes important for host defense against infection, and also mediate anti-tumor responses. NK cell effector functions are triggered by ligand-mediated engagement of activating receptors and by cytokine stimulation. Human NK cells may be divided into developmental and functional CD56bright and CD56dim subsets with distinct biology. Both NK subsets have constitutive expression of CD122 (IL-2/15Rβ) and CD132 (γc), allowing for responsiveness to IL-15 and high doses (1 nM) of IL-2. The high affinity heterotrimeric IL-2 receptor is formed by CD122, CD132, and CD25 (IL-2Rα), allowing for responses to very low (10 pM) concentrations of IL-2. Previous studies demonstrated that resting CD56bright, but not CD56dim, NK cells constitutively express a functional high-affinity IL-2Rαβγ, as picomolar concentrations of IL-2 result in proliferation and co-stimulate IFN-γ production. We hypothesized that cytokine activation, which occurs at the site of an inflammatory response or interaction with a priming dendritic cell, may induce the expression of CD25 on CD56dimNK cells, allowing for enhanced responsiveness to IL-2. Methods. Purified normal donor NK cells (>95% CD56+CD3-) were cultured for 16h in media containing various cytokines, including IL-15 (100ng/mL) + IL-18 (50ng/mL), IL-15 (100ng/mL) + IL-12 (10ng/mL) or low dose IL-15 (1ng/mL) as a control (to support survival). Following stimulation, cells were washed and analyzed for surface expression of CD25 by antibody staining and flow cytometry. To assess the functional capacity of an induced IL-2Rα chain, NK cells were purified and treated with cytokines as above, washed extensively and replated in media devoid of all cytokines. At 3d after initial stimulation, cultures were briefly (15min) stimulated with IL-2 (10pM, 100pM and 1nM) or IL-15 (100ng/mL) to induce phosphorylation of STAT5. Cells were immediately fixed, permeablized, and assessed for intracellular phosphoSTAT5 by flow cytometry. Results. Pretreatment with IL-15 + IL-18 or IL-12 + IL-18, but no single cytokine, induced marked upregulation of CD25 on both CD56bright as well as CD56dim NK cells, with 93.6 ±1.9% of CD56bright and 97.7 ±1.2% (n=4) of CD56dim NK cells positive for CD25 expression immediately following the initial 16h stimulation with IL-15+18, compared to 20.7 ±6.8 and 3.6 ±1.4% for control treated (low dose IL-15) CD56bright and CD56dim NK. In preliminary experiments, CD25 appears to be markedly upregulated at the mRNA level, following treatment with IL-15+18. While kinetic analyses revealed that the absolute surface receptor expression was maximal at time points early after stimulation, 75.8 ±4.1% of CD56bright and 84.9 ±5.7% (n=4) of CD56dimNK cells pretreated with IL-15+18 retained CD25 surface expression at 7d post-stimulation. Importantly, CD25 induced on both CD56dim and CD56bright NK cells resulted in a signaling-competent high affinity IL-2 receptor, as stimulation with low dose IL-2 at 3d following initial cytokine pre-activation revealed increased production of phosphoSTAT5, versus control treated NK cells. The greatest enhancement was noted in CD56dim NK cells, showing an 8-fold increase in responsiveness to low dose (10pM) IL-2 stimulation (48.2±7.9% pSTAT5+ in IL-15+18 pretreated cells vs. 5.8±2.4% in control treated cells, p<0.02). Cytokine-pretreated CD56bright NK cells demonstrated no enhancement in phosphoSTAT5 following stimulation with low dose IL-2 (10pM) (27.6 ±8.3% in IL-15+18 pretreated vs. 26.0 ±6.4% in control) at this time-point. However, these results are in agreement with published data, which describe expression of CD25 on resting CD56bright but not CD56dimNK cells. Conclusions. Here, we report the induction of CD25, and a signal-competent high-affinity component of the IL-2 receptor, on CD56dim human NK cells, following cytokine pre-activation. These results have implications for the function of both CD56bright and CD56dim NK cells in the context of inflammation and potential for cross-talk with IL-2-producing T cells during an adaptive immune response. In addition, since rhIL-2 is clinically available and used following NK cell adoptive transfer in leukemia patients, these data provides a rationale for low dose IL-2 following allogeneic NK cell pre-activation with combinations of IL-15, IL-12, and IL-18. Disclosures: No relevant conflicts of interest to declare.

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Promising Preliminary Activity of Optimized Affinity, CD38 CAR NK Cells Generated Using a Non-Viral Engineering Approach in Gene Edited Cord Blood Derived NK Cells for the Treatment of Multiple Myeloma

Blood ◽

10.1182/blood-2021-151379 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 4793-4793

Author(s):

Rohit Duggal ◽

Sumit Sen Santara ◽

Myra Gordon ◽

Aoife Kilgallon ◽

David Hermanson ◽

...

Keyword(s):

Multiple Myeloma ◽

Cell Surface ◽

Cord Blood ◽

Nk Cells ◽

Nk Cell ◽

Surface Expression ◽

Cell Surface Expression ◽

Feeder Layer ◽

Guide Rnas ◽

Rnp Complex

Abstract CD38 is a multifunctional cell surface protein that is frequently overexpressed on malignant plasma cells as well as on immune suppressive cells within the tumor microenvironment and constitutes a validated immunotherapeutic target in the treatment of multiple myeloma (MM). At ONK Therapeutics we are developing a gene edited, cord blood-derived NK (CBNK) cell product targeting CD38 for treatment of patients with relapsed and/or refractory MM. The product will be generated using a workflow shown in Figure 1A. This involves starting with cord blood that is processed for NK expansion using a clinically validated, Epstein Barr Virus-transformed lymphoblastoid cell line (EBV-LCL) feeder layer. The NK cells would undergo genetic engineering that involves gene editing followed by a non-viral chimeric antigen receptor (CAR) introduction process mediated by the TcBuster (TcB) DNA transposon system (Biotechne). This is followed by a second round of expansion on the EBV-LCL feeder layer resulting in a characterized NK cell product that can then be cryopreserved. In order to develop protocols for optimizing the best transfection efficiencies using the Maxcyte ATx instrument, GFP mRNA (TriLink) was used for transfecting CBNK cells using different electroporation programs. High transfection efficiency was obtained using all programs (Figure 1B.), with the best from program NK4. Since the product employs an optimized affinity second generation anti CD38 CAR (Stikvoort et al., Hemasphere 2021) which could also target CD38 expressed on neighbouring activated NK cells, it is imperative to knock out (KO) the cell surface expression of CD38 on the CAR-NK cells. To achieve this we carried out CRISPR Cas9 based KO studies of CD38 (Figure 1C. left top), using guide RNAs targeting CD38 (Synthego) in the form of a ribonucleoprotein (RNP) complex with Cas9. CBNK cells were transfected using the Maxcyte ATx instrument and CD38 cell surface expression monitored. As shown in Figure 1C. (left top), complete CD38 KO was achieved 11 days post transfection. ONK Therapeutics is actively involved in targeting and downregulating the negative regulator of cytokine signalling, cytokine inducible SH2-containing protein (CIS), which is encoded by the CISH gene, as part of their CBNK products. It has been demonstrated that in addition to facilitating greater cytokine signalling, CISH KO also confers greater metabolic capacity to NK cells resulting in their increased persistence (Daher et al., Blood 2021). Therefore, ONK Therapeutics have evaluated CISH KO in CBNK cells (Figure 1C, top right) using the same scheme that was used for the CD38 KO. Guide RNAs in the form of a RNP complex with Cas9 (Synthego) were transfected into CBNK cells and intracellular CIS protein levels monitored over time. Almost complete KO was attained by 9 days post transfection. In order to dial in CISH KO as part of the product, we further carried out a simultaneous KO of CD38 and CISH, in addition to individual KO of CD38 or CISH (Fig 1C, bottom). Simultaneous multiplexing of the CD38 and CISH KOs resulted in efficient double KO (DKO) . The extent of knock down leading to KO in the DKO setting was very similar to that of individual gene KOs. We then introduced the anti CD38 CAR as part of a transposon that could be transposed by TcB transposase in CBNK cells. After DKO of CD38 and CISH in CBNK cells, the transposon DNA and mRNA for transposase were electroporated. CAR expression was detected 4-5 days post transposition (Figure 1D) with more than 50% of cells expressing the anti CD38 CAR. These CAR expressing CBNK cells were then tested for functionality in a co-culture kill assay against the CD38 positive MM cell line, RPMI8226, which was engineered to express firefly luciferase. In a 4 hour killing assay, robust killing of the RPMI8226 cells was achieved by the CAR-CBNK cells with an EC 50 ten times lower (more potent) than that of mock electroporation control CBNK cells. To our knowledge this is the first successful expression of an anti CD38 CAR in cord-derived NK cells, and with a double CD38/CISH KO, using non-viral CAR insertion approaches. Current work is focusing on designing and developing a manufacturing-ready workflow for this potential product and further examining the effects of CAR NK cell activity in a DKO setting where both KOs contribute to improved metabolism and potentially NK cell persistence, as well as exploring the added benefit of a DR5 TRAIL variant to enhance cytotoxicity. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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Induction Therapy With Continuous Alternate-Day Low Dose Lenalidomide Combined With Low-Dose Prednisone In Octogenarian Multiple Myeloma Patients

Blood ◽

10.1182/blood.v122.21.5394.5394 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5394-5394

Author(s):

Luca Pezzullo ◽

Bianca Serio ◽

Raffaele Fontana ◽

Idalucia Ferrara ◽

Mariarosaria Sessa ◽

...

Keyword(s):

Multiple Myeloma ◽

Nk Cells ◽

Low Dose ◽

Nk Cell ◽

Conflicts Of Interest ◽

Performance Status ◽

Progressive Increase ◽

Hematologic Toxicity ◽

Dose Prednisone

Abstract About 30% of patients with newly diagnosed multiple myeloma (NDMM) are older than 75 years. Immunomodulatory drugs (IMIDs) have improved response rates and outcomes of NDMM, except for patients older than 75 years more vulnerable to side effects of IMIDs because of their frailty and comorbidities. We evaluated efficacy, toxicity and health-related quality of life (HRQOL) associated with continuous alternate-day low dose lenalidomide (LD-R, 10 mg on alternate days) and low dose prednisone (15 mg/day) (LD-RP) in 7 octogenarian NDMM patients (5 males and 2 females) with a median age of 82 years (range 80-87). All octogenarian patients had IgG MM, except 1 oligosecretory lambda chain MM; all were in Durie-Salmon stage III, except 1 in stage II, and had poor WHO performance status (median: 2, range 1-3). Patients were evaluated at baseline and every 6 months for HRQOL according to MM-specific questionnaire QLQ-MY20 of European Organisation for Research and Treatment of Cancer (EORTC). All patients received aspirin thromboprophylaxis, 57% of them requiring from diagnosis erythropoietin and zoledronic acid treatment. In these 7 octogenarian NDMM patients completing at least three months of therapy, the overall response rate (ORR) was 86%, including 1 complete remission (CR), 2 very good partial remission (VgPR) and 3 PR. After a median follow-up of 12 months (range 3-24), the quality of response improved with continuous LD-RP treatment with a cumulative median reduction in monoclonal protein levels of 85% (range 20-100%); none of the patients required discontinuation of treatment secondary to specific hematologic and/or extra-hematologic toxicity. In addition, QLQ MY-20 questionnaires revealed that 70% of patients treated with continuous LD-RP reported improvements of QOL scores. Two out of 7 octogenarian patients died (1 for progression after 12 months and 1 for sepsis no treatment-related), and 2-year overall survival and progression-free survival estimates were 41% and 75%, respectively. Noteworthy, all patients treated with continuous alternate-day LD-RP showed a progressive increase in the percentage of CD3+ CD56+ NK cells during the first 6 months of LD-RP therapy reaching a plateau maintained until +12 months after initiation of therapy: the median percentage of NK cells was 4% before LD-RP treatment versus 10%, 13%, 30%, 31%, and 27% at +1, +3, +6, +9 and +12 months, respectively. Mean fold increase of NK cells during LD-RP therapy was 1.5, 2.5, and 6.5 at +1, +3 and +6 months, respectively. Progressive increase of NK cells was concomitantly associated with reduction in tumor-linked monoclonal immunoglobulin in all patients and increased circulating NK cells further support that this drug may mediate its anti-MM effect, at least in part by modulating NK-cell number and function. Our data provide evidence that continuous alternate-day low dose lenalidomide is a manageable and effective frontline treatment for octogenarian NDMM patients and increases circulating NK cells. These preliminary results require further validation in prospective larger studies. Disclosures: No relevant conflicts of interest to declare.

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Multiparameter Flow Cytometry Analysis of Peripheral Blood T, NK and Dendritic Cells From High-Risk Smoldering Multiple Myeloma Patients Treated with Lenalidomide and Dexamethasone

Blood ◽

10.1182/blood.v116.21.1906.1906 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1906-1906

Author(s):

Bruno Paiva ◽

Lucía López-Corral ◽

María-Belén Vidriales ◽

Luis Ignacio Sánchez Abarca ◽

Miguel T. Hernandez ◽

...

Keyword(s):

Cell Cycle ◽

Multiple Myeloma ◽

Flow Cytometry ◽

T Cells ◽

High Risk ◽

Nk Cells ◽

Nk Cell ◽

Multiparameter Flow Cytometry ◽

Cd8 Cells

Abstract Abstract 1906 Lenalidomide is an immunomodulatory agent that interacts with different components of the immune system by altering cytokine production, regulating T cells and increasing NK cell cytotoxicity. In multiple myeloma (MM), lenalidomide is approved for use in combination with dexamethasone in patients who have received at least one prior therapy. Recent observations have shown that dexamethasone enhances the anti-myeloma effect of lenalidomide; however, dexamethasone may also antagonize the immunomodulatory properties of lenalidomide. In the present study we evaluated by multiparameter flow cytometry (MFC) peripheral blood (PB) T, NK and dendritic (plasmacytoid, myeloid and monocytic) cells (DC) from high-risk smoldering MM (SMM) patients, defined by the presence of at least 2 of the 3 following criteria at diagnosis: bone marrow plasma cell (BMPC) infiltration ≥10%; and/or high M-component (IgG≥30g/L or IgA≥20g/L or B-J Protein>10g/L); and/or ≥95% myelomatous-PC/BMPC and immune paresis. SMM patients were treated according to the QuiReDex trial (NCT 00480363): an induction phase of nine four-week cycles of lenalidomide plus dexamethasone (LenDex) followed by maintenance with lenalidomide until disease progression. In this ongoing study, immunophenotypic data is available in 53 patients at diagnosis (baseline), 30 after 3 cycles of LenDex and 22 at the end of induction therapy (9th cycle). Here we will focus on the 22 cases with information at the 3 time points. For MFC analysis, PB samples were stained using a four-color direct immunofluorescence technique that allowed the quantification and characterization of T, NK and DC cells, including cell cycle analysis. The percentage of PB T cells in total PB cellularity was stable from baseline vs 3 vs 9 cycles of LenDex (22% vs 21% vs 21%; respectively, NS), with similar results also obtained for T CD4 (12% vs 11% vs 9%; respectively, NS) and T CD8 (8% vs 6% vs 8%; respectively, NS) cells. NK cells were slightly increased after 9 cycles of LenDex for both the CD56dim (4.1%, 3.4% and 6%; respectively; NS) and CD56bright (0.05%, 0.04% and 0.15%; respectively; NS) NK cell compartments. Similarly, the percentage of DC slightly increased along treatment, especially for plasmacytoid DC (0.2% at baseline vs 0.4% after 9 cycles; p=0.09). However, when a more detailed immunophenotypic characterization of T and NK cells was carried out significant differences emerged following LenDex treatment (Figure 1A). Accordingly, after 3 and 9 cycles of LenDex both T CD4 and CD8 cells showed increased expression of activation markers such as CD69 (p=.03), CD25 (p=.02 and NS, respectively), CD54 (p<.001), CD28 (p≤.03) and CD120b (p≤.01), together with increased production of IFNγ (p=.03) and IL-2 (p=.1 and p=.008, respectively). Interestingly, after induction therapy an up-regulation of chemokine receptors related to the Th1 (CCR5; p<.001) but also Th2 (CCR4; p≤.002) immune response was detectable in CD4 and CD8 T cells. T CD4 cells displayed a clear maturation into a central memory phenotype following LenDex treatment (38% at baseline vs 50% and 66% at 3 and 9 cycles, respectively; p<.001) while T CD8 cells displayed an increased effector memory phenotype (44% vs 59% vs 62%; p=.004). Further analysis showed increased expression of HLA-DR (p≤.008), the antibody-dependent cell-mediated cytotoxicity associated receptor CD16 (p≤.03), and the adhesion molecules CD11a (p’.006) and CD11b (p≤.004) both on NK (CD56dim and CD56bright) and T cells. No consistent changes were observed in other NK cell receptors, such as CD94 and the immunoglobulin like receptors CD158a, CD161, NKB1 (3DL1) and NKAT2 (2DL3). Concerning cell cycle analysis, the percentage of cells in S-phase was significantly increased from baseline vs 3 vs 9 cycles of LenDex in T CD4 (0.05% vs 0.15% vs 0.16%; p<.001), CD8 (0.05% vs 0.11% vs 0.23%; p<.001) and NK cells (0.09% vs 0.17% vs 0.20%; p=.001). Finally, an unsupervised cluster analysis of the overall immunophenotypic profile obtained after 9 cycles of LenDex (Figure 1B) was able to discriminate two groups of patients (A and B). Interestingly, within the group with higher activation profile (A) 50% of patients achieved ≥VGPR vs 23% in group B (p=.2). In summary, these preliminary results show that in high risk SMM patients the combination of lenalidomide and dexamethasone modulates PB T and NK cells, with increased activation status that may contribute to disease control. Disclosures: Off Label Use: Lenalidomide is not approved for the treatment of smoldering multiple myeloma. De La Rubia:Janssen-Cilag: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Rosiñol:Celgene: Honoraria. Oriol:Celgene: Consultancy; Janssen-Cilag: Consultancy; Novartis: Consultancy. Hernández:Celgene: Honoraria. de Arriba:Janssen-Cilag: Honoraria; Celgene: Honoraria. Mateos:Celgene: Honoraria. San Miguel:Janssen-Cilag: Honoraria; Celgene: Honoraria, Speakers Bureau.

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Forced Fucosylation With ASC-101 Enhances The Binding Of Ex Vivo Expanded Human NK Cells To E-Selectin: A Novel Method To Improve The Homing Of Adoptively Transferred NK Cells To The Bone Marrow In Patients With Hematological Malignancies

Blood ◽

10.1182/blood.v122.21.4499.4499 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4499-4499 ◽

Cited By ~ 2

Author(s):

Toshihiro Onishi ◽

Maria Berg ◽

Robert Reger ◽

Leonard Miller ◽

Steve Wolpe ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Stem Cell ◽

Nk Cells ◽

Hematological Malignancies ◽

Nk Cell ◽

Ex Vivo ◽

Binding Capacity ◽

Surface Expression ◽

Selectin Ligands

Introduction The ability of adoptively infused NK cells to home and traffic to the microenvironment where the tumor resides may be a critical determinant of their ability to mediate clinically meaningful anti-tumor effects. The initial step in leukocyte emigration from post-capillary venules, referred to as “tethering”, is a low-affinity interaction between leukocyte ligands with selectins expressed on endothelial cells. Since E-selectin is constitutively expressed on endothelium of skin and bone marrow in humans, leukocyte recruitment to bone marrow is thought to be largely dependent on E-selectin-binding. Among E-selectin ligands, only ligands bearing sialyl Lewis X with a terminal fucose (fucosylated) are functional forms that actively bind to E-selectin. One of the pitfalls of ex vivo NK cell expansion for adoptive infusion in humans is that expanded NK cells express predominantly non-fucosylated E-selectin ligands. We hypothesized that ex vivo fucosylation could enhance the binding capacity of E-selectin ligands on NK cells improving their homing into bone marrow where hematological malignancies reside. Methods CD56+/CD3- NK cells were isolated from normal human subjects by immuno-magnetic bead selection and were expanded ex vivo over 7-21 days by co-culturing with irradiated EBV-LCL feeder cells in IL-2 containing medium. Expanded NK cells were incubated for 30 minutes at room temperature with GDP-fucose and alpha1,3 fucosyltransferase-VI (ASC-101). The levels of fucosylation were determined by CLA surface expression measured by flow cytometry using the antibody HECA-452. After fucosylation, NK cells were analyzed by flow cytometry to assess for phenotype changes, viability and stability of fucosylation. Chromium release assays were performed to assess NK cell cytotoxicity against tumor cells. To determine whether fucosylated NK cells had enhanced binding to E-selectin, the binding capacity of NK cells to human recombinant E-selectin/Fc Chimera protein was evaluated by flow cytometry. Results Expanded human NK cells had low levels of baseline fucosylation, ranging from only 10-25%. Expanded NK cells were successfully fucosylated with ASC-101 in a dose-dependent manner (figure); the MFI of CLA on NK cells peaked at 25 ug/ml of ASC-101, with nearly 100% of NK cells being fucosylated (CLA positive). Fucosylation did not affect NK cell viability nor was it associated with changes in NK cell phenotype including surface expression of CD16, CD56, KIR2DL1, KIR2DL2/3, KIR3DL1, NKG2A, NKG2D, TRAIL, perforin, or granzymes A/B. Ex vivo cell culture showed fucosylation was sustained at nearly 100% for 48 hours, but then rapidly declined returning to baseline levels by 96 hours. NK cell cytotoxicity against tumor targets including K562 cells and myeloma cells was preserved and unaffected by fucosylation. Fucosylation significantly enhanced the binding capacity of NK cells to human E-selectin. Further, NK cell binding to recombinant human E-selectin/Fc chimera protein directly correlated with the degree of NK cell fucosylation (figure), which was dose-dependent on the ASC-101 concentration. The effects of forced fucosylation on the ability of human NK cells to home to the bone marrow following adoptive transfer into immuno-deficient mice is currently being explored. Conclusion Expanded NK cells primarily express non-glycosylated ligands for E-selectin, potentially limiting their ability to home to the bone marrow following adoptive transfer in humans with hematological malignancies. Ligands for E-selectin on the surface of expanded NK cells can be glycosylated ex vivo rapidly and to high degrees using ASC-101, significantly enhancing their ability to bind E-selectin. These data suggest forced fucosylation of NK cells could be used as a novel approach to improve the antitumor effects of adoptive NK cell infusions in patients with hematological malignancies. Disclosures: Miller: America Stem Cell Inc: Employment. Wolpe:American Stem Cell, Inc: Employment. Koh:America Stem Cell Inc: Employment.

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CS1 Is Expressed on Myeloma Cells from Early Stage, Late Stage, and Drug-Treated Multiple Myeloma Patients, and Is Selectively Targeted by the HuLuc63 Antibody.

Blood ◽

10.1182/blood.v108.11.660.660 ◽

2006 ◽

Vol 108 (11) ◽

pp. 660-660 ◽

Cited By ~ 5

Author(s):

Susann Szmania ◽

Balaji Balasa ◽

Priyangi Malaviarachchi ◽

Fenguhuang Zhan ◽

Yongsheng Huang ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Protein Expression ◽

Nk Cells ◽

Late Stage ◽

Plasma Cells ◽

Nk Cell ◽

Early Stage ◽

High Dose ◽

Myeloma Cells

Abstract Introduction: One-third of multiple myeloma (MM) patients exhibit high-risk features such as abnormal cytogenetics, high LDH, amplification of CKS1-B or spiked expression of MAF, MAF-B or FGFR3. While not affecting complete response rates, median durations of event-free and overall survival, even with high-dose melphalan-based tandem autotransplants of such patients, do not exceed 24 mo and 36 mo, compared to 60 mo and ≥ 90 mo for the remainder. Monoclonal antibody (mAb)-mediated therapy may target a chemotherapy-resistant myeloma cell pool. CS1 (CD2 subset 1, CRACC, SLAMF7), a member of the CD2 family of cell surface glycoproteins, exhibits high-level expression on primary myeloma cells, indicating that CS1 is a potential target for treatment in MM. Methods: Gene expression was assessed using an Affymetrix GeneChip array. Protein expression was measured by flow cytometry, and immunohistochemistry (IHC), using HuLuc63, a novel humanized anti-CS1 mAb. HuLuc63-mediated lysis of myeloma cells via antibody dependent cellular cytotoxicity (ADCC) was measured by 51Cr-release. Results: CS1 mRNA was detected in >95% of CD138+ purified plasma cells from >95% of healthy donors, newly diagnosed myeloma patients, and those with relapsed myeloma (Fig. 1a). CS1 remained highly expressed in patients following VDTPACE treatment, albeit at a reduced level. CS1 expression was also high following bortezomib (Velcade®) treatment, with a subset of patients showing increased expression post-treatment. CS1 protein expression on primary myeloma cells was confirmed by flow cytometry, while IHC analysis of normal tissues revealed anti-CS1 staining primarily on CD138+ tissue plasma cells. Finally, we determined that HuLuc63 could induce killing of myeloma cells using purified allogeneic NK cells (Fig. 1b). Blocking the Fc receptor greatly reduced this activity indicating an ADCC mechanism. Killing of myeloma targets was also observed in autologous systems suggesting that HuLuc63 can overcome KIR-mediated NK cell inhibition of autologous NK cells. In summary, we observed high mRNA and protein expression of CS1 in myeloma from early stage, late stage, and treated patients, and showed enhanced lysis of myeloma cells in vitro with HuLuc63. Our data support the potential clinical utility of CS1-targeted therapy. HuLuc63 will be entering a phase I clinical trial for advanced myeloma patients. Figure 1a. CS1 mRNA is highly expressed in CD138+ purified plasa cells. Figure 1a. CS1 mRNA is highly expressed in CD138+ purified plasa cells. Figure 1b. CS1 antibody enhances killing of myeloma cells by allo - NK cells. Figure 1b. CS1 antibody enhances killing of myeloma cells by allo - NK cells.

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Direct Binding of Human NK Cell Natural Cytotoxicity Receptor NKp44 to the Surfaces of Mycobacteria and Other Bacteria

Infection and Immunity ◽

10.1128/iai.00870-07 ◽

2008 ◽

Vol 76 (4) ◽

pp. 1719-1727 ◽

Cited By ~ 91

Author(s):

Semih Esin ◽

Giovanna Batoni ◽

Claudio Counoupas ◽

Annarita Stringaro ◽

Franca Lisa Brancatisano ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Cell Subset ◽

Surface Expression ◽

Binding Assays ◽

Igg Antibody ◽

Direct Stimulation ◽

Activating Receptors ◽

Accessory Cells

ABSTRACT Our previous studies demonstrated that Mycobacterium bovis bacillus Calmette-Guérin (BCG) can directly interact with human NK cells and induce the proliferation, gamma interferon production, and cytotoxic activity of such cells without the need for accessory cells. Thus, the aim of the present study was to identify the putative receptor(s) responsible for the recognition of BCG by human NK cells and potentially involved in the activation of NK cells. To this end, we first investigated the surface expression of three NK cell-activating receptors belonging to the natural cytoxicity receptor (NCR) family on highly purified human NK cells upon in vitro direct stimulation with BCG. An induction of the surface expression of NKp44, but not of NKp30 or NKp46, was observed after 3 and 4 days of in vitro stimulation with live BCG. The NKp44 induction involved mainly a particular NK cell subset expressing the CD56 marker at high density, CD56bright. In order to establish whether NKp44 could directly bind to BCG, whole BCG cells were stained with soluble forms of the three NCRs chimeric for the human immunoglobulin G (IgG) Fc fragment (NKp30-Fc, NKp44-Fc, NKp46-Fc), followed by incubation with a phycoerythrin (PE)-conjugated goat anti-human IgG antibody. Analysis by flow cytometry of the complexes revealed a higher PE fluorescence intensity for BCG incubated with NKp44-Fc than for BCG incubated with NKp30-Fc, NKp46-Fc, or negative controls. The binding of NKp44-Fc to the BCG surface was confirmed with immunogold labeling using transmission electron microscopy, suggesting the presence of a putative ligand(s) for human NKp44 on the BCG cell wall. Similar binding assays performed on a number of gram-positive and gram-negative bacteria revealed a pattern of NKp44-Fc binding restricted to members of the genus Mycobacterium, to the mycobacterium-related species Nocardia farcinica, and to Pseudomonas aeruginosa. Altogether, the results obtained indicate, for the first time, that at least one member of the NCR family (NKp44) may be involved in the direct recognition of bacterial pathogens by human NK cells.

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Impact of β2 integrin deficiency on mouse natural killer cell development and function

Blood ◽

10.1182/blood-2010-10-315457 ◽

2011 ◽

Vol 117 (10) ◽

pp. 2874-2882 ◽

Cited By ~ 18

Author(s):

Karine Crozat ◽

Céline Eidenschenk ◽

Baptiste N. Jaeger ◽

Philippe Krebs ◽

Sophie Guia ◽

...

Keyword(s):

Cell Surface ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Surface Expression ◽

Cell Maturation ◽

Cell Surface Expression ◽

Mcmv Infection ◽

Β2 Integrin

Abstract Natural killer (NK) cells are innate immune cells that express members of the leukocyte β2 integrin family in humans and mice. These CD11/CD18 heterodimers play critical roles in leukocyte trafficking, immune synapse formation, and costimulation. The cell-surface expression of one of these integrins, CD11b/CD18, is also recognized as a major marker of mouse NK-cell maturation, but its function on NK cells has been largely ignored. Using N-ethyl-N-nitrosourea (ENU) mutagenesis, we generated a mouse carrying an A → T transverse mutation in the Itgb2 gene, resulting in a mutation that prevented the cell-surface expression of CD18 and its associated CD11a, CD11b, and CD11c proteins. We show that β2 integrin–deficient NK cells have a hyporesponsive phenotype in vitro, and present an alteration of their in vivo developmental program characterized by a selective accumulation of c-kit+ cells. NK-cell missing-self recognition was partially altered in vivo, whereas the early immune response to mouse cytomegalovirus (MCMV) infection occurred normally in CD18-deficient mice. Therefore, β2 integrins are required for optimal NK-cell maturation, but this deficiency is partial and can be bypassed during MCMV infection, highlighting the robustness of antiviral protective responses.

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542 Expansion of cytotoxic NK Cells from PBMCs using individualized cytokine combination

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2020-sitc2020.0542 ◽

2020 ◽

Vol 8 (Suppl 3) ◽

pp. A578-A578

Author(s):

Andreia Maia ◽

Joana Lerias ◽

Markus Maeurer ◽

Mireia Castillo-Martin

Keyword(s):

Flow Cytometry ◽

Nk Cells ◽

Natural Killer ◽

Peripheral Blood ◽

Blood Donors ◽

Nk Cell ◽

Tumour Cells ◽

List Type ◽

Mhc I ◽

Cytokine Combination

BackgroundAdoptive immunotherapy relies on the use of T-cells to target tumour cells, through Major Histocompatibility Complex (MHC) Class I recognition(1). However, many tumours display alterations in the MHC-I pathway, a well-described immune evasion mechanism(2). Natural Killer (NK) cells recognize transformed cells independently from the presence of MHC-I and may be a reliable therapeutic option for patients with altered tumour MHC-I expression. The source of NK cells may be autologous or allogeneic and NK cells are also clinically relevant recipients of transgenic receptors (TCRs or antibodies) targeting tumour cells. NK cells have been categorized according to their CD56 and CD16 surface expression into different subpopulations: cytotoxic (CD56+CD16+) and regulatory (CD56brightCD16-)(3). Expanding cytotoxic NK cells is challenging, since the frequency of NK cells is low in peripheral blood(4) and there is also – at this point – not an optimal expansion protocol available.The goal of this project is to determine the best cytokine combination that facilitates expansion of cytotoxic NK cells that either target tumor cells directly or serve as recipients for transgenic receptors.MethodsPeripheral Blood Mononuclear Cells (PBMCs) were extracted using Ficoll methodology from blood donors and cultured in T25 flasks with Cell Genix Medium supplemented with 10% human serum and antibiotics. NK cells were expanded supplemented with feeder cells (ratio 1:1) and different cytokine combinations (1000 U/mL of IL-2, 10 U/ml of IL-12, 180 U/mL of IL-15 and/or 1 U/mL of IL-21) during 20 days. The immunophenotype of expanded NK cells was analyzed at days 0, 5, 10, 15 and 20 by flow cytometry. The cytotoxicity of NK cells was measured by a CD107a Assay or by a Total Cytotoxicity and Apoptosis Assay at days 10 and 20. Thirteen different cytokine combinations were tested.Results4/13 cytokine combinations produced a statistically significant increase of the absolute number of NK cells with a higher percentage of cytotoxic NK cells (figure 1). However, induction of cytotoxicity was not associated with a strong NK cell expansion. The regulatory NK cells subset (CD56brightCD16-) showed the highest percentage of CD107a-expressing cells, more than the CD56+CD16+, the most cytotoxic subpopulation of NK cells.Abstract 542 Figure 1Representative percentage of NK cells in total lymphocytes (A), CD56+CD16+ subpopulation in total NK cells (B), and CD56brightCD16- subpopulation amongst total NK cells (C) at different time points (5, 10, 15 and 20 days) expanded from PBMCs* p-value < 0.05ConclusionsThis work shows that we are able to grow and efficiently expand NK cells from PBMCs with different cytokine combinations leading to clinically relevant NK cell numbers as well as cytotoxic functions. This enables to produce NK cell products for therapy and as recipients for transgenic tumor antigen-specific receptors.AcknowledgementsThe authors would like to thank the Champalimaud Foundation Biobank, the Vivarium Facility and the Flow Cytometry Platform of the Champalimaud Centre for the Unknown.Ethics ApprovalThis study was approved by the Champalimaud Foundation Ethics Committee and by the Ethics Research Committee of NOVA Medical School of NOVA University of Lisbon.ConsentWritten informed consent was obtained from the blood donors to use their samples for research purposes.ReferencesRosenberg SA, Restifo NP, Yang JC, Morgan RA, Mark E. Adoptive cell transfer: a clinical path to effective cancer immunotherapy. Nat Rev Cancer 2008;8(4):299–308.Aptsiauri N, Ruiz-Cabello F, Garrido F. The transition from HLA-I positive to HLA-I negative primary tumors: the road to escape from T-cell responses. Curr Opin Immunol 2018;51:123–32.Di Vito C, Mikulak J, Mavilio D. On the way to become a natural killer cell. Front Immunol. 2019;10(August):1–15.Zotto G Del, Antonini F, Pesce S, Moretta F, Moretta L. Comprehensive phenotyping of human PB NK Cells by Flow Cytometry. 2020;1–9.

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Role and Modulation of NK Cells in Multiple Myeloma

Hemato ◽

10.3390/hemato2020010 ◽

2021 ◽

Vol 2 (2) ◽

pp. 167-181

Author(s):

Marie Thérèse Rubio ◽

Adèle Dhuyser ◽

Stéphanie Nguyen

Keyword(s):

Multiple Myeloma ◽

Monoclonal Antibodies ◽

Nk Cells ◽

Tumor Cells ◽

Nk Cell ◽

Ex Vivo ◽

Killer Cell ◽

Proteasome Inhibitors ◽

Disease Evolution ◽

Cell Functions

Myeloma tumor cells are particularly dependent on their microenvironment and sensitive to cellular antitumor immune response, including natural killer (NK) cells. These later are essential innate lymphocytes implicated in the control of viral infections and cancers. Their cytotoxic activity is regulated by a balance between activating and inhibitory signals resulting from the complex interaction of surface receptors and their respective ligands. Myeloma disease evolution is associated with a progressive alteration of NK cell number, phenotype and cytotoxic functions. We review here the different therapeutic approaches that could restore or enhance NK cell functions in multiple myeloma. First, conventional treatments (immunomodulatory drugs-IMids and proteasome inhibitors) can enhance NK killing of tumor cells by modulating the expression of NK receptors and their corresponding ligands on NK and myeloma cells, respectively. Because of their ability to kill by antibody-dependent cell cytotoxicity, NK cells are important effectors involved in the efficacy of anti-myeloma monoclonal antibodies targeting the tumor antigens CD38, CS1 or BCMA. These complementary mechanisms support the more recent therapeutic combination of IMids or proteasome inhibitors to monoclonal antibodies. We finally discuss the ongoing development of new NK cell-based immunotherapies, such as ex vivo expanded killer cell immunoglobulin-like receptors (KIR)-mismatched NK cells, chimeric antigen receptors (CAR)-NK cells, check point and KIR inhibitors.

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