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Elotuzumab (Elo) is an IgG1 monoclonal antibody targeting SLAMF7 (CS1, CRACC, and CD319), which is highly expressed on multiple myeloma (MM) cells, natural killer (NK) cells, and subsets of other leukocytes. By engaging with FcγRIIIA (CD16), Elo promotes potent NK cell–mediated antibody-dependent cellular cytotoxicity (ADCC) and macrophage-mediated antibody-dependent cellular phagocytosis (ADCP) toward SLAMF7+ MM tumor cells. Relapsed/refractory MM patients treated with the combination of Elo, lenalidomide, and dexamethasone have improved progression-free survival. We previously showed that Elo enhances NK cell activity via a costimulation mechanism, independent of CD16 binding. Here, we further studied the effect of Elo on cytotoxicity of CD16-negative NK-92 cells. Elo, but not other SLAMF7 antibodies, uniquely enhanced cytotoxicity mediated by CD16-negative NK-92 cells toward SLAMF7+ target cells. Furthermore, this CD16-independent enhancement of cytotoxicity required expression of SLAMF7 containing the full cytoplasmic domain in the NK cells, implicating costimulatory signaling. The CD16-independent costimulation by Elo was associated with increased expression of NKG2D, ICAM-1, and activated LFA-1 on NK cells, and enhanced cytotoxicity was partially reduced by NKG2D blocking antibodies. In addition, an Fc mutant form of Elo that cannot bind CD16 promoted cytotoxicity of SLAMF7+ target cells by NK cells from most healthy donors, especially if previously cultured in IL2. We conclude that in addition to promoting NK cell–mediated ADCC (CD16-dependent) responses, Elo promoted SLAMF7–SLAMF7 interactions in a CD16-independent manner to enhance NK cytotoxicity toward MM cells.

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Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multiplex data from patient tissue, three-dimensional coculturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. We found that NetG1+ cancer-associated fibroblasts (CAF) support PDAC survival, through a NetG1-mediated effect on glutamate/glutamine metabolism. Also, NetG1+ CAFs are intrinsically immunosuppressive and inhibit natural killer cell–mediated killing of tumor cells. These protumor functions are controlled by a signaling circuit downstream of NetG1, which is comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally, blocking NetG1 with a neutralizing antibody stunts in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC.Significance:This study demonstrates the feasibility of targeting a fibroblastic protein, NetG1, which can limit PDAC tumorigenesis in vivo by reverting the protumorigenic properties of CAFs. Moreover, inhibition of metabolic proteins in CAFs altered their immunosuppressive capacity, linking metabolism with immunomodulatory function.See related commentary by Sherman, p. 230.This article is highlighted in the In This Issue feature, p. 211

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Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multiplex data from patient tissue, three-dimensional coculturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. We found that NetG1+ cancer-associated fibroblasts (CAF) support PDAC survival, through a NetG1-mediated effect on glutamate/glutamine metabolism. Also, NetG1+ CAFs are intrinsically immunosuppressive and inhibit natural killer cell–mediated killing of tumor cells. These protumor functions are controlled by a signaling circuit downstream of NetG1, which is comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally, blocking NetG1 with a neutralizing antibody stunts in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC. Significance: This study demonstrates the feasibility of targeting a fibroblastic protein, NetG1, which can limit PDAC tumorigenesis in vivo by reverting the protumorigenic properties of CAFs. Moreover, inhibition of metabolic proteins in CAFs altered their immunosuppressive capacity, linking metabolism with immunomodulatory function. See related commentary by Sherman, p. 230. This article is highlighted in the In This Issue feature, p. 211

Elotuzumab (Elo) is an IgG1 monoclonal antibody targeting SLAMF7 (CS1, CRACC, and CD319), which is highly expressed on multiple myeloma (MM) cells, natural killer (NK) cells, and subsets of other leukocytes. By engaging with FcγRIIIA (CD16), Elo promotes potent NK cell–mediated antibody-dependent cellular cytotoxicity (ADCC) and macrophage-mediated antibody-dependent cellular phagocytosis (ADCP) toward SLAMF7+ MM tumor cells. Relapsed/refractory MM patients treated with the combination of Elo, lenalidomide, and dexamethasone have improved progression-free survival. We previously showed that Elo enhances NK cell activity via a costimulation mechanism, independent of CD16 binding. Here, we further studied the effect of Elo on cytotoxicity of CD16-negative NK-92 cells. Elo, but not other SLAMF7 antibodies, uniquely enhanced cytotoxicity mediated by CD16-negative NK-92 cells toward SLAMF7+ target cells. Furthermore, this CD16-independent enhancement of cytotoxicity required expression of SLAMF7 containing the full cytoplasmic domain in the NK cells, implicating costimulatory signaling. The CD16-independent costimulation by Elo was associated with increased expression of NKG2D, ICAM-1, and activated LFA-1 on NK cells, and enhanced cytotoxicity was partially reduced by NKG2D blocking antibodies. In addition, an Fc mutant form of Elo that cannot bind CD16 promoted cytotoxicity of SLAMF7+ target cells by NK cells from most healthy donors, especially if previously cultured in IL2. We conclude that in addition to promoting NK cell–mediated ADCC (CD16-dependent) responses, Elo promoted SLAMF7–SLAMF7 interactions in a CD16-independent manner to enhance NK cytotoxicity toward MM cells.

Pancreatic ductal adenocarcinoma (PDAC) has a poor 5-year survival rate and lacks effective therapeutics. Therefore, it is of paramount importance to identify new targets. Using multi-plex data from patient tissue, three-dimensional co-culturing in vitro assays, and orthotopic murine models, we identified Netrin G1 (NetG1) as a promoter of PDAC tumorigenesis. NetG1+ cancer-associated fibroblasts (CAFs) supported PDAC survival, through a NetG1 mediated effect on glutamate/glutamine metabolism. NetG1+ CAFs were intrinsically immunosuppressive and inhibited NK cell mediated killing of tumor cells. These pro-tumor functions were controlled by a signaling circuit downstream to NetG1, which was comprised of AKT/4E-BP1, p38/FRA1, vesicular glutamate transporter 1, and glutamine synthetase. Finally blocking NetG1 with a neutralizing antibody stunted in vivo tumorigenesis, suggesting NetG1 as potential target in PDAC.SignificancePDAC is a devastating disease lacking effective therapies. A major hallmark of PDAC is desmoplasia, characterized by the expansion of CAFs and their extracellular matrix, creating a unique microenvironment that limits blood-supplied nutrition and is highly immunosuppressive. A better understanding of the role of CAFs in PDAC may lead to the identification of new targets for therapeutic intervention. Here, we uncovered roles for NetG1 in CAFs to promote tumorigenesis. NetG1 was important for two major CAF functions: the metabolic support of PDAC cells and the intrinsic immunosuppressive capacity of CAFs. Our results helped clarify the role that CAFs play in PDAC, by defining CAF phenotypes through NetG1 expression. Moreover, we established a link between CAF driven metabolism and their intrinsic immunosuppressive capacity, and identified a signaling circuit that governs NetG1 functions. Finally, we demonstrated the therapeutic potential of inhibiting NetG1 in vivo by limiting tumorigenesis in mice with a neutralizing antibody, illustrating that targeting stromal NetG1 could be an attractive therapeutic approach.

Elotuzumab is a humanized therapeutic monoclonal antibody directed to the surface glycoprotein SLAMF7 (CS1, CRACC, CD319), which is highly expressed on multiple myeloma (MM) tumor cells. Improved clinical outcomes have been observed following treatment of MM patients with elotuzumab in combination with lenalidomide or bortezomib. Previous work showed that elotuzumab stimulates NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC), via Fc-domain engagement with FcγRIIIa (CD16). SLAMF7 is also expressed on NK cells, where it can transmit stimulatory signals. We tested whether elotuzumab can directly activate NK cells via ligation with SLAMF7 on NK cells in addition to targeting ADCC through CD16. We show that elotuzumab strongly promoted degranulation and activation of NK cells in a CD16-dependent manner, and a non-fucosylated form of elotuzumab with higher affinity to CD16 exhibited enhanced potency. Using F(ab')2 or Fc-mutant forms of the antibody, the direct binding of elotuzumab to SLAMF7 alone could not stimulate measurable CD69 expression or degranulation of NK cells. However, the addition of soluble elotuzumab could costimulate calcium signaling responses triggered by multimeric engagement of NKp46 and NKG2D in a CD16-independent manner. Thus, while elotuzumab primarily stimulates NK cells through CD16, it can also transduce effective "trans"-costimulatory signals upon direct engagement with SLAMF7, since these responses did not require direct co-engagement with the activating receptors. Trans-costimulation by elotuzumab has potential to reduce activation thresholds of other NK cell receptors engaging with their ligands on myeloma target cell surfaces, thereby potentially further increasing NK cell responsiveness in patients.

Pancreatic ductal adenocarcinoma (PDAC) is predicted to soon become the second killer cancer in the USA. One of the hallmarks of PDAC is desmoplasia, which consists of an expansion in activated stellate cells, known as cancer associated fibroblasts (CAFs), and remodeling of their extracellular matrix. Desmoplasia is a major culprit for the unique PDAC microenvironment, which both collapses blood vessels restricting nutrients and fosters immunosuppression. Crosstalk between CAFs, PDAC, and immune cells, within this microenvironment, is not fully understood. To study the functions of CAFs on PDAC development, we first performed a microarray comparing patient matched normal pancreatic stellate cells and CAFs cultured using our stromal 3D system. We identified proteins upregulated in CAFs that have not been previously implicated in desmoplasia. To understand the functional role of these proteins, CRISPR/Cas9 mediated deletion of target proteins was undertaken and cells were subjected to a number of co-culture assays, with PDAC cells and NK cells. Assays consisted of material transfer, cell engagement, proliferation, survival, NK cell activation and PDAC killing. Syngeneic/orthotopic in vivo models, RNAseq analyzes and quantitative multi-plex microscopy, on pathological samples, were also used. We uncovered the ectopic expressions of NetrinG1 (NG1), a neural pre-synaptic protein, in CAFs, and of NG1’s post-synaptic binding partner, NGL1, in PDAC cells. Using our 3D system, we observed that heterotypic CAF-to-PDAC interactions, via NG1/NGL1 engagement, are critical for providing survival advantages to nutrient deprived PDAC cells and for CAFs to protect PDAC from NK cell-driven elimination. Mechanistically, we uncovered that PDAC starvation is overcome by NG1 expressing CAFs that provide nutrition via material transfer. Further, knockout of NG1, in CAFs, significantly reduced the production of immunosuppressive cytokines, such as IL-6, IL-8, and TGF-β. Moreover, loss of NG1 from CAFs resulted in anti-tumor NK cell activation. Unexpectedly, we observed a link between glutamate metabolism in CAFs and the inactivation of anti-tumor NK cells, and this was dependent on CAFs’ NG1 expression. These results were confirmed in an orthotopic PDAC mouse model in which ablation of NGL1, in syngeneic mouse PDAC cells, significantly halted tumor growth. RNAseq comparing CON or NG1+ CAFs vs. knockout NG1- CAFs rendered changes in neural, metabolic and fibrous signature pathways. Translationally, we saw clear expression of stromal NG1 only in PDAC affected pancreata. Overall, this study identifies two novel targets for PDAC; a cancer that lacks effective therapies. Inhibition of the NG1/NGL1 axis, constitutes a potential future therapeutic approach, as it alters CAF mediated metabolism and reverts immunosuppression, stunting PDAC growth in vitro and in vivo. Citation Format: Ralph Francescone, Débora Barbosa Vendramini-Costa, Janusz Franco-Barraza, Jessica Wagner, Alex Muir, Linara Gabitova, Tatiana Pazina, Tiffany Luong, Neelima Shah, Dustin Rollins, Ruchi Malik, Sapna Gupta, Roshan Thapa, Diana Restifo, Allison Lau, Yan Zhou, Kathy Q. Cai, Harvey Harvey Hensley, Emmanuelle Nicolas, Warren D. Kruger, Karthik Devarajan, Siddharth Balachandran, Wafik S. El-Deiry, Matthew Vander Heiden, Kerry Campbell, Igor Astsaturov, Edna Cukierman. NG1/NGL1 engagement supports PDAC development via CAF to PDAC nutrition and CAF-regulated immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2038.

The natural cytotoxicity receptor (NCR) family is constituted by NKp46, NKp44, and NKp30 in humans, which are expressed mainly on natural killer (NK) cells and are encoded by the ncr1, ncr2, and ncr3 genes, respectively. NCRs have classically been defined as activating receptors that trigger cytotoxicity and cytokine responses by NK cells upon engaging with ligands on tumor cells. Several new findings, however, have challenged this model and identified alternative mechanisms regulating the function of NCRs. Recent reports indicate that ligand matters, since the interaction of NKp44 with distinct ligands on target cells can either activate or inhibit NK cells. Also, the NCRs have been found to interact with distinct specificities to various heparan sulfate glycosaminoglycans, which are complex polysaccharides found in extracellular matrix or on cell surface heparan sulfate proteoglycans (HSPGs). The NCRs can engage with HSPGs in trans as a co-ligand on the target cells or in cis on the NK cell surface to regulate receptor–ligand interactions and NK cell activation. A number of splice variants of ncr2 and ncr3 have also been identified, and a predominant expression of certain variants results in inhibitory signaling through NKp44 and NKp30. Several recent studies have found that the selective expression of some of these inhibitory splice variants can significantly influence outcome in the contexts of cancer, infection, and pregnancy. These findings establish that NCR functions are more diverse than originally thought, and better understanding of their splice variant expression profiles and ligand interactions are needed to establish their functional regulation in the context of human health.

CONTEXT: Elotuzumab is an immunoglobulin G1 monoclonal antibody targeting SLAMF7. Elotuzumab promotes potent NK cellu2013mediated antibody-dependent cellular cytotoxicity via CD16, resulting in killing of SLAMF7+ MM cells, which is enhanced when combined with lenalidomide. Elotuzumab may also enhance NK cell activity via a CD16-independent costimulation mechanism. OBJECTIVE: To characterize how elotuzumab affects interactions between NK and MM cells to enhance NK cellu2013mediated cytotoxicity. METHODS: We generated 2 MM cell lines (MM.1R, RPMI8226) modified to express low/high levels of SLAMF7, and the SKOV3 ovarian adenocarcinoma cell line transduced to express SLAMF7. A SLAMF7-deficient human NK-92 cell line was generated using CRISPR/Cas9 technology. Variants of this SLAMF7-deficient line and an NK-92 line expressing high levels of SLAMF7 that expressed or lacked CD16 were also generated. Cytotoxicity was measured using CytoTox 96u00ae (Promega) and xCELLigenceu00ae real-time cell analysis (ACEA Biosciences) platforms. A human SLAMF7 extracellular/T-cell receptor [TCR]-zeta intracellular fusion construct transfected into a 3A9 mouse T-cell line was used as a reporter of SLAMF7 engagement by measuring interleukin-2 production.RESULTS: Addition of elotuzumab strongly enhanced cytotoxicity of CD16-expressing SLAMF7+ NK-92 cells against SLAMF7+ MM and SKOV3 cells. Elotuzumab also substantially boosted cytotoxicity by CD16-deficient SLAMF7+ parental NK-92 cells toward SLAMF7+ RPMI8226 and SKOV3 cells. Knockout of SLAMF7 on parental NK-92 cells, however, abrogated elotuzumab-mediated cytotoxicity toward SLAMF7+ SKOV3 targets. Using a SLAMF7/TCR-zetau2013expressing reporter cell line and plate-bound recombinant SLAMF7, we found elotuzumab, but not other anti-SLAMF7 antibodies, promoted notable reporter activity, suggesting it may facilitate/enhance SLAMF7u2013SLAMF7 interactions. CONCLUSIONS: Elotuzumab has an additional function as an NK cellu2013activating antibody. SLAMF7 naturally engages with itself in homotypic interactions. Elotuzumab uniquely promoted NK cellu2013mediated cytotoxicity in a CD16-independent manner, but only if both NK and target cells expressed SLAMF7. This suggests that elotuzumab can facilitate/enhance SLAMF7u2013SLAMF7 interactions between NK and MM cells. As elotuzumab is clinically used in combination with lenalidomide, additional studies are needed to understand the impact of combination treatment on elotuzumab-mediated SLAMF7u2013SLAMF7 interactions. SLAMF7 expression on NK cells warrants further investigation as a potential biomarker for elotuzumab efficacy.Funding: BMSPrevious presentation: EHA 2018

Elotuzumab (Elo) is an IgG1 monoclonal antibody targeting SLAMF7 (CS1, CRACC, CD319) protein, which is highly and uniformly expressed on multiple myeloma cells. Improved survival has been observed in multiple myeloma patients treated with Elo in combination with immunomodulatory drugs such as dexamethasone and lenalidomide. Previous work showed that the main mechanism of Elo action in vitro is CD16-mediated antibody dependent cellular cytotoxicity (ADCC) via Fc interaction with CD16 (FcγRIIIa) receptor on NK cells. However, SLAMF7 is also expressed on NK cells where it transmits an activating signal. We hypothesized that Elo can directly activate NK cells via SLAMF7 ligation. Utilizing non-fucosylated Elo that has higher affinity to CD16 and Elo mutant that has an Fc region mutation that abrogates binding to CD16, we confirmed that Elo promotes NK cell activation and degranulation in a CD16-dependent manner. Specifically, non-fucosylated Elo had higher potency, whereas Elo mutant did not stimulate degranulation or CD69 expression. To test for co-stimulatory effects of Elo and Elo mutant we measured calcium signaling responses triggered by those antibodies in combination with antibodies to engage the activating receptors NKp46 and NKG2D. Elo or Elo mut alone had no effect on calcium signaling, but when added in combination with multimeric engagement of NKp46 and NKG2D both antibodies co-stimulated calcium signaling responses. We conclude that Elo stimulates NK cells via interaction with CD16, but direct engagement with SLAMF7 can also transduce co-activating signals to potentiate calcium signals generated through NKp46 and NKG2D in a CD16-independent manner. Our results provide evidence that direct engagement with SLAMF7 by Elo has the potential to reduce the threshold of NK cell activation triggered through other activating receptors. Citation Format: Tatiana Pazina, Ashley Mentlik James, Alexander W. MacFarlane, Natalie A. Bezman, Robert F. Graziano, Michael D. Robbins, Adam D. Cohen, Kerry S. Campbell. Elotuzumab can costimulate NK cell responses by engaging SLAMF7 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2998. doi:10.1158/1538-7445.AM2017-2998

We developed a novel detection method for osteopontin (OPN), a new biomarker for prostate cancer, by attaching a genetically engineered single chain variable fragment (scFv) protein with high binding affinity for OPN to a carbon nanotube field-effect transistor (NTFET). Chemical functionalization using diazonium salts is used to covalently attach scFv to NT-FETs, as confirmed by atomic force microscopy, while preserving the activity of the biological binding site for OPN. Electron transport measurements indicate that functionalized NT-FET may be used to detect the binding of OPN to the complementary scFv protein. A concentration-dependent increase in the source-drain current is observed in the regime of clinical significance, with a detection limit of approximately 30 fM. The scFv-NT hybrid devices exhibit selectivity for OPN over other control proteins. These devices respond to the presence of OPN in a background of concentrated bovine serum albumin, without loss of signal. Based on these observations, the detection mechanism is attributed to changes in scattering at scFv protein-occupied defect sites on the carbon nanotube sidewall. The functionalization procedure described here is expected to be generalizable to any antibody containing an accessible amine group, and to result in biosensors appropriate for detection of corresponding complementary proteins at fM concentrations., Comment: 11 pages, 5 figures, appears in ACS Nano

Introduction: Elotuzumab is a humanized monoclonal antibody that binds specifically to Signaling Lymphocyte Activation Molecule Family Member 7 (SLAMF7; CS1), a glycoprotein highly expressed on the surface of multiple myeloma (MM) cells and on subsets of immune cells, including natural killer cells. SLAMF7 also exists as a soluble form (sSLAMF7) and has been detected in the serum of patients with MM at statistically significant higher levels than healthy individuals. Although the mechanism of action of elotuzumab involves the direct activation of natural killer cells and the initiation of antibody-dependent cell-mediated cytotoxicity of SLAMF7-expressing myeloma cells, its effects on sSLAMF7 have not been reported. The biological significance of sSLAMF7 has not been described in MM; however, it has been suggested that sSLAMF7 may be associated with disease stage. In order to assess the pharmacodynamic and predictive utility of this biomarker, we investigated levels of serum sSLAMF7 at baseline and on treatment (Cycle 2 Day 1 [C2D1]) across several elotuzumab clinical studies (HuLuc63-1703, CA204-009, and CA204-011) in both smoldering MM and relapsed/refractory MM. Additionally, associations between changes in sSLAMF7 and pharmacokinetic parameters were investigated. Lastly, associations between sSLAMF7 and markers of disease burden were also assessed. Methods: Two enzyme-linked immunosorbent assays were developed to detect sSLAMF7 in the presence of elotuzumab: the total assay (TA) detected all forms of sSLAMF7 (both unbound and elotuzumab-bound sSLAMF7) and the free assay (FA) detected only unbound sSLAMF7. Serum was collected from peripheral blood before initiation of treatment (C1D1) and during treatment (C2D1) and assessed for levels of sSLAMF7 by the TA and FA. Soluble component (non-cellular) from bone marrow aspirates were obtained prior to any therapy to assess the relationship between concentrations of sSLAMF7 in matched marrow and blood samples. Results: As previously reported, these data confirm sSLAMF7 expression in patients with MM. The TA demonstrated a significant dose- and time-dependent increase in sSLAMF7 following elotuzumab treatment alone or in combination with either bortezomib/dexamethasone (E-B/d) or lenalidomide/dexamethasone (E-L/d); this increase was not observed in patients treated with bortezomib/dexamethasone (B/d) only. The increase in total sSLAMF7 observed in the TA demonstrated elotuzumab pharmacodynamic specificity and likely reflected stabilization of the protein in the serum. The FA detected a significant decrease in free sSLAMF7 (at C2D1) in both elotuzumab- and non-elotuzumab-treated patients, with the greatest reduction observed in elotuzumab-treated patients. The decrease in free sSLAMF7 detected using the FA, regardless of treatment, suggest the potential use of this biomarker as a surrogate for disease burden. Additionally, the extent of the C2D1 changes in free and total sSLAMF7 following elotuzumab-containing regimens demonstrated a trend for association with increased depth of response (comparing progressive disease/stable disease/minimal response vs partial response vs very good partial response/complete response). Baseline and C2D1 levels of serum sSLAMF7 showed a trend for an inverse relationship with progression-free survival (for E-L/d) but this was not statistically significant. Baseline sSLAMF7 did demonstrate a positive relation with the percentage of bone marrow plasma cells. At C2D1, with the dose of 10 mg/kg, the mean elotuzumab concentration was 337.1 µg/mL (N=38; CV of 51.6%), which was significantly higher than the mean free sSLAMF7 concentration 0.255 ng/mL (N=37; CV of 200%). In addition, E-L/d treatment demonstrated the greatest reduction of free sSLAMF7 (more than 80% of patients treated with E-L/d reached >75% of free sSLAMF7 reduction) compared with that observed for E-B/d and B/d. Conclusions: Together, these data suggest that sSLAMF7 may be a surrogate marker of disease burden in MM, and early changes in sSLAMF7 may provide an indication of likelihood of response. Additional analysis of sSLAMF7 is ongoing and future studies will help us to further understand the use of this biomarker for prognostic or predictive purposes. Study supported by: Bristol-Myers Squibb Disclosures Postelnek: Bristol-Myers Squibb: Employment. Off Label Use: Elotuzumab is an investigational agent being studied for the treatment of multiple myeloma.. Sheridan:AbbVie Biotherapeutics: Employment, Equity Ownership. Keller:AbbVie Biotherapeutics: Employment, Equity Ownership. Sheng:Bristol-Myers Squibb: Employment, Equity Ownership. Poulart:Bristol-Myers Squibb: Employment. Robbins:Bristol-Myers Squibb: Employment, Other: shareholder.

Background: Monoclonal antibodies (mAbs) are an emerging therapeutic class for MM patients (pts). Elotuzumab, a mAb in late-phase clinical development, targets the SLAMF7 receptor expressed highly on MM cells. While its primary mechanism of action is through CD16-mediated ADCC, elotuzumab can also directly activate SLAMF7-expressing NK cells. Gaining a greater understanding of phenotypic and functional changes in NK cells over the course of the disease, and how these changes impact capacity for ADCC, may help identify profiles that can better select pts likely to benefit from elotuzumab or other mAb therapies. Methods: We prospectively performed a comprehensive flow cytometry-based analysis of lymphocyte subsets, focusing on expression of NK cell activating and inhibitory receptors, activation and maturation markers, and degranulation in 30 MM pts (12 newly-diagnosed (ND), 18 relapsed/refractory (RR)) and 19 aged-matched healthy donors (HD). Over 140 immune parameters were analyzed, with differences in expression between HD and pt subsets compared by Wilcoxon rank-sum test. We analyzed correlations between expression of certain markers with each other, and with elotuzumab-induced NK cell degranulation against MM cell targets (MM1R) in a 2-hour co-culture assay. We also compared NK cell parameters in blood and bone marrow (BM) from pts with matched samples available. Results: Within the blood, there was no difference in relative NK cell frequency between the groups, and little difference phenotypically between HD and ND pt NK cells, except for decreased DNAM1 expression in ND. In contrast, in comparison to HD, CD56dim NK cells in RR pts were less mature with a higher CD56bright to CD56dim NK cell ratio and reduced expression of the terminal differentiation/maturation markers, CD57 and KLRG1. RR pts also showed increased expression of the activation marker CD69 on all NK cells, and their CD56dim NK cells had increased levels of the natural cytotoxicity receptors, NKp30 and NKp46 and decreased expression of activating receptors DNAM1 and NKG2D. SLAMF7 expression was also increased in RR pts, but only on the CD56bright subset. Consistent changes in NK cell expression of checkpoint/co-stimulatory molecules (eg. PD-1, Tim3, LAG3, CD137) were not seen. Despite these phenotypic changes, no significant differences between groups were noted for elotuzumab-induced ADCC against MM1R targets, as measured by CD107a degranulation by CD56dim NK cells, with significant variability noted within groups. Interestingly, the expression levels of SLAMF7 on CD56dim NK cells directly correlated with CD16 levels, particularly within RR pts (Fig.), suggesting cooperative interactions between these receptors that may be beneficial in MM patients treated with elotuzumab. In addition, degranulation toward elotuzumab-treated MM1R targets was significantly associated with surface expression levels of both SLAMF7 and CD16 on the CD56dim NK cells. The status of NK cells was also compared between matching blood and BM samples from ND (n=7) and RR (n=8) pts. NK cell phenotype and degranulation in blood and BM were similar in ND pts, but in RR pts, expression of CD69 and SLAMF7 were higher on BM-derived NK cells, and CD56dim NK cells from BM demonstrated greater degranulation toward elotuzumab-treated MM1R targets. DNAM1 expression was reduced, but NKG2D, NKp30, and NKp46 were upregulated on various NK cell populations in BM from RR pts compared to peripheral blood. Conclusions: Taken together, our data indicate that NK cells in RR MM pts had increased activation, reduced maturation status, and distinct changes in activating receptor expression levels that are often further enhanced in the BM microenvironment. Furthermore, CD56dim NK cells in many RR pts had parallel increased expression levels of CD16 and SLAMF7, which correlated with enhanced degranulation toward elotuzumab-treated MM target cells. The fact that these changes are seen primarily in RR pts rather than untreated ND pts implies a significant impact of disease evolution and prior therapy on the NK cell compartment, and supports further exploration of these parameters as potential biomarkers of activity of elotuzumab and other therapeutic mAbs in myeloma. Figure 1. Figure 1. Disclosures Campbell: Bristol-Meyers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding. Cohen:Bristol-Meyers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees.

Multiple myeloma (MM) is a malignancy of plasma cells characterized by a monoclonal expansion of tumor cells in bone marrow and accumulation of a monoclonal protein in blood and urine, which ultimately leads to end-stage organ damage. Despite advances in therapy, MM remains an incurable disease. Recent investigations have revealed a new therapeutic target for MM, SLAMF7 (CS1), which is expressed at high levels on MM tumor cells. SLAMF7 is also found on NK, CD8+ T, CD4+ T and activated B cells. Elotuzumab (HuLuc63) is a humanized IgG1 anti-SLAMF7 antibody, which can facilitate killing of primary MM cells by NK cells via antibody-dependent cellular cytotoxicity (ADCC) and has shown promise in treating relapsed MM. A key question in understanding elotuzumab’s mechanism of action is whether it can directly activate NK cells via SLAMF7 binding, in addition to Fc-mediated activation via binding of CD16 (FcγRIIIA). We have examined the in vitro effect of elotuzumab on NK cells in fresh human peripheral blood from healthy donors and MM patients incubated with or without MM cell lines that have varied levels of SLAMF7 surface expression. Cytotoxic degranulation of NK cells was quantified by staining for CD107a (marker of degranulation) after 2 hours, and direct effects of elotuzumab on NK cells were assessed by measuring upregulation of the CD69 activation marker after 24 hours of antibody exposure. Degranulation of fresh primary NK cells toward SLAMF7-bearing MM target cell lines was stimulated by addition of elotuzumab. Intensity of the degranulation response was dependent upon the concentration of elotuzumab and the expression level of SLAMF7 on the MM target cells. While elotuzumab alone could stimulate low-level degranulation of NK cells in fresh PBL from healthy donors, the degranulation response was stronger if SLAMF7-expressing MM target cells were added in combination with elotuzumab. Addition of F(ab’)2 elotuzumab or an Fc mutant form of elotuzumab that cannot bind CD16 did not stimulate degranulation, implying that binding of the antibody to SLAMF7 on NK cells and MM cells, without concomitant engagement of NK cell Fc receptors, is insufficient to trigger degranulation. Soluble intact elotuzumab stimulated CD69 expression and degranulation by fresh primary NK cells in the absence of MM target cells, which peaked at 1 µg/ml and only modestly increased up to 100 µg/ml elotuzumab. In the same assays, we only observed modest stimulation of CD69 expression using soluble F(ab’)2 or Fc mutant elotuzumab. In contrast, plate-bound F(ab’)2 elotuzumab stimulated robust CD69 expression, indicating that the antibody can induce direct stimulation of NK cells through SLAMF7, but strong stimulation requires aggregation of the antibody. In soluble form, a non-fucosylated version of elotuzumab with greater affinity for CD16 stimulated significantly stronger degranulation and CD69 expression than intact elotuzumab, both in the presence or absence of MM target cells, and with both high and low SLAMF7 expression on target cells. Importantly, elotuzumab was found to induce similar degranulation by NK cells from freshly isolated peripheral blood of MM patients as compared to healthy donors. Taken together, our data support the hypothesis that binding of soluble elotuzumab to SLAMF7 can weakly activate NK cells, but strong activation and degranulation require antibody aggregation or engagement of the Fc domain with CD16. Elotuzumab variants with enhanced CD16 binding, such as a non-fucosylated version, can augment ADCC against myeloma cell lines, and warrant further study as a potential strategy to improve clinical efficacy in MM patients. Disclosures Cohen: Celgene: Member, Independent Response Adjudication Committee Other; Janssen: Advisory Board, Advisory Board Other; Bristol-Myers Squibb: Advisory Board, Advisory Board Other, Research Funding; Onyx: Advisory Board, Advisory Board Other. Campbell:Bristol-Myers Squibb: Advisory Board Other, Research Funding; Janssen: Advisory Board, Advisory Board Other; Conkwest: Consultancy, Patented NK-92 cell lines, Patented NK-92 cell lines Patents & Royalties, Research Funding.