Trispecific Antibodies for Selective CD16A-Directed NK-Cell Engagement in Multiple Myeloma

Abstract Development of antibody scaffolds to directly engage cytotoxic effector cells such as T-cells for therapeutic applications is limited by the scarcity of surface antigens which are expressed exclusively on tumor cells and show limited or no expression on non-malignant cells. We have therefore designed a novel antibody format to selectively retarget effector cell cytotoxicity to tumor cells co-expressing two surface antigens. NK-cells play an important role in the innate immune response to multiple myeloma (MM) and are known to contribute to the efficacy of novel therapeutics. We, therefore, utilized a MM-based model system to generate proof-of concept data demonstrating antibody-mediated NK-cell retargeting to cell lines co-expressing two MM-expressed surface antigens with increased selectivity ('dual-targeting'). B-cell maturation antigen (BCMA/CD269) is widely considered to be a promising target antigen for antibody-based therapies of MM due to its almost universal expression on patient myeloma cells and its restricted surface expression on cells outside of the haematological lineage. However, low levels of expression on healthy tissue, including skin, has been reported, which could account for potential side effects associated with BCMA-targeted antibody therapies due to effector cell activation in these organs. To increase selectivity of antibody-induced, effector cell-mediated cytotoxicity towards malignant tissue, we developed a trispecific antibody format capable of selectively engaging NK-cells through bivalent binding to CD16A (FcγRIIIa) and monovalent binding to both BCMA and CD200, a second MM-expressed surface antigen found in the majority of MM patients. Using an in vitro model system, we demonstrated that binding to BCMA+/CD200+ cell lines and the resulting increase in avidity leads to preferential lysis of antigen double-positive cells compared with antigen single-positive cells. These data suggest that dual-targeting may increase the therapeutic window compared to approaches targeting only one antigen, thereby improving safety of BCMA-directed antibody therapeutics for MM. In addition to the MM-based model system used here, the novel trispecific antibody scaffolds we have developed may be adapted to alternative target combinations within MM or in other tumor indications. Moreover, they could be used to target phenotypically distinct tumor cell clones to induce deeper and more prolonged antitumor responses. Consequently, dual-targeting of effector cells to tumors using the described antibody technology could also be applied to increase safety of T-cell engaging antibodies in the absence of exclusively tumor-expressed target antigens. Disclosures Gantke: Affimed GmbH: Employment. Weichel:Affimed GmbH: Employment. Reusch:Affimed: Employment, Patents & Royalties: Patents. Ellwanger:Affimed GmbH: Employment. Fucek:Affimed GmbH: Employment. Griep:AbCheck s.r.o.: Employment. Molkenthin:AbCheck s.r.o.: Employment. Kashala:Affimed Inc.: Employment. Treder:Affimed: Employment.

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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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Lenalidomide Strongly Enhances Natural Killer (NK) Cell Mediated Antibody-Dependent Cellular Cytotoxicity (ADCC) of Rituximab Treated Non-Hodkin’s Lymphoma Cell Lines In Vitro.

Blood ◽

10.1182/blood.v108.11.3714.3714 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3714-3714 ◽

Cited By ~ 2

Author(s):

Lei Wu ◽

Peter Schafer ◽

George Muller ◽

David Stirling ◽

J. Blake Bartlett

Keyword(s):

Nk Cells ◽

Tumor Cells ◽

Cell Lines ◽

Nk Cell ◽

Dependent Manner ◽

Activation Markers ◽

Early Results ◽

Ifn Γ ◽

Dose Dependent

Abstract Lenalidomide (Revlimid® is approved for the treatment of transfusion-dependent patients with anemia due to low- or intermediate-1-risk MDS associated with a del 5q cytogenetic abnormality with or without additional cytogenetic abnormalities, and in combination with dexamethasone is for the treatment of multiple myeloma patients who have received at least one prior therapy. Encouraging early results suggest a potential for clinical efficacy in B cell non-Hodgkin’s lymphoma (NHL). Potential mechanisms of action include anti-angiogenic, anti-proliferative and immunomodulatory activities. Lenalidomide has been shown to enhance Th1-type cytokines and T cell and NK cell activation markers in patients with advanced cancers. Furthermore, lenalidomide has been shown to enhance rituximab-mediated protection in a SCID mouse lymphoma model in vivo. We have utilized an in vitro ADCC system to assess the ability of lenalidomide to directly enhance human NK cell function in response to therapeutic antibodies, such as rituximab (chimeric anti-CD20 mAb). Isolated NK cells produced little or no IFN-γ in response to IgG and/or IL-2 or IL-12. However, pre-treatment of NK cells with lenalidomide greatly enhanced IFN-γ production by NK cells in a dose-dependent manner. In a functional ADCC assay, NHL cell lines (Namalwa, Farage & Raji) were pre-coated with rituximab and exposed to NK cells pre-treated with lenalidomide in the presence of either exogenous IL-2 or IL-12. After 4 hours in culture the viability of the tumor cells was assessed. Lenalidomide consistently and synergistically increased the killing of tumor cells in a dose-dependent manner and up to >4-fold compared to rituximab alone. Rituximab alone had only a small effect in this model and there was no killing of cells in the absence of rituximab. The presence of either exogenous IL-2 or IL-12 was required to see enhanced killing by lenalidomide. In cancer patients lenalidomide has been shown to increase serum IL-12 levels and is also known to induce IL-2 production by T cells in vitro. Potential mechanisms for enhanced ADCC include increased signaling through NK FCγ receptors and/or IL-2 or IL-12 receptors. However, we found that these receptors are unaffected by lenalidomide, although downstream effects on NK signaling pathways are likely and are being actively investigated. In conclusion, we have shown that lenalidomide strongly enhances the ability of rituximab to induce ADCC mediated killing of NHL cells in vitro. This provides a strong rationale for combination of these drugs in patients with NHL and CLL.

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Loss of CD20 Expression and Exhaustion of Effector Cells Limit ADCC in CLL Patients Treated with Rituximab.

Blood ◽

10.1182/blood.v114.22.1610.1610 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1610-1610 ◽

Cited By ~ 1

Author(s):

Berengere Vire ◽

Justin SA Perry ◽

Elinor Lee ◽

Lawrence S Stennett ◽

Leigh Samsel ◽

...

Keyword(s):

Nk Cells ◽

Effector Cell ◽

Cell Function ◽

Nk Cell ◽

Cell Surface Expression ◽

Target Cells ◽

Effector Cells ◽

Cell Functions ◽

Anti Cd20

Abstract Abstract 1610 Poster Board I-636 A major mechanism how the chimeric anti-CD20 monoclonal antibody rituximab (RTX) depletes B-cells is antibody-dependent cellular cytotoxicity (ADCC). ADCC has been modeled in-vitro and in mouse models. However, investigations on ADCC directly in patients treated with RTX are scarce. Recent efforts have focused on improving ADCC through modifications in the Fc binding portion of novel antibodies or through stimulation of effector cell functions with GM-CSF. A more detailed understanding of ADCC as a therapeutic process is needed to optimize such strategies and to identify biomarkers of improved efficacy. Here we report a comprehensive analysis of ADCC in previously untreated CLL patients during the first two RTX infusions (375mg/m2) given in combination with fludarabine every 4 weeks. Following the initial infusion of RTX the absolute lymphocyte count (ALC) decreased by a median of 74% at 2h, followed by a partial recrudescence of cells so that by 24h the median decrease in ALC reached 39% (n=11). ADCC is mediated by effector cells that include NK cells, monocytes/macrophages, and granulocytes. First, we investigated changes in NK cell function: consistent with NK cell activation we found an increase in CD69 at 2, 6 and up to 24h (median 4.2-fold, p=0.005, n=10) after RTX administration and increased expression of the degranulation marker CD107a/b (median 1.9-fold, p<0.001, n=5) and down-regulation of perforin expression (median decrease 63%, p<0.001, n=5) at 4h from treatment start. Activation of NK cells is triggered by the engagement of CD16/FcγRIIIa by RTX coated CLL cells. Interestingly, CD16 expression on NK cells was rapidly lost, already apparent at 2h and maximal at 6h from the start of the RTX infusion (median decrease 82%, p=0.02, n=10) and was not completely recovered by 24h. We also found a significant decrease in expression of CD16 on granulocytes (78%, p<0.001, n=5) but an increase in monocytes (3.9-fold, p<0.001, n=5). In addition to loss of CD16, we found that the cytotoxic capacity of the effector cells was rapidly exhausted: in an oxidative-burst assay, monocytes showed a significant decrease in the production of reactive oxygen species 4h after initiation of RTX infusion (median 60% decrease, p=0.043) and at 6h from the start of the RTX infusion NK cell-mediated killing of K562 target cells was reduced by half (p<0.001, n=3). Interestingly, both the acute reaction to RTX infusions that manifest as a cytokine release syndrome and changes in effector cell function peaked during the first hours of the RTX infusion. We hypothesized that this might be due to the process of CD20 shaving, a rapid and pronounced decrease of CD20 cell surface expression modeled in-vitro and in mice as the result of a mechanism called trogocytosis that relies on the direct and rapid exchange of cell membrane fragments and associated molecules between effectors and target cells (Beum, J Immunol, 2008). First, we used western blot analysis of total CD20 protein in CLL cells and found a rapid loss of CD20 that was apparent already at 2h resulting in virtually complete loss of expression at 24h. Next, we used ImageStream technology to directly visualize ADCC interactions in-vivo. We indeed detected transfer of CD20 from CLL cells to NK cells and monocytes, resulting in complete CD20 loss in circulating CLL cells. While we detected transfer of CD20 into both cell types, monocytes were much more engaged in trogocytosis than NK cells. Consistently, 4h post RTX infusion we found a significant increase in intracellular RTX in granulocytes and monocytes using intracellular staining for human IgG. CD20 shaving appears to be of particular importance given that immunohistochemical analyses revealed that persistent disease in the bone marrow aspirates after 4 cycles of RTX treatment was mostly CD20 negative. Collectively, our results identify loss of CD20 from CLL cells by trogocytosis and exhaustion of immune effector mechanisms as limitations for anti-CD20 immunotherapy. These data identify possible avenues for improving CD20 mediated immunotherapy and characterize endpoints on which different anti-CD20 antibodies can be compared. Given that trogocytosis appears to be a common occurrence our findings likely have general importance to immunotherapy of hematologic malignancies. Disclosures No relevant conflicts of interest to declare.

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Enhancing Natural Killer (NK) Cell Mediated Killing of Non-Hodgkin's Lymphoma.

Blood ◽

10.1182/blood.v114.22.2706.2706 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2706-2706 ◽

Cited By ~ 4

Author(s):

Shivani Srivastava ◽

Hailin Feng ◽

Shuhong Zhang ◽

Jing Liang ◽

Patrick Squiban ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Tumor Cells ◽

Mhc Class I ◽

Median Survival ◽

Nk Cell ◽

Normal Donor ◽

Natural Cytotoxicity ◽

Raji Cells ◽

Effector Cells

Abstract Abstract 2706 Poster Board II-682 Follicular lymphoma is incurable with the current chemo- or chemoimmunotherapy with median survival of 8–12 years. Relapse free survival after each subsequent therapy steadily decreases, resulting in an expected median survival of 4.5 years following initial relapse. Hence new treatment strategies are needed. Natural killer (NK) cells are important effector cells in mediating the anti-lymphoma effect of rituximab. Indeed, antibody-dependent cell-mediated cytotoxicity (ADCC) is a major mechanisms of action of rituximab with NK cells being important effector cells. However, in addition to ADCC, NK cells also exert natural cytotoxicity against tumor cells, which is modulated by a balance of inhibitory and activating signals through NK cell receptors. NK cell function is inhibited when their inhibitory killer immunoglobulin-like receptors (KIR) are ligated by their cognate MHC class I antigens on tumor targets. The novel agent IPH2101 (1-7F9) is a fully human monoclonal antibody directed against KIR2DL receptor that blocks the interaction of KIR with its HLA-C ligands breaking NK cell tolerance to autologous tumor cells. We investigated whether the combination of the IPH2101and Rituximab will augment the NK cell mediated cytotoxicity against CD20+ lymphoma targets as compared to rituximab alone. Raji cells are human CD20+ Burkitt lymphoma cell line cells that expresses HLA-A*03,- (ligand to inhibitory KIR3DL2); -B*71[Bw6] (no inhibitory KIR-Ligand) and -Cw*03,w*04 (group 1 and 2 of HLA-C ligands to inhibitory KIR2DL2/3 and KIR2DL1), and were chosen for study because they have HLA-C antigens that ligate the inhibitory KIR2DL2/3 and KIR2DLI receptors, making them a good target to test our hypothesis of inhibiting inhibitory KIR. NK cells were isolated from normal donor PBMC (peripheral blood mononuclear cells) with the Miltenyi NK isolation Kit. Using LDH release based cytotoxicity assay, we show (Figure 1) that the treatment of target Raji cells with Rituximab significantly enhanced natural cytotoxicity of the purified NK cells against Raji cells. IPH2101alone treatment of NK cells also significantly enhanced the cytotoxicity of Raji cells, however, the combination of IPH2101treated NK cells against Rituximab treated Raji cells significantly enhanced cytotoxicity beyond that observed with each agent alone. Effector: Target (E:T) ratios of 14:1 or less, from more than 5 random donors showed similar results indicating a synergistic, or at least and additive effect ( representative experiment shown Figure 1) . In these experiments purified NK cells were treated with 30ug/ml of IPH2101for 30 min and Raji targets were treated with 0.1-30ug/ml of Rituximab for 30 min. NK cells in the presence or absence of IPH2101were co-cultured with Raji cells in the presence or absence of Rituximab for 4 hour in a 96 well plate. NK cytotoxicity was assessed with an LDH release based assay. Our results suggest that there is a positive cooperation between natural cytotoxicity mediated through KIR-MHC blockade and that mediated by ADCC. Indeed, wee have shown that the blockade of KIR-MHC class I interaction by anti-KIR blocking antibody (IPH2101) augments the cytotoxicity of freshly isolated normal donor NK cells against CD20+ lymphoma cell lines as compared to rituximab alone, providing a rationale for the clinical investigation of the combination of IPH2101 (1-7F9) and rituximab in non-Hodgkin's lymphoma Disclosures: Squiban: Innate pharma: Employment.

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Synergistic Action of the Human Inhibitory KIR Antibody IPH2102, and the Human CD38 Antibody Daratumumab to Enhance the Lysis of Primary Multiple Myeloma (MM) Cells in the Bone Marrow Mononuclear Cells (MNCs) From Myeloma Patients

Blood ◽

10.1182/blood.v118.21.1865.1865 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1865-1865

Author(s):

Inger S. Nijhof ◽

Michel de Weers ◽

Pascale Andre ◽

Berris van Kessel ◽

Henk M. Lokhorst ◽

...

Keyword(s):

Multiple Myeloma ◽

Monoclonal Antibody ◽

Bone Marrow ◽

Nk Cells ◽

Tumor Cells ◽

Research Funding ◽

Mononuclear Cells ◽

Nk Cell ◽

Human Monoclonal Antibody ◽

Cell Activity

Abstract Abstract 1865 Despite significant improvements in the treatment of multiple myeloma (MM), this progressive malignancy of antibody-producing clonal plasma cells is still considered incurable. New innovative treatments need to be developed to improve long term outcomes. Recent successes of CD20 antibodies in the clinical lymphoma management indicate that targeted immunotherapy can represent a powerful therapeutical strategy for hematological malignancies. Towards developing a similar strategy for MM, we have recently generated a novel human monoclonal antibody, daratumumab (DARA), which targets the CD38 molecule expressed at high levels on MM cells. We have demonstrated that DARA mediates the lysis of CD38+ MM cells via direct apoptosis, complement mediated lysis and antibody-dependent cell mediated cytotoxicity (ADCC). Natural killer (NK) cells appeared important effector cells mediating the ADCC effect. Since NK cell activity against tumor cells is regulated by the balance of signals generated by inhibitory or activating receptors of NK cells (KIRs), we now explored whether blocking the inhibitory KIRs would improve the NK cell mediated DARA dependent lysis of MM cells. Thus, we evaluated the potential benefits of combining DARA with a novel human anti KIR monoclonal antibody, IPH2102, which blocks the inhibitory KIR2DL1/2/3 receptors (HLA-C specific KIRs), and has been shown to augment NK cell function against MM cells. We recently developed FACS-based ex vivo MM cell lysis assays, in which DARA-dependent NK cell-mediated lysis of MM cells can be directly measured in bone marrow MNCs, thus without separating the malignant cells from autologous NK cells and other accessory cells. Using these, we investigated whether the addition of IPH2102 would augment the DARA dependent lysis of MM cells. As expected, DARA induced lysis of MM cells in bone marrow MNCs isolated from MM patients (n=10). Mean lysis at 10 μg/ml DARA was 27.6% (range 11.3–48.1%). IPH2102 showed little or no lysis of MM cells (at 0.3, 1, 3 and 10 μg/ml) in this setting. The combination of 10 μg/ml IPH2102 with 3 and 10 μg/ml DARA significantly enhanced cytotoxicity against primary MM tumor cells compared to DARA alone (p=0.013 and p=0.028 respectively). Mean lysis of MM tumor cells at 10 μg/ml DARA and 10 μg/ml IPH2102 was 38%. These data confirm our previous findings that NK-cell mediated killing is an important mechanism of action of DARA. We demonstrate a clear synergy between DARA and IPH2102 to achieve effective lysis of MM cells directly in the bone marrow MNC of MM patients, indicating that complementary effects may be achieved by combining IPH2102 and DARA in clinical MM management. Disclosures: Weers: Genmab: Employment. Andre:Innate Pharma: Employment. Lokhorst:Genmab: Research Funding. Parren:Genmab: Employment. Morel:Innate Pharma: Employment. Mutis:Genmab: Research Funding.

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Pluripotent Stem Cell-Derived Human Natural Killer Cells with Potent Anti-Multiple Myeloma Activity,

Blood ◽

10.1182/blood.v118.21.4034.4034 ◽

2011 ◽

Vol 118 (21) ◽

pp. 4034-4034

Author(s):

David A. Knorr ◽

Zhenya Ni ◽

Allison Bock ◽

Vijay G. Ramakrishnan ◽

Shaji Kumar ◽

...

Keyword(s):

Stem Cells ◽

Multiple Myeloma ◽

Stem Cell ◽

Nk Cells ◽

Natural Killer ◽

Tumor Cells ◽

Adoptive Immunotherapy ◽

Target Cell ◽

Peripheral Blood ◽

Nk Cell

Abstract Abstract 4034 Natural Killer (NK) cells are lymphocytes of the innate immune system with anti-viral and anti-cancer activity. Over the past decade, they have gained interest as a promising cellular source for use in adoptive immunotherapy for the treatment of cancer. Most notably, NK cells play an important role in the graft-vs-tumor effect seen in allogeneic hematopoietic stem cell transplantation (allo-HSCT), and a better understanding of NK cell biology has translated into improved transplant outcomes in acute myelogenous leukemia (AML). Small studies have demonstrated a role for NK cell activity in multiple myeloma (MM) patients receiving allo-HSCT. Investigators have also utilized haplo-identical killer immunoglobulin-like receptor (KIR) mismatched NK cells for adoptive immunotherapy in patients with multiple myeloma (MM). Our group has focused on the development of NK cells from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) as a novel starting source of lymphocytes for immunotherapy. We have previously demonstrated potent anti-tumor activity of hESC-derived NK cells in vitro and in vivo against a variety of different targets. We have also shown that iPSC-derived NK cells from a variety of different somatic cell starting sources posses potent anti-tumor and anti-viral activity. Here, we demonstrate hESC- and iPSC-derived NK cell development in a completely defined, feeder-free system that is amenable to clinical scale-up. These cultures contain a pure population of mature NK cells devoid of any T or B cell contamination, which are common adverse bystanders of cellular products isolated and enriched from peripheral blood. Our cultures are homogenous for their expression of CD56 and express high levels of effector molecules known to be important in anti-MM activity, including KIR, CD16, NKG2D, NKp46, NKp44, FasL and TRAIL. We have now tested the activity of hESC- and iPSC-derived NK cells against MM tumor cells in order to provide a universal source of lymphocytes for adoptive immunotherapy in patients with treatment refractory disease. We find that similar to peripheral blood NK cells (PB-NK), hESC- and iPSC-derived NK cells are cytotoxic against 3 distinct MM cell lines in a standard chromium release cytotoxicity assay. Specifically, activated PB-NK cells killed 48.5% of targets at 10 to 1 effector to target ratios, whereas hESC (46.3%) and iPSC (42.4%) derived NK cells also demonstrated significant anti-MM activity. Also, hESC- and iPSC-derived NK cells secrete cytokines (IFNγ and TNFα) and degranulate as demonstrated by CD107a surface expression in response to MM target cell stimulation. When tested against freshly isolated samples from MM patients, hESC- and IPSC-derived NK cells respond at a similar level as activated PB-NK cells, the current source of NK cells used in adoptive immunotherapy trials. These MM targets (both cell lines and primary tumor cells) are known to express defined ligands (MICA/B, DR4/5, ULBP-1, BAT3) for receptors expressed on NK cells as well as a number of undefined ligands for natural cytotoxicity receptors (NCRs) and KIR. As these receptor-ligand interactions drive the anti-MM activity of NK cells, we are currently evaluating expression of each of these molecules on the surface of both the effector and target cell populations. Not only do hESC- and iPSC-derived NK cells provide a unique, homogenous cell population to study these interactions, they also provide a genetically tractable source of lymphocytes for improvement of the graft-vs-myeloma effect and could be tailored on a patient specific basis using banks of hESC-or iPSC-derived NK cells with defined KIR genotypes for use as allogeneic or autologous effector cells. Disclosures: No relevant conflicts of interest to declare.

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Decitabine Or 5-Azacitidine Pre-Treatment Does Not Compromise The Novel Fc-Engineered Antibody Mab 33.1-Mediated ADCC Against Primary AML Blasts - A Rationale For Combination Therapy

Blood ◽

10.1182/blood.v122.21.3959.3959 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3959-3959

Author(s):

Shun He ◽

Carolyn Cheney ◽

Susan P. Whitman ◽

Jianhua Yu ◽

Sumithira Vasu ◽

...

Keyword(s):

Nk Cells ◽

Cell Line ◽

Cell Lines ◽

Nk Cell ◽

Hl60 Cell ◽

Adcc Activity ◽

Term Survival ◽

Effector Cells ◽

Blast Cells ◽

Pre Treatment

Abstract Introduction Acute Myeloid leukemia (AML) in patients older than 60 years is a devastating diagnosis with long-term survival rates of 10%. Elderly patients have poor survival both due to chemoresistance and presence of concomitant comorbidities rendering them ineligible for induction chemotherapy. Hence novel treatment options are warranted in this patient population. Promising activity of monoclonal antibodies such as alemtuzumab and rituximab for chronic lymphocytic leukemia (CLL) and rituximab for lymphomas has raised the potential use of antibody therapies in AML. CD33 is expressed on greater than 90% of AML blast cells while absent from all non-hematopoietic tissues. Hence CD33 is a viable target for antibody-based therapeutics in AML. Here, we tested the ex vivo efficacy of the mAb 33.1, a fully human anti-CD33 antibody Fc-engineered for increased binding to Fcγ receptors on AML cell lines and primary AML blasts. The goals of this study are to evaluate 1) the efficacy of mAb33.1 on purified allogeneic and autologous natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) against primary AML Blasts; 2) to evaluate efficacy of mAb 33.1 in combination with azanucleosides (i.e. decitabine, 5-azacitidine) that are currently used in AML therapy on NK cell-mediated ADCC against primary AML blasts; and 3) to correlate the levels of surface expression of CD33 on AML blasts to the mAb 33.1 mediated ADCC. Methods mAb 33.1 mediated NK cell activation was determined by NK degranulation as determined by CD107a induction, and ADCC was determined by standard 4-hour 51Cr-release assay. An AML cell line HL60 and a total of 15 AML blast samples were used as targets in this study. NK cells enriched from normal donor PBMC (for allogeneic assays) or sorted from AML blast samples (for autologous assays) were used as effector cells. Results The mAb 33.1 induced potent ADCC activity (>40%) compared to control non-Fc engineered antibody at the concentration of 10 μg/ml in the HL60 cell line. For the AML blasts, mAb 33.1 mediated significantly higher ADCC activity when compared to the control antibody (p<0.05). The relative cytotoxicity mediated by mAb 33.1 varied among different patients, ranging from 4.4% to 65.8%. Subsequent quantification of CD33 showed that there is a positive correlation between ADCC activity and the number of surface CD33 molecules on the AML blasts. Induction of CD107a expression was also observed in both allogeneic and autologous NK cells when the blasts were labeled with mAb 33.1. Pre-treatment of the NK cells and/or target blasts with decitabine or 5-azacitidine for 48hrs, did not alter the mAb 33.1 mediated ADCC activity or CD107 induction. Conclusion mAb33.1 mediated potent ADCC activity and NK activation against AML cell lines and primary AML blasts. Both autologous and allogeneic NK cell-mediated ADCC against primary blast cells from AML patients was observed. The level of NK cell-mediated ADCC was positively associated with the levels of the surface CD33 expression on target AML blasts. Pre-treatment of either AML blasts and/or NK effector cells with Decitabine or 5-azacitidine did not compromise mAb 33.1-mediated ADCC. These pre-clinical studies support further clinical development of mAb 33.1 in combination with relevant anti-AML therapies such as decitabine or 5-azacitidine in patients with CD33 expression. Disclosures: Heider: boehringer-ingelheim: Employment.

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The PD-1/PD-L1 axis modulates the natural killer cell versus multiple myeloma effect: a therapeutic target for CT-011, a novel monoclonal anti–PD-1 antibody

Blood ◽

10.1182/blood-2010-02-271874 ◽

2010 ◽

Vol 116 (13) ◽

pp. 2286-2294 ◽

Cited By ~ 459

Author(s):

Don M. Benson ◽

Courtney E. Bakan ◽

Anjali Mishra ◽

Craig C. Hofmeister ◽

Yvonne Efebera ◽

...

Keyword(s):

Multiple Myeloma ◽

Immune Response ◽

Nk Cells ◽

Natural Killer ◽

Tumor Cells ◽

Cell Function ◽

Nk Cell ◽

Killer Cell ◽

Cell Immune Response ◽

Cell Versus

Abstract T-cell expression of programmed death receptor-1 (PD-1) down-regulates the immune response against malignancy by interacting with cognate ligands (eg, PD-L1) on tumor cells; however, little is known regarding PD-1 and natural killer (NK) cells. NK cells exert cytotoxicity against multiple myeloma (MM), an effect enhanced through novel therapies. We show that NK cells from MM patients express PD-1 whereas normal NK cells do not and confirm PD-L1 on primary MM cells. Engagement of PD-1 with PD-L1 should down-modulate the NK-cell versus MM effect. We demonstrate that CT-011, a novel anti–PD-1 antibody, enhances human NK-cell function against autologous, primary MM cells, seemingly through effects on NK-cell trafficking, immune complex formation with MM cells, and cytotoxicity specifically toward PD-L1+ MM tumor cells but not normal cells. We show that lenalidomide down-regulates PD-L1 on primary MM cells and may augment CT-011's enhancement of NK-cell function against MM. We demonstrate a role for the PD-1/PD-L1 signaling axis in the NK-cell immune response against MM and a role for CT-011 in enhancing the NK-cell versus MM effect. A phase 2 clinical trial of CT-011 in combination with lenalidomide for patients with MM should be considered.

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Combination Therapy with Daratumumab and CAR-NK Targeting CS1 for Multiple Myeloma

Blood ◽

10.1182/blood.v128.22.1342.1342 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1342-1342 ◽

Cited By ~ 3

Author(s):

Yibo Zhang ◽

Lichao Chen ◽

Yufeng Wang ◽

Xinxin Li ◽

Tiffany Hughes ◽

...

Keyword(s):

Multiple Myeloma ◽

Monoclonal Antibody ◽

T Cell ◽

Combination Therapy ◽

Nk Cells ◽

Tumor Cells ◽

Cell Activation ◽

Nk Cell ◽

Car T ◽

Ifn Γ

Abstract Daratumumab (Dara), a targeted therapy utilizing a monoclonal antibody against CD38, and its combination with other are becoming a new standard of care treatment in multiple myeloma (MM). Recently, chimeric antigen receptor (CAR) T cell immunotherapy has been successful in the clinic for the treatment of leukemia and lymphoma. Our preliminary data suggest that both CS1-CAR T cells and CS1-CAR NK cells are effective in eradicating MM cells in vitro and in vivo (Chu et al., 2014, Leukemia and Chu et al., 2014, Clinical Cancer Research). In this study, we investigated the combination therapy with Dara and CS1-CAR NK cells for the treatment of relapsed MM. We first showed that that in MM patients, CD38brightCD138─CD34─CD20+CD27+ MM cancer stem-like cells (CSCs) express CS1 at levels much higher than any other cells, and are susceptible to being eradicated by CS1-CAR NK cells. However, CD34+hematopoietic stem cells from bone marrow of healthy donors do not express CS1. These data suggest that CS1-CAR NK cells can target MM CSCs, and thus may prevent relapse of MM, as ample evidence shows that relapsed or recurrent tumor cells are derived from CSCs. We also demonstrated that CD38 is highly expressed on NK and MM cells. Dara triggered IFN-γ and GZMB expression (p< 0.01) in primary human NK cells, even in the absence of crosslinking with tumor cells. Interestingly, the increase IFN-γ expression can be validated in the CD16 (+) haNK-92 (high-affinity natural killer cells), but not in the parental NK-92 cell line. Blocking the recognition between CD16 and Dara (an IgG1 mAb) with an Fc blocking Ab completely impaired Dara-induced IFN-γ and GZMB expression, indicating that Dara-induced NK cell activation is CD16-dependent. Mechanistically, Dara significantly induced phosphorylation of NFkB and STAT1, indicating that Dara induces IFN-γ and GZMB in NK cells, which may occur through CD16 and be mediated downstream by STAT1 and NFkB. We also found that Dara failed to stimulate GZMB and IFN-γ expression in CD38(-) CD16(+) NK cells, while successful in stimulating CD38(+) CD16(+) NK cells, indicating that Dara induces NK cell activation, which requires not only the binding between CD16 and Fc fragment of Dara, but also the CD38 signaling pathway. Furthermore, we found that Dara mediated cytotoxicity of NK cells against MM cells through antibody-dependent cell-mediated cytotoxicity (ADCC) against CD38-positive (e.g., MM1.S), but not CD38-negative (e.g., U266), which can be blocked by CD16 blocking Ab. Moreover, Dara displays ADCC effects in CD16(+) NK cells but not CD16(-) NK cells. When CD16(+) NK cells were armed with the CS1-CAR, ADCC is still observed against CD38(+) MM cells at low effector to target ratios, i.e., Dara still enhances cytotoxicity of CS1-CAR NK cells, which already have enhanced cytotoxicity. We observed that Dara-induced NK cell ADCC against CD38(+) MM MM1.S cells led to increased T cell proliferation and activation in a co-culture system including dendritic cells. This effect was not observed when MM U266 cells were included as the NK cell target. Out data are consistent with that recent discovery by DiLillo and Ravetch showing that engagement of monoclonal antibody can induce an antitumor vaccine effects (David J et al., Cell, 2015). To tested Dara affects NK cell survival, immunoblotting was performed with anti-cleaved Caspase-3 and anti-cleaved PARP-1 antibodies. We demonstrated that apoptotic activity was increased in both CD16(+)NK cells (primary NK and haNK-92) and parental CD16(-)NK-92 cells treated with Dara for 24 h in a dose-dependent manner. Unlike Dara's positive effects on CD16(+) NK cells (i.e. stimulating IFN-γ production and ADCC), induction of apoptosis seems to be CD16-independent, as parental NK-92 cells, which are CD16(-), also showed an increased levels of apoptosis induced by Dara. We are testing whether the apoptosis induction is dependent on the antigen for Dara, because as mentioned above, both primary NK cells, and modified as well as unmodified NK-92 cells, that were CD38 (+). In conclusion, our study demonstrates that the combination of Dara and CS1-CAR NK cells, which target two different tumor-associated antigens, both of which have potent anti-MM efficacy, may show additive or synergistic effects; however due to the positive and negative effects of Dara on NK cells, sequential treatment rather than a concomitant treatment modality should be considered. Disclosures No relevant conflicts of interest to declare.

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Hypersialylation Protects Multiple Myeloma Cells from NK Cell-Mediated Immunosurveillance and This Can be Overcome By Targeted Desialylation Using a Sialyltransferase Inhibitor

Blood ◽

10.1182/blood-2019-126613 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 138-138

Author(s):

John Daly ◽

Subhashis Sarkar ◽

Alessandro Natoni ◽

Robert Henderson ◽

Dawn Swan ◽

...

Keyword(s):

Multiple Myeloma ◽

Sialic Acid ◽

Nk Cells ◽

Board Of Directors ◽

Cell Lines ◽

Immune Evasion ◽

Research Funding ◽

Nk Cell ◽

Advisory Committees

Introduction: Evading Natural Killer (NK) cell-mediated immunosurveillance is key to the development of Multiple Myeloma (MM). Recent attention has focused on the role of hypersialylation in facilitating immune-evasion of NK cells. Abnormal cell surface sialylation is considered a hallmark of cancer and we have implicated hypersialylation in MM disease progression. Certain sialylated glycans can act as ligands for the sialic acid-binding immunoglobulin-like lectin (Siglec) receptors expressed by NK cells (Siglec-7 and Siglec-9). These ITIM motif-containing inhibitory receptors transmit an inhibitory signal upon sialic acid engagement. We hypothesized that desialylation of MM cells or targeted interruption of Siglec expression could lead to enhanced NK cell mediated cytotoxicity of MM cells. Methodology: MM cells were treated with the sialidase neuraminidase prior to co-culture with primary NK (PNK) cells. MM cells were treated with 300µM 3Fax-Neu5Ac (sialyltransferase inhibitor) for 3 days prior to co-cultures with PNK cells. PNK cells were expanded, IL-2 activated (500U/ml) overnight, or naïve (resting). Primary MM samples/MM cell lines were screened with Siglec-7/9 chimeras (10µg/ml). PNK (IL-2 activated) cells were stained with anti-Siglec-7 and anti-Siglec-9 antibodies. Siglec-7 was targeted for knockout (KO) using the CRISPR/Cas9 system, a pre-designed guideRNA and the MaxCyteGT transfection system. MM cells were treated with 10µg/ml of Daratumumab prior to co-culture with expanded PNK cells. Results: Using recombinant Siglec-7/9 chimeras a panel of MM cell lines (MM1S, RPMI-8226, H929, JJN3 and U266) were shown to express ligands for Siglec-7 and Siglec-9 (>85%, n=3). Primary MM cells isolated from BM of newly diagnosed (n=3) and relapsed patients (n=2) were also shown to express Siglec-7 ligands (72.5±17.5%, 36.5% respectively). PNK cells express Siglec-7 and Siglec-9 (94.3±3.3% and 61±8.8% respectively, n=6). Desialylation of the MM cell lines JJN3 and H929 using neuraminidase significantly enhanced killing of MM cells by healthy donor (HD) derived PNK cells (expanded, IL-2 activated and naïve, n=7) at multiple effector:target (E:T) cell ratios. Furthermore, de-sialylation of JJN3 and H929 using neuraminidase resulted in increased NK cell degranulation (CD107α expression), compared to a glycobuffer control (n=7). De-sialylation, using 300µM 3Fax-Neu5Ac, resulted in strongly enhanced killing of MM1S by expanded HD-derived PNK cells at multiple E:T ratios (n=5, p<0.01 at 0.5:1, p<0.001 at 1:1, p<0.01 at 2.5:1). Furthermore, CD38 expression on H929 MM cells significantly increased after treatment with 300µM 3Fax-Neu5Ac for 3 days (p<0.01, n=3). In a cytotoxicity assay, expanded PNK cell-mediated antibody dependent cellular cytotoxicity (ADCC) of H929 MM cells pre-treated with Daratumumab (anti-CD38 moAb) and 3Fax-Neu5Ac was significantly higher than H929 cells pre-treated with Dara (p<0.05 at 0.5:1, p<0.01 at 1:1) or 3Fax-Neu5Ac (p<0.01 at 0.5:1, p<0.01 at 1:1) alone (n=5). Using CRISPR/Cas9, over 50% complete KO of Siglec-7 was observed on expanded PNK cells, yet did not result in enhanced NK cell-mediated cytotoxicity against either H929 or JJN3 (n=7). Siglec-9 KO using CRISPR/Cas9 is ongoing. Discussion: Hypersialylation of MM cells facilitates immune evasion and targeted removal of sialic acid strongly enhances the cytotoxicity of NK cells against MM. However, to date the role of Siglecs remains inconclusive. Nevertheless, our data suggest that targeted desialylation is a novel therapeutic strategy worth exploring in MM. In particular, upregulation of CD38 provides a strong rationale for combinatory strategies employing targeted desialylation with CD38 moAbs such as Daratumumab, with the goal of maximizing ADCC. Disclosures Sarkar: Onkimmune: Research Funding. O'Dwyer:Onkimmune: Equity Ownership, 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, Research Funding; BMS: Research Funding; GlycoMimetics Inc: Research Funding; AbbVie: Consultancy.

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