scholarly journals Natural Killer (NK) Cell Expression of CD2 as a Predictor of Serial Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC)

Antibodies ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 54
Author(s):  
Jennifer J.-J. Tang ◽  
Alexander P. Sung ◽  
Michael J. Guglielmo ◽  
Lydia Navarrete-Galvan ◽  
Doug Redelman ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Target Cells ◽  
Dependent Cell ◽  
Cd20 Antibody ◽  
Daudi Cells ◽  
Serial Killing ◽  
Anti Cd20 ◽  
Cell Expression ◽  
Perforin Expression

NK cell ADCC supports monoclonal antibody anti-tumor therapies. We investigated serial ADCC and whether it could be predicted by NK phenotypes, including expression of CD16A, CD2 and perforin. CD16A, the NK receptor for antibodies, has AA158 valine or phenylalanine variants with different affinities for IgG. CD2, a costimulatory protein, associates with CD16A and can augment CD16A-signaling. Pore-forming perforin is essential for rapid NK-mediated killing. NK cells were monitored for their ADCC serial killing frequency (KF). KF is the average number of target cells killed per cell by a cytotoxic cell population. KF comparisons were made at 1:4 CD16pos NK effector:target ratios. ADCC was toward Daudi cells labeled with 51Cr and obinutuzumab anti-CD20 antibody. CD16A genotypes were determined by DNA sequencing. CD2, CD16A, and perforin expression was monitored by flow cytometry. Serial killing KFs varied two-fold among 24 donors and were independent of CD16A genotypes and perforin levels. However, high percentages of CD2pos of the CD16Apos NK cells and high levels of CD16A were associated with high KFs. ROC analysis indicated that the %CD2pos of CD16Apos NK cells can predict KFs. In conclusion, the extent of serial ADCC varies significantly among donors and appears predictable by the CD2posCD16Apos NK phenotype.

Negative Impact Of KIR/HLA Interactions On NK Cell Activity Can Be Overcome By The Glycoengineered Antibody GA101 (Obinutuzumab)

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 376-376
Author(s):  
Grzegorz Terszowski ◽  
Christian Klein ◽  
Jakob Passweg ◽  
Martin Stern
Keyword(s):  
B Cell ◽  
Nk Cells ◽  
Target Cell ◽  
Cell Activation ◽  
Nk Cell ◽  
Negative Impact ◽  
Target Cells ◽  
Cd107a Expression ◽  
Cd20 Antibody ◽  
Anti Cd20

Abstract Antibody dependent cellular cytotoxicity (ADCC) is one of the mechanisms by which therapeutic antibodies mediate tumor cell killing. The anti-CD20 antibody rituximab is the current standard of care in the treatment of B-cell lymphomas. GA101, a novel anti-CD20 antibody, contains a glycoengineered Fc-portion allowing approximately 10-fold greater affinity to FcgR3A, the Fc-IgG receptor expressed on the majority of natural killer (NK) cells. NK cell function is also regulated by inhibitory killer-cell immunoglobulin-like receptors (KIR), which interact with HLA class I antigens (2DL1-HLA-C2; 2DL2/3-HLA-C1, 3DL1-HLA-Bw4). The KIR/HLA interaction during NK cell development leads to the acquisition of full effector function in the “licensing” process, but also provides one of the main mechanisms of NK cell tolerance. The present study analyzed how KIR/HLA interactions influence ADCC, and whether there are differences between conventional and glycoengineered antibodies. We analyzed the activation (in terms of the degranulation measured by the CD107a expression) and killing capacity of KIR-positive NK cells induced by rituximab, GA101, and the parental non-Fc modified (wild-type) GA101wt. Target cells included HLA-negative B-cell lymphoma lines or B-cell lines expressing one or more HLA molecules. We confirmed previous observations that the licensing status affects the potential for rituximab-induced ADCC (degranulation against HLA-deficient 721.221 in licensed cells 35 ± 4% versus 19 ± 3% of unlicensed cells, p<0.01); and that KIR/HLA interactions strongly and selectively inhibit the response to targets expressing cognate HLA ligands (e.g. CD107a expression in KIR3DL1+ NK cells 17 ± 3% against 721.221-Bw4 cells, compared to 32 ± 4% against 721.221, p<0.01). Next, we analyzed rituximab-induced NK cell activation in donors expressing one, two, or three KIR ligands after co-incubation with target B-cell lines expressing corresponding HLA molecules. These experiments showed that the inhibitory effect during target cell encounter dominates over the activating effect of NK cell licensing, which leads to unlicensed NK cells being the strongest effectors of ADCC with rituximab (Figure, Panel A). We next compared the effect of the KIR/HLA interaction on rituximab-, GA101wt- and GA101-induced ADCC. GA101 largely compensated the hyporesponsiveness of unlicensed cells and NK cell activation was independent of the presence of HLA KIR ligands on target cells (Figure, Panel A). Finally, we addressed the question of how levels of NK cell degranulation correspond to target cell elimination. Correlation between CD107a expression and target cell elimination was excellent for all antibodies (Figure Panel B). GA101 induced the highest level of activation and the most effective target elimination. In contrast to rituximab and GA101wt, no negative impact of KIR/HLA interaction on degranulation or target cell elimination could be detected for GA101. In summary, we show that KIR/HLA interactions are relevant for ADCC with rituximab, with the negative impact during target cell encounter dominating over the positive effect of licensing. In contrast, the novel glycoengineered GA101 antibody overrides the negative signals derived from the KIR/HLA interaction and activates all NK cell subsets. These data suggest that the Fc-modification to enhance ADCC can be an effective strategy to augment the efficacy of therapeutic monoclonal antibodies by recruiting NK cells irrespective of their inhibitory KIR expression. Disclosures: Terszowski: Roche: Research Funding. Klein:Roche Glycart AG: Employment. Stern:Roche: Research Funding.

scholarly journals Antibody Afucosylation Augments CD16-Mediated Serial Killing and IFNγ Secretion by Human Natural Killer Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Alexandros Karampatzakis ◽  
Petr Brož ◽  
Camille Rey ◽  
Björn Önfelt ◽  
Gabriela Dos Santos Cruz De Matos ◽  
...  
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Nk Cell ◽  
Time Lapse ◽  
Cell Interactions ◽  
Receptor Expression ◽  
Target Cells ◽  
Aqueous Environment ◽  
Serial Killing ◽  
Anti Cd20

One mechanism by which monoclonal antibodies (mAb) help treat cancer or autoimmune disease is through triggering antibody-dependent cellular cytotoxicity (ADCC) via CD16 on Natural Killer (NK) cells. Afucosylation is known to increase the affinity of mAbs for CD16 on NK cells and here, we set out to assess how mAb afucosylation affects the dynamics of NK cell interactions, receptor expression and effector functions. An IgG1 version of a clinically important anti-CD20 mAb was compared to its afucosylated counterpart (anti-CD20-AF). Opsonization of CD20-expressing target cells, 721.221 or Daudi, with anti-CD20-AF increased NK cell cytotoxicity and IFNγ secretion, compared to anti-CD20. The afucosylated mAb also caused a more rapid and greater loss of CD16 from NK cell surfaces. Loss of CD16 has recently been shown to be important for NK cell detachment and sequential engagement of multiple target cells. Here, live-cell time-lapse microscopy of individual cell-cell interactions in an aqueous environment and a three-dimensional matrix, revealed that anti-CD20-AF induced more rapid killing of opsonized target cells. In addition, NK cells detached more quickly from target cells opsonized with anti-CD20-AF compared to anti-CD20, which increased engagement of multiple targets and enabled a greater proportion of NK cells to perform serial killing. Inhibition of CD16 shedding with TAPI-0 led to reduced detachment and serial killing. Thus, disassembly of the immune synapse caused by loss of cell surface CD16 is a factor determining the efficiency of ADCC and antibody afucosylation alters the dynamics of intercellular interactions to boost serial killing.

scholarly journals 894 The bispecific innate cell engagers AFM13 (CD30/CD16A) and AFM24 (EGFR/CD16A) increase the fraction of tumor target-responsive NK cells and boost serial killing

2021 ◽  
Vol 9 (Suppl 3) ◽  
pp. A938-A938
Author(s):  
Chiara Zambarda ◽  
Karolin Guldevall ◽  
Chiara Zambarda ◽  
Karolin Guldevall ◽  
Christian Breunig ◽  
...  
Keyword(s):  
Nk Cells ◽  
Single Cell ◽  
Tumor Cells ◽  
Cell Biology ◽  
Nk Cell ◽  
Biological Evaluation ◽  
Target Cells ◽  
List Type ◽  
Tumor Target ◽  
Serial Killing

BackgroundThe use of bispecific natural killer (NK) cell engagers has emerged as a successful strategy for immune cell activation and killing of tumor cells through antibody-dependent cellular cytotoxicity (ADCC). Among these, tetravalent, bispecific innate cell engagers (ICE®) with specificity for the activating receptor CD16A selectively triggering innate responses from NK cells or macrophages represent the most clinically advanced concept. The CD30/CD16A specific ICE® AFM13, has shown efficacy in patients with CD30+ lymphomas as monotherapy1 and combination therapy with check-point inhibitors2 and most recently in combination with adoptive NK cell therapy.3 The EGFR/CD16A specific ICE® AFM24, targeting a variety of solid tumors like colorectal, or lung cancer with a unique mode of action independent of EGFR signaling inhibition, is currently evaluated in an ongoing Ph1/2a clinical study.MethodsWe used a microchip-based screening with single cell resolution4 to elucidate the dynamic responses of individual NK cells towards tumor target cells upon treatment with AFM13 or AFM24.ResultsWe found that AFM13 and AFM24 mediated potent activation of NK cells, leading to increased responsive cytotoxic NK cells and significantly increased the number of NK cells that exerted engagement with multiple target cells rendering these NK cells serial killers. Strikingly, bispecific ICE® molecules triggered stronger cytotoxic responses compared to monoclonal antibodies. One suggested strategy to boost killing by NK cells is to use molecular inhibitors or protein constructs that prevent shedding of CD16.5 However, previous results have shown that this can lead to impaired detachment from target cells, reducing the capacity for an individual NK cell to form serial contacts to target cells.6 We observed that the elevated NK cell killing induced by ICE® molecules was largely conserved when cells were treated with the shedding inhibitor Batimastat. Analysis of the functional dynamics of NK cells revealed that inhibition of CD16 shedding prevented NK cell detachment from target cells, resulting in cell cluster formation. This might strongly impact targeting of distant tumor cells by an individual NK cell thus limiting its anti-tumoral activity.ConclusionsIn conclusion, we show that both AFM13 and AFM24 increase the fraction of tumor-target responsive NK cells and boost serial killing of target cells by individual NK cells. Based on these data, ICE® molecules can be characterized as potent anti-tumoral agents leveraging the enormous potential of NK cells while maintaining crucial features of NK cell biology.AcknowledgementsWe thank members of the Önfelt lab for their valuable help and feedback.ReferencesSawas A, Elgedawe H, Vlad G, Lipschitz M, Chen P-H, Rodig SJ, et al. Clinical and biological evaluation of the novel CD30/CD16A tetravalent bispecific antibody (AFM13) in relapsed or refractory CD30-positive lymphoma with cutaneous presentation: a biomarker phase Ib/IIa study (NCT03192202). Blood 2018;132(Supplement 1):2908–2908.Bartlett NL, Herrera AF, Domingo-Domenech E, Mehta A, Forero-Torres A, Garcia-Sanz R, et al. A phase 1b study of AFM13 in combination with pembrolizumab in patients with relapsed or refractory Hodgkin lymphoma. Blood 2020. Blood 2020;136(21):2401–2409.Kerbauy LN, Marin ND, Kaplan M, Banerjee PP, Berrien-Elliott MM, Becker-Hapak M, et al. Combining AFM13, a bispecific CD30/CD16 antibody, with cytokine-activated blood and cord blood–derived NK cells facilitates CAR-like responses against CD30 + malignancies. Clin Cancer Res Epub 2021.Guldevall K, Brandt L, Forslund E, Olofsson K, Frisk TW, Olofsson PE, et al. Microchip screening platform for single cell assessment of NK cell cytotoxicity. Front Immunol 2016;7:119.Romee R, Foley B, Lenvik T, Wang Y, Zhang B, Ankarlo D, et al. NK cell CD16 surface expression and function is regulated by a disintegrin and metalloprotease-17 (ADAM17). Blood 2013;121(18):3599–608.Srpan K, Ambrose A, Karampatzakis A, Saeed M, Cartwright ANR, Guldevall K, et al. Shedding of CD16 disassembles the NK cell immune synapse and boosts serial engagement of target cells. J Cell Biol 2018;217(9):3267–83.Ethics ApprovalThis work was performed with NK cells from healthy anonymous blood donors, which requires no ethical permit according to local regulations.

Preclinical Development of Edit-201, a Multigene Edited Healthy Donor NK Cell with Enhanced Anti-Tumor Function and Superior Serial Killing Activity in an Immunosuppressive Environment

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 33-33
Author(s):  
Christopher M Borges ◽  
Kevin Wasko ◽  
Jared M Nasser ◽  
Kelly Donahue ◽  
Amanda Pfautz ◽  
...  
Keyword(s):  
Cell Growth ◽  
Nk Cells ◽  
Target Cell ◽  
Nk Cell ◽  
Hematologic Malignancies ◽  
Target Cells ◽  
Publicly Traded ◽  
Killing Assay ◽  
Serial Killing

Natural killer (NK) cells distinguish tumor from healthy tissue via multiple mechanisms, including recognition of stress ligands and loss of MHC class I expression. For example, KIR mismatching enables allogenic NK cells to respond to MHC positive tumors in a similar manner to MHC negative tumors, making allogeneic NK cell therapy a promising approach for broad oncology indications. Accordingly, allogenic human HD-NK cells, including gene-modified cells, have demonstrated an impressive safety and efficacy profile when administered to patients with advanced hematologic malignancies. However, effector function of allogeneic NK cells can be diminished by the lack of functional persistence, as well as tumor-intrinsic immunosuppressive mechanisms, such as production of TGF-β. To this end, we developed a next-generation allogeneic NK cell therapy using CRISPR-Cas12a gene editing to enhance NK cell function through knockout of the genes CISH and TGFBR2. Both single and simultaneous targeting (DKO) of TGFBR2 and CISH in NK cells using CRISPR-Cas12a produced in/dels at both targets in greater than 80% of NK cells, with greater than 90% of edited NK cells viable at 72 hours post-editing. Importantly, we find that DKO NK cells do not phosphorylate the SMAD2/3 protein downstream of the TGF-b receptor complex and demonstrate increased phosphorylation of pSTAT3 and pSTAT5 upon IL-15 stimulation, consistent with protein level knockout of TGFBR2 and CISH. To determine whether DKO NK cells exhibited superior function relative to control NK cells, we first measured the ability of DKO NK cells to kill Nalm6 cells (adult B cell ALL) relative to unedited control NK cells. We find that in the presence of exogenous TGF-b, DKO NK cells demonstrate improved cytotoxicity against Nalm6 tumor targets by delaying tumor re-growth in comparison to control NK cells. To better characterize the ability of DKO NK cells to kill tumor cells, we developed an in vitro serial killing assay. In this long-duration assay, up to 30 days, control and DKO NK cells (grown in the presence of IL-15) were challenged every 48 hours with a new bolus of Nalm6 tumor targets. Both DKO and unedited NK cells control Nalm6 target cell growth for greater than 18 days (9 additions of new Nalm6 target cells), demonstrating a surprising ability for the same NK cells to serially kill new Nalm6 target cells for a prolonged period of time in vitro. We find that DKO NK cells produce higher levels of IFN-γ and TNF-α relative to control NK cells over the duration of the entire serial killing assay, suggesting that DKO NK cells can continue to produce these inflammatory cytokines even after serial killing. As many tumors, including hematologic malignancies, have high concentrations of TGF-β in their microenvironments, we next tested the ability of DKO NK cells to control the growth of Nalm6 cells in our serial killing assay in the presence of TGF-b. 10ng/mL TGF-β was added at the start of the assay as well as at each addition of new Nalm6 target cells. We observed that control NK cells fail to restrict Nalm6 target cell growth beyond 4 days (after 1 addition of new Nalm6 target cells) whereas DKO NK cells control Nalm6 target cell growth for greater than 18 days (after 9 additions of new Nalm6 target cells). Similar to the serial killing assay without TGF-b, we find that DKO NK cells produce higher concentrations of IFN-γ and TNF-α relative to control NK cells over the duration of the entire serial killing assay. Broadening our repertoire of target cells beyond B cell malignancies is now in progress, including the AML-like cell lines HL-60 and THP-1, the multiple myeloma cell line RPMI 8226, and various solid tumor targets. In summary, using CRISPR-Cas12a we demonstrated highly efficient gene editing of primary human NK cells at two unique targets designed to augment NK cell anti-tumor activity across a variety of malignancies. Most significantly, we demonstrate sustained anti-tumor serial-killing activity in the presence of the potent immunosuppressive cytokine TGF-β. Together, the increased overall effector function of CISH/TGFBR2 DKO primary human NK cells and their ability to serial kill, support their development as a potent allogeneic cell-based medicine for cancer. This potential medicine, termed EDIT-201, is being advanced to clinical study. Disclosures Borges: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wasko:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Nasser:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Donahue:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Pfautz:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Antony:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Leary:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Sexton:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Morgan:Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Wong:Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

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

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1610-1610 ◽  
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.

GA101-Coated Target Cells Are More Effective Than Rituximab-Coated Target Cells at Activating NK Cells When Complement Is Present.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2707-2707 ◽  
Author(s):  
Britnie Spaunhorst ◽  
George J Weiner
Keyword(s):  
B Cells ◽  
Nk Cells ◽  
Mononuclear Cells ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Autologous Serum ◽  
Target Cells ◽  
Type I ◽  
Raji Cells ◽  
Anti Cd20

Abstract Abstract 2707 Poster Board II-683 Rituximab has had a major impact on the treatment of B cell malignancies. The mechanisms responsible for mediating the anti-tumor effects of rituximab are complex. For example, complement can have both positive and negative effects on the ability of rituximab to induce target cell lysis. In particular, we recently reported that rituximab-mediated complement activation results in C3b deposition on the rituximab Fc. C3b then impedes interaction between rituximab and NK cell CD16, thereby limiting NK cell activation and ADCC. GA101 is a type II anti-CD20 monoclonal antibody that mediates enhanced direct cell death induction. It has significantly reduced CDC activity compared to type I anti-CD20 antibodies such as rituximab. In addition, GA101 was engineered to mediate increased ADCC (Umana et al., ASH 2007). The current studies were designed to assess whether the decreased ability of GA101 to activate complement results in an enhanced ability of GA101 to activate NK cells when complement is present. Peripheral blood mononuclear cells (PBMCs) were obtained from normal donors and added to Raji cells (Burkitt lymphoma cell line) at a 1:1 ratio. Various concentrations of rituximab or GA101 were added along with media, 20% autologous serum or 20% heat-inactivated autologous serum (heated to 57°C for 30 minutes). Samples were cultured for 20 hours. NK cell (CD3−, CD56+) activation, as determined by phenotypic changes, was evaluated by flow cytometry based on prior studies demonstrating that downmodulation of CD16, and upregulation of CD54 and CD69 are reproducible surrogates for mAb-induced NK activation and ADCC. Raji cells coated with either rituximab or GA101 were able to activate NK cells when cultures were performed in media alone or with heat-inactivated serum (left panel). In contrast, serum blocked the ability of rituximab to activate NK cells, but not the ability of GA101 to activate NK cells (right panel). Similar results were found when upregulation of CD69 or downmodulation of CD16 were evaluated as markers of NK activation and using PBMCs from two other donors. We conclude that the presence of complement does not limit the ability of GA101-coated target B cells to activate NK cells. This is in contrast to rituximab-coated target B cells which are unable to activate NK cells in the presence of serum. These results suggest that the decreased ability of GA101 to fix complement could, paradoxically, enhance the efficacy of GA101 by resulting in enhanced activation of NK cells and increased ADCC. Disclosures: No relevant conflicts of interest to declare.

Reactive Oxygen Species Produced By Myeloid Cells Block CD20-Antibody Dependent NK Cell-Mediated Cytotoxicity Against Chronic Lymphocytic Leukemia Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3638-3638
Author(s):  
Olle Werlenius ◽  
Johan Aurelius ◽  
Maria Simpanen ◽  
Rebecca E Riise ◽  
Ali A Akhiani ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Nk Cells ◽  
Nk Cell ◽  
Myeloid Cells ◽  
Oxidase Inhibitor ◽  
Lymphocytic Leukemia ◽  
Ros Production ◽  
Nadph Oxidase Inhibitor ◽  
Cd20 Antibody ◽  
Anti Cd20

Abstract Introduction: The addition of the anti-CD20 antibodies rituximab or ofatumumab to chemotherapy has improved the outcome for patients with chronic lymphocytic leukemia (CLL), but the efficacy of anti-CD20 monoclonal antibodies (mAbs) as single agents is limited. Immunosuppressive reactive oxygen species (ROS), produced by myeloid cells, including monocytes, may be a limiting factor for the efficacy of ADCC-dependent therapy. With the aim of reducing a potentially inhibitory role of ROS on mAb-dependent NK cell cytotoxicity against CLL cells, we performed in vitro cytotoxicity experiments in the presence of monocytes and the ROS formation inhibitor histamine dihydrochloride (HDC). Methods: After informed consent, blood samples were collected from Binet stage A CLL patients seen at Sahlgrenska University Hospital, Gothenburg, Sweden. NK cells, monocytes and CLL cells were isolated from PBMCs by immunomagnetic negative selection or flow cytometry. ROS production by monocytes was assessed by isoluminol-enhanced chemiluminescense upon stimulation with the chemotactic peptide fMLF in the presence or absence of CLL cells, rituximab (Roche), ofatumumab (GSK), HDC or the NADPH oxidase inhibitor DPI. NK cell death was assessed by flow cytometry after over-night culture of monocytes and lymphocytes. Autologous ADCC assays were performed with NK cells, monocytes and CFSE-labeled CLL cells in the presence or absence of rituximab, ofatumumab, HDC, catalase and interleukin-2 (IL-2). CLL cell death was determined by flow cytometry after live/dead staining. The study was approved by the local ethical review board of Gothenburg, and conducted in accordance with the declaration of Helsinki. Results: Rituximab and ofatumumab both triggered extracellular ROS production by monocytes. ROS production was blocked by the NADPH oxidase inhibitor DPI. Furthermore, monocytes induced ROS-dependent apoptosis in adjacent NK cells. In the cytotoxicity experiments, rituximab-mediated NK cell dependent cytotoxicity against CLL cells was significantly inhibited by the presence of autologous monocytes. This suppression, as well as the induction of NK cell apoptosis, were dependent on ROS, since both HDC and catalase significantly increased cytotoxicity and rescued NK cells from monocyte-induced apoptosis. Expectedly, IL-2 augmented NK cell-mediated ADCC. Conclusion: The results imply that CLL patients harbor immunosuppressive myeloid cells that reduce CD20-antibody dependent NK cell-mediated cytotoxicity against leukemic cells by producing ROS. We propose that treatment with HDC, preferably in conjunction with NK cell stimulatory cytokines, may improve anti-CD20-based immunotherapy in CLL. Disclosures No relevant conflicts of interest to declare.

scholarly journals CD19 Target Activated Natural Killer (CD19.TaNK) Cellular Therapy: A Novel immunotherapeutic Approach to the Treatment of Non-Hodgkin Lymphoma (NHL)

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4174-4174
Author(s):  
Ravi Dashnamoorthy ◽  
Afshin Beheshti ◽  
Saheli Sarkar ◽  
Pooja Sabhachandani ◽  
Frank C. Passero ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Lines ◽  
Nk Cell ◽  
Resistant Cell ◽  
Cytolytic Activity ◽  
Lymphoma Cells ◽  
Cd20 Antibody ◽  
Anti Cd20 ◽  
Targeted Immunotherapy ◽  
Cd20 Antibodies

Abstract Background: Continued improvement in the treatment of NHL is desired, especially via the incorporation of 'targeted' immunotherapy agents. This is especially important in B-cell NHL (bNHL) as resistance to rituximab anti-CD20 antibody, and now second-generation antibodies (e.g., obinutuzumab), may occur. Activated NK-92 (aNK-92) is a continuously growing cell line consisting of "pure" (100%) activated NK cells. These cells were subsequently bioengineered to express human anti-CD19 chimeric antigen receptor (CAR) recognizing CD19+ B cells. The goal of this project was to investigate the specificity and the efficacy of a novel 'off the shelf' targeted immunotherapy, CD19.TaNK, in a multitude of B-cell NHL cell lines, including anti-CD20 antibody resistant cell lines. Methods: Using gene expression profiling, Gene Set Enrichment Analysis and Ingenuity Pathway Analysis, we first investigated the expression of NK activation and inhibitory ligands in varied lymphoma cells. The bNHL cell lines, SUDHL10 (DLBCL), L540, L428 (Hodgkin lymphoma), HF1 (follicular), Raji (Burkitt's), and Mino (mantle cell) were purchased from ATCC and maintained in RPMI1640 medium. aNK and CD19.TaNK were supplied by NantKwest, Inc and were maintained in Myelocult supplemented with recombinant human IL-2 (500IU/ml). NK cell mediated cytotoxicity was determined using lactate dehydrogenase (LDH) release glucose-6-phosphate dehydrogenase (G6PD) release (aCella-tox assay). Briefly, 10,000 target bNHL cells were co-cultured with effector NK cells, at clinically relevant effector to target ratios (E:T 1:1-10:1) for 4 hours, and the supernatant was assayed for LDH or G6PD release. Percent cytotoxicity was determined based on the experimental levels of LDH or G6PD release from NK mediated bNHL cell lysis compared to maximum LDH or G6PD release from target cells. To determine if resistance to anti-CD20 antibodies would interfere with sensitivity to CD19.TaNK therapy, rituximab and obinutuzumab resistant bNHL cell lines (SUDHL4, SHUDHL10, and Raji) were established; cells were exposed to incremental increasing concentrations of antibody drugs (5-20μg/ml) over a period of 8 weeks. CD19, CD20 and CD30 expression in bNHL cells was determined by flow cytometry. Additionally, the efficacy of primary NK cells were determined against CD20 monoclonal antibody sensitive and resistant cell lines utilizing droplet microfluidics based assessment. Results: We observed that bNHL cell lines expressed a multitude of ligands associated with stimulating NK cell activity, while expression of inhibitory ligands was minimal. This indicates that NK cell interaction with bNHL cells is predicted to lead to overall robust antitumor immune response (Figure). Using LDH and G6PD release assays in bNHL cell lines, we observed increased cytolytic activity in an E:T ratio dependent manner, with Raji and L428 cells being the most sensitive to CD19.TaNK at 1:1 E:T ratio. Development of resistance to anti-CD20 antibodies (rituximab and obinutuzumab) resulted in significantly decreased down regulation of CD20, but not CD19 or CD30, as detected by flow cytometry. After direct contact with primary NK cells, we observed that rituximab resistant SUDHL10 cells were poorly sensitive (7%), while in rituximab sensitive cells, there was 22% cell loss. Moreover, at 4 hours using CD19.TaNK therapy (1:5 ratios), there was marked cytolytic activity with consistent high LDH release seen across all bNHL cell lines without differences noted regardless of rituximab or obinutuzumab resistance (ie, SUDHL4, SHUDHL10, and Raji). These results were further confirmed using live cell video microscopy measuring the cytolytic activity of CD19.TaNK versus bNHL cells. Conclusion: We identified that bNHL cells contain high expression levels of NK activation ligands and low amounts of inhibitory ligands and that CD19.TaNK immunotherapy had potent single-agent anti-tumor activity against a spectrum of bNHL cells. Furthermore, CD19.TaNK maintained high cytolytic activity in bNHL cells resistant to standard CD20 antibody therapy, which were poorly sensitive to innate NK cells. Ongoing analyses include systems biology studies to determine potential biologic mechanisms of activity of CD19.TaNK therapy as well as well as to help guide optimum combinatorial therapy. Figure Expression of NK activation and inhibitory ligands in lymphoma cells. Figure. Expression of NK activation and inhibitory ligands in lymphoma cells. Disclosures Boissel: NantKwest, Inc.: Employment. Evens:Takeda: Other: Advisory board.

Ublituximab, an Optimized Anti-CD20 Monoclonal Antibody, Demonstrates Greater NK-Mediated ADCC Than Rituximab in Waldenstrom's Macroglobulinemia Patients Supporting a Therapeutic Strategy with Ublituximab

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1654-1654 ◽  
Author(s):  
Magali Le Garff-Tavernier ◽  
Linda Herbi ◽  
Christophe de Romeuf ◽  
Jean-Francois Prost ◽  
Patrice Debré ◽  
...  
Keyword(s):  
B Cells ◽  
Nk Cells ◽  
Research Funding ◽  
Nk Cell ◽  
Patent Application ◽  
Surface Expression ◽  
Target Cells ◽  
Raji Cells ◽  
Anti Cd20 ◽  
Functional Capacities

Abstract Abstract 1654 Background: Anti-CD20 monoclonal antibody (mAb) therapy is an important therapeutic option in the treatment of Waldenström's Macroglobulinemia (WM), exhibiting an ORR up to 55% when used as monotherapy. NK cells are involved in mAb therapy by an antibody-dependent cellular cytotoxicity (ADCC) mechanism through their FcγRIIIa (CD16) receptor. Ublituximab (TGTX-1101 or LFB-R603) is a novel, glycoengineered chimeric anti-CD20 mAb that has a high affinity for FcγRIIIa (CD16) receptors. In this study, we evaluate the ADCC functional capacities of NK cells in the presence of ublituximab compared to rituximab. Patients and Methods: Blood samples from 37 un-treated, or without ongoing treatment WM patients and from 30 age-matched healthy donors (Ctl) were collected to quantify CD16 expression (clone 3G8, Quantibrite) on NK cells and/or to measure their functional capacities. Patients were divided in two groups relative to the presence (WM clone+) or absence (WM clone-) of blood clonal B cells. NK cell degranulation was assessed by the surface expression of CD107a on NK cells after incubation of PBMC with or without Raji CD20+ target cells in the presence of anti-CD20 mAbs at 10 and 1,000 ng/ml. ADCC experiments were performed using a chromium assay with purified NK cells and autologous B cells or Raji target cells, in the presence of anti-CD20 mAbs at 1 and 100 ng/ml. Statistical analyses were performed with Prism 5 software. Intergroup comparisons were assessed with the nonparametric Krustal–Walis test, with the Dunns postanalysis test. Results: In the presence of Raji cells, at low concentration, a significantly greater amount of CD107a expression was observed with ublituximab compared to rituximab (P<0.01), regardless of patient's groups. In contrast, at the highest concentration, similar effects were obtained with both anti-CD20 mAbs. These results were confirmed by ADCC. In the presence of Raji cells, a high level of ADCC (>40%) was detected at low concentration of ublituximab and remained stable at 100 ng/ml. In contrast, with rituximab the highest concentration was necessary to reach similar efficacy. In the presence of autologous B cells, degranulation assays revealed that none of the NK cells from WM clone- patients exhibited degranulation, irrespectively of the anti-CD20 mAb or its concentration. More importantly, NK cells of 3/8 WM clone+ patients exhibited CD107a+ NK cells in the presence of both concentrations of ublituximab. In contrast, with rituximab only 1/8 patients expressed CD107a+ NK cells, and only at the highest concentration. Of note, similar frequency and cell-surface expression level of CD16 on NK cells were detected in both patient groups. Importantly, these data were confirmed by ADCC. In the presence of autologous purified B cells from WM clone- patients, absence or low levels of ADCC were detected, irrespective of the concentration and the anti-CD20 mAb used. Interestingly, in WM clone+ patients, ADCC was detected in all of the 5 tested patients with ublituximab, but only in 2/5 patients with rituximab, and at the highest concentration. These results are confirmed by polyfunctionality assays of NK cells against Raji cells (assessment of CD107a degranulation and intracellular production of IFN-γ and TNF-α). Conclusion: These results show that ublituximab is more efficient than rituximab in inducing ADCC at low doses, in the presence of Raji cells. More importantly, our results suggest that ublituximab could be more efficient than rituximab both to induce NK cell degranulation and ADCC in the presence of autologous peripheral tumor cells. These findings highlight a new putative role of this optimized anti-CD20 mAb in the control of WM, and prompt further investigations in a large cohort of WM patients. A Phase I/II trial with single agent ublituximab in patients with rituximab relapsed/refractory NHL, including WM patients is currently ongoing. Disclosures: Le Garff-Tavernier: LFB: 2012 filed patent application Other, Research Funding. Herbi:LFB: Employment, Research Funding. de Romeuf:LFB: 2012 filed patent application Other, Employment. Prost:LFB: Employment. Urbain:LFB: Employment. Leblond:LFB: 2012 filed patent application Other. Vieillard:LFB: 2012 filed patent application Other. Merle-Béral:LFB: 2012 filed patent application Other, Research Funding.

Therapeutic Effects of ALT-803, an IL-15 Superagonist, in Combination with Anti-CD20 Chimeric Antigen Receptor Modified Expanded Natural Killer CELLS Against Pediatric Burkitt Lymphoma (BL)

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3085-3085 ◽  
Author(s):  
Yaya Chu ◽  
Fangyu Lee ◽  
Janet Ayello ◽  
Brian Hang ◽  
Melanie Zhang ◽  
...  
Keyword(s):  
Natural Killer Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Peripheral Blood ◽  
Burkitt Lymphoma ◽  
Nk Cell ◽  
Target Cells ◽  
Killer Cells ◽  
Anti Cd20

Abstract Background: The outcome for children with Burkitt lymphoma (BL)has improved significantly but for patients who relapse, the prognosis is dismal due to chemo-immunotherapy resistance (Cairo et al, JCO, 2012, Cairo et al, Blood, 2007). NK cells are bone marrow-derived cytotoxic lymphocytes that play a major role in the rejection of tumors. A variety of activating and inhibitory receptors on the NK cell surface are engaged to regulate NK cell activities and to discriminate target cells from other healthy 'self' cells. However, NK therapy is limited by several factors, including small numbers of active NK cells in unmodified peripheral blood, lack of tumor targeting specificity, and multiple mechanisms of tumor escape of NK cell immunosurveillance. Our group has successfully modified expanded peripheral blood Natural Killer cells (exPBNK) with an anti-CD20 CAR to target rituximab sensitive/resistant CD20+ BL cells in vitro and in NSG mice (Chu/Cairo, et al, Can Imm Res 2015). However, the short lifespan/persistence of adoptively transferred NK cells has limited the therapeutic efficacy. ALT-803 (Altor BioScience Corporation) is a superagonist of an IL-15 variant bound to an IL-15Rα-Fc fusion with enhanced IL-15 biological activity (Zhu et al. 2009 J Immunol), longer half-life and increased potency (Han, et al. Cytokine. 2011). It is currently in several clinical trials in patients with variety of cancers such as refractory indolent non-Hodgkin's lymphoma (NCT02384954). Objective: We hypothesize that ALT-803, IL-15 superagonist complex, promotes exPBNK persistence and significantly enhances the cytotoxicity of anti-CD20 CAR exPBNK against CD20+ BL. Method: PBMCs were expanded with lethally irradiated K562-mbIL21-41BBL cells (Dean Lee et al, PLoS One, 2012). CD56+ CD3- exPBNK cells were isolated using Miltenyi NK cell isolation kit. Anti-CD20-4-1BB-CD3 ζ mRNA (CAR mRNA) was producedin vitro and nucleofected into exPBNK as we have previously described (Chu/Cairo, et al, Can Imm Res 2015). ALT-803 was provided by Altor BioScience Corporation. ExPBNK cells were cultured with 0.35ng/ml or 3.5ng/ml ALT-803. NK proliferation was monitored with MTS assays. NK receptors expression and cytotoxicity were examined by flow cytometry (Chu/Cairo, et al, ASH 2014). NK resistant BL cells Raji and Daudi were used as target cells. Results: % CD56+ CD3- PBNK cells were significantly increased compared to media alone at day 14 (mean 81.85% vs 14.91%, n=3, p<0.001) when co-cultured with the irradiated feeder cell K562-mbIL21-41BBL. The absolute NK numbers were enhanced with irradiated K562-mbIL21-41BBL cells as feeders compared to IL-2 alone after normalized to the INPUT NK cell numbers (mean 2247 fold±293.7 vs 0.516 fold±0.225, n=3, p<0.001) at day 14. Different doses of ALT-803 or IgG were added to the culture medium of purified expanded exPBNK. Proliferation assays were performed at day 3, 7,11, and 17. ALT-803 significantly promoted exPBNK proliferation and persistence compared to IgG in vitro in a dose-dependent manner (A490 reading at 3.5ng/ml dose: ALT803 vs IgG=0.3383+0.009 vs 0.0987+0.0007, P<0.0001 at d17). And ALT-803 significantly enhanced exPBNK cytotoxicity against NK resistant BL cells: Raji (ALT803 vs IgG= 49.54%+2.7% vs 5.99+0.34%, p<0.001, E:T=10:1) and Daudi (ALT803 vs IgG= 63.73%+3.09% vs 2.58+1.96%, p<0.001, E:T=10:1). It also maintained the highcytoxicity of exPBNK at d4, d10 and d18 against Raji (E:T=10:1, d4 vs d10 vs d18=62.07% vs 49.54% vs 61.47%) and against Daudi (E:T=10:1, d4 vs d10 vs d18=76.02% vs 63.73% vs 55%) by maintaining the activating receptors expression such as NKp30, NKp44, and NKp46. Further-more, we demonstrated ALT-803 significantly enhanced the cytotoxicity of anti-CD20 CAR modified exPBNK against Raji (CAR vs MOCK= 81.19%+0.35% vs 66.19+0.94%, p<0.001, E:T=10:1) and Daudi (CAR vs MOCK= 91.41%+0.45% vs 80.56+1.07%, p<0.001, E:T=10:1) compared to mock modified exPBNK. ALT-803 also significantly enhanced the cytotoxicity of anti-CD20 CAR modified exPBNK against NK resistant BL cells: Raji and Daudi compared to anti-CD20 CAR modified exPBNK maintained in medium without ALT803 (Fig.1). Conclusions: ALT-803 maintained the cytotoxicity of exPBNK and in vitro persistence and significantly enhanced anti-CD20 CAR exPBNK cytotoxicity against pediatric NK resistant BL. The in vivo effect of ALT-803 on CAR exPBNK using humanized NSG models is under investigation. Disclosures Wong: Altor BioScience Corporation: Employment, Other: stockholder of Altor Bioscience Corporation. Lee:Intrexon, Ziopharm, Cyto-Sen: Equity Ownership.

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