R603: A New Low Dose Efficient Anti-CD20 Immunotherapy for CLL Patients ?

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4155-4155
Author(s):  
Magali Le Garff-Tavernier ◽  
Julie Decocq ◽  
Christophe de Romeuf ◽  
Jean-Luc Teillaud ◽  
Charles-Antoine Dutertre ◽  
...  
Keyword(s):  
B Cells ◽  
Nk Cells ◽  
Low Dose ◽  
Surface Expression ◽  
Raji Cells ◽  
Healthy Donors ◽  
The One ◽  
Anti Cd20 ◽  
Functional Capacities

Abstract Immunotherapy with monoclonal antibodies (mAbs), such as anti-CD20, is used in CLL treatment and represents a promising approach for achieving MRD eradication. Given their FcγRIIIa expression, NK cells are known to be involved in mAb therapy. We previously conducted a complete NK cells phenotypic expertise and functional assays including cytotoxicity against K562 cell line and antibody-dependent cellular cytotoxicity (ADCC) with rituximab, showing no major differences between NK cells from CLL patients and NK cells from healthy donors. We are now interested in functional capacities of NK cells in presence or not of a new anti-CD20 mAb: R603, a chimeric anti-CD20 mAb exhibiting a low fucose content as described for EMAB-6 mAb (C. de Romeuf et al, BJH 2008) in comparison to rituximab. To assess the degranulation of NK cells from CLL patients in response to anti-CD20 mAbs, we examined the surface expression of CD107a (percentage of CD107a+ NK cells) after co-incubation of PBMC from untreated CLL patients (n=8) with Raji cells (E/T ratio: 1/1) at 2 concentrations of each anti-CD20 mAb (10 and 1,000 ng/ml). At the higher mAb dose (1,000 ng/ml), R603 related degranulation of CLL NK cells (median (m): 43.6%; range (r): 27.0–79.8) was similar to the one obtained with rituximab (m: 38.9%; r: 22.4–75.2). At the lower dose (10 ng/ml), R603 related degranulation of CLL NK cells (m: 45.7%; r: 28.7–79.2) was similar to the one obtained with the high mAb concentration, contrary to rituximab related degranulation which was significantly decreased (m: 14.1%; r: 1.4–45.4) (p<0.0001). These results are emphasized by ADCC chromium assay performed with purified CLL NK cells (E/T ratio: 5/1 and 10/1) against Raji cells and with or without anti-CD20 mAbs (at 3 doses: 1, 10 and 1,000 ng/ml). R603 related ADCC levels were high whatever the mAb concentration, contrary to rituximab related ADCC levels which were very low at 1 ng/ml and only reached R603 ADCC levels at 1,000 ng/ml. Similar results were obtained with healthy donors. Without addition of Raji cells (CLL PBMC + mAb), at the lower dose (10 ng/ml), none of the NK cells from CLL patients exhibited degranulation with rituximab, contrary to R603 where 5/8 CLL patients exhibited degranulation (cut-off: more than 10% of CD107a+ NK cells). At the higher dose (1,000 ng/ml), NK cells from 6/8 CLL patients with rituximab and from 7/8 CLL patients with R603 showed degranulation and R603 related degranulation levels (m: 32.3%, r: 0.8–51.0) were significantly superior to rituximab related degranulation levels (m: 12.1%, r: 0.1–30.6) (p=0.0005). These results showed that R603 in the presence of CLL B cells might induce CLL NK degranulation. In conclusion, NK cells from CLL patients appeared to be capable of being efficient in anti-CD20 immunotherapy by the ADCC pathway. Moreover, R603 a new anti-CD20 mAb, induced at low dose a significantly higher in vitro ADCC against Raji cells and autologous CLL B cells, compared to rituximab. This R603 mAb feature may be useful in therapeutic strategy for CLL patients.

Can NK Cells Play a Role in Anti-CD20 Immunotherapy for CLL Patients?.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3103-3103
Author(s):  
Magali Le Garff-Tavernier ◽  
Christophe De Romeuf ◽  
Jean-Luc Teillaud ◽  
Julie Decocq ◽  
Charles-Antoine Dutertre ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Hla Class I ◽  
Drug Candidate ◽  
Raji Cells ◽  
Healthy Donors ◽  
Direct Cytotoxicity ◽  
Anti Cd20 ◽  
Functional Capacities

Abstract Immunotherapy with monoclonal antibodies (mAbs), such as anti-CD20, is used in CLL treatment and represents a promising approach for achieving MRD eradication. Given their FcγRIIIa expression, NK cells are known to be involved in mAb therapy. However, little is known about characteristics of NK cells in CLL patients. For 29 untreated CLL patients and 18 healthy donors, we conducted a complete phenotypic expertise and/or we measured functional capacities of NK cells in presence or not of 2 anti-CD20 mAbs: rituximab or EMAB-6, an optimized chimeric anti-CD20 mAb exhibiting a low fucose content (C. De Romeuf et al, submitted). Four-color FACS analyses were performed on fresh blood cells from 19 CLL patients and 10 healthy donors. NK cells were analyzed on CD3-CD19-CD56+ lymphocyte subset by staining with antibodies against: CD16 (3G8), CD69, activating (NKp30, NKp44, NKp46, NKG2C and NKG2D) and inhibitory (p58a, p58b, p70, NKG2A, ILT2 and LAIR-1) cytotoxicity receptors. We showed that absolute numbers of NK cells were identical or increased in CLL patients (median (m): 410/mm3, range (r): 123–2034) compared to healthy donors (m: 127/mm3, r: 14–314). Immunophenotyping of CLL NK cells revealed a CD16 heterogenous expression. Although no difference in other NK markers expression appeared in patients with normal or high expression of CD16, an heterogeneity and a diminution of activatory cytotoxicity receptors expression were observed on low CD16 NK cells (6/19). In addition, functional tests including NK cell direct cytotoxicity and/or antibody-dependent cellular cytotoxicity (ADCC) were performed simultaneously on NK-enriched PBMC from 10 CLL patients and 8 healthy donors. NK cell direct cytotoxicity was evaluated in a standard 4h 51Cr-release assay against the HLA class-I deficient K562 cell line. NK cells direct cytotoxicity of CLL patients (m: 47%, r: 20–78) was preserved and comparable to that of healthy donors (m: 55%, r: 26–61). Furthermore, preliminary ADCC experiments were performed on 51Cr Raji cells with or without anti-CD20 mAbs at the single dose of 1 μg/ml. Under these conditions, rituximab related ADCC levels obtained with CLL NK cells (m: 45%, r: 25–96) were equal or superior to those obtained with healthy donors NK cells (m: 30%, r: 25–58). EMAB-6 related ADCC levels obtained with CLL NK cells (m: 57%, r: 34–98) were, as for rituximab, also equal or superior to those obtained with healthy donors NK cells (m: 45%, r: 30–61). A moderate enhanced ADCC was observed with EMAB-6 in CLL NK cells or in healthy donors as compared to rituximab, which is in agreement with previous observations (C. De Romeuf et al, submitted). In conclusion, NK cells from CLL patients appeared to be capable of being efficient in anti-CD20 immunotherapy: they are quantitatively and qualitatively similar to those observed in healthy donors, except in a subgroup of patients with low CD16 NK cells, which need to be more investigated. Integrity of ADCC pathway is very encouraging for mAb treatment. Moreover, EMAB-6, an optimized anti-CD20 mAb, induced a higher in vitro ADCC against Raji cells and could be a promising drug candidate for the treatment of CLL.

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.

Defective Triggering of NK Cells Results in Primary CLL Cells Resistance to Cytotoxicity,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3876-3876
Author(s):  
Caroline Veuillen ◽  
Jerome Rey ◽  
Rémy Castellano ◽  
Florence Orlanducci ◽  
Françoise Mallet ◽  
...  
Keyword(s):  
B Cells ◽  
Nk Cells ◽  
Nk Cell ◽  
K562 Cells ◽  
Surface Expression ◽  
Resting Cells ◽  
Nk Receptors ◽  
Healthy Donors

Abstract Abstract 3876 Chronic lymphocytic leukemia (CLL) remains an incurable disease except after allogenic transplantation. Natural killer (NK) cells are one of the main effectors of immune surveillance involved in tumor control. Alterations of NK cells functions have been well characterized in myeloid malignancies. However the role of NK cells in immune escape of CLL in less known and controversial. Here we describe extensive phenotypic and functional characterization of NK cells and primary CLL cells and their interactions in vitro and in vivo. Twenty eight untreated CLL patients, twenty four age-matched healthy donors and ten AML patients were enrolled in the study. We have previously shown that expression and function of NK cell-triggering receptors is defective in AML. We then assessed the phenotypic and functional properties of NK cells from CLL patients. Unlike the results found in AML, no significant differences were observed in term of activating receptors, NKp46, DNAM-1, NKG2D, 2B4 and CD16. Only the natural cytotoxicity receptor (NCR) NKp30 was weakly decreased compared to healthy donors (p=0.0107). There wasn't any difference in the expression of inhibitory receptors CD158a, b, e, ILT2 and NKG2A. Looking at the spontaneous NK-mediated cytotoxicity, CLL NK cells displayed a cytolytic activity similar to that of healthy donors against K562 cell line. To further evaluate the functional consequences of the decreased expression of NKp30, mAb redirected killing assays was performed against P815 cell lines. The NK cells killing was slightly lower in CLL patients compared to healthy donors when anti-NKp30 was used although no difference could be observed with anti-NKp46 and anti-CD16. All these results supported that NK cells cytotoxicity should be effective in CLL. We then studied the susceptibility of CLL B cells to allogenic NK killing both in vitro and in vivo. Unlike AML cells and K562 cells, CLL cells were resistant to NK cytotoxicity mediated by resting cells. Exogenous stimulation of allogenic NK cells with IL2 and IL15 restored partially CLL killing, which was nevertheless still lower than AML blasts and K562 cells killing (p=0.0288 and <0.0001 respectively). Murine xenotransplantation model using NOD/SCID g null (NSG) mice allowed us to study the anti-leukemic capacity of purified NK cells after activation with IL2. We didn't observe any clearance of CLL cells after allogenic NK cell injection while CLL and NK cells were checked to be present in blood, bone marrow, spleen and liver. These experiments confirmed the CLL resistance to NK-mediated killing. To investigate the potential mechanisms of this resistance, we analyzed the surface expression of ligands for activating and inhibitory NK receptors on CLL cells. CLL cells displayed poor expression of ligands for activating NK receptors MICA/B, ULBP1-3, PVR, nectin-2 and CD54. Interestingly, this profile of surface expression was similar to that of normal B cells except a slight increase of ULBP3 expression on CLL cells. Regarding ligands for inhibitory NK receptors, HLA-class I molecules were significantly down-regulated while HLA-E tended to be up-regulated on CLL cells compared to normal B cells. Finally, we tested ADCC in order to overcome the resistance of CLL cells to NK killing: the presence of rituximab increased significantly CLL lysis. Of note, priming of NK cells with IL2+IL15 still increased CLL cytotoxicity (p<0.0001). Our findings demonstrate that primary CLL cells are resistant to NK mediated killing. This defect is mainly due to the lack of ligands for NK receptors on CLL cells surface leading to deficient triggering of NK cells. However NK cells of CLL patients are fully competent. Attempts to optimize NK cell therapy for treatment of CLL will require overcoming the low immunogenicity of B-CLL cells. Our xenograft model provides the tools for such preclinical development. 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.

Individuals Expressing FcγRIIIA-158 V/V and V/F Show Increased NK Cell Surface Expression of FcgRIIIA (CD16), Rituximab Binding, and Demonstrate Higher Levels of ADCC Activity in Response to Rituximab.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 776-776 ◽  
Author(s):  
Evdoxia Hatjiharissi ◽  
Daniel Ditzel Santos ◽  
Lian Xu ◽  
Sigitas Verselis ◽  
Michael Modica ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Nk Cells ◽  
Nk Cell ◽  
Basic Mechanism ◽  
Surface Expression ◽  
Receptor Expression ◽  
Cell Surface Expression ◽  
Protein Levels ◽  
Healthy Donors ◽  
Anti Cd20

Abstract The homozygous expression of phenylalanine (F/F) at codon 158 of FcγRIIIa (CD16) is associated with inferior clinical responses to the anti-CD20 monoclonal antibody rituximab in patients with indolent non-Hodgkin’s lymphoma, in contrast to higher response rates observed for Waldenstrom’s macroglobulinemia patients expressing at least one valine (V/F, V/V) and follicular NHL patients who are homozygous for valine (V/V). We attempted elucidate the potential basic mechanism(s) behind these clinical observations by analyzing all the potential implications of this polymorphism among the 3 polymorphic subgroups at FcγRIIIA-158 (F/F, V/F, and V/V). We therefore used peripheral blood isolated natural killer (NK) cells from a pool of 52 unrelated healthy donors who were genotyped for FcγRIIIA-158. We evaluated allele-specific differences for FcγRIIIa gene expression by quantitative RT-PCR and demonstrated higher transcript levels among V/V (23.2 ng/mL) versus V/F (6.7 ng/mL) and F/F (6.2 ng/mL) (p&lt;0.0001). We then determined protein levels for CD16 in the same subgroup of donors using quantitative flow cytometry. The number of CD16 receptors per NK cell was 105,947, 94,863, and 69,130 for the V/V, V/F and F/F donors, respectively and was significantly higher among donors who expressed at least one valine (V/V and V/F) versus F/F (p=0.033). We next determined rituximab binding affinity to NK cells from V/V, V/F and F/F donors following incubation at concentrations of 10–200 ug/ml, and use of an indirect competitive assay with the anti-CD16 monoclonal antibody 3G8 (Cancer Research 64:4664). Rituximab binding to NK cells was higher among donors expressing at least one valine at all concentrations evaluated, with mean rituximab binding (defined as % of inhibition of mean fluorescence intensity for 3G8) as follow: V/V (72%); V/F (53%); and FF (37%); (p=0.017). Lastly, we assessed rituximab dependent NK cell mediated cytotoxicity (ADCC) using CD20 expressing ARH-77 B-cells and observed higher levels of ADCC killing among V/V (82%) and V/F (80%) versus F/F (23%) donors at an effector: target cell ratio of 20:1. Taken together, these studies suggest that individuals who express at least one valine at FcγRIIIA-158 might have better clinical outcomes to rituximab therapy on the basis of increased FcγRIIIA-158 receptor expression, rituximab binding and ADCC mediated killing of tumor cells.

scholarly journals IL-2 Induces Selective Activation of Helios-Positive Regulatory T Cells and CD56bright NK Cells in Vitro and in Patients with Chronic Gvhd Receiving Low-Dose IL-2 Therapy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 919-919 ◽  
Author(s):  
Masahiro Hirakawa ◽  
Tiago R Matos ◽  
John Koreth ◽  
Edouard Forcade ◽  
Jennifer Whangbo ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Board Of Directors ◽  
Cd8 T Cells ◽  
Low Dose ◽  
Advisory Committees ◽  
Low Concentrations ◽  
Healthy Donors

Abstract Introduction: CD4+ FoxP3+ CD25+ regulatory T cells (Treg) play a central role in the maintenance of immune tolerance and prevention of chronic graft-versus-host disease (cGVHD) after allogeneic stem cell transplantation (SCT). Treg constitutively express high-affinity interleukin-2 (IL-2) receptors and murine models have established that IL-2 is a critical homeostatic regulator of Treg in vivo. We previously reported that daily administration of low-dose IL-2 in patients with cGVHD induces selective expansion of Treg and NK cells and results in clinical improvement in approximately 50% of patients. However, the mechanisms responsible for these selective effects and the influence of IL-2 therapy on other lymphocytes have not been established due to the limited resolution of traditional cell analytic methods such as flow cytometry. Methods: Single cell mass cytometry (CyTOF) with a panel of 33 markers was used to simultaneously examine the phenotypic and functional effects of low-dose IL-2 on lymphocyte populations in vitro and in vivo. The analytic panel included 22 cell surface markers to identify distinct T, B and NK cell subsets and 11 intracellular markers to measure functional status and activation of specific signaling pathways. viSNE, a cytometry analysis tool, was used to visualize high-dimensional cytometry data on a two-dimensional map. Results: In unstimulated lymphocytes from healthy donors, constitutive expression of CD25 (IL-2Ra) at high levels was restricted to Treg and CD56bright NK cells. Central memory (CM) and effector memory (EM) subsets of conventional CD4 T cells (Tcon) and CM CD8 T cells expressed low levels of CD25. Within the Treg population, the highest expression of CD25 was closely associated with expression of Helios transcription factor. Helios+ Treg also express higher levels of FoxP3, HLA-DR and CD95 and lower levels of BCL2 compared to Helios- Treg. To examine responses to IL-2, we stimulated peripheral blood mononuclear cells (PBMC) from healthy donors with IL-2 for 15 min in vitro (Figure 1). At low IL-2 concentrations (1 to 10 IU/ml), pSTAT5 was preferentially activated in Treg. Notably, pSTAT5 activation was more robust in memory Treg than naïve Treg and in Helios+ Treg than Helios- Treg. In addition, we observed activation of pSTAT5 in CD56bright NK cells at low concentrations of IL-2 (10 IU/ml). Higher IL-2 concentrations (100-1000 IU/ml) were required to activate pSTAT5 in Tcon, CD8 T cells and CD56dim NK cells. At high IL-2 concentrations, pSTAT5 was activated in all Treg, NK, Tcon and CD8 subsets. To examine the response to IL-2 in vivo, we examined PBMC from 14 patients with chronic GVHD receiving daily low-dose IL-2 using the same CyTOF panel of markers. Without additional in vitro stimulation, pSTAT5 expression was increased preferentially in Helios+ Treg. Peak pSTAT5 expression occurred 1 week after starting IL-2 and decreased with continued IL-2 therapy. Similarly, increased expression of FoxP3, CD25, HLA-DR and Ki67 occurred primarily in Helios+ Treg with peak expression at 1 week. At later time points during IL-2 therapy, changes in Treg included increased expression of CD95, CTLA4, PD-1, BIM and BCL2. Although there was no activation of pSTAT5 in CD4 Tcon and CD8 T cells, expression of PD-1 increased in effector memory subsets of Tcon and CD8 T cells 1 week after starting IL-2 therapy. Selective expansion of CD56bright NK cells was also noted, with peak activation at 1 week. No other changes were noted in Tcon, CD8 T cells and B cells. All changes observed during IL-2 therapy returned to baseline levels 4 weeks after treatment was stopped. However, examination of PBMC from 8 patients who received continuous daily low-dose IL-2 therapy for approximately 1 year showed that all of the changes noted above persisted during extended therapy. Conclusion: Comprehensive analysis of T, B and NK cells from healthy donors revealed that low concentrations of IL-2 result in selective activation of Helios+ Treg and CD56bright NK cells. Higher concentrations of IL-2 are required for activation of CD4 Tcon, CD8 T cells and CD56dim NK cells. Identical populations are activated in patients with cGVHD receiving daily low-dose IL-2 and these functional effects persist during extended IL-2 therapy. Although the function of Helios transcription factor is not well defined, Helios expression identifies those Treg most primed to respond to low concentrations of IL-2 in vitro and in vivo. Disclosures Armand: Infinity Pharmaceuticals: Consultancy; Merck: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding. Antin:Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Gentium S.p.A.: Membership on an entity's Board of Directors or advisory committees. Soiffer:Gentium SpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Immunoliposomes Incorporated with Humanized Monoclonal Antibody, Milatuzumab, Induce Cell Death in CLL by Retention of the CD74 Receptor On the Surface of B Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 721-721 ◽  
Author(s):  
Erin K Hertlein ◽  
Georgia Triantafillou ◽  
Joshua Hessler ◽  
Xiaoli Zhang ◽  
David Jarjoura ◽  
...  
Keyword(s):  
T Cells ◽  
Cell Death ◽  
B Cells ◽  
Cell Surface ◽  
Nk Cells ◽  
Surface Expression ◽  
Receptor Internalization ◽  
Parp Cleavage ◽  
Fluorescent Intensity

Abstract Abstract 721 Chronic lymphocytic leukemia (CLL) is a progressive disease for which most patients require treatment. Therapy with monoclonal antibodies (mAbs) such as rituximab (anti-CD20) and alemtuzumab (anti-CD52) have improved the outcomes of patients with CLL; however, patients display varied levels of response to these agents and many develop resistance. We describe here a humanized mAb, milatuzumab (Immunomedics, Inc.), against CD74. The CD74 antigen has several ligands, including macrophage inhibitory factor, MIF, whose binding promotes internalization and nuclear localization with concomitant activation of NF-κB. Milatuzumab is currently in clinical trials in multiple myeloma and CLL; however, the mechanism by which this antibody induces apoptosis is still unknown. We demonstrate surface expression of CD74 on CLL and circulating normal CD19+ B cells, but no expression on T cells. Milatuzumab-induced cytotoxicity occurs very rapidly, with 70% cell survival at 4 h and 45% at 24 h, which is superior to that observed with the CD20 antibody rituximab. No toxicity was detected in NK and T cells treated with milatuzumab. The mode of cell death induced by milatuzumab was found to be independent of caspase activation, with no detectable caspase-3 processing, PARP cleavage, or inhibition of death with the pan-caspase inhibitor, BocD-fmk. In vitro, the addition of a-Fc crosslinking antibody is necessary for milatuzumab-induced cell death. However, previous reports noted milatuzumab does not mediate antibody-dependent cellular cytotoxicity (ADCC) in lymphoma cell lines and this was verified in CLL using peripheral blood mononuclear cells, NK cells, and granulocytes as effector cells. Thus, milatuzumab-mediated death appears to be mediated directly through interaction with CD74 and effects on signal transduction. We further investigated the role of anti-Fc crosslinking antibody in mediating milatuzumab-induced cell death, and found that upon milatuzumab treatment in the presence of anti-Fc, CLL cells aggregated in culture. Furthermore, the mean fluorescent intensity (MFI) of CD74 on the cell surface increased, which did not occur following treatment with anti-Fc alone, milatuzumab alone, or anti-Fc along with other anti-B-cell mAbs such as rituximab. This suggests that milatuzumab, when crosslinked, promotes the maintenance of CD74 on the cell surface and thereby prevents receptor internalization and subsequent signaling. However, association with Fc receptors on other cells in the microenvironment may not be sufficient to mediate this effect, as indicated by the lack of ADCC. Furthermore, in vitro co-culture of NK cells in the presence of milatuzumab did not induce the same cellular aggregation and elevated CD74 surface expression as seen with the anti-Fc antibody. These results indicate that milatuzumab treatment in vivo may not be as efficient as suggested by the in vitro studies without a sufficient method to sequester CD74 on the cell surface. We tested this hypothesis by incorporating milatuzumab into liposomes (Mila-ILP). Our results show that this conjugate mediates the same receptor aggregation on the cell surface as milatuzumab combined with anti-Fc crosslinking antibody. This effect was not evident with incorporation of irrelevant immunoglobulin into liposomes (IgG-ILP). Furthermore, Mila-ILP induced the same degree of cell death as observed previously with in vitro antibody crosslinking. Therefore, the incorporation of milatuzumab into liposomes induces cell death independent of other cell types in the microenvironment. Together, these data describe a mechanism of milatuzumab-induced cytotoxicity in CLL, and encourage the clinical application of milatuzumab-immunoliposome-based therapy in CLL and other CD74-positive lymphoid malignancies. Disclosures: Goldenberg: Immunomedics: Employment, Equity Ownership.

scholarly journals AB0024 IN VITRO CHARACTERIZATION OF CENERIMOD, A POTENT AND SELECTIVE SPHINGOSINE 1-PHOSPHATE RECEPTOR 1 (S1P1) MODULATOR IN PREVENTING MIGRATION OF NON-ACTIVATED AND ACTIVATED PRIMARY HUMAN B CELLS IN THE PRESENCE OR ABSENCE OF GLUCOCORTICOIDS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 1046.1-1046
Author(s):  
L. Schlicher ◽  
P. Kulig ◽  
M. Murphy ◽  
M. Keller
Keyword(s):  
B Cells ◽  
B Cell ◽  
B Lymphocytes ◽  
Cell Activation ◽  
Surface Expression ◽  
Cell Surface Expression ◽  
B Cell Activation ◽  
Human B Cells ◽  
Circulating Lymphocytes

Background:Cenerimod is a potent, selective, and orally active sphingosine 1-phosphate receptor 1 (S1P1) modulator that is currently being evaluated in a Phase 2b study in patients with systemic lupus erythematosus (SLE) (NCT03742037). S1P1 receptor modulators sequester circulating lymphocytes within lymph nodes, thereby reducing pathogenic autoimmune cells (including B lymphocytes) in the blood stream and in inflamed tissues. Extensive clinical experience has become available for the nonselective S1P receptor modulator fingolimod in relapsing forms of multiple sclerosis, supporting this therapeutic concept for the treatment of autoimmune disorders.Objectives:Although the effect of S1P-receptor modulators in reducing peripheral B cells is well documented1,2, the role of the S1P1 receptor on this cell type is only incompletely understood. In this study, the mode of action of cenerimod on primary human B cells was investigated in a series of in vitro experiments, including S1P1 receptor cell surface expression and chemotaxis towards S1P. Moreover, S1P1 expression following B cell activation in vitro was studied. As glucocorticoids (GC) are frequently used in the treatment of patients with autoimmune disorders including SLE, the potential influence of GC on the mode of action of cenerimod was evaluated.Methods:Primary human B lymphocytes from healthy donors were isolated from whole blood. In one set of experiments, cells were treated with different concentrations of cenerimod to measure S1P1 receptor internalization by flow cytometry. In a second set of experiments, isolated B cells were activated using different stimuli or left untreated. Cells were then analysed for S1P1 and CD69 cell surface expression and tested in a novel real-time S1P-mediated migration assay. In addition, the effect of physiological concentrations of GCs (prednisolone and prednisone) on cenerimod activity in preventing S1P mediated migration was tested.Results:In vitro, cenerimod led to a dose-dependent internalization of the S1P1 receptor on primary human B lymphocytes. Cenerimod also blocked migration of nonactivated and activated B lymphocytes towards S1P in a concentration-dependent manner, which is in line with the retention of lymphocytes in the lymph node and the reduction of circulating lymphocytes observed in the clinical setting. Upon B cell activation, which was monitored by CD69 upregulation, a simultaneous downregulation of S1P1 expression was detected, leading to less efficient S1P-directed cell migration. Importantly, physiological concentrations of GC did not affect the inhibitory activity of cenerimod on B cell migration.Conclusion:These results show that cenerimod, by modulating S1P1, blocks B lymphocyte migration towards its natural chemoattractant S1P and demonstrate compatibility of cenerimod with GC. These results are consistent with results of comparable experiments done previously using primary human T lymphocytes.References:[1]Nakamura M et al., Mult Scler. 2014 Sep; 20(10):1371-80.[2]Strasser DS et al., RMD Open 2020;6:e001261.Disclosure of Interests:None declared

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

2008 ◽  
Vol 76 (4) ◽  
pp. 1719-1727 ◽  
Author(s):  
Semih Esin ◽  
Giovanna Batoni ◽  
Claudio Counoupas ◽  
Annarita Stringaro ◽  
Franca Lisa Brancatisano ◽  
...  
Keyword(s):  
Nk Cells ◽  
Nk Cell ◽  
Cell Subset ◽  
Surface Expression ◽  
Binding Assays ◽  
Igg Antibody ◽  
Direct Stimulation ◽  
Activating Receptors ◽  
Accessory Cells

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

scholarly journals Stem Cell Factor Enhances Interleukin-2–Mediated Expansion of Murine Natural Killer Cells In Vivo

Blood ◽  
1997 ◽  
Vol 90 (9) ◽  
pp. 3647-3653 ◽  
Author(s):  
Todd A. Fehniger ◽  
William E. Carson ◽  
Ewa Mrózek ◽  
Michael A. Caligiuri
Keyword(s):  
Nk Cells ◽  
Cell Expansion ◽  
Stem Cell Factor ◽  
Low Dose ◽  
Nk Cell ◽  
Interleukin 2 ◽  
Innate Immune ◽  
Ifn Γ

Abstract The administration of low dose interleukin-2 (IL-2) results in a selective expansion of natural killer (NK) cells in vivo, and promotes the differentiation of NK cells from hematopoietic precursor cells in vitro. We have previously shown that stem cell factor (SCF ), the ligand to the c-kit tyrosine kinase receptor, enhances IL-2–induced NK cell proliferation and differentiation in vitro. Here, we investigated the effects of SCF plus IL-2 delivered to mice in vivo. Eight-week-old C57BL/6 mice were treated with a continuous subcutaneous infusion of IL-2 (1 × 104 IU/d) plus a daily intraperitoneal dose of SCF (100 μg/kg/d), IL-2 alone, SCF alone, or vehicle alone for 8 weeks. The in vivo serum concentration of IL-2 ranged between 352 ± 12.0 pg/mL and 606 ± 9.0 pg/mL, achieving selective saturation of the high affinity IL-2 receptor, while the peak SCF serum concentration was 296 ± 13.09 ng/mL. Alone, the daily administration of SCF had no effect on the expansion of NK cells. The continuous infusion of IL-2 alone did result in a significant expansion of NK1.1+CD3− cells compared to mice treated with placebo or SCF. However, mice treated with both SCF and IL-2 showed an increase in the absolute number of NK cells that was more than twofold that seen with IL-2 alone, in the spleen (P ≤ .005), bone marrow (P ≤ .025), and blood (P < .05). NK cytotoxic activity against YAC-1 target cells was significantly higher for mice treated with SCF plus IL-2, compared to mice treated with IL-2 alone (P ≤ .0005). Interferon-γ (IFN-γ) production in cytokine-activated splenocytes was also greater for the SCF plus IL-2 group, over IL-2 treatment alone (P ≤ .01). The effect of SCF plus IL-2 on NK cell expansion was likely mediated via NK cell precursors, rather than mature NK cells. In summary, we provide the first evidence that SCF can significantly enhance expansion of functional NK cells induced by the prolonged administration of low dose IL-2 in vivo. Since the NK cell is a cytotoxic innate immune effector and a potent source of IFN-γ, this therapeutic strategy for NK cell expansion may serve to further enhance innate immune surveillance against malignant transformation and infection in the setting of cancer and/or immunodeficiency.

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