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 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.

scholarly journals Cyclin-Dependent Kinases (CDK) Signaling Blockade Potentiates NK Cell Mediated Cytotoxicity Against Acute Myelogenous Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4538-4538
Author(s):  
Hyun Don Yun ◽  
Dawn Schirm ◽  
Martin Felices ◽  
Jeffrey S. Miller ◽  
Craig E. Eckfeldt
Keyword(s):  
Flow Cytometry ◽  
Drug Treatment ◽  
Nk Cells ◽  
Cell Activation ◽  
Nk Cell ◽  
Myelogenous Leukemia ◽  
Target Cells ◽  
Dead Cell ◽  
Cd107a Expression ◽  
Anti Cancer

Abstract Dinaciclib, a CDK 1, 2, 5, and 9 inhibitor, has been shown to have potent anti-cancer activity against several malignancies. Our group previously demonstrated a direct RALB-dependent proapoptotic activity of dinaciclib against acute myeloid leukemia (AML) (Oncogene (2017) 36, 3263-3273). Furthermore, there is emerging evidence that dinaciclib can influence the recognition and killing of cancer cells by the immune system suggesting that it could have multimodal anti-cancer activities. As natural killer (NK) cells have a critical role in antineoplastic cytotoxicity, we hypothesized that dinaciclib may influence NK cell mediated cytotoxicity against human AML cells. To address this hypothesis, we evaluated the effects of dinaciclib on the NK cytotoxicity against human AML target cells and expression of known regulators of NK cell function. Specifically, we treated THP-1 and KG-1 cells with vehicle or dinaciclib at 5-20 nM for 24 hours, then washed the AML targets and added purified NK cells from PBMCs of healthy donors at an effector: target (E:T) ratio of 2:1. After 24 hours, viable AML target cells were enumerated using Fixable Aqua Dead Cell Stain and AccuCheck Counting Beads (Thermo Fisher Scientific) by flow cytometry. Relative NK cell target killing (%) was calculated by comparing the percentage of AML targets killed with drug treatment combined with donor NK cells relative to drug treatment alone in the absence of NK cells (Relative NK killing %). We assessed NK cell activation by measuring intracellular interferon-ɣ production and CD107a expression as well as NK ligand expression on dinaciclib-treated AML targets using flow cytometry. Dinaciclib treatment of AML targets enhanced the relative NK killing % for both KG-1 and THP-1 AML targets compared to control treatment (for KG-1, 45.6% vs 14.5%, p=0.05; for THP-1, 63.7% vs 31.7%, p=0.01) (Figure 1A, 1B) and was associated with enhanced NK cell degranulation as measured by CD107a expression (DMSO group, 8.3% vs dinaciclib 20nM group, 17.7%, p=0.002) and inflammatory cytokine production as measured by intracellular interferon-ɣ expression (DMSO group, 10.4% vs dinaciclib 20nM group 17.3%, p=0.01) (Figure 1C, 1D) supporting the ability of dinaciclib to sensitize AML targets for NK cell-based immunotherapy. To investigate potential mechanisms that promote NK cell activation by dinaciclib-treated AML targets, we assessed expression of several key NK cell ligands on AML targets and found that dinaciclib treatment led to decreased expression of inhibitory ligands including HLA class I, CD112, CD155 and increased expression in activating ligands such as TRAILR1, CD48 (Figure 1E, 1F) providing potential mechanistic insights. Notably, preliminary studies using a primary, patient-derived AML sample treated with dinaciclib resulted in a similar increase in the proportion of CD107a+ NK cells compared to the control group (44.7% increase, p=0.02, Figure 1G, 1H). While the detailed mechanisms for our findings remain to be determined, this is the first report to our knowledge that inhibiting CDK signaling can sensitize AML targets to NK cell based immunotherapy, a completely novel treatment approach. Further studies are ongoing to investigate the potential for combined targeted therapy with dinaciclib and adoptive NK cell therapy including patient derived xenograft (PDX) mice to validate its translational potential. Disclosures Felices: GT Biopharma: Research Funding.

scholarly journals Recognition of virus-infected cells by natural killer cell clones is controlled by polymorphic target cell elements.

1993 ◽  
Vol 178 (3) ◽  
pp. 961-969 ◽  
Author(s):  
M S Malnati ◽  
P Lusso ◽  
E Ciccone ◽  
A Moretta ◽  
L Moretta ◽  
...  
Keyword(s):  
T Cells ◽  
Nk Cells ◽  
Natural Killer ◽  
Target Cell ◽  
Nk Cell ◽  
Class I ◽  
Target Cells ◽  
Cell Recognition ◽  
Infected Cells ◽  
Nk Cell Recognition

Natural killer (NK) cells provide a first line of defense against viral infections. The mechanisms by which NK cells recognize and eliminate infected cells are still largely unknown. To test whether target cell elements contribute to NK cell recognition of virus-infected cells, human NK cells were cloned from two unrelated donors and assayed for their ability to kill normal autologous or allogeneic cells before and after infection by human herpesvirus 6 (HHV-6), a T-lymphotropic herpesvirus. Of 132 NK clones isolated from donor 1, all displayed strong cytolytic activity against the NK-sensitive cell line K562, none killed uninfected autologous T cells, and 65 (49%) killed autologous T cells infected with HHV-6. A panel of representative NK clones from donors 1 and 2 was tested on targets obtained from four donors. A wide heterogeneity was observed in the specificity of lysis of infected target cells among the NK clones. Some clones killed none, some killed only one, and others killed more than one of the different HHV-6-infected target cells. Killing of infected targets was not due to complete absence of class I molecules because class I surface levels were only partially affected by HHV-6 infection. Thus, target cell recognition is not controlled by the effector NK cell alone, but also by polymorphic elements on the target cell that restrict NK cell recognition. Furthermore, NK clones from different donors display a variable range of specificities in their recognition of infected target cells.

Thermal stresses reduce natural killer cell cytotoxicity

1995 ◽  
Vol 79 (3) ◽  
pp. 732-737 ◽  
Author(s):  
S. J. Won ◽  
M. T. Lin
Keyword(s):  
Nk Cells ◽  
Natural Killer ◽  
Target Cell ◽  
Nk Cell ◽  
Cell Activity ◽  
Cortisol Concentration ◽  
Target Cells ◽  
Nk Cell Activity ◽  
Colonic Temperature ◽  
Effector Target

The effects of different ambient temperatures (Ta) on the splenic natural killer (NK) cell activity, effector-target cell conjugation activity, and NK cell numbers were assessed in male inbred C3H/HeNCrj mice (7–10 wk old). The splenic NK cytotoxic activities were examined in a 4-h 51Cr release assay in mouse spleen cells that were obtained 1, 2, 4, 8, or 16 days after exposure to Ta of 22, 4, or 35 degrees C. The percentage of conjugating lymphocytes was calculated by counting the number of single lymphocytes bound to single target cells per 400 effector cells. The numbers of NK cells were expressed by the percentage of 5E6-positive cells. The 5E6 identifies only a subset of NK cells. It was found that the splenic NK cell activity, the effector-target cell conjugation activity, or the NK cell number began to fall 1 day after cold (Ta 4 degrees C) or heat (Ta 35 degrees C) stress. After a 16-day period of either cold or heat exposure, the fall in the splenic NK cell activity, the effector-target cell conjugation activity, or the number of 5E6-positive subsets of NK cells was still evident. Compared with those of the control group (Ta 22 degrees C), the cold-stressed mice had higher adrenal cortisol concentration and lower colonic temperature, whereas the heat-stressed animals had higher adrenal cortisol concentration and higher colonic temperature during a 16-day period of thermal exposure. However, neither cold nor heat stress affected both the body weight gain and the spleen weight in our mice.

scholarly journals Understanding the Synergy of NKp46 and Co-Activating Signals in Various NK Cell Subpopulations: Paving the Way for More Successful NK-Cell-Based Immunotherapy

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 753 ◽  
Author(s):  
Loris Zamai ◽  
Genny Del Zotto ◽  
Flavia Buccella ◽  
Sara Gabrielli ◽  
Barbara Canonico ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Response ◽  
Cell Activation ◽  
Nk Cell ◽  
High Sensitivity ◽  
Leukemic Cells ◽  
Target Cells ◽  
Hematopoietic Stem ◽  
Oncologic Patients ◽  
The Way

The NK cell population is characterized by distinct NK cell subsets that respond differently to the various activating stimuli. For this reason, the determination of the optimal cytotoxic activation of the different NK cell subsets can be a crucial aspect to be exploited to counter cancer cells in oncologic patients. To evaluate how the triggering of different combination of activating receptors can affect the cytotoxic responses of different NK cell subsets, we developed a microbead-based degranulation assay. By using this new assay, we were able to detect CD107a+ degranulating NK cells even within the less cytotoxic subsets (i.e., resting CD56bright and unlicensed CD56dim NK cells), thus demonstrating its high sensitivity. Interestingly, signals delivered by the co-engagement of NKp46 with 2B4, but not with CD2 or DNAM-1, strongly cooperate to enhance degranulation on both licensed and unlicensed CD56dim NK cells. Of note, 2B4 is known to bind CD48 hematopoietic antigen, therefore this observation may provide the rationale why CD56dim subset expansion correlates with successful hematopoietic stem cell transplantation mediated by alloreactive NK cells against host T, DC and leukemic cells, while sparing host non-hematopoietic tissues and graft versus host disease. The assay further confirms that activation of LFA-1 on NK cells leads to their granule polarization, even if, in some cases, this also takes to an inhibition of NK cell degranulation, suggesting that LFA-1 engagement by ICAMs on target cells may differently affect NK cell response. Finally, we observed that NK cells undergo a time-dependent spontaneous (cytokine-independent) activation after blood withdrawal, an aspect that may strongly bias the evaluation of the resting NK cell response. Altogether our data may pave the way to develop new NK cell activation and expansion strategies that target the highly cytotoxic CD56dim NK cells and can be feasible and useful for cancer and viral infection treatment.

scholarly journals Potential Role of NK Cells in the Pathogenesis of Inflammatory Bowel Disease

2011 ◽  
Vol 2011 ◽  
pp. 1-6 ◽  
Author(s):  
Praveen K. Yadav ◽  
Chi Chen ◽  
Zhanju Liu
Keyword(s):  
Inflammatory Bowel Disease ◽  
Nk Cells ◽  
Proinflammatory Cytokines ◽  
Bowel Disease ◽  
Cell Activation ◽  
Potential Role ◽  
Nk Cell ◽  
Target Cells ◽  
Intestinal Epithelial ◽  
Inflammatory Bowel

NK cells are a major component of the innate immune system and play an important role in the tissue inflammation associated with autoimmune diseases such as inflammatory bowel disease (IBD). NK cells are unique in bearing both stimulatory and inhibitory receptors specific for MHC class I molecules, and their function is regulated by a series of inhibiting or activating signals. The delicate balance between activation and inhibition that decides NK cell final action provides an opportunity for their possible modulatory effect on specific therapeutic settings. Intestinal NK cells are phenotypically distinct from their counterparts in the blood and resemble “helper” NK cells, which have potentially important functions both in promoting antipathogen responses and in the maintenance of intestinal epithelial homeostasis. NK cell activities have been found to be significantly below normal levels in both remissive and active stages of IBD patients. However, some proinflammatory cytokines (e.g., IL-15, IL-21, and IL-23) could potently induce NK cell activation to secret high levels of proinflammatory cytokines (e.g., IFN-γ and TNF) and promote the cytolytic activities against the target cells. This paper provides the characteristics of intestinal NK cells and their potential role in the pathogenesis of IBD.

scholarly journals Specificity of HLA class I antigen recognition by human NK clones: evidence for clonal heterogeneity, protection by self and non-self alleles, and influence of the target cell type.

1993 ◽  
Vol 178 (4) ◽  
pp. 1321-1336 ◽  
Author(s):  
V Litwin ◽  
J Gumperz ◽  
P Parham ◽  
J H Phillips ◽  
L L Lanier
Keyword(s):  
Nk Cells ◽  
Mhc Class I ◽  
Target Cell ◽  
Nk Cell ◽  
Hla Class I ◽  
Class I ◽  
Target Cells ◽  
Clonal Heterogeneity ◽  
Multiple Alleles ◽  
Wide Range

Prior studies using polyclonal populations of natural killer (NK) cells have revealed that expression of certain major histocompatibility complex (MHC) class I molecules on the membrane of normal and transformed hematopoietic target cells can prevent NK cell-mediated cytotoxicity. However, the extent of clonal heterogeneity within the NK cell population and the effect of self versus non-self MHC alleles has not been clearly established. In the present study, we have generated more than 200 independently derived human NK cell clones from four individuals of known human histocompatibility leukocyte antigens (HLA) type. NK clones were analyzed for cytolytic activity against MHC class I-deficient Epstein Barr virus (EBV) transformed B lymphoblastoid cell lines (B-LCL) stably transfected with several HLA-A, -B, or -C genes representing either self or non-self alleles. All NK clones killed the prototypic HLA-negative erythroleukemia K562 and most lysed the MHC class I-deficient C1R and 721.221 B-LCL. Analysis of the panel of HLA-A, -B, and -C transfectants supported the following general conclusions. (a) Whereas recent studies have suggested that HLA-C antigens may be preferentially recognized by NK cells, our findings indicate that 70% or more of all NK clones are able to recognize certain HLA-B alleles and many also recognize HLA-A alleles. Moreover, a single NK clone has the potential to recognize multiple alleles of HLA-A, HLA-B, and HLA-C antigens. Thus, HLA-C is not unique in conferring protection against NK lysis. (b) No simple patterns of HLA specificity emerged. Examination of a large number of NK clones from a single donor revealed overlapping, yet distinct, patterns of reactivity when a sufficiently broad panel of HLA transfectants was examined. (c) Both autologous and allogeneic HLA antigens were recognized by NK clones. There was neither evidence for deletion of NK clones reactive with self alleles nor any indication for an increased frequency of NK clones recognizing self alleles. (d) With only a few exceptions, protection conferred by transfection of HLA alleles into B-LCL was usually not absolute. Rather a continuum from essentially no protection for certain alleles (HLA-A*0201) to very striking protection for other alleles (HLA-B*5801), with a wide range of intermediate effects, was observed. (e) Whereas most NK clones retained a relatively stable HLA specificity, some NK clones demonstrated variable and heterogeneous activity over time. (f) NK cell recognition and specificity cannot be explained entirely by the presence or absence of HLA class I antigens on the target cell.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Enhanced NK Cell Activation, Cytotoxicity and Ex-Vivo Expansion (EvE) of Cryopreserved Cord Blood (CB) Natural Killer (NK) Cells: Potential Role for CB NK Cells in Adoptive Cellular Immunotherapy (ACI).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 726-726
Author(s):  
Janet Ayello ◽  
Julia Nemiroff ◽  
Prakash Satwani ◽  
Carmella van de Ven ◽  
Evan Shereck ◽  
...  
Keyword(s):  
Nk Cells ◽  
Cell Activation ◽  
Nk Cell ◽  
Ex Vivo ◽  
Granzyme B ◽  
Cellular Immunotherapy ◽  
Common Mechanism ◽  
Short Term ◽  
Cd107a Expression ◽  
Short Term Culture

Abstract CD56+ NK subsets exhibit differential NK receptors (NKR ) such as NCR profiles including killer-Ig-like receptors (KIR), C-lectin (NKG2) and natural cytoxicity receptors (NCR) involved with tumor target recognition (Farag et al Blood, 2002). NK cell activation and NK mediated cytolysis is induced by several NKRs such as NCR (i.e. NKp44, NKp46) and NKG2 surface receptors like NKG2D (Moretta et al, Curr Opinion in Immunol, 2004). Target cell killing by activated NK cells via the granule-dependent pathway is a common mechanism of NK and CTLs and degranulation is followed by the expression of lysosomal-associated membrane protein-1 [LAMP-1] on the cell surface (Penack et al, Leukemia, 2005). CB is limited by the absence of available donor effector cells (NK, CTL, LAK and NKT cells) for infusion after UCBT (Cairo, et al, Transfusion, 2005). We have demonstrated the ability to EvE CB in short-term culture (48 hrs) with IL-2, IL-7, IL-12 and anti-CD3 (ABCY) cryopreserved, thawed, recryopreserved, rethawed and EvE (CTCTE) CB with significant increase in CD3−/16+/56+ bright/dim subsets expressing KIR3DL1, KIR2DL1/S1, KIR2DL2 and CD94/NKG2a (Ayello/Cairo et al BBMT, 2006). In this study, we compared short-term culture (48 hrs) with prolonged cultures (4 to 10 days) on expansion, expression of NCR, NKG2, KIR and cytolytic ability and mechanisms in CTCTE CB. Rethawed nonadherent CB cells were cultured (2–10 days) in serum-free media alone or with anti-CD3 (50 ng/ml), IL-2 (5 ng/ml), IL-7 (10 ng/ml) and IL-12 (10 ng/ml) [ABCY]. NKR expression (CD94, NKG2D, Nkp44 and KIR2DS4), intracellular perforin, granzyme B activity and LAMP-1 receptor (CD107a) expression were determined by flow cytometry. Cytoxicity was measured by europium release assay and tumor targets used were K562, Daudi, neuroblastoma (SHSY5Y) and AML (Kasumi-1) at a 20:1 E:T ratio. C-lectin activating receptor CD94/NKG2D was increased at day 7 vs 2 following ABCY EvE (41.4±0.43 vs 23.7±2.%, p&lt;0.001). Significant increases were seen in activating KIR2DS4 at day 10 vs 2 in ABCY in both CD3−/16+/56+dim and bright subsets (16.9±0.4 vs 2.1±0.2% and 22.3±0.3 vs 0.9± 0.2%, p&lt;0.001, respectively). In contrast, NCR expression in CD3−/16+/56+dim NKp44 subset was significantly decreased at day 10 vs 2 of EvE CB in ABCY (15.2±0.7 vs 27.2±0.7%, p&lt;0.001). Granzyme B expression was increased from day 2 to 10 (25.8± vs 45.1± 1.7%, p&lt;0.001) yet perforin was decreased in EvE CB in ABCY at day 7 vs 2 (68.3±2.19 vs 84.3±1.3%, p&lt;0.001). CD107a expression was significantly increased at day 7 vs 2 in ABCY EvE CB (12.95±1.47 vs 69.34±2.22%, p&lt;0.001). In addition, significant increases in cytolytic activity was demonstrated at day 7 vs 2 of EvE CB cells in ABCY against tumor targets K562 (71.5±±0.81 vs 53.8±3.9%, p&lt;0.001), Daudi (63.9±0.73 vs 31.8±1.8%, p&lt;0.001), SYSY5Y (76.8±6.5 vs 57.5±3.4%, p&lt;0.05) and Kasumi-1 (56.6.5±0.4 vs 38±1.1%, p&lt;0.001). In summary, CB MNC may be thawed at time of CB transplantation, recryopreserved, rethawed at a later date, EvE and activated for up to 10 days to yield significantly increased cytotolytic activity against NHL, AML and neuroblastoma with increased expression of NK KAR KIR2DS4 and granzyme B, LAMP-1 degranulation (NK activation) but decreased NK C-lection CD94/NKG2D, NCR NKp44 and perforin expression.

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.

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