scholarly journals FRI0009 IMMUNO-PHENOTYPIC ANALYSIS OF PERIPHERAL BLOOD MONONUCLEAR CELLS IN RHEUMATOID ARTHRITIS PATIENTS TREATED WITH E6011, A HUMANIZED ANTI-FRACTALKINE MONOCLONAL ANTIBODY

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 575.2-576
Author(s):  
T. Yamada ◽  
J. Kakuta ◽  
E. Fusaoka-Nishioka ◽  
J. I. Ito ◽  
N. Yasuda ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
T Cells ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Initial Treatment ◽  
Inadequate Response ◽  
Color Flow ◽  
Research Institute

Background:Fractalkine (FKN) and its solo receptor CX3CR1 are deeply involved in the pathogenesis of rheumatoid arthritis (RA). FKN is expressed on vascular endothelium, while CX3CR1 is expressed on peripheral blood leukocytes such as monocytes, macrophages, NK cells, effector CD8+T cells and a minor fraction of CD4+T cells. E6011, a novel humanized anti-FKN monoclonal antibody (mAb), is under clinical development in RA.Objectives:In order to continuously assess the E6011 pharmacodynamics by monitoring the alteration of peripheral blood immune cells, including CX3CR1-expressed cell populations, a series of multi-color flow cytometry (FCM) was conducted before and during the course of the E6011 treatment of active RA patients in phase 2 clinical trial.Methods:Immuno-phenotypic changes were explored by FCM during the E6011 administration in 190 Japanese RA patients with inadequate response to MTX (NCT02960438). Patient’s peripheral blood were drawn into fixative tube (Cyto-Chex® BCT, Streck) at each clinics and thereafter transported to the FCM facility at KAN Research Institute, Inc. within 30 hours after the blood collection to operate the FCM analysis by standardized method. Immuno-phenotyping was carried out by multi colors flow cytometry (BD FACSCantoIITM, BD LSRFortessaTM, BD Biosciences).Results:Based on these determined conditions, CX3CR1 expression on monocytes, NK cells and a part of CD8+and CD4+T cells were confirmed in this method. Interestingly, during the E6011 treatment, the proportion of CD16+monocytes, which highly express CX3CR1 within whole monocytes, were significantly decreased at 2 week after initial treatment from the baseline (E6011:p< 0.001, placebo:p> 0.48) and sustained up to 24 week, while that of CD16-monocytes were increased. The reduction of the frequency of CX3CR1+cells in NK cells, CD4+and CD8+T cells were not observed, but in some certain populations like CX3CR1-expressed CD56+CD16+NK cells and terminal differentiated effector CD8+T cells, the frequency of CX3CR1+cells in these populations tended to increase from the baseline at 2 week and kept increasing up to 24 week by the E6011 treatment.Conclusion:E6011 significantly decreased the proportion of CD16+monocytes in whole monocytes. Our results indicated that the reduction of CD16+monocytes after initial treatment might be a sensitive marker of E6011 in peripheral blood, possibly reflecting mechanism of action of E6011, since the CD16+monocytes highly express CX3CR1.References:[1]Tanaka Y, et al., Mod Rheumatol (2018) 28, 58-65Disclosure of Interests:Tomohiro Yamada Employee of: KAN Research Institute, Inc,, Jungo Kakuta Employee of: KAN Research Institute, Inc., Eri Fusaoka-Nishioka Employee of: KAN Research Institute, Inc., Jun-ichi Ito Employee of: EISAI, Nobuyuki Yasuda Employee of: KAN Research Institute, Inc., Tetsu Kawano: None declared, Toshio Imai: None declared

scholarly journals Risk Factors and Changes of Peripheral NK and T Cells in Pulmonary Interstitial Fibrosis of Patients with Rheumatoid Arthritis

2019 ◽  
Vol 2019 ◽  
pp. 1-6
Author(s):  
Na-Lin Lai ◽  
Wen Jia ◽  
Xia Wang ◽  
Jing Luo ◽  
Guang-Ying Liu ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Risk Factors ◽  
Flow Cytometry ◽  
T Cells ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Interstitial Fibrosis ◽  
High Titer ◽  
Pulmonary Interstitial Fibrosis ◽  
The Absolute

Objective. The absolute and relative changes of peripheral NK and T subsets are unclear in rheumatoid arthritis (RA) associated with pulmonary interstitial fibrosis (RA-ILD). To investigate the clinical risk factors, especially the changes of lymphocyte subsets, in RA-ILD in order to make early diagnosis and achieve prevention of the pulmonary interstitial lesions. Methods. A total of 100 RA and 100 RA-ILD patients were enrolled. Rheumatoid factor, anti-cyclic citrulline peptide antibody, erythrocyte sedimentation rate, immunoglobulin, and C-reactive protein were examined. The percentage and absolute number of NK, T, B, Treg, Th1, Th2, and Th17 cells in peripheral blood were determined by flow cytometry. Results. RA-ILD is more common in older and male RA patients and/or those with higher autoantibody titers. Flow cytometry showed that the absolute and relative numbers of CD56+ NK cells were significantly higher in RA-ILD (280.40 ± 180.51 cells/μl vs. 207.66 ± 148.57 cells/μl; 16.62 ± 8.56% vs. 12.11 ± 6.47%), whereas the proportion of T cells and CD4+ T cells was lower in peripheral blood of RA-ILD patients (69.82 ± 9.30%; 39.44 ± 9.87 cells/μl) than that in RA patients (74.45 ± 8.72%; 43.29 ± 9.10 cells/μl). Conclusions. The occurrence of RA-ILD is closely related to the older male patients with high titer of various self-antibodies. Imbalance of CD3−CD56+ NK cells and T cells with other subsets were found in RA-ILD patients, which, together with older age, male, and high levels of autoantibodies should be considered as risk factors of pulmonary interstitial lesions.

Flow Cytometric Detection of the Expression of CXCR4 on CD34+ Cells and the Distribution of Lymphocyte Subsets in Allogeneic Hematological Grafts.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5195-5195
Author(s):  
Lulu Lu ◽  
Yongping Song ◽  
Baogen Ma ◽  
Xiongpeng Zhu ◽  
Xudong Wei ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
Cord Blood ◽  
Peripheral Blood ◽  
Lymphocyte Subsets ◽  
Color Flow ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Mobilized Peripheral Blood

Abstract Background and objectives: Normal human bone marrow (BM), cord blood (CB) and mobilized peripheral blood (MPB) are the most commonly used sources for allogeneic hematopoietic stem cell transplantation (HSCT). The aim of this study was to detect the expression of CXCR4 on CD34+ cells and to assess the distribution of lymphocyte subsets in each type allograft. Methods: CD34+ cells were separated from BM (n=30), CB (n=30) and MPB (n=30) by the CD34 MultiSort Kit immunomagnetic bead system. The expression of CXCR4 on CD34+cells was assayed by double color flow cytometry. The lymphocyte subsets in each type of allograft were detected by three-color flow cytometry. The groups of monoclonal antibodies were used as the following: CXCR4-PE/CD34−Pecy5, CD8−FITC/CD4−R-PE/CD3−TC, CD45RA-FITC/CD45RO-PE/CD4−Pecy5, CD45RA-FITC/CD45RO-PE/CD8−Pecy5, and CD3−FITC/CD16+56-PE. Isotype-specific antibodies were used as controls. Results: The expression of CXCR4 of cord blood and mobilized peripheral blood CD34+ cells was lower than that of bone marrow cells (BM 40.21%±6.72%, CB 20.93%±3.96%, MPB 20.93%±3.96%, P &lt;0.05). The difference between cord blood and mobilized peripheral blood was not significant (P&gt;0.05). The CD3+CD8low and CD3+CD4−CD8low subsets were higher in BM than that of CB and MPB (BM 8.61%±1.40%, CB 3.31%±0.88%, MPB 5.11%±0.76%,P&lt;0.01). The relative frequencies of the naïve CD45RA+ CD45RO− phenotype among CD4+ and CD8high T cells were highest in CB, and it was higher in MPB than in BM grafts (BM 28.09%±4.52%, 41.86 %±3.31%; CB83.83%±12.24%, 86.69%±6.12%; MPB 43.58%±4.54%, 57.64%±4.77%, P&lt;0.01). Naïve T cells (CD45RA+ CD45RO−) were mobilized preferentially compared to memory T cells (CD45RA− CD45RO+)(P &lt;0.01); The relative frequencies of NKT (CD3+CD16+56+) among lymphocytes were lower in CB than that in BM and MPB (CB 0.77±0.19, BM4.15±1.10, MPB 4.13±0.84, P&lt;0.01). Conclusion: BM, CB and MPB allografts differ widely in cellular makeup of CD34+ cells and lymphocyte subsets, which are associated with the distinct characteristics after allogeneic HSCT from different allogeneic hematological sources.

Tigit, CD226 and PD-L1/PD-1 Are Highly Expressed By Marrow-Infiltrating T Cells in Patients with Multiple Myeloma

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2102-2102 ◽  
Author(s):  
Mahesh Yadav ◽  
Cherie Green ◽  
Connie Ma ◽  
Alberto Robert ◽  
Andrew Glibicky ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Bone Marrow ◽  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Nk Cells ◽  
Cd8 T Cells ◽  
Cd4 T Cells ◽  
Plasma Cells ◽  
Color Flow

Abstract Introduction:TIGIT (T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif [ITIM] domain) is an inhibitory immunoreceptor expressed by T and natural killer (NK) cells that is an important regulator of anti-tumor and anti-viral immunity. TIGIT shares its high-affinity ligand PVR (CD155) with the activating receptor CD226 (DNAM-1). We have recently shown that TIGIT blockade, together with PD-L1/PD-1 blockade, provides robust efficacy in syngeneic tumor and chronic viral infection models. Importantly, CD226 blockade abrogates the benefit of TIGIT blockade, suggesting additional benefit of TIGIT blockade through elaboration of CD226-mediated anti-tumor immunity, analogous to CTLA-4/CD28 regulation of T-cell immunity. Whether TIGIT and CD226 are expressed in patients with multiple myeloma (MM) and how TIGIT expression relates to PD-L1/PD-1 expression is unknown. Here we evaluate expression of TIGIT, CD226, PD-1 and PD-L1 in patients with MM to inform novel immunotherapy combinations. Methods:We performed multi-color flow cytometry (n = 25 patients), and multiplex qRT-PCR (n = 7) on bone marrow specimens from patients with MM to assess expression of TIGIT, CD226, PD-1, and PD-L1 on tumor and immune cells. Cells were stained with fluorescently conjugated monoclonal antibodies to label T cells (CD3, CD4, CD8), NK cells (CD56, CD3), plasma cells (CD38, CD45, CD319, CD56), inhibitory/activating receptors (PD-1, TIGIT, PD-L1, CD226), and an amine-reactive viability dye (7-AAD). Stained and fixed cells were analyzed by flow cytometry using BD FACSCanto™ and BD LSRFortessa™. Results:TIGIT, CD226 and PD-L1/PD-1 were detectable by flow cytometry in all patients with MM who were tested, with some overlapping and distinct expression patterns. TIGIT was commonly expressed by marrow-infiltrating CD8+ T cells (median, 65% of cells), CD4+ T cells (median, 12%) and NK cells. In contrast, CD226 was more commonly expressed by marrow-infiltrating CD4+ T cells (median, 74%) compared with CD8+ T cells (median, 38%). PD-1 was expressed by marrow-infiltrating CD8+ T cells (median 38%) and CD4+ T cells (median, 16%). TIGIT was co-expressed with PD-1 on CD8+ T cells (67%-97% TIGIT+ among PD-1+), although many PD-1-negative CD8+ T cells also expressed TIGIT (39%-78% of PD-1-negative). PD-L1 was also expressed by CD8+ (median, 23%) and CD4+ (median, 8%) T cells in addition to MM plasma cells (median, 95%), albeit with significantly lower intensity on T cells compared with plasma cells. The expression of TIGIT and PD-L1 mRNA was highly correlated (R2 = 0.80). Analysis of PVR expression will also be presented. Conclusions: TIGIT, CD226, PD-1, and PD-L1 were commonly expressed in MM bone marrow, but with different patterns. Among CD8+ T cells, the frequency of TIGIT+ T cells was almost twice that of PD-1+ T cells, whereas the majority of CD4+ T cells expressed CD226. TIGIT blockade may complement anti-PD-L1/PD-1 immunotherapy by activating distinct T-cell/NK-cell subsets with synergistic clinical benefit. These results provide new insight into the immune microenvironment of MM and rationale for targeting both the PD-L1/PD-1 interaction and TIGIT in MM. Disclosures Yadav: Genentech, Inc.: Employment. Green:Genentech, Inc.: Employment. Ma:Genentech, Inc.: Employment. Robert:Genentech, Inc.: Employment. Glibicky:Makro Technologies Inc.: Employment; Genentech, Inc.: Consultancy. Nakamura:Genentech, Inc.: Employment. Sumiyoshi:Genentech, Inc.: Employment. Meng:Genentech, Inc.: Employment, Equity Ownership. Chu:Genentech Inc.: Employment. Wu:Genentech: Employment. Byon:Genentech, Inc.: Employment. Woodard:Genentech, Inc.: Employment. Adamkewicz:Genentech, Inc.: Employment. Grogan:Genentech, Inc.: Employment. Venstrom:Roche-Genentech: Employment.

scholarly journals A Novel P-Selectin Glycoprotein Ligand-1 Monoclonal Antibody Recognizes an Epitope Within the Tyrosine Sulfate Motif of Human PSGL-1 and Blocks Recognition of Both P- and L-Selectin

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 154-164 ◽  
Author(s):  
Karen R. Snapp ◽  
Han Ding ◽  
Kristin Atkins ◽  
Roger Warnke ◽  
Francis W. Luscinskas ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
T Cells ◽  
B Cells ◽  
Nk Cells ◽  
Flow Cytometric Analysis ◽  
Lymphoid Cells ◽  
B Cell Differentiation ◽  
Tyrosine Sulfation ◽  
Color Flow ◽  
Cos Cells

Interactions between P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) mediate the earliest “rolling” of leukocytes on the lumenal surface of endothelial cells at sites of inflammation. Previously, PSGL-1 has been shown to be the primary mediator of interactions between neutrophils and P-selectin, but studies on the ability of PSGL-1 to mediate interactions between P-selectin and other subsets of leukocytes have yielded variable and conflicting results. A novel IgG monoclonal antibody (MoAb) to human PSGL-1 was generated, and the specificity of this MoAb was confirmed by both flow cytometric analysis and Western blotting of cells transfected with human PSGL-1. This newly developed MoAb, KPL1, inhibited interactions between P-selectin expressing COS cells and either HL60 cells, neutrophils, or lymphocytes. Furthermore, KPL1 completely inhibited interactions between P-selectin and either purified CD4 T cells or neutrophils in a flow assay under physiological conditions, but had no effect on interactions of T cells or neutrophils with E-selectin. In addition, KPL1 blocked interactions between lymphoid cells transfected with L-selectin and COS cells expressing PSGL-1. The KPL1 epitope was mapped to a site within a consensus tyrosine sulfation motif of PSGL-1, previously shown to be essential for interaction with P-selectin and now shown to be essential for interaction with L-selectin, and to be distinct from the epitope identified by the PL1 function blocking anti-PSGL-1 MoAb. Two-color flow cytometry of normal leukocytes showed that while natural killer (NK) cells (CD16+), monocytes, CD4 and CD8 T cells, and α/β and γ/δ T cells were uniformly positive for PSGL-1, B cells expressed low levels of the KPL1 epitope. This low level of KPL1 staining was also observed immunohistologically in germinal centers, which had no detectable KPL1 staining, whereas T-cell areas (interfollicular region) were positive for KPL1. Interestingly, plasma cells in situ and interleukin-6–dependent myeloma cell lines were KPL1+. Thus, PSGL-1 is expressed on essentially all blood neutrophils, NK cells, B cells, T cells, and monocytes. Variation in tyrosine sulfation during B-cell differentiation may affect the ability of B cells to interact with P- and L-selectin.

CD4+CD28+ T Lymphoctye Subsets Decreased in Healthy Donors after G-CSF Mobilization.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3922-3922
Author(s):  
Jianyu Weng ◽  
Xin Du ◽  
Jianjun Zhang ◽  
Suijin Wu ◽  
Zesheng Lu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Peripheral Blood ◽  
Allogeneic Transplantation ◽  
Peripheral Blood Stem Cells ◽  
Color Flow ◽  
Cd8 Cells ◽  
Healthy Donors ◽  
Blood Stem Cells ◽  
Safety Considerations

Abstract Objective Allogeneic transplantation of recombinant human granulocyte colony-stimulating factor (rhG-CSF) -mobilized peripheral blood stem cells (PBSCs) is now being increasingly performed, but safety considerations for hematologically normal PBSC donors have not been fully addressed. The effection of G-CSF on donors’ lymphocytes have not been defined clearly. Our study was to detect and analyze the phenotypical and functional properties of lymphocytes from allo-PBSC donors treated with recombinant human G-CSF by flow cytometry. Methods Thirty-four HLA-identical sibling donors (13male, 21female; median age, 35 years) were treated by subcutaneous injection with rhG-CSF at a dose of 5 μg/kg twice daily for 4–6 consecutive days to mobilize HSCs to the peripheral blood. Leukapheresis was performed using a continuous flow blood cell separator (COBE Spectra, Lakewood, CO) on 1 to 2 consecutive days beginning on day 4 of rhG-CSF administration. Donor blood samples, which were obtained before the first administration of G-CSF (pre-G), on day 4 of G-CSF administration (post-G), and one week after the last rhG-CSF were analyzed by 3-color flow cytometry. Monoclonal antibodies included: CD3, CD4, CD8, CD20, CD16, CD56, CD25, CD69, CD45RA, CD45RO, CD28, CD95. Results Absolute counts of lymphocytes, B cells, T cells, CD4+ and CD8+cells post-G were significantly elevated two more times than pre-G (P&lt;0.05), but these changes recovered when the last G-CSF administrated one week later. The percentage of CD3+, CD4+, CD8+ and the ratio of CD4 and CD8 T cells had no significantly difference between pre-G and post-G. The percentage of CD4+CD25+, CD4+CD45RO+ and CD4+CD28+ T cells were significantly decreased post-G (45.26±9.68% to 37.34±11.12%; 65.53±13.74% to 52.32±13.47%; 94.75±4.02% to 91.74±8.72%, P=0.00329, 0.0003 and 0.0947, respectively). We found that CD4+CD28+ T cells(91.77 ± 6.15, P=0.00218) were still lower than pre-G when stopped administrate G-CSF one week later, while others changes have recovered to the Pre-G level. Conclusion: The study shows that CD4+CD28+ T lymphoctye subsets were still lower than pre-G, although most of other change recover one week later after mobilization

scholarly journals OMIP‐064: A 27‐Color Flow Cytometry Panel to Detect and Characterize Human NK Cells and Other Innate Lymphoid Cell Subsets, MAIT Cells, and γδ T Cells

2020 ◽  
Vol 97 (10) ◽  
pp. 1019-1023 ◽  
Author(s):  
Nina Hertoghs ◽  
Katharine V. Schwedhelm ◽  
Kenneth D. Stuart ◽  
Margaret Juliana McElrath ◽  
Stephen C. De Rosa
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Nk Cells ◽  
Lymphoid Cell ◽  
Γδ T Cells ◽  
Innate Lymphoid Cell ◽  
Color Flow ◽  
Mait Cells

scholarly journals High Levels of CD38+ NK Cells Contribute to Immune Imbalance of Th1/Th2 and Th17/Treg in Rheumatoid Arthritis

2020 ◽  
Author(s):  
Hongxing Wang ◽  
Kehua Fang ◽  
Xiaotian Chang
Keyword(s):  
Rheumatoid Arthritis ◽  
Flow Cytometry ◽  
T Cells ◽  
Synovial Fluid ◽  
Nk Cells ◽  
Treg Cell ◽  
Cd4 T Cells ◽  
Th17 Cell ◽  
Ifn Γ ◽  
Cell Proportion

Abstract Background Increased CD38 expression and CD38+ cell proportion as well as their importance had been reported in rheumatoid arthritis (RA).Methods The proportion of lymphocyte subtypes in RA patients and rats with collagen-induced arthritis (CIA) was examined using flow cytometry. CD38+ NK cells, CD38+ NKT cells and CD4+ T cells as well as mononuclear cells (MNCs) depleted of CD38+ cells were isolated from RA synovial fluid using flow cytometry and cocultured in transwell apparatus.Results This study detected a significantly increased CD38+ NK cell proportion and a decreased CD38+ NKT cell proportion in RA peripheral blood and synovial fluid. The CD38+ NK/CD38+ NKT ratio was positively correlated with the disease activity. A similar result was observed in CIA rats. When CD38+ NK cells were cocultured with MNCs, the Treg cell proportion in MNCs and IL-10 level significantly decreased, and Th17 cell proportion and IFN-γ level increased. When the CD38+ NK cells were pretreated with monoclonal anti-CD38 antibody, Treg cell proportion and IL-10 level significantly increased, and the Th17 cell proportion and IFN-γ and IL-6 level decreased. When CD38+ NK cells were cocultured with CD4+ T cells, the Th1/Th2 and Th17/Treg ratios significantly increased, and mTOR signaling was activated in the cells. When the CD38+ NK cells were pretreated with the anti-CD38 antibody, the opposite result was obtained. Coculturing CD38+ NKT cells with MNCs or CD4+T cells showed opposite results. The anti-CD38 antibody also significantly increased TGF-β expression in the CD38+ NK cells.Conclusions Our results suggest that a high CD38+ NK and low CD38+ NKT proportion in RA elevates Th1/Th2 and Th17/Treg ratio to contribute to the pathogenesis.

CD19 Targeting of Lymphoid Malignancies by Novel Fc-Domain Engineered Monoclonal Antibody.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3725-3725
Author(s):  
Farrukh Awan ◽  
Rosa Lapalombella ◽  
Rossana Trotta ◽  
Jonathan P Butchar ◽  
Bo Yu ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Nk Cells ◽  
Nk Cell ◽  
Wild Type ◽  
Color Flow ◽  
Mechanistic Insight ◽  
Insight Into

Abstract Abstract 3725 Poster Board III-661 CD19 is a lineage-specific B-cell antigen, expressed at a high density on CLL cells, that contributes to B-cell receptor signaling but to date has not been effectively targeted with therapeutic monoclonal antibodies. XmAb5574 is a novel engineered anti-CD19 monoclonal antibody with a modified Fc-domain designed to enhance binding of FcγRIIIa that is predominately expressed on Natural Killer (NK)-cells. Utilizing freshly isolated chronic lymphocytic leukemia (CLL) patient B-cells we demonstrate that XmAb5574 lacks significant internalization seen with other anti-CD19 antibodies [maximum internalization for XmAb5574 was only 27.9% at 30-minutes (95%CI 14.5%, 41.4%)], thereby enhancing its ability to induce potent antibody-dependent cellular cytotoxicity (ADCC). Annexin V/PI flow cytometry analysis revealed that XmAb5574 mediates modest direct cytotoxicity not significantly different from Rituximab (0.6% increase, 95%CI -10.5%, 11.7%, p=0.91), and no complement mediated cytotoxicity (CDC) against primary CLL B-cells. Multi-color flow cytometry and monocyte derived macrophages (MDM) were used to assess XmAb5574 antibody dependent cellular phagocytosis (ADCP) against CLL cells and revealed no significant impact of the Fc-domain modification on MDM induced ADCP against CLL cells as compared to the wild type parental anti-CD19 antibody (12.37% vs. 10.51%, p=0.58). Interestingly, utilizing NK-cells and CLL cells isolated from normal donors and CLL patients, and employing autologous and allogeneic effector-target (E:T) conditions, XmAb5574 was found to mediate significantly higher ADCC when compared to the control humanized anti-CD19 non-engineered antibody (26.9% higher at E:T 25:1, p=0.0004 for allogeneic conditions, and 23.6% higher, p=0.004 for autologous conditions). ADCC mediated by XmAb5574 was also significantly higher as compared to Rituximab (33.5% higher at E:T 25:1, p<0.0001 for allogeneic conditions and 27.1% higher, p=0.004 for autologous conditions), a therapeutic antibody widely utilized in the treatment of CLL, hence confirming the functional in vitro efficacy and utility of the Fc-domain modification. By using inhibitor studies we further provide mechanistic insight into the XmAb5574–dependent ADCC mediated by NK-cells through a Granzyme B dependent mechanism. XmAb5574 also enhanced NK-cell activation as exhibited by an increased phosphorylation of Erk1/2 downstream of Fcγ receptor. The enhancement of subsequent cytolytic and secretory function was shown by the measurement CD107a up regulation on the surface of NK-cells (19.4% increase, p=0.005, as compared to wild type anti-CD19 antibody), and interferon-gamma release as measured by ELISA assays (6.4 times higher, p=0.007, as compared to wild type anti-CD19 antibody). Notably, enhanced NK-cell mediated ADCC observed with XmAb5574 against primary CLL B-cells could be augmented further by treatment with Lenalidomide (17.9% higher, p=0.04). These findings provide strong pre-clinical evidence for further clinical development of XmAb5574, both as monotherapy and in combination with Lenalidomide, for the therapy of CLL and related CD19+ B-cell malignancies. We also provide mechanistic insight into the utility and feasibility of Fc-domain engineering of specific antibodies, which will enhance their efficacy through an increased ability to recruit the innate immune system to more effectively control tumor progression. Disclosures: Desjarlais: Xencor: Employment.

scholarly journals A Novel P-Selectin Glycoprotein Ligand-1 Monoclonal Antibody Recognizes an Epitope Within the Tyrosine Sulfate Motif of Human PSGL-1 and Blocks Recognition of Both P- and L-Selectin

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 154-164 ◽  
Author(s):  
Karen R. Snapp ◽  
Han Ding ◽  
Kristin Atkins ◽  
Roger Warnke ◽  
Francis W. Luscinskas ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
T Cells ◽  
B Cells ◽  
Nk Cells ◽  
Flow Cytometric Analysis ◽  
Lymphoid Cells ◽  
B Cell Differentiation ◽  
Tyrosine Sulfation ◽  
Color Flow ◽  
Cos Cells

Abstract Interactions between P-selectin and P-selectin glycoprotein ligand-1 (PSGL-1) mediate the earliest “rolling” of leukocytes on the lumenal surface of endothelial cells at sites of inflammation. Previously, PSGL-1 has been shown to be the primary mediator of interactions between neutrophils and P-selectin, but studies on the ability of PSGL-1 to mediate interactions between P-selectin and other subsets of leukocytes have yielded variable and conflicting results. A novel IgG monoclonal antibody (MoAb) to human PSGL-1 was generated, and the specificity of this MoAb was confirmed by both flow cytometric analysis and Western blotting of cells transfected with human PSGL-1. This newly developed MoAb, KPL1, inhibited interactions between P-selectin expressing COS cells and either HL60 cells, neutrophils, or lymphocytes. Furthermore, KPL1 completely inhibited interactions between P-selectin and either purified CD4 T cells or neutrophils in a flow assay under physiological conditions, but had no effect on interactions of T cells or neutrophils with E-selectin. In addition, KPL1 blocked interactions between lymphoid cells transfected with L-selectin and COS cells expressing PSGL-1. The KPL1 epitope was mapped to a site within a consensus tyrosine sulfation motif of PSGL-1, previously shown to be essential for interaction with P-selectin and now shown to be essential for interaction with L-selectin, and to be distinct from the epitope identified by the PL1 function blocking anti-PSGL-1 MoAb. Two-color flow cytometry of normal leukocytes showed that while natural killer (NK) cells (CD16+), monocytes, CD4 and CD8 T cells, and α/β and γ/δ T cells were uniformly positive for PSGL-1, B cells expressed low levels of the KPL1 epitope. This low level of KPL1 staining was also observed immunohistologically in germinal centers, which had no detectable KPL1 staining, whereas T-cell areas (interfollicular region) were positive for KPL1. Interestingly, plasma cells in situ and interleukin-6–dependent myeloma cell lines were KPL1+. Thus, PSGL-1 is expressed on essentially all blood neutrophils, NK cells, B cells, T cells, and monocytes. Variation in tyrosine sulfation during B-cell differentiation may affect the ability of B cells to interact with P- and L-selectin.

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