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Zhong Wu Guo - One of the best experts on this subject based on the ideXlab platform.
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Chemical Synthesis of GPI Glycan-Peptide Conjugates by Traceless Staudinger Ligation.
Organic letters, 2017Co-Authors: Sanyong Zhu, Zhong Wu GuoAbstract:A new strategy has been developed for GPI glycan–peptide conjugate synthesis based upon a traceless Staudinger reaction between a peptide phosphinothioester and a GPI glycan azide. The strategy was first studied and optimized with simple peptides and GPI glycans, which offered excellent yields of the desired conjugates in both organic and aqueous solvents. It was then used to successfully synthesize an analogue of the human CD52 Antigen containing the whole CD52 peptide sequence and the conserved trimannose motif of all GPI anchors.
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Total synthesis of a glycosylphosphatidylinositol anchor of the human lymphocyte CD52 Antigen
Chemistry (Weinheim an der Bergstrasse Germany), 2011Co-Authors: Srinivas Burgula, Benjamin M. Swarts, Zhong Wu GuoAbstract:The first total synthesis of a glycosylphosphatidylinositol (GPI) anchor bearing a polyunsaturated arachidonoyl fatty acid is reported. This lipid is found in mammalian GPIs that do not undergo lipid remodeling, a process that has important implications in the localization and function of GPI-anchored proteins. Incorporation of the oxidation- and reduction-sensitive arachidonoyl lipid in the target GPI was accomplished by using the para-methoxybenzyl (PMB) group for permanent hydroxyl group protection, which featured a selective, rapid, and efficient global deprotection protocol. The flexibility of this synthetic strategy was further highlighted by the inclusion of two additional GPI core structural modifications present in the GPI anchor of the human lymphocyte CD52 Antigen.
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Synthesis and CD structural studies of CD52 peptides and glycopeptides
Carbohydrate research, 2008Co-Authors: Benjamin M. Swarts, Yu Cheng Chang, Zhong Wu GuoAbstract:Abstract The syntheses of five natural and N-terminal acetylated peptides and glycopeptides of the CD52 Antigen are described. Solid phase peptide synthesis was employed in the construction of the target compounds from Fmoc-protected commercial amino acids and synthetic glycan–asparagine conjugates. Circular dichroism studies of the synthetic targets showed that they exist as random coils in solution, and no significant change in secondary structure was observed when the CD52 peptide was either acetylated at the N-terminus or glycosylated at the Asn 3 residue with a disaccharide or a fucose-containing branched trisaccharide.
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Convergent synthesis of a fully phosphorylated GPI anchor of the CD52 Antigen.
Organic letters, 2007Co-Authors: Zhong Wu GuoAbstract:A fully phosphorylated GPI anchor (1) of the CD52 Antigen was synthesized by a highly convergent strategy. After a trimannose and a phospholipidated pseudodisaccharide were prepared separately, they were coupled together to form the GPI core, which was then phosphorylated to introduce two phosphoethanolamine moieties in one step to afford CD52 GPI in its fully protected form. Finally, global deprotection of the product resulted in 1.
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First total synthesis of a GPI-anchored peptide.
The Journal of organic chemistry, 2003Co-Authors: Jie Xue, Ning Shao, Zhong Wu GuoAbstract:A GPI-anchored dipeptide of sperm CD52 Antigen was prepared through a convergent synthesis. First, the dipeptide with its C-terminus free and the GPI with its nonreducing end phosphoethanolamine bearing a free amino group were synthesized separately. Then, the two building blocks were coupled with use of EDC/HOBt as the condensation reagent. Finally, the GPI-anchored peptide was deprotected to give the target molecule 1.
Geoff Hale - One of the best experts on this subject based on the ideXlab platform.
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CD52 Antigen as a target for immunotherapy
Transplantation Reviews, 2003Co-Authors: Geoff HaleAbstract:T he Antigenic epitope CD52 is expressed on the surface membranes of peripheral blood lymphocytes, monocytes, and macrophages, as well as the epithelial lining of the male reproductive system. The structural features of the CD52 Antigen, specifically its small size, lateral mobility, and closeness to the cell membrane, as well as the sheer abundance of the Antigen (CD52 is expressed on 5% of the lymphocyte surface), make it an ideal target for antibody-mediated killing1 (Fig 1). Alemtuzumab (CAMPATH 1H; ILEXTM Oncology, Inc [ILEX], San Antonio, TX), a humanized monoclonal antibody against the CD52 epitope, has a relatively low affinity and requires a concentration of approximately 50 g/mL to achieve maximum binding2 (Fig 2). Nevertheless, it is an extremely potent agent for depletion of lymphocytes in vivo, and a short course of treatment with as little as 40 mg (total) gives rapid and long-lasting depletion of lymphocytes, particularly CD4 cells, in transplant patients.3,4 The potent activity of alemtuzumab appears to be primarily initiated through cell-mediated killing (antibody-dependent cell-mediated cytotoxicity), which is maximal at concentrations as low as 10 ng/mL, whereas optimum activation of complement-mediated lysis requires concentrations of 10 g/mL5 (Fig 3). In patients with chronic lymphocytic leukemia, the indication for which alemtuzumab is approved, both the route of administration and the tumor burden influence the time necessary to achieve peak serum levels. The time to reach peak serum antibody levels is shorter with intravenous administration than with subcutaneous administration, and the blood levels reached after intravenous administration are higher in patients who have a good clinical response. In contrast, serum levels among bone marrow transplant recipients were more consistent among patients, presumably because they did not have a substantial burden of CD52 tumor cells.4 Pharmacokinetic analysis of serum from bone marrow transplant recipients shows that the half-life of alemtuzumab is in the range of 1 to 3 weeks. Therapeutic antibodies have long been used to mediate immunosuppression and prevent acute rejection in transplant recipients. An issue with antibody therapy, however, is the development of antiantibodies that can limit efficacy. Antithymocyte globulin is currently the most commonly used T-cell– depleting agent for induction therapy in kidney transplant recipients. Although antithymocyte globulin has been associated with high graft survival rates ( 98%) and low infection rates ( 10%), the potential for development of xenogeneic antibodies is high ( 78%). Muromonab anti-CD3 (OKT3) is less immunogenic; however, human antimouse antibody development is possible because of its murine origin. Because alemtuzumab is a humanized From the Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom. Address reprint requests to Geoff Hale, PhD, Therapeutic Antibody Centre, Old Road, Headington, Oxford OX3 7JT, United Kingdom. © 2003 Elsevier Inc. All rights reserved. 0955-470X/03/1704-0000$30.00/0 doi:10.1016/S0955-470X(03)00073-9 Figure 1. Structure of the CD52 Antigen showing the closeness of the Campath epitope to the lipid anchor. (Modified and reprinted with permission from Hale G. The CD52 Antigen and development of the CAMPATH antibodies. Cytotherapy 2001; 3:137. Published by Taylor & Francis.)
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Alemtuzumab in stem cell transplantation
Medical Oncology, 2002Co-Authors: Geoff HaleAbstract:Alemtuzumab, otherwise called CAMPATH-1H, is a humanized monoclonal antibody directed against the CD52 Antigen of lymphocytes. It is one of a family of CAMPATH-1 antibodies that have been used over the last 20 yr in stem cell transplantation for depletion of donor and recipient T-cells to prevent graft-versus-host disease and graft rejection. Clinical trials have been carried out by a cooperative group of physicians and the protocols have gradually evolved as different problems have been identified and overcome. T-cell depletion carries risks of reducing graft-versus-tumor effects and increasing susceptibility to virus reactivation, but offers significant benefits in terms of reduced mortality and morbidity from graft-versus-host disease (GVHD). Two protocols are currently favored: (1) simple addition of CAMPATH-1H to the stem cell infusion and (2) administration in vivo prior to the transplant—especially in the context of nonmyeloablative conditioning regimens. Both of these give excellent control of GVHD with minimal graft rejection. Contrary to earlier expectations, a short course of posttransplant cyclosporin A (CyA) appears to further reduce transplant-related mortality, mainly by a reduction in late deaths from infection. These results need to be consolidated by means of prospective clinical trials.
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Pharmacokinetics of CAMPATH-1H: assay development and validation
Journal of Immunological Methods, 2001Co-Authors: Peppy Rebello, Geoff HaleAbstract:CAMPATH-1H is a humanised monoclonal antibody against the CD52 Antigen which is being developed for treatment of chronic lymphocytic leukaemia (CLL), autoimmune disease and prevention of transplant rejection. Measurement of antibody serum levels is important for optimising dose regimens but difficult owing to the low concentration compared with normal human IgG. After consideration of various methods, a suitable assay was developed based on indirect immunofluorescence. Test samples were incubated with target cells (HUT-78, a human T cell line) and the CAMPATH-1H was detected by binding of a fluorescent-labelled anti-human Ig using a flow cytometer. Robustness of the assay was demonstrated under a range of experimental conditions. Because of the low affinity of CAMPATH-1H, only a weak signal was seen at low concentrations. The limit of detection was 0.15 microg/ml and the limit of quantitation was 0.25 microg/ml. Since serum samples were diluted at least 1:2, the lowest concentration which can be measured in patient serum was 0.5 microg/ml. The overall precision (coefficient of variation) was +/-13% and the overall accuracy (bias) was +9%. There was a low incidence of false-positive results (
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The CD52 Antigen and development of the CAMPATH antibodies.
Cytotherapy, 2001Co-Authors: Geoff HaleAbstract:Correspondence to: Geoff Hale, Therapeutic Antibody Centre, Old Road, Headington, Oxford OX3 7JT, UK. Introduction The first CD52 Abs were isolated in 1980 during a search for Abs that could kill lymphocytes by activating human complement [1]. This was motivated by the thought that they should be ideal for removal of T cells, which were believed to cause GvHD. Other T-cell specific MAbs, such as OKT3, had already been suggested for this purpose, but they did not activate human effector systems (such as complement) to the extent necessary to destroy sufficient cells. These Abs could kill cells with rabbit serum, because natural inhibitors such as decay accelerating factor and CD59 do not protect against heterologous complement. Nevertheless, it was perceived that this would introduce an undesirable source of variability and expense. Few MAbs are efficient activators of lysis by autologous complement, but several were isolated from a fusion to create rat hybridomas making antibodies against human lymphocytes. All of them were directed against the same Antigen, which is now defined as CD52.
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Cross-linking of the CAMPATH-1 Antigen (CD52) triggers activation of normal human T lymphocytes.
International immunology, 1995Co-Authors: Wendy C. Rowan, Geoff Hale, John Tite, S BrettAbstract:The CAMPATH-1 (CD52) Antigen is a 21-28 kDa glycopeptide which is highly expressed on lymphocytes and macrophages and is coupled to the membrane by a glycosylphosphatidylinositol (GPI) anchoring structure. The function of this molecule is unknown. However, it is an extremely good target for complement-mediated attack and antibody-mediated cellular cytotoxicity. The humanized CAMPATH-1H antibody, which is directed against CD52, is very efficient at mediating lymphocyte depletion in vivo, and is currently being used in clinical trials for lymphoid malignancy and rheumatoid arthritis. It is therefore important to examine the functional effects of this antibody on different lymphocyte sub-populations. Because several other GPI-linked molecules expressed on the surface of T lymphocytes are capable of signal transduction resulting in cell proliferation, we have investigated whether the CAMPATH-1 Antigen can also mediate these effects. In the presence of phorbol esters and cross-linking anti-Ig antibodies, mAbs specific for CD52 induced proliferation and lymphokine production in highly purified resting CD4+ and CD8+ T lymphocytes. The rat IgG2c YTH 361.10 anti-CD52 antibody, however, was able to activate resting CD4+ and CD8+ T cells directly without cross-linking or phorbol myristate acetate in the absence of Fc-bearing cells. Anti-CD52 antibodies also augmented the anti-CD3 mediated proliferative response of CD4+ and CD8+ T cells when the two antibodies were co-immobilized onto the same surface or cross-linked in solution by the same second antibody. Both CD4+ CD45RA and CD4+ CD45RO T cells were stimulated to proliferate by anti-CD52 antibodies in the presence of appropriate co-stimulatory factors. Anti-CD52 mAbs did not, however, synergize with anti-CD2 or CD28 mAb to induce CD4+ T cell proliferation. The activation of CD4+ T cells by anti-CD52 antibodies was inhibited by cyclosporin A, suggesting a role for the calcineurin-dependent signal transduction pathways. Although CD52 could transduce a signal in T cells, anti-CD52 antibodies did not inhibit Antigen-specific or polyclonal T cell responses, suggesting this molecule does not play an essential co-stimulatory role in normal T cell activation.
S Brett - One of the best experts on this subject based on the ideXlab platform.
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ANTIBODY SELECTION AGAINST CD52 PRODUCES A PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA PHENOTYPE IN HUMAN LYMPHOCYTES BY A NOVEL MECHANISM
Biochemical Journal, 1997Co-Authors: Vanessa C. Taylor, S Brett, Martin J. Sims, Mark C. FieldAbstract:The CD52 Antigen is a lymphocyte glycoprotein with an extremely short polypeptide backbone and a single N-linked glycan, and it is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. Treatment of rheumatoid arthritis patients with CAMPATH-1H, a humanized monoclonal antibody against CD52, resulted, in a small number of cases, in the appearance and persistence of CD52-negative T cells. Similarly, CD52-negative B cells emerged following in vitro treatment of a CD52-positive human B cell line with CAMPATH-1H. Both the B and T CD52-negative cells were also found to be defective in surface expression of other GPI-anchored proteins. Biochemical analysis revealed a severe defect in the synthesis of a mature GPI precursor in both the B and T cell lines. Therefore the phenotype of these CD52-negative B and T cells closely resembles that of lymphocytes from patients with paroxysmal nocturnal haemoglobinuria (PNH), in which the first step of the GPI-biosynthetic pathway, i.e. synthesis of GlcNAc-phosphatidylinositol, is blocked. In all cases studied to date, this defect maps to a mutation of the phosphatidylinositolglycan class A (PIG-A) structural gene. We therefore amplified the PIG-A gene from both the GPI-negative B and T cells by PCR and determined the nucleotide sequence. No differences from the wild-type sequence were detected; therefore a classical PNH mutation cannot be responsible for the GPI-biosynthesis defect in these cell lines. Significantly, the GPI-negative phenotype of the B cells was reversible upon separation of the positive and negative cells, resulting in a redistribution to a mixed population with either CD52-positive or -negative cells, whereas populations of 100% CD52-negative T cells were stably maintained during culture. Therefore, whereas the GPI-biosynthesis deficiency in the T cell lines may be due to a mutation in another gene required by the GPI-biosynthetic pathway, the reversible nature of this block in the B cell lines suggests a less direct cause, possibly an alteration in a regulatory factor. Overall, these data demonstrate that the PNH phenotype can be generated without a mutation in the PIG-A structural gene, and thereby identify a novel mechanism for the development of GPI deficiency.
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repopulation of blood lymphocyte sub populations in rheumatoid arthritis patients treated with the depleting humanized monoclonal antibody campath 1h
Immunology, 1996Co-Authors: S Brett, G Baxter, H Cooper, J M Johnston, J Tite, N RapsonAbstract:Patients with severe rheumatoid arthritis who had failed treatment with conventional therapies were treated with a course of five or 10 daily intravenous infusions of CAMPATH-1H, a humanized antibody against the CD52 Antigen, resulting in profound depletion of peripheral blood mononuclear cells. During the subsequent 18 months, lymphocytes were analysed for sub-populations by fluorescence-activated cell sorter (FACS) and for proliferation in response to polyclonal T-cell stimulation with anti-CD3 or staphylococcal enterotoxin B (SEB). Treatment resulted in almost complete depletion of lymphocytes from the blood followed by gradual repopulation. CD16+ natural killer (NK) cells and CD14+ monocytes returned to pretreatment levels within 1-2 months. CD19+ B cells returned to within 50% of pre-treatment levels by day 66 and to within normal range by day 150, whereas CD8+ T cells recovered to 50% of pretreatment levels by day 66, but did not show any further increase during the rest of the study period. The most profound effects were on the CD4+ T lymphocyte sub-population, as the mean CD4+ count did not increase above 20% of pre-treatment level at any time during the study period (550 days), at all the doses tested. The T cells which initially repopulated the blood 1-2 months after treatment, nearly all expressed the activation markers human leucocyte Antigen (HLA)-DR and CD45RO, although the percentage of T cells expressing these molecules gradually declined to normal levels over time. Proliferative responses to polyclonal T-cell stimulation (anti-CD3 and SEB) were also significantly reduced in the first few months after treatment, but recovered to pre-treatment levels by day 250. The relationship between these observations and the clinical response is discussed.
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Repopulation of blood lymphocyte sub‐populations in rheumatoid arthritis patients treated with the depleting humanized monoclonal antibody, CAMPATH‐1H
Immunology, 1996Co-Authors: S Brett, G Baxter, H Cooper, J M Johnston, J Tite, N RapsonAbstract:Patients with severe rheumatoid arthritis who had failed treatment with conventional therapies were treated with a course of five or 10 daily intravenous infusions of CAMPATH-1H, a humanized antibody against the CD52 Antigen, resulting in profound depletion of peripheral blood mononuclear cells. During the subsequent 18 months, lymphocytes were analysed for sub-populations by fluorescence-activated cell sorter (FACS) and for proliferation in response to polyclonal T-cell stimulation with anti-CD3 or staphylococcal enterotoxin B (SEB). Treatment resulted in almost complete depletion of lymphocytes from the blood followed by gradual repopulation. CD16+ natural killer (NK) cells and CD14+ monocytes returned to pretreatment levels within 1-2 months. CD19+ B cells returned to within 50% of pre-treatment levels by day 66 and to within normal range by day 150, whereas CD8+ T cells recovered to 50% of pretreatment levels by day 66, but did not show any further increase during the rest of the study period. The most profound effects were on the CD4+ T lymphocyte sub-population, as the mean CD4+ count did not increase above 20% of pre-treatment level at any time during the study period (550 days), at all the doses tested. The T cells which initially repopulated the blood 1-2 months after treatment, nearly all expressed the activation markers human leucocyte Antigen (HLA)-DR and CD45RO, although the percentage of T cells expressing these molecules gradually declined to normal levels over time. Proliferative responses to polyclonal T-cell stimulation (anti-CD3 and SEB) were also significantly reduced in the first few months after treatment, but recovered to pre-treatment levels by day 250. The relationship between these observations and the clinical response is discussed.
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Cross-linking of the CAMPATH-1 Antigen (CD52) triggers activation of normal human T lymphocytes.
International immunology, 1995Co-Authors: Wendy C. Rowan, Geoff Hale, John Tite, S BrettAbstract:The CAMPATH-1 (CD52) Antigen is a 21-28 kDa glycopeptide which is highly expressed on lymphocytes and macrophages and is coupled to the membrane by a glycosylphosphatidylinositol (GPI) anchoring structure. The function of this molecule is unknown. However, it is an extremely good target for complement-mediated attack and antibody-mediated cellular cytotoxicity. The humanized CAMPATH-1H antibody, which is directed against CD52, is very efficient at mediating lymphocyte depletion in vivo, and is currently being used in clinical trials for lymphoid malignancy and rheumatoid arthritis. It is therefore important to examine the functional effects of this antibody on different lymphocyte sub-populations. Because several other GPI-linked molecules expressed on the surface of T lymphocytes are capable of signal transduction resulting in cell proliferation, we have investigated whether the CAMPATH-1 Antigen can also mediate these effects. In the presence of phorbol esters and cross-linking anti-Ig antibodies, mAbs specific for CD52 induced proliferation and lymphokine production in highly purified resting CD4+ and CD8+ T lymphocytes. The rat IgG2c YTH 361.10 anti-CD52 antibody, however, was able to activate resting CD4+ and CD8+ T cells directly without cross-linking or phorbol myristate acetate in the absence of Fc-bearing cells. Anti-CD52 antibodies also augmented the anti-CD3 mediated proliferative response of CD4+ and CD8+ T cells when the two antibodies were co-immobilized onto the same surface or cross-linked in solution by the same second antibody. Both CD4+ CD45RA and CD4+ CD45RO T cells were stimulated to proliferate by anti-CD52 antibodies in the presence of appropriate co-stimulatory factors. Anti-CD52 mAbs did not, however, synergize with anti-CD2 or CD28 mAb to induce CD4+ T cell proliferation. The activation of CD4+ T cells by anti-CD52 antibodies was inhibited by cyclosporin A, suggesting a role for the calcineurin-dependent signal transduction pathways. Although CD52 could transduce a signal in T cells, anti-CD52 antibodies did not inhibit Antigen-specific or polyclonal T cell responses, suggesting this molecule does not play an essential co-stimulatory role in normal T cell activation.
N Rapson - One of the best experts on this subject based on the ideXlab platform.
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repopulation of blood lymphocyte sub populations in rheumatoid arthritis patients treated with the depleting humanized monoclonal antibody campath 1h
Immunology, 1996Co-Authors: S Brett, G Baxter, H Cooper, J M Johnston, J Tite, N RapsonAbstract:Patients with severe rheumatoid arthritis who had failed treatment with conventional therapies were treated with a course of five or 10 daily intravenous infusions of CAMPATH-1H, a humanized antibody against the CD52 Antigen, resulting in profound depletion of peripheral blood mononuclear cells. During the subsequent 18 months, lymphocytes were analysed for sub-populations by fluorescence-activated cell sorter (FACS) and for proliferation in response to polyclonal T-cell stimulation with anti-CD3 or staphylococcal enterotoxin B (SEB). Treatment resulted in almost complete depletion of lymphocytes from the blood followed by gradual repopulation. CD16+ natural killer (NK) cells and CD14+ monocytes returned to pretreatment levels within 1-2 months. CD19+ B cells returned to within 50% of pre-treatment levels by day 66 and to within normal range by day 150, whereas CD8+ T cells recovered to 50% of pretreatment levels by day 66, but did not show any further increase during the rest of the study period. The most profound effects were on the CD4+ T lymphocyte sub-population, as the mean CD4+ count did not increase above 20% of pre-treatment level at any time during the study period (550 days), at all the doses tested. The T cells which initially repopulated the blood 1-2 months after treatment, nearly all expressed the activation markers human leucocyte Antigen (HLA)-DR and CD45RO, although the percentage of T cells expressing these molecules gradually declined to normal levels over time. Proliferative responses to polyclonal T-cell stimulation (anti-CD3 and SEB) were also significantly reduced in the first few months after treatment, but recovered to pre-treatment levels by day 250. The relationship between these observations and the clinical response is discussed.
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Repopulation of blood lymphocyte sub‐populations in rheumatoid arthritis patients treated with the depleting humanized monoclonal antibody, CAMPATH‐1H
Immunology, 1996Co-Authors: S Brett, G Baxter, H Cooper, J M Johnston, J Tite, N RapsonAbstract:Patients with severe rheumatoid arthritis who had failed treatment with conventional therapies were treated with a course of five or 10 daily intravenous infusions of CAMPATH-1H, a humanized antibody against the CD52 Antigen, resulting in profound depletion of peripheral blood mononuclear cells. During the subsequent 18 months, lymphocytes were analysed for sub-populations by fluorescence-activated cell sorter (FACS) and for proliferation in response to polyclonal T-cell stimulation with anti-CD3 or staphylococcal enterotoxin B (SEB). Treatment resulted in almost complete depletion of lymphocytes from the blood followed by gradual repopulation. CD16+ natural killer (NK) cells and CD14+ monocytes returned to pretreatment levels within 1-2 months. CD19+ B cells returned to within 50% of pre-treatment levels by day 66 and to within normal range by day 150, whereas CD8+ T cells recovered to 50% of pretreatment levels by day 66, but did not show any further increase during the rest of the study period. The most profound effects were on the CD4+ T lymphocyte sub-population, as the mean CD4+ count did not increase above 20% of pre-treatment level at any time during the study period (550 days), at all the doses tested. The T cells which initially repopulated the blood 1-2 months after treatment, nearly all expressed the activation markers human leucocyte Antigen (HLA)-DR and CD45RO, although the percentage of T cells expressing these molecules gradually declined to normal levels over time. Proliferative responses to polyclonal T-cell stimulation (anti-CD3 and SEB) were also significantly reduced in the first few months after treatment, but recovered to pre-treatment levels by day 250. The relationship between these observations and the clinical response is discussed.
Geoffrey Hale - One of the best experts on this subject based on the ideXlab platform.
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1 9 a structure of the therapeutic antibody campath 1h fab in complex with a synthetic peptide Antigen
Journal of Molecular Biology, 1999Co-Authors: Leo C James, Geoffrey Hale, Herman Waldmann, A C BloomerAbstract:CAMPATH-1 antibodies have a long and successful history in the treatment of leukaemia, autoimmune disease and transplant rejection. The first antibody to undergo "humanisation", CAMPATH-1H, permits treatment with limited patient anti-globulin response. It recognises the CD52 Antigen which is a small glycosylphosphatidylinositol(GPI)-anchored protein expressed on lymphocytes and mediates cell depletion. We present the 1.9 A structure of the CAMPATH-1H Fab complexed [corrected] with an analogue of the Antigenic determinant of CD52. Analysis of the CAMPATH-1H binding site reveals that in contrast to most antibodies CDR L3 plays a dominant role in Antigen binding. Furthermore CDR H3, which is essential for effective Antigen recognition in most antibodies, participates in only two main-chain interactions in CAMPATH-1H. The CAMPATH-1H binding site is highly basic; ionic interaction with the enthanolamine phosphate of the CD52 GPI anchor has long been hypothesised to be important in Antigen binding. The structure reveals a number of important specific ionic interactions, including Lys53H but not Lys52bH as had previously been suggested. Prolonged treatment with CAMPATH-1H can lead to patient anti-idiotype responses which may be exacerbated by the unusually high number of basic residues in the antibody. This suggests that a strategy where redundant basic residues are replaced with neutral counterparts may be effective in further reducing the immunogenicity of this versatile and widely used antibody.
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transient increase in symptoms associated with cytokine release in patients with multiple sclerosis
Brain, 1996Co-Authors: Thibault Moreau, Geoffrey Hale, Herman Waldmann, Alasdair Coles, M G Wing, John D Isaacs, Alastair CompstonAbstract:Summary Fourteen patients with multiple sclerosis were treated with the humanized monoclonal antibody CAMPATH-IH which targets the CD52 Antigen present on all lymphocytes and some monocytes; four also received anti-CD4 antibody. Lymphopaenia developed rapidly and was sustained for at least 1 year. In 12 patients, the first infusion of antibody was characterized by significant exacerbation or re-awakening of pre-existing symptoms lasting several hours. These clinical effects of antibody treatment correlated with increased levels of circulating cytokines. Peak levels of tumour necrosis factor (TNF)-α and interferon (IFN)-γ occurred at 2 h, whereas the rise in interleukin-6 (IL-6) was significantly delayed and peaked at 4 h after starting antibody treatment. There was a decline in CH50, indicating complement activation. The neurological symptoms could not be attributed directly to pyrexia and were not provoked (in one patient) by an artificial rise in temperature. In the remaining two patients, a single pre-treatment with intravenous methylprednisolone (500 mg) prevented both the transient increase in neurological symptoms and the cytokine release. Our results, involving 14 intensively studied patients treated with humanized monoclonal antibodies, suggest that soluble immune mediators contribute to symptom production in multiple sclerosis; the mechanism remains uncertain but, on the available evidence, we favour the interpretation that cytokines directly affect conduction through partially demyelinated pathways.
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synthetic peptide mimotope of the campath 1 CD52 Antigen a small glycosylphosphatidylinositol anchored glycoprotein
Immunotechnology, 1995Co-Authors: Geoffrey HaleAbstract:Abstract Background: CAMPATH-1 (CD52) antibodies are among the most powerful and specific lympholytic agents in humans and have numerous potential applications for human therapy. The CD52 Antigen is a GPI-anchored glycoprotein with an exceptionally short peptide sequence of only 12 amino acids and a single, complex, N-linked oligosaccharide. Antibodies bind to the deglycosylated Antigen and to a proteolytic fragment, but not to the synthetic peptide alone. Objectives: To characterise the Antigenic epitope more precisely and to construct a synthetic analogue. Such an analogue would be useful for assay and purification of the therapeutic CAMPATH-1 antibodies as well as for studies of the antibody-Antigen binding site. Study Design: Collections of synthetic peptides based on the natural sequence were screened with a panel of CD52 antibodies. Results and Conclusion: A synthetic peptide composed of the natural C-terminal amino acids plus two additional residues was found to mimic the Antigen with sufficient affinity to be useful for a variety of assays and for construction of an affinity matrix for antibody purification. Systematic mutation of this peptide enabled the definition of the critical residues for antibody binding, which will be of great help in building a model of the antibody-Antigen interaction. Peptide mimotopes synthesised using a natural sequence as a starting point, rather than a completely random library, may be useful in many other similar circumstances.