Journal articles on the topic 'Rec mAb'
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1
Jacques, Y., B. Le Mauff, A. Godard, D. Olive, J. F. Moreau, and J. P. Soulillou. "Regulation of interleukin 2 receptor expression on a human cytotoxic T lymphocyte clone, synergism between alloantigenic stimulation and interleukin 2." Journal of Immunology 136, no. 5 (March 1, 1986): 1693–99. http://dx.doi.org/10.4049/jimmunol.136.5.1693.
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Abstract A human T cell clone (termed 40.2.6) established from a rejected human kidney allograft has been studied for its ability to express membrane IL 2 receptors in response to antigen (irradiated cells from the graft's donor) and recombinant IL 2 (rec-IL 2). On antigenic stimulation, the 40.2.6 clone produced low levels (0.15 U/ml) of IL 2 (peak at 24 hr) and incorporated (3H)thymidine (peak at 48 hr). This incorporation was strongly enhanced on addition of rec-IL 2 and was inhibited by the 33B31 antibody, an anti-human IL 2 receptor monoclonal antibody (Mab). The 125I-labeled 33B31 Mab has been used to quantify the density of IL 2 receptors on 40.2.6 cells. Cells not re-exposed to antigen or rec-IL 2 had a level of 33B31-binding sites which declined rapidly (10% of starting value after 2 days). This level remained much more stable when rec-IL 2 (1 U/ml) was present in the medium (80% at day 2). Antigen induced a three- to eightfold increase in the level of 33B31-binding sites which peaked at 24 hr and then declined. When a similar antigenic stimulation was performed in the presence of rec-IL 2 (1 U/ml), the level of 33B31-binding sites peaked at a higher value (eight- to 20-fold increase at day 2), and its subsequent decline was slower. These potentiating effects of rec-IL 2 were dose-dependent and occurred at low concentrations corresponding to the saturation by rec-IL 2 of high affinity IL 2 receptor sites. Finally, high affinity IL 2 receptors, as measured by the binding of 35S-labeled rec-IL 2, were found to be similarly up-regulated by antigen and rec-IL 2. Together, our results obtained on a monoclonal human T cell population with highly purified rec-IL 2 demonstrate that rec-IL 2 and antigen act in synergy to induce the expression of both high and low affinity membrane IL 2 receptors.
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Schweigel, Monika, Hi-Sung Park, Benjamin Etschmann, and Holger Martens. "Characterization of the Na+-dependent Mg2+ transport in sheep ruminal epithelial cells." American Journal of Physiology-Gastrointestinal and Liver Physiology 290, no. 1 (January 2006): G56—G65. http://dx.doi.org/10.1152/ajpgi.00014.2005.
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This study examines the routes by which Mg2+ leaves cultured ovine ruminal epithelial cells (REC). Mg2+-loaded (6 mM) REC were incubated in completely Mg2+-free solutions with varying Na+ concentrations, and the Mg2+ extrusion rate was calculated from the increase of the Mg2+ concentration in the incubation medium determined with the aid of the fluorescent probe mag-fura 2 (Na+ salt). In other experiments, REC were also studied for the intracellular free Mg2+ concentration ([Mg2+]i; using mag-fura 2), the intracellular Na+ concentration (using Na+-binding benzofuran isophthalate), the intracellular cAMP concentration ([cAMP]i; using an enzyme-linked immunoassay), and Na+/Mg2+ exchanger existence [using a monoclonal antibody (mAb) raised against the porcine red blood cell Na+/Mg2+ exchanger]. Mg2+-loaded REC show a Mg2+ efflux that was strictly dependent on extracellular Na+. The Mg2+ extrusion rate increased from 0.018 ± 0.009 in a Na+-free medium to 0.73 ± 0.3 mM·l cells−1·min−1 in a 145 mM Na+ medium and relates to extracellular Na+ concentration ([Na+]e) according to a typical saturation kinetic ( Km value for [Na+]e = 24 mM; maximal velocity = 11 mM·l cells−1·min−1). Mg2+ efflux was reduced by imipramine (48%) and increased after application of dibutyryl-cAMP (55%) or PGE2 (17%). These effects are completely abolished in Na+-free media. Furthermore, an elevation of [cAMP]i led to an [Mg2+]i decrease that amounted to 375 ± 105 μM. The anti-Na+/Mg2+ exchanger mAb inhibits Mg2+ extrusion; moreover, it detects a specific 70-kDa immunoreactive band in protein lysates of ovine REC. The data clearly demonstrate that a Na+/Mg2+ exchanger is existent in the cell membrane of REC. The transport protein is the main pathway (97%) for Mg2+ extrusion and can be assumed to play a considerable role in the process of Mg2+ absorption as well as the maintenance of the cellular Mg2+ homeodynamics.
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Bessos, Hagop, Pamela Brown, Gholamreza Anani Sarab, Shirley Gibson, Michael Moss, Robert N. Barker, and Stanislaw J. Urbaniak. "Purification of Anti-HPA-1a Directly from Plasma Using Immobilised Recombinant HPA-1a Enables Determination of Antibody Affinity by Surface Plasmon Resonance Technology in Neonatal Alloimmune Thrombocytopenia." Blood 112, no. 11 (November 16, 2008): 3402. http://dx.doi.org/10.1182/blood.v112.11.3402.3402.
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Abstract Background: Most severe cases of neonatal alloimmune thrombocytopenia (NAIT) in the Caucasian population are due to anti-HPA-1a. The precise influences of antibody amount or affinity on severity of NAIT remain to be elucidated. In an earlier study, we assessed the interaction between anti-HPA-1a and HPA-1a antigen by surface plasmon resonance technology (SPRT) using IgG fractions recovered from antibody (ab) positive HPA-1b maternal sera using protein-A chromatography. However, high background in some of the IgG fractions prohibited the determination of antibody affinity (Bessos et al, Trans Apher Sci, In Press). The aim of this study was to investigate the affinity of anti-HPA-1a purified directly from plasma using immobilised HPA-1a antigen. Methods: Native glycoprotein (GP) IIb/IIIa was extracted and purified from HPA-1a1a platelet donors, while recombinant PSI domain of HPA-1a GPIIIa (rec-HPA-1a) was obtained by conventional cloning techniques. CNBr activated Sepharose 4B was coupled either with native GPIIb/IIIa or with rec-HPA-1a and used for the immunopurification of anti-HPA-1a directly from 4 female plasma samples positive for anti-HPA-1a ab. The plasma samples were pre-adsorbed with non-coupled Sepharose 4B prior to mixing with immobilised HPA-1a antigen. The acid eluted abs were dialysed, concentrated and assessed by ELISA (using HPA-1a GPIIb/IIIa coated plates), silver staining (using reduced gels) and SPRT-Biacore X, using CM5 sensor chips bound with HPA-1a1a or HPA-1b1b antigen at 600–800 response units (RU). GPIIIa AP3 monoclonal ab (Mab) & CamTran 007 recombinant anti-HPA-1a (r-anti-1a) were used as controls in SPRT. Affinity and dissociation constants (Ka and Kd respectively) of abs tested in serial doubling dilutions were determined using the computer software BIAevaluation 3.2 RC1 (Biacore) - Langmuir 1:1 mathematical model. Results: Control AP3 Mab bound to both HPA-1a & -1b chips in SPRT yielding respective Ka values of 2x105 & 1.7x104, and respective Kd values of 2.6x10−3 & 6.7x10−5; while control r-anti-1a bound specifically to HPA-1a chips yielding Ka and Kd of 2.9x105 & 8.3x10−4. Antibodies purified from plasma using immobilised rec-HPA-1a demonstrated higher binding in the ELISA and purer samples by silver staining (IgG heavy and light chains) compared to abs purified using immobilised native GP. This was reflected in SPRT where anti-HPA-1a purified using immobilised native GP exhibited high background that prohibited Ka and Kd determinations, whereas abs purified using immobilised rec-HPA- 1a bound specifically to HPA-1a chips, enabling Ka and Kd determinations which ranged from 6.9x103 to 7.9x104 for Ka, and from 1.3x10−4 to 3.4x10−4 for Kd. Anti-HPA-1a purified by immobilised rec-HPA-1a also interacted specifically with CM 5 sensor chips bound with rec-HPA-1a but not rec-HPA-1b, although with lower affinity constants compared to chips bound with native HPA-1a GP, yielding Ka of 4.2x102 to 9.1x103, & Kd of 3.4x10−3 to 8.6x10−5. Conclusion: This is the first report of the direct purification of anti-HPA-1a from plasma by immobilised HPA-1a antigen. Our study shows that immunopurification of plasma using immobilised rec-HPA-1a yields pure anti-HPA-1a antibodies that bind specifically in SPRT to chips coupled with both native or recombinant HPA-1a, allowing determination of antibody affinity and dissociation constants. This novel development would enable much needed analysis of anti-HPA-1a binding mechanisms in NAIT involving patients with different disease outcomes.
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Hadley, G. A., S. T. Bartlett, C. S. Via, E. A. Rostapshova, and S. Moainie. "The epithelial cell-specific integrin, CD103 (alpha E integrin), defines a novel subset of alloreactive CD8+ CTL." Journal of Immunology 159, no. 8 (October 15, 1997): 3748–56. http://dx.doi.org/10.4049/jimmunol.159.8.3748.
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Abstract The interaction of CD8+CTL with epithelial layers is an important but poorly defined aspect of organ allograft rejection. We herein report that CD103 (formerly alpha E integrin), a known receptor for the epithelial cell-specific ligand E-cadherin, is expressed by a major subset of CD8 + CTL elicited in response to allogeneic renal epithelial cells (REC). In contrast, CD103 was expressed poorly on CD8 + CTL generated in the conventional manner by stimulation with allogeneic leukocytes, although expression could be dramatically up-regulated by supplementing cultures with REC or exogenous TGF-beta 1. That TGF-beta controls the expression of CD103 on CD8+ CTL was further supported by the capacity of anti-TGF-beta mAb to block the generation of such cells in anti-REC cultures. Clonal analyses of anti-REC cultures revealed that individual CD8+ CTL clones were discretely CD103+ or CD103-, nd maintained their respective phenotypes independently of the cell type used for clonal restimulation. In a mouse model of graft-vs-host disease, 16.4 +/- 2.7% of CD8 cells that infiltrated host kidneys were CD103+ (n = 4). CD8 kidney-infiltrating lymphocytes were predominantly of donor origin and displayed an activated/memory phenotype (CD62L-, CD44high), consistent with expression of CD103 on a CD8 effector subset elicited in vivo following allogeneic transplantation. Taken together, the present data demonstrate that CD103 identifies a novel CD8 effector subset and, moreover, that such cells may comprise a significant component of the response to allogeneic tissues. The potential for CD103+ CTL as an important effector mechanism in organ allograft rejection, and more generally, as a mechanistic basis for tissue-specific immune phenomena, is discussed.
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Peyrat, M. A., F. Davodeau, I. Houde, F. Romagné, A. Necker, C. Leget, J. P. Cervoni, N. Cerf-Bensussan, H. Vié, and M. Bonneville. "Repertoire analysis of human peripheral blood lymphocytes using a human V delta 3 region-specific monoclonal antibody. Characterization of dual T cell receptor (TCR) delta-chain expressors and alpha beta T cells expressing V delta 3J alpha C alpha-encoded TCR chains." Journal of Immunology 155, no. 6 (September 15, 1995): 3060–67. http://dx.doi.org/10.4049/jimmunol.155.6.3060.
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Abstract V delta 3 usage and combinatorial expression of V gamma and V delta regions was studied on peripheral T cells with a novel V delta 3-specific mAb (p11.10b), generated against a soluble V gamma 9V delta 3 TCR. V delta 3+ cells represented the vast majority of V delta 1/V delta 2- gamma delta T cells within peripheral blood and mucosal lymphocytes. No preferential V gamma region expression was noted within V delta 3+ cells, but the frequency of V gamma 9+ cells was significantly lower among V delta 3+ than among V delta 1+ or V delta 2+ PBL. Phenotypic analysis of cultured V delta 3+ cells sorted with p11.10b mAb revealed the presence of T lymphocytes with unusual phenotypes. First, cells carrying two distinct surface TCR delta-chains, recognized by both V delta 1- and V delta 3-specific mAbs, were detected in most T cell lines, though at frequencies much lower than that of dual gamma expressors, indicating that allelic exclusion of delta genes is more tightly regulated than that of gamma genes. Moreover, a significant fraction of V delta 3+ cells were recognized by C beta- but not C delta-specific mAbs. Molecular analysis of V delta 3+C beta+ clones revealed the presence of V delta 3J alpha C alpha transcripts in all of them. Given the peculiar location of the V delta 3 gene between the delta Rec/psi J alpha elements, those observations formally demonstrate that activation of rearrangements with J alpha elements is not necessarily preceded by a delta Rec/psi J alpha-mediated deletion of the delta locus on the same chromosome.
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Kamenova, I., S. Dallot, V. Bozkova, and S. Milusheva. "First Report of the Plum Pox Virus Recombinant Strain on Peach in Bulgaria." Plant Disease 95, no. 10 (October 2011): 1320. http://dx.doi.org/10.1094/pdis-05-11-0405.
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Plum pox virus (PPV) causes sharka, the most damaging viral disease of stone fruit species. Seven distinct PPV strains are known; PPV-M, PPV-D, and PPV-Rec are the most common (3). PPV-Rec is a unique recombinant (3) between PPV-M and PPV-D and has been reported from plum, apricot, Japanese plum, myrobalan, and blackthorn in eastern and central Europe, but has never been found in peach as a single natural infection (2). A survey was conducted during spring 2009 in eight peach orchards located in the southwest, southeast, and south central regions of Bulgaria to assess the incidence of PPV infection. A total of 98 leaf samples from individual trees showing PPV-like symptoms were collected and analyzed by triple-antibody sandwich (TAS)-ELISA with the universal monoclonal antibody (MAb) 5B (Agritest, Valenzano, Italy). Sixty one samples reacted positive for PPV (optical density 0.161 to 1.267) and these samples were further analyzed with PPV-M (AL) and PPV-D (4DG5) specific MAbs (1). All 61 samples reacted positively with PPV-M specific MAbs. To distinguish PPV-M and PPV-Rec strains, which are serologically identical, immunocapture (IC)-reverse transcription (RT)-PCR was carried out with PPV-M (CIP-M: 5′-GTC GCA GCA TTT GTA GCC CTT GTT-3′, CIP-MR: 5′-CCA ACA CGT TAA CGC CAT GCT TCA-3′) and PPV-D (CIP-D: 5′-ATG ATG CTG TTT GAC TCG GAG CGA-3′, CIP-DR: 5′-TCG CAA CTG CTT GCA CAC ATT CTC-3′) specific primers targeting the 6K1-CI genomic region. A PCR fragment of ~880 bp amplified with PPV-M specific primers obtained from 59 samples confirmed that these were PPV-M isolates. However, the remaining two samples (both coming from infected tress located in two different orchards in the southwest region) yielded a 468-bp PCR fragment with PPV-D specific primers, suggesting that these two samples belonged to PPV-Rec strain. These samples together with controls of PPV-M, PPV-D, and PPV-Rec strains were further analyzed by RT-PCR using mD5/mM3 primers spanning the recombination breakpoint (4). Both peach samples and the PPV-Rec strain control produced a single 605-bp PCR product. The two peach amplicons were purified and sequenced directly with the same primers. The nucleotide (nt) sequences obtained were 100% identical to each other. BLAST analysis of the two samples with PPV-Rec (No. AF421118.1) showed maximum nt identity of 98%. Percent maximum nt identity with PPV-M (No. AY324837.1) and PPV-D (No. AB576062.1) were 93 and 87%, respectively. The deduced amino acid sequences of the two isolates were 98% identical to PPV-Rec (No. No. AF421118.1), 93% identical to PPV-M (No. M92280.1), and 84% identical to PPV-D (No. AB576062.1). Analyzed samples were further transmitted from the diseased trees to peach seedlings (GF 305) by chip-budding in a greenhouse during the fall of 2009. Six months later, faint vein clearing on the leaves of inoculated seedlings was observed. The presence of PPV was confirmed by TAS-ELISA and PPV-Rec presence was shown by IC-RT-PCR (mD5/mM3 primers). One of the generated 605-bp products was sequenced and showed 100% nt identity with the isolate used for inoculation. To our knowledge, this is the first identification of PPV-Rec strain in naturally infected peach trees, a finding that calls for further large-scale investigations of PPV-Rec incidence in peach in Bulgaria. References: (1) M. Cambra et al. OEPP/EPPO Bull. 24:569, 1994. (2) S. Dallot et al. Acta Hortic. 781:227, 2008. (3). M. Glasa et al. J. Gen. Virol. 85:2671, 2004. (4) Z. Šubr et al. Acta Virol. 48:173, 2004.
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Barth, Matthew J., Gopichand Pendurti, Ping-Chiao Tsai, Cory Mavis, Pavel Klener, Myron S. Czuczman, and Francisco Hernandez-Ilizaliturri. "Ofatumumab (OFA) Is a Novel Anti-CD20 Monoclonal Antibody (mAb) with Improved Anti-Tumor Activity in Vitro and in Vivo in Mantle Cell Lymphoma (MCL) Pre-Clinical Models." Blood 120, no. 21 (November 16, 2012): 2757. http://dx.doi.org/10.1182/blood.v120.21.2757.2757.
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Abstract Abstract 2757 MCL is typically characterized by an aggressive clinical course and inevitable development of refractory disease despite early intervention that often includes: immunotherapy (e.g., rituximab), multi-agent induction chemotherapy and consolidation with high dose chemotherapy and autologous stem cell transplant in first remission. Residual disease at the time of stem cell collection is an important cause for treatment failure. There is a need to evaluate more potent anti-CD20 mAbs capable to kill lymphoma cells with low CD20 surface levels. In Burkitt's lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL) pre-clinical models we previously demonstrated that OFA was more potent than rituximab (RIT) in vitro and in vivo. In order to characterize the activity of OFA against MCL, we evaluated the activity of OFA against cytarabine (Ara-C)-sensitive (eg. Mino, Jeko-1, Rec-1, HBL-2, Granta and Z-138); –resistant MCL cell lines (eg. MinoAraCR, Jeko-1AraCR, Rec-1AraCR, HBL-2AraCR and GrantaAraCR); and primary tumor cells derived from MCL patients (n=2). Antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) were measured by standard 51Cr release assays in MCL exposed to OFA, RIT or isotype control. OFA vs. RIT direct anti-proliferative effects were measured in by alamar blue reduction assay. Apoptosis following in vitro exposure to OFA or RIT was detected by caspase 3/PARP cleavage. Patient tumor cells were isolated from biopsy specimens by negative selection using magnetic beads and incubated with OFA or RIT +/− human serum as a complement source. Cell viability was determined at 48 hours by CellTiterGlo assay. Surface CD20 and the complement inhibitory proteins (CIPs) (CD55 and CD59) density in MCL cell lines was determined by flow cytometry (Image stream) and compared to BL or DLBCL cell lines. For in vivo experiments 6–8 week-old SID mice were inoculated subcutaneously with 5×106 matrigel suspended Z-138 cells. Upon tumor engraftment, mice were assigned to RIT (10mg/kg), OFA (10mg/kg) or control groups. Tumor growth curves were calculated for each group. Mice were sacrificed if tumor size reached >2cm in any dimension. After 6 months, survival was analyzed by Kaplan-Meier analysis and compared by log-rank test. OFA induced significantly higher levels of CDC associated cell lysis compared to RIT in almost all MCL cell lines tested (10/11) (Mino: 53.2% vs 0.2%; MinoAraCR: 72.6% vs. 0.6%; Jeko-1: 33.4% vs. 9.8%; Jeko-1AraCR: 38.3% vs. 2.8%; REC-1: 17% vs 3%; Rec-1AraCR: 7.8% vs. 0.2%; HBL-2: 27.1% vs. 19.2%; HBL-2AraCR: 86.6% vs. 72.2%; GrantaAraCR: 17% vs 0.9%; Z-138: 56.4% vs. 0.65%; all p-values <0.05). No differences in RIT or OFA mediated ADCC or direct signaling was observed. As previously noted in BL and DLBCL models, OFA was capable of inducing a higher degree of CMC even at low CD20 levels in contrast to RIT. In vivo, OFA slowed tumor growth, and prolonged survival in Z-138 bearing SCID mice compared to RIT (median survival for RIT was 127 days vs. not reached for OFA treated animals; p<0.05). Our data suggest that, OFA is more potent than RIT against Ara-C-sensitive and –resistant MCL cells in vitro, delays tumor growth and prolongs survival compared to RIT in an in vivo MCL SCID mouse model, and retains CDC activity despite low CD20 and high CIP surface expression levels. OFA appears to be a promising mAb targeting CD20 in MCL and is undergoing clinical testing in the front-line setting (NCT01527149). Disclosures: Czuczman: Genmab: Unrestricted Research Grant Other.
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Alinari, Lapo, Beth Christian, Bo Yu, Jungook Shin, Erin K. Hertlein, Rosa Lapalombella, Fengting Yan, et al. "Co-Treatment with Milatuzumab (Anti-CD74 mAb) and Rituximab (Anti-CD20 mAb) Results in the Induction of Mantle Cell Lymphoma Cell Death That Is Dependent On Actin Polymerization and Inhibition of NF-Kb." Blood 114, no. 22 (November 20, 2009): 1694. http://dx.doi.org/10.1182/blood.v114.22.1694.1694.
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Abstract Abstract 1694 Poster Board I-720 Mantle cell lymphoma (MCL) is an incurable B-cell malignancy and patients with this disease have limited therapeutic options. Despite the success of rituximab in treatment of B-cell lymphoma, its use as a single agent or in combination with chemotherapy in MCL has demonstrated modest activity; thus, novel strategies are needed. CD74 is an integral membrane protein expressed on malignant B cells and is implicated in promoting survival and growth, making it an attractive therapeutic target. The humanized anti-CD74 monoclonal antibody (mAb), milatuzumab, (Immunomedics) has shown promising preclinical activity against several human B-cell lymphoma cell lines, but has not been studied in MCL. Since rituximab and milatuzumab target distinct antigens lacking known association, we explored a combination strategy with these mAbs in MCL cell lines, patient samples, and in a preclinical model of MCL. Flow cytometric analysis shows that 6 different MCL cell lines (Mino, JeKo, SP53, Rec-1, Hbl2, Granta-519) and MCL patient primary tumor cells, express variable levels of CD74, with Mino cells showing highest level and Rec-1 the lowest. Incubation of the 6 MCL cell lines and primary cells (7 patients) with immobilized milatuzumab (5 μg/ml) and rituximab (10 μg/ml) resulted in mitochondrial depolarization and in statistically significant enhanced induction of apoptosis determined by Annexin V/PI and flow cytometry. The combination of both agents resulted in additive induction of apoptosis that was caspase independent in 5 MCL cell lines (synergistic in JeKo cells) and in primary cells, at 8, 24 and 48 hours. Importantly, while sensitivity to milatuzumab depends on the level of CD74 expression, the combination of milatuzumab and rituximab was able to induce enhanced cell death in all MCL cell lines and MCL primary cells, regardless of antigen density. We demonstrated that the combination of milatuzumab and rituximab induced enhanced apoptosis in a caspase-independent fashion with no apparent involvement of apoptotic key regulatory proteins such as Bax, Bcl-2, Bcl-Xl and Mcl-1. However, changes in the nuclear level of p65 were observed with either drug alone and with the combination, starting as early as 4 hours after treatment. The association of CD74 with MHC class II led us to explore pro-death mechanisms that become operable during HLA-DR-specific mAb treatment of lymphoma cells (Ivanov A et al., J Clin Invest 2009). We therefore investigated the role of actin polymerization by addition of cytochalasin D and latrunculin B, inhibitors of actin polimerization, prior to treatment with milatuzumab and/or rituximab. These studies showed that milatuzumab-induced MCL cell (Jeko and Mino) death was dependent on actin polymerization. To examine the in vivo activity of rituximab and milatuzumab, a preclinical model of human MCL using the SCID (CB17 scid/scid) mouse depleted of NK cells with TMβ1 mAb (anti-murine IL2Rb) was used. In this model, i.v. injection of 40×106 JeKo cells results in disseminated MCL 3 weeks after engraftment. The primary end-point was survival, defined as the time to develop cachexia/wasting syndrome or hind limb paralysis. Ten mice/group were treated starting at day 15 post-engraftment with intraperitoneal trastuzumab mAb control (300 μg qod), milatuzumab (300 μg qod), rituximab (300 μg qod), or a combination of milatuzumab and rituximab. The mean survival for the combination-treated group was 44.5 days (95%CI:39,51), compared to 28 days for trastuzumab-treated mice (95% CI:24,30), 33.5 days for the milatuzumab-treated mice (95% CI:28,36), and 38 days for the rituximab-treated mice (95%CI:36,42). The combination treatment prolonged survival of this group compared to trastuzumab control (P<0.0001), milatuzumab (P<0.0001) or rituximab (P=0.03). No overt toxicity from milatuzumab or the combination regimen was noted. These preliminary results provide justification for further evaluation of milatuzumab and rituximab in combination in MCL. Disclosures Off Label Use: Milatuzumab for Mantle Cell Lymphoma Treatment. Goldenberg:Immunomedics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties.
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Nayak, Ramesh, Prosenjit Sen, Charles Esmon, Usha Pendurthi, and L. Vijaya Mohan Rao. "Endothelial Cell Protein C Receptor Cellular Localization and Trafficking." Blood 112, no. 11 (November 16, 2008): 1015. http://dx.doi.org/10.1182/blood.v112.11.1015.1015.
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Abstract Endothelial cell protein C receptor (EPCR) is the cellular receptor for protein C (PC) and activated protein C (APC). Recent studies from others and us showed that EPCR also acts as a cellular binding site for factor VII (FVII) and activated factor VII (FVIIa). Although much is know about the biochemistry and pathophysiological importance of EPCR, little is know how EPCR interaction with its ligands on cell surfaces affects its expression and facilitate internalization of the ligands. The present study was undertaken to characterize cellular localization and trafficking of EPCR and investigate how FVIIa or APC binding to EPCR influences these processes. The studies employed two cell model systems - primary cultured human umbilical endothelial cells (HUVEC) and CHO cells stably transfected with EPCR. Cellular localization of EPCR and its trafficking was analyzed by immunofluorescence confocal microscopy or monitoring the expression of EPCR tagged with green fluorescence protein (GFP). EPCR endocytosis was evaluated by biotinylation of cell surface proteins with NHS-SS- biotin, followed by monitoring the protection of biotinylated EPCR from a membrane-impermeable reducing agent. FVIIa and APC internalization and recycling was evaluated by monitoring the uptake/release of 125I-labeled ligands or following the intracellular routing of fluorescent dye (AF488)-conjugated ligands added to EPCR expressing cells. Data from these studies showed that a majority of EPCR is localized on the cell surface and distributed in a patchy and punctuate manner. Immunostaining of HUVEC and CHO-EPCR cells with EPCR mAb and caveolin-1 antibodies showed a high degree of co-localization of EPCR and caveolin-1. Depletion of cholesterol from the plasma membrane, which disrupts caveolae, by ß methyl cyclodextrin reduced the extent EPCR and caveloin-1 colocalization. These data indicate EPCR on the cell surface predominantly localizes in caveolae. Inside the cell, EPCR is mainly localized in a small perinuclear structure, which is the site of centrosome. Colocalization of EPCR with tubulin (a marker for centrosome) and rab 11 (a marker of recycling compartment, REC) revealed that EPCR is localized actually in the REC and not in the centrosome. A small fraction of EPCR is also localized in endosomes as evident from colocalization of EPCR with EEA1 and Rab5, early endosome markers. Chasing the cell surface biotinylated proteins showed no significant increase in the biotinylated EPCR in the intracellular pool of proteins, which indicate that EPCR is not actively endocytosed constitutively or that the internalized EPCR is immediately recycled back to the cell surface. FVIIa or APC binding to EPCR promoted the EPCR endocytosis. The endocytosed receptors were first observed in proximity of the plasma membrane after 10 min and by 30 to 60 min most of the endocytosed EPCR was accumulated in the REC. The internalized FVIIa or APC appeared to follow the same route of the endocytosed EPCR. Proteolytically inactive FVIIa or APC behaved same as FVIIa or APC in promoting EPCR endocytosis and its trafficking. EPCR-dependent FVIIa or APC internalization is a dynamin-dependent process as the inhibition of the GTPase activity of dynamin by a specific inhibitor (Dynasore) completely abrogated their internalization and accumulation in the REC. Additional studies revealed that disruption of coated-pit pathway by potassium depletion blocked the endocytosis of transferrin, a classic marker for endocytosis via clathrin-dependent coated-pit pathway, but had no effect on EPCR-dependent FVIIa or APC internalization. In contrast, disruption of caveolae by cholesterol depletion blocked the internalization of FVIIa but not transferrin. Rab11 dominant negative mutant form (S25N) prevented the passage of the endocytosed EPCR or the internalized FVIIa or APC into the REC. After peaking at 30 min, the amount of both the ligands and the receptor in the REC gradually decreased, and some of the internalized ligand re-appeared at the cell surface. Overall the data provided herein suggest that FVIIa or APC binding to EPCR, independent of their protease activity, promotes EPCR endocytosis via the dynamin-dependent caveolar pathway and the activation of rab11 by GTP is required for exit of the endocytosed receptor or the ligands from sorting endosomes to the recycling compartment.
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Barth, Matthew John, Gopichand Pendurti, Cory Mavis, Natalie Czuczman, Jospeh J. Skitzki, Francisco J. Hernandez-Ilizaliturri, and Myron Stefan Czuczman. "Preclinical activity of ofatumumab (OFA) in mantle cell lymphoma (MCL) in vitro, ex vivo, and in vivo models." Journal of Clinical Oncology 30, no. 15_suppl (May 20, 2012): e18537-e18537. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.e18537.
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e18537 Background: MCL is characterized by an aggressive clinical course and inevitable development of refractory disease despite early intervention that often includes: immunotherapy (e.g., rituximab), multi-agent induction chemotherapy and consolidation with high dose chemotherapy and autologous stem cell transplant in first remission. OFA is a fully human anti-CD20 mAb targeting a novel membrane-proximal epitope on CD20. To characterize the activity of ofatumumab in MCL, we conducted pre-clinical studies in cell lines, primary tumor cells derived from MCL patients and a MCL bearing severe combined immunodeficiency (SCID) mouse model. Methods: Antibody-dependent cellular cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) assays were performed in 51Cr labeled Mino, Jeko, Rec-1 and Z-138 cells comparing RTX or OFA. Primary tumor cells were exposed ex vivo to OFA or RTX with human serum, differences in cell viability were determined by Cell Titer Glo assay. Expression of CD20 and complement inhibitory proteins (CIPs) CD55 and CD59 was determined by Imagestream analysis and Western blot. SCID mice were inoculated SQ with 10x106 Z-138 cells. Once tumors were established, mice were assigned to observation versus 4 doses of either OFA or RTX, and anti-tumor activity was measured by changes in tumor volume. Results: OFA induced higher CDC than RTX in all MCL cell lines tested (Mino: 65.9% vs. 0.5% ; Jeko 43.9% vs. 13.3% ; Rec-1 25.4% vs. 4.7% ; Z-138: 56.4% vs. 0.65%). No differences in ADCC were noted between OFA and RTX. In primary tumor cells, OFA and RTX demonstrated similar activity. CD20 levels were similar in all MCL cell lines tested. Of interest, CIP expression in MCL cell lines was higher when compared to other NHL cell lines, explaining differences observed between OFA and RTX. In vivo OFA was more effective in slowing tumor progression than RTX. Conclusions: Our data suggest OFA is more potent than RTX against MCL pre-clinical models. In addition and as expected, OFA exhibits potent CDC despite high expression of CIP. Our results support the evaluation of ofatumumab in future prospective clinical trials for patients with MCL.
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Repetto-Llamazares, Ada H. V., Roy Hartvig Larsen, Landsverk Kirsti, Trond Stokke, Bergthora Eiriksdottir, Roman Generalov, and Jostein Dahle. "Treatment with 177 lu-HH1 Increases CD20 Expression in Non-Hodgkin Lymphoma Cells in Vitro and In Vivo." Blood 126, no. 23 (December 3, 2015): 3245. http://dx.doi.org/10.1182/blood.v126.23.3245.3245.
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Abstract Immunotherapy (IT) with the anti-CD20 monoclonal antibody rituximab in combination with chemotherapy has resulted in significantly improved response rate and survival in patients with various types of CD20 positive B-cell lymphoproliferative disorders. To be effective, rituximab depends on selective expression of a sufficient number of CD20 antigens per cell. Treatment with rituximab alone or in combination with chemotherapy can, however, result in disappearance of the CD20 expression, which may result in reduced clinical effect of subsequent CD20 targeted treatments. We have discovered that treatment of NHL in vitro and in vivo with the anti-CD37 antibody radionuclide conjugate (ARC) 177Lu-DOTA-HH1 (177Lu-HH1 or Betalutin™) results in an upregulation of the CD20 antigen expression, and therefore represents a rationale for a combination treatment with both agents. The in vitro expression of CD20 in Burkitt's Lymphoma, Daudi, cells 1-7 days after treatment with 177Lu-HH1 increased up to 120 % when compared with cells treated with unlabeled mAb, while Ramos (Burkitt's Lymphoma) and Rec-1 (Mantle Cell Lymphoma) cells showed 10 to 30 % increase, indicating a variation of the antigen upregulation in vitro with different cell lines. An upregulation of CD20 at the same order of magnitude was observed when cells where treated with similar absorbed radiation doses of external beam radiation. Treatment of nude mice with Ramos xenografts with 177Lu-HH1 resulted in a 3 times higher uptake of radiolabeled rituximab in tumor xenografts 5 days after start of treatment than in mice treated with unlabeled HH1 (p < 0.05) while uptake in normal organs was similar in both treatment groups (p > 0.05). SCID mice with intravenously injected Rec-1 cells were treated with NaCl, 100 mg rituximab, 40 MBq/kg 177Lu-HH1 or with the combination of 40 MBq/kg 177Lu-HH1 followed with 100 mg rituximab 5 days later. The combination of 177Lu-HH1 and rituximab resulted in significantly improved survival as compared with NaCl or rituximab alone, and a strong therapeutic gain as compared with 177Lu-HH1 alone (Table 1). In conclusion, 177Lu-HH1 treatment seems to improve uptake of rituximab and increase tumor suppression when used prior to anti-CD20 monoclonal antibody targeting in preclinical models. The reason for the upregulation of CD20 is probably related to the oxidative stress induced by the ARC-treatment, which will be evaluated in further studies. If the upregultation of CD20 is confirmed in clinical studies this effect could affect the way ARC and CD20 immunotherapy would be used in the future. Table 1. Therapy experiment groups and result Group Median ± SD Surviving fraction at the end of the study % Increase in symptom free survival compared to control NaCl + NaCl 64 ± 2 0.1 ---- NaCl + Rituximab 75 ± 10 0.3 15.4 177 Lu-HH1 + NaCl 92 ± 14 * 0.3 43.8 177 Lu-HH1 + Rituximab > 132 * 0.7 > 106.3 *Significantly different from NaCl + NaCl group (p < 0.01) Disclosures Repetto-Llamazares: Nordic Nanovector ASA: Employment, Equity Ownership. Larsen:Nordic Nanovector ASA: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Stokke:Nordic nanovector ASA: Equity Ownership. Generalov:Nordic Nanovector ASA: Employment. Dahle:Nordic Nanovector ASA: Employment, Equity Ownership.
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Oosterwijk, E., G. N. Van Muijen, J. C. Oosterwijk-Wakka, and S. O. Warnaar. "Expression of intermediate-sized filaments in developing and adult human kidney and in renal cell carcinoma." Journal of Histochemistry & Cytochemistry 38, no. 3 (March 1990): 385–92. http://dx.doi.org/10.1177/38.3.1689337.
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We studied the distribution of intermediate-sized filaments in developing and adult kidneys and renal cell carcinoma (RCC) by indirect immunohistochemistry, using a pan-cytokeratin mouse monoclonal antibody (MAb), chain-specific anti-cytokeratin MAb, and anti-vimentin and anti-desmin MAb, to resolve controversy concerning intermediate-sized filament expression in the kidney. With the pan-cytokeratin MAb, cytokeratin expression was detectable in all stages of nephron development, starting with expression in the renal vesicles, the progenitors of the glomeruli, proximal tubules, Henle's loop, and part of the distal tubules. Using chain-specific anti-cytokeratin MAb, cytokeratin 8 and 18 expression was demonstrated in all developmental structures of the nephron, whereas cytokeratin 19 expression was more complex. None of the nephrogenic blastema cells from which the renal vesicles arise expressed cytokeratins. Transient expression of vimentin and cytokeratin 19 was observed in differentiating collecting ducts and proximal tubule cells at the S-shaped stage of nephron development, respectively. In RCC, cytokeratin expression closely resembled that of the mature proximal tubule, i.e., RCC cells expressed cytokeratins 8 and 18. However, in a subset of RCC additional cytokeratin 19 expression was noted. In addition, all except one RCC showed co-expression of cytokeratins and vimentin.
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Kmiec, E. B., A. Cole, and W. K. Holloman. "The REC2 gene encodes the homologous pairing protein of Ustilago maydis." Molecular and Cellular Biology 14, no. 11 (November 1994): 7163–72. http://dx.doi.org/10.1128/mcb.14.11.7163.
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Amino acid sequence analysis has established that the homologous pairing protein of Ustilago maydis, known previously in the literature as rec1, is encoded by REC2, a gene essential for recombinational repair and meiosis with regional homology to Escherichia coli RecA. The 70-kDa rec1 protein is most likely a proteolytic degradation product of REC2, which has a predicted mass of 84 kDa but which runs anomalously during sodium dodecyl sulfate-gel electrophoresis with an apparent mass of 110 kDa. To facilitate purification of the protein product, the REC2 gene was overexpressed from a vector that fused a hexahistidine leader sequence onto the amino terminus, enabling isolation of the REC2 protein on an immobilized metal affinity column. The purified protein exhibits ATP-dependent DNA renaturation and DNA-dependent ATPase activities, which were reactions characteristic of the protein as purified from cell extracts of U. maydis. Homologous pairing activity was established in an assay that measures recognition via non-Watson-Crick bonds between identical DNA strands. A size threshold of about 50 bp was found to govern pairing between linear duplex molecules and homologous single-stranded circles. Joint molecule formation with duplex DNA well under the size threshold was efficiently catalyzed when one strand of the duplex was composed of RNA. Linear duplex molecules with hairpin caps also formed joint molecules when as few as three RNA residues were present.
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Tazawa, Hirofumi, Toshimitsu Irei, Yuka Tanaka, Yuka Igarashi, Hirotaka Tashiro, and Hideki Ohdan. "Blockade of invariant TCR-CD1d interaction specifically inhibits antibody production against blood group A carbohydrates." Blood 122, no. 15 (October 10, 2013): 2582–90. http://dx.doi.org/10.1182/blood-2012-02-407452.
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Key PointsAdministration of anti-mouse CD1d blocking mAb prior to A-RBC immunization abolished IL-5 production and anti-A Ab production in mice. In human peripheral blood mononuclear cell–NOD/SCID mice, administration of anti-human CD1d mAb prior to A-RBC immunization completely inhibited anti-A Ab production.
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Kmiec, E. B., A. Cole, and W. K. Holloman. "The REC2 gene encodes the homologous pairing protein of Ustilago maydis." Molecular and Cellular Biology 14, no. 11 (November 1994): 7163–72. http://dx.doi.org/10.1128/mcb.14.11.7163-7172.1994.
Full textAbstract:
Amino acid sequence analysis has established that the homologous pairing protein of Ustilago maydis, known previously in the literature as rec1, is encoded by REC2, a gene essential for recombinational repair and meiosis with regional homology to Escherichia coli RecA. The 70-kDa rec1 protein is most likely a proteolytic degradation product of REC2, which has a predicted mass of 84 kDa but which runs anomalously during sodium dodecyl sulfate-gel electrophoresis with an apparent mass of 110 kDa. To facilitate purification of the protein product, the REC2 gene was overexpressed from a vector that fused a hexahistidine leader sequence onto the amino terminus, enabling isolation of the REC2 protein on an immobilized metal affinity column. The purified protein exhibits ATP-dependent DNA renaturation and DNA-dependent ATPase activities, which were reactions characteristic of the protein as purified from cell extracts of U. maydis. Homologous pairing activity was established in an assay that measures recognition via non-Watson-Crick bonds between identical DNA strands. A size threshold of about 50 bp was found to govern pairing between linear duplex molecules and homologous single-stranded circles. Joint molecule formation with duplex DNA well under the size threshold was efficiently catalyzed when one strand of the duplex was composed of RNA. Linear duplex molecules with hairpin caps also formed joint molecules when as few as three RNA residues were present.
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Kerr, L., C. Huntoon, J. Donohue, P. J. Leibson, N. H. Bander, T. Ghose, S. J. Luner, R. Vessella, and D. J. McKean. "Heteroconjugate antibody-directed killing of autologous human renal carcinoma cells by in vitro-activated lymphocytes." Journal of Immunology 144, no. 10 (May 15, 1990): 4060–67. http://dx.doi.org/10.4049/jimmunol.144.10.4060.
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Abstract Tumor cell lysis can be enhanced significantly in vitro when heteroconjugate (HC) antibodies (anti-CD3 x anti-tumor mAb) are used to specifically direct lymphocyte effector cells to the tumor cell target. In order to effectively utilize HC antibodies in an immunotherapy protocol, methods must be identified for the optimum expansion, activation, and retargeting of lymphocyte-effector populations from cancer patients. In this study, we have compared the proliferative responses of different normal and renal cell carcinoma (RCC) patient lymphocyte preparations (PBL, tumor-infiltrating lymphocytes) stimulated in vitro for periods up to 12 days with a variety of growth factor combinations (anti-CD3, rIL-2, rIL-4). These activated lymphocyte preparations were then tested in vitro for their ability to kill RCC tumor cells and tumor cell lines in the presence of HC preparations (anti-CD3 mAb covalently linked to mAb reactive to different RCC tumor-associated Ag). RCC patient PBL cultured with anti-CD3 plus rIL-2 for 12 days resulted in a 3- to 160-fold expansion of effector cells. These cells, as well as tumor infiltrating lymphocytes, when retargeted with appropriate HC antibodies were capable of mediating high levels of killing of autologous tumor cells. No constitutive autologous anti-tumor cell response was detected in the absence of added HC antibodies. Of the five anti-RCC mAb tested (A6H, K29, K20, UR07, and URO 3), HC containing URO 3 x anti-CD3 and K20 x anti-CD3 elicited the highest level of tumor cell lysis by the activated lymphocyte effector cells. Together these results demonstrate that HC antibodies may be a useful imunotherapeutic reagent for directing the killing of RCC tumor cells by autologous lymphocytes.
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Oosterwijk-Wakka, J. C., S. Lafferty-Van Eijk, P. M. M. Van Hauten, R. J. A. Maas, P. K. J. D. De Jonge, H. Dolstra, P. F. A. Mulders, and E. Oosterwijk. "Combination of mAb cG250 with natural killer cells for RCC treatment." European Urology 79 (June 2021): S756. http://dx.doi.org/10.1016/s0302-2838(21)00934-9.
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Cohen, Adam D., Nikoletta Lendvai, Sacha Gnjatic, Achim A. Jungbluth, Stephane Bertolini, Linda Pan, Ralph Venhaus, et al. "Recombinant (rec) MAGE-A3 Protein Immunotherapy and Peripheral Blood Lymphocyte (PBL) Reconstitution Induce Strong Antigen-Specific Humoral and Cellular Immune Responses in Patients Undergoing Autologous Stem Cell Transplantation (ASCT) for Consolidation of Multiple Myeloma (MM)." Blood 124, no. 21 (December 6, 2014): 1184. http://dx.doi.org/10.1182/blood.v124.21.1184.1184.
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Abstract BACKGROUND: MAGE-A3 is an immunogenic tumor-associated antigen detected in 1/3 of newly diagnosed MM patients, and confers a poor prognosis, making it a rational target for immunotherapy. We previously reported (Cohen et al, ASH 2013, #154) that pre- and post-ASCT administration of recMAGE-A3 + AS15 adjuvant (containing MPL, QS21, and CpG7909) and infusion of vaccine-primed autologous peripheral blood lymphocytes (PBL) in the early post-ASCT period had an acceptable safety profile and induced robust antibody responses against MAGE-A3. We now report our initial cellular immune response data, and update the humoral response and clinical outcome data. METHODS: The composition of recMAGE-A3 +AS15 and the immunization schedule (Fig. 1) for this pilot study have been described (ASH 2013, #154). Antibody responses were assessed by ELISA. CD4 and CD8 T cell responses were assessed by ELISpot and intracellular cytokine release assays after in vitro re-stimulation with MAGE-A3 overlapping peptide pools or controls and autologous antigen-presenting cells. Clinical responses were determined by IMWG criteria. M3H67 mAb (specific for MAGE-A3 and homologous MAGE-A family members) was used for assessing expression in MM cells by immunohistochemistry (IHC). RESULTS: Thirteen patients enrolled (med. age 56; 45% high-risk cytogenetics; 42% ISS II/III). All had MAGE-A+ myeloma cells and had achieved at least VGPR following induction. Twelve of 12 (100%) subjects tested to date developed high-titer (1:104-106) antibodies against MAGE-A3 that persisted to at least 1 year post-SCT. These titers were 10-100-fold higher than those seen in a prior study in lung cancer patients with recMAGE-A3 + AS02b, an older adjuvant lacking CpG7909 (PNAS 2008; 105:1650). Epitope mapping identified at least 7 distinct MAGE-A3 epitopes clustering in the hydrophobic regions from aa. 1-100 and 220-300. Isotyping and IgG subclass analysis demonstrated IgG class switching in all patients, with IgG1 and IgG3 subclasses most prevalent. Peripheral blood T cell responses have been evaluated in 3 subjects to date. All had MAGE-A3-specific CD4 responses by IFNγ ELISpot starting as early as d+31 after ASCT, with significant expansion after booster vaccinations and persistence through 1 year post-ASCT. Intracellular cytokine staining confirmed a polyfunctional, Th1-biased CD4 T cell response (IFNγ+, TNFα+, IL5-) in all 3 patients. No CD8 responses against MAGE-A3 have been detected to date. Clinical response assessments were as follows: there were 12 VGPR and 1 CR at enrollment, 7 VGPR and 6 CR (3 stringent CR) at 3 months (mos.) post-ASCT, and 3 VGPR and 5 CR (4 sCR) at 1 year post-ASCT, with 4 patients relapsing at or before 1 year, and 1 not yet evaluable. With a median follow-up of 19 mos. (range 6-32), 6 patients have relapsed (estimated median PFS is 24 mos.) and 1 died of progressive MM. There was no difference between progressors and non-progressors with regard to cytogenetics, baseline MAGE-A expression, antibody titers, hematologic response, or use of lenalidomide maintenance (n=4). MAGE-A expression was assessed by IHC in 3 relapse bone marrow biopsies, and all were negative. CONCLUSIONS: RecMAGE-A3 immunotherapy and PBL reconstitution is well-tolerated, feasible, and induces antibody and Th1-biased CD4 T cell responses, but not CD8 responses, in the setting of ASCT for MM. Cellular immune assessments are ongoing. The magnitudes of antibody and CD4 responses appear greater than those seen historically with older formulations of recMAGE-A3 in other cancers, despite significant immune compromise after ASCT, suggesting a benefit from the new AS15 adjuvant formulation, or from immunization and autologous PBL transfer in the peri-ASCT setting, or both. The loss of MAGE-A3 expression in relapsing patients implies antigen-specific immune selective pressure even in the absence of CD8 T cell responses, and also suggests that combination strategies aimed at limiting immune escape (eg multi-antigen vaccines) should be investigated. Clinical outcomes are promising for this high-risk patient population. These results support advanced phase clinical trials to investigate clinical efficacy of recMAGE-A3 vaccine immunotherapy in MM. Figure 1 Figure 1. Disclosures Cohen: Onyx Pharmaceuticals: Advisory Board, Advisory Board Other; Bristol-Myers Squibb: Advisory Board, Advisory Board Other, Research Funding; Janssen: Advisory Board, Advisory Board Other; Celgene: Member, Independent Response Adjudication Committee Other. Bertolini:Ludwig Institute for Cancer Research: Employment. Pan:Ludwig Institute for Cancer Research: Employment. Venhaus:Ludwig Institute for Cancer Research: Employment. Fellague-Chebra:GlaxoSmithKline: Employment. Gruselle:GlaxoSmithKline: Employment.
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Miller, R. D., M. J. Caulfield, and C. E. Calkins. "Expression and regulation of a recurrent anti-erythrocyte autoantibody idiotype in spleen cells from neonatal and adult BALB/c mice." Journal of Immunology 148, no. 8 (April 15, 1992): 2452–55. http://dx.doi.org/10.4049/jimmunol.148.8.2452.
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Abstract BALB/c spleen cells depleted of CD8+ T cells generate an autoantibody response to mouse RBC (MRBC) when cultured 5 days in the presence of syngeneic RBC. More than 80% of the cells secreting anti-MRBC antibody are blocked by an antiidiotypic mAb that recognizes the G8 Id. This G8 Id was originally identified in an autoimmune NZB mouse derived anti-MRBC mAb and later characterized as a dominant Id in NZB anti-MRBC autoantibodies. Furthermore, the CD8+ regulatory T cells that control this autoimmune response in BALB/c mice are specifically eliminated by cytotoxic treatment with the G8 mAb + C, suggesting that the regulatory cells recognize the G8 Id. Spleen cells from neonatal BALB/c mice, which lack those regulatory cells can generate an in vitro antibody response to MRBC without depletion of CD8+ cells. More than 80% of these AFC were also found to express the G8 Id. We propose that Id determinants on autoantibodies that are produced neonatally induce Id-specific regulatory cells that maintain peripheral tolerance to self-RBC throughout the life of normal animals.
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Oosterwijk, E., N. H. Bander, C. R. Divgi, S. Welt, J. C. Wakka, R. D. Finn, E. A. Carswell, S. M. Larson, S. O. Warnaar, and G. J. Fleuren. "Antibody localization in human renal cell carcinoma: a phase I study of monoclonal antibody G250." Journal of Clinical Oncology 11, no. 4 (April 1993): 738–50. http://dx.doi.org/10.1200/jco.1993.11.4.738.
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PURPOSE To define the imaging and biodistribution characteristics of iodine 131-labeled monoclonal antibody (mAb) G250 (131I-mAbG250), which recognizes a cell-surface antigen expressed by human renal cell carcinoma (RCC). PATIENTS AND METHODS G250 is a cell-surface antigen recognized by mAbG250 expressed by RCC but not detected in normal kidney. Clear-cell RCC, the most frequent form of RCC, shows homogeneous expression of G250, whereas non-clear-cell RCC and cancers derived from other organs generally do not express G250. Expression in normal tissues is highly restricted and limited to large bile ducts and gastric epithelium. 131I-mAbG250 was administered intravenously (IV) to 16 patients with RCC 7 to 8 days before surgery at five dose levels, with at least three patients entered at each dose level. RESULTS Clear tumor images were observed in 12 patients with G250-positive tumors and in one of three patients with G250-negative tumors. Imaged lesions in the peritoneal cavity were confirmed at surgery. The smallest lesion visualized was 8 mm in diameter. The specificity of 131I-mAbG250 localization to tumor tissue was established by radioactivity measurements, autoradiography, and immunohistochemistry of biopsied tissues, and technetium 99-human serum albumin blood-flow studies. The fraction of the injected 131I-mAbG250 dose per gram tumor (%ID/g tumor) localized in G250-positive tumors showed a broad range, but reached levels as high as 0.02% to 0.12%. CONCLUSION 131I-mAbG250 localized specifically to G250 antigen-positive RCC and seems to have considerable potential as an imaging agent in RCC patients. 131I-mAbG250 uptake in the tumors, relative as well as absolute, are among the highest reported for tumor biopsies obtained 8 days after IV mAb administration. Based on the specific localization and high accumulation, mAb G250 may have therapeutic potential.
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Edberg, J. C., E. Wright, and R. P. Taylor. "Quantitative analyses of the binding of soluble complement-fixing antibody/dsDNA immune complexes to CR1 on human red blood cells." Journal of Immunology 139, no. 11 (December 1, 1987): 3739–47. http://dx.doi.org/10.4049/jimmunol.139.11.3739.
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Abstract We have used direct binding isotherm analyses to measure the association constant (Ka) and number of binding sites for the binding of prepared complement-fixing antibody (Ab)/dsDNA immune complexes (IC) to human red blood cells (RBC). In order to generalize this study we have examined the binding reaction for a number of different anti-dsDNA Ab (from systemic lupus erythematosus plasmas), complement sources, RBC donors, and dsDNA sizes. The affinity of the IC for the RBC is quite high, and the Ka values fall within a narrow range (5 to 14 X 10(10) liter/mol). Similarly, the limiting stoichiometries for the number of IC bound per RBC were between 40 and 91. The very high affinity and limiting stoichiometries both suggest that the IC bind to the RBC via multiple contacts with clusters of complement receptor type 1 (CR1). Furthermore, we have used three specific monoclonal AB (mAb) to quantitate CR1 on human RBC in the presence and absence of bound IC. One of these Ab, mAb 1B4, is blocked from binding to the RBC if IC are previously bound, and we have used this observation to verify the multivalent nature of the interaction of complement-fixing IC with CR1 on human RBC.
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Wagstaff, J. E., S. Klapholz, C. S. Waddell, L. Jensen, and R. E. Esposito. "Meiotic exchange within and between chromosomes requires a common Rec function in Saccharomyces cerevisiae." Molecular and Cellular Biology 5, no. 12 (December 1985): 3532–44. http://dx.doi.org/10.1128/mcb.5.12.3532.
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We used haploid yeast cells that express both the MATa and MAT alpha mating-type alleles and contain the spo13-1 mutation to characterize meiotic recombination within single, unpaired chromosomes in Rec+ and Rec- Saccharomyces cerevisiae. In Rec+ haploids, as in diploids, intrachromosomal recombination in the ribosomal DNA was detected in 2 to 6% of meiotic divisions, and most events were unequal reciprocal sister chromatid exchange (SCE). By contrast, intrachromosomal recombination between duplicated copies of the his4 locus occurred in approximately 30% of haploid meiotic divisions, a frequency much higher than that reported in diploids; only about one-half of the events were unequal reciprocal SCE. The spo11-1 mutation, which virtually eliminates meiotic exchange between homologs in diploid meiosis, reduced the frequency of intrachromosomal recombination in both the ribosomal DNA and the his4 duplication during meiosis by 10- to greater than 50-fold. This Rec- mutation affected all forms of recombination within chromosomes: unequal reciprocal SCE, reciprocal intrachromatid exchange, and gene conversion. Intrachromosomal recombination in spo11-1 haploids was restored by transformation with a plasmid containing the wild-type SPO11 gene. Mitotic intrachromosomal recombination frequencies were unaffected by spo11-1. This is the first demonstration of a gene product required for recombination between homologs as well as recombination within chromosomes during meiosis.
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Brossay, L., G. Paradis, A. Pépin, W. Mourad, L. Coté, and J. Hébert. "Idiotype and anti-anti-idiotype antibodies to Neisseria gonorrhoeae lipooligosaccharides with bactericidal activity but no cross-reactivity with red blood cell antigens." Journal of Immunology 151, no. 1 (July 1, 1993): 234–43. http://dx.doi.org/10.4049/jimmunol.151.1.234.
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Abstract A panel of murine mAb against three different epitopes of Neisseria gonorrhoeae lipooligosaccharides (LOS) was developed. Only one of these, 3G5, displays bactericidal activity against all in vitro serum-resistant strains of N. gonorrhoeae. Evidence suggests that sialylation, which could occur in vivo, modifies some LOS epitopes in such a way that the strains become resistant to bactericidal activity and are no longer recognized by specific antibodies. The epitope recognized by our bactericidal mAb is not affected by sialylation as shown by immunoblot analysis. We also provided evidence that the 3G5 epitope is different from RBC precursor Ag, since our mAb did not induce RBC agglutination. Since LOS induce an immune response in a T cell-independent fashion and are highly toxic, they cannot be used for immunization. Use of anti-idiotypic antibodies (aId) could be a way to bypass these difficulties. Therefore, in the present study, aId were produced in rabbits and rendered idiotype-specific by appropriate adsorption. These aId specifically bind to the relevant Id but not to LOS, and inhibit only the binding of anti-LOS mAb (3G5) to LOS preparations from N. gonorrhoeae in a dose-response fashion. The specificity of our aId for the binding site of anti-LOS mAb is suggested by the binding inhibition of affinity-purified aId to Id by LOS. In addition, the capacity of aId to inhibit bactericidal activity of this anti-LOS mAb and the idiotypic cross-reactivity between rat and mouse anti-LOS antibodies support this point. Finally, the elicitation of anti-LOS activity with bactericidal activity upon immunization of naive mice with aId confirms the internal image properties of the aId. These data suggest that a bactericidal mAb suitable for immunoprotection was obtained, and the production of aId opens the door for development of a vaccine.
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Jokiranta, T. S., and S. Meri. "Biotinylation of monoclonal antibodies prevents their ability to activate the classical pathway of complement." Journal of Immunology 151, no. 4 (August 15, 1993): 2124–31. http://dx.doi.org/10.4049/jimmunol.151.4.2124.
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Abstract Biotinylation of mAb has become a standard procedure for a variety of applications that exploit the specific high affinity interaction between biotin and avidin. In the present study, we investigated how biotinylation of mAb affects their ability to sensitize target cells to C-dependent lysis in vitro. mAb were biotinylated by cross-linking biotin covalently with an N-succinimidyl ester to the epsilon-amino groups of lysine residues. Human RBC were treated with two rat mAb, either alone or together: one against glycophorin A (YTH89.1), another against CD59 (protectin; YTH53.1), an inhibitor of the membrane attack complex of C. Melanoma cells (G361) were attacked by a mouse mAb (27A) against an O-acetylated GD3 ganglioside. As compared with the nonbiotinylated mAb, the biotinylated forms of all the investigated mAb were much weaker in causing classical C pathway-mediated lysis of the target cells. Biotinylation did not reduce the ability of the mAb to bind to their Ag, nor of the anti-CD59 mAb to neutralize the C lysis-restrictive effect of CD59. In binding assays using 125I-labeled C1q, significantly less C1q bound to the biotinylated anti-glycophorin-A and anti-CD59 mAb than to the nonbiotinylated mAb. These data show that biotinylated antibodies do not activate the classical C pathway because binding of C1q to the antibody Fc-regions is blocked.
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Wunderlich, Mark, Kevin A. Link, Fu-Sheng Chou, and James C. Mulloy. "Constitutive Activation of Rac GTPases Is Induced by Cooperating Mutations in a Human Model of MLL-AF9 Leukemia." Blood 114, no. 22 (November 20, 2009): 2959. http://dx.doi.org/10.1182/blood.v114.22.2959.2959.
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Abstract Abstract 2959 Poster Board II-935 Retroviral transduction of primary human umbilical cord blood CD34+ cells with a gene encoding MLL-AF9 (MA9) efficiently promotes immortalization in vitro and development of leukemia in a xenotransplant model. MA9 cells remain cytokine dependent with a strict requirement for Flt3 signaling. Additionally, the cells are hypersensitive to inhibition of the RAC GTPases. In this current study, we have examined a potential link between these pathways. We found that MA9 cells proliferate in culture media containing Flt3 Ligand (FL) as the sole cytokine, indicating that Flt3 signaling is sufficient for growth and survival. Furthermore, introduction of a mutant Flt3-ITD gene in these cells (MA9-ITD cells) not only promotes cytokine independent growth, but also significantly decreases leukemia latency in xenotransplant experiments. Consistent with the persistent requirement for active Rac signaling, we found that MA9 cells stimulated with FL induce the activated form of Rac (Rac-GTP) while MA9-ITD cells contain constitutively high levels of Rac-GTP, even after serum starvation and in the absence of all cytokines. Interestingly, we found that expression of the activated mutant NRas-G12D in MA9 cells (MA9-NRas cells) also leads to constitutive Rac activation, cytokine independent growth, and decreased leukemia latency upon transplantation into immunodeficient mice. Taken together, these results indicate that RAC activation may be a common integrating pathway by which secondary mutations cooperate in the progression towards leukemia. Consistent with this notion, both MA9-ITD and MA9-NRas cells remain sensitive to Rac inhibition with a small molecule Rac inhibitor or shRNA knockdown of Rac1 or Rac2. These data indicate that Rac signaling remains critical for MA9 survival and proliferation even in the context of additional mutations and highlights the therapeutic potential for targeting Rac. Disclosures: No relevant conflicts of interest to declare.
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Fossati-Jimack, Liliane, Luc Reininger, Yves Chicheportiche, Raphael Clynes, Jeffrey V. Ravetch, Tasuku Honjo, and Shozo Izui. "High Pathogenic Potential of Low-Affinity Autoantibodies in Experimental Autoimmune Hemolytic Anemia." Journal of Experimental Medicine 190, no. 11 (December 6, 1999): 1689–96. http://dx.doi.org/10.1084/jem.190.11.1689.
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To assess the potency of low-affinity anti–red blood cell (RBC) autoantibodies in the induction of anemia, we generated an immunoglobulin (Ig)G2a class-switch variant of a 4C8 IgM anti–mouse RBC autoantibody, and compared its pathogenic potential with that of its IgM isotype and a high-affinity 34-3C IgG2a autoantibody. The RBC-binding activity of the 4C8 IgG2a variant was barely detectable, at least 1,000 times lower than that of its IgM isotype, having a high-binding avidity, and that of the 34-3C IgG2a monoclonal antibody (mAb). This low-affinity feature of the 4C8 mAb was consistent with the lack of detection of opsonized RBCs in the circulating blood from the 4C8 IgG2a–injected mice. However, the 4C8 IgG2a variant was highly pathogenic, as potent as its IgM isotype and the 34-3C IgG2a mAb, due to its capacity to interact with Fc receptors involved in erythrophagocytosis. In addition, our results indicated that the pentameric form of the low-affinity IgM isotype, by promoting the binding and agglutination of RBCs, is critical for its pathogenic activity. Demonstration of the remarkably high pathogenic potency of low-affinity autoantibodies, if combined with appropriate heavy chain effector functions, highlights the critical role of the Ig heavy chain constant regions, but the relatively minor role of autoantigen-binding affinities, in autoimmune hemolytic anemia.
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Caulfield, M. J., and D. Stanko. "A pathogenic monoclonal antibody, G8, is characteristic of antierythrocyte autoantibodies from Coombs'-positive NZB mice." Journal of Immunology 148, no. 7 (April 1, 1992): 2068–73. http://dx.doi.org/10.4049/jimmunol.148.7.2068.
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Abstract With age, NZB mice develop anti-RBC autoantibodies resulting in the development of autoimmune hemolytic anemia. We now have evidence that this spontaneous autoantibody response consists of antibodies that are similar in specificity and Id expression to a pathogenic autoantibody (G8) that was cloned from an autoimmune NZB mouse. Similar to autoantibodies eluted from Coombs'-positive mouse E (MRBC), the G8 mAb recognizes native (unmodified) MRBC but not RBC from other species. Interestingly, G8 and four additional mAb bind with a higher titer to bromelain-treated MRBC than to native MRBC. Nucleotide sequence analysis reveals, however, that unlike "natural" antibodies that react solely with bromelain-MRBC, G8 is encoded by a J558 VH gene and a V kappa 12,13 L-chain gene. Thus G8 is clearly distinct from antibodies to bromelain-MRBC which are encoded by unrelated V genes. Instead, the sequence of the G8 VH chain was found to be nearly identical to that of an anti-DNA mAb derived from an MRL-lpr/lpr mouse. The results suggest Coombs'-positive autoantibodies from NZB mice are not derived from "natural" antibodies, but rather, consist of a restricted set of autoantibodies expressing the G8 IdX.
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Wagstaff, J. E., S. Klapholz, C. S. Waddell, L. Jensen, and R. E. Esposito. "Meiotic exchange within and between chromosomes requires a common Rec function in Saccharomyces cerevisiae." Molecular and Cellular Biology 5, no. 12 (December 1985): 3532–44. http://dx.doi.org/10.1128/mcb.5.12.3532-3544.1985.
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We used haploid yeast cells that express both the MATa and MAT alpha mating-type alleles and contain the spo13-1 mutation to characterize meiotic recombination within single, unpaired chromosomes in Rec+ and Rec- Saccharomyces cerevisiae. In Rec+ haploids, as in diploids, intrachromosomal recombination in the ribosomal DNA was detected in 2 to 6% of meiotic divisions, and most events were unequal reciprocal sister chromatid exchange (SCE). By contrast, intrachromosomal recombination between duplicated copies of the his4 locus occurred in approximately 30% of haploid meiotic divisions, a frequency much higher than that reported in diploids; only about one-half of the events were unequal reciprocal SCE. The spo11-1 mutation, which virtually eliminates meiotic exchange between homologs in diploid meiosis, reduced the frequency of intrachromosomal recombination in both the ribosomal DNA and the his4 duplication during meiosis by 10- to greater than 50-fold. This Rec- mutation affected all forms of recombination within chromosomes: unequal reciprocal SCE, reciprocal intrachromatid exchange, and gene conversion. Intrachromosomal recombination in spo11-1 haploids was restored by transformation with a plasmid containing the wild-type SPO11 gene. Mitotic intrachromosomal recombination frequencies were unaffected by spo11-1. This is the first demonstration of a gene product required for recombination between homologs as well as recombination within chromosomes during meiosis.
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Bhat, N. M., M. M. Bieber, C. J. Chapman, F. K. Stevenson, and N. N. Teng. "Human antilipid A monoclonal antibodies bind to human B cells and the i antigen on cord red blood cells." Journal of Immunology 151, no. 9 (November 1, 1993): 5011–21. http://dx.doi.org/10.4049/jimmunol.151.9.5011.
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Abstract We describe two independently derived human mAb, A6(H4C5) and 216, initially selected for their reactivity to the lipid A domain of bacterial LPS, which also react with the following Ag: the i Ag present on cord RBC, a ligand on human B lymphocytes, and to certain autoantigens, defining these mAb as polyreactive. Both mAb have specific affinity for a carbohydrate epitope consisting minimally of a disaccharide with an acyl substitution at the 2-carbon position. Structural examination of the diverse Ag recognized by the two antibodies reveals the presence of this carbohydrate structure required for antibody binding. A6(H4C5) and 216 are IgM in isotype, but differ in their L chain expression. Molecular analysis shows that both the mAb are encoded by a highly conserved VH4 gene, designated VH4-21. This gene encodes a number of autoantibodies, particularly cold agglutinins. Specific recognition of lipid A and of a carbohydrate epitope on B lymphocytes by the two human mAb suggests a dual function for the highly conserved VH4-21 gene in antibacterial response and in B cell development and regulation.
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Wei, Junping, Mark Wunderlich, Chad Harris, Benjamin Mizukawa, Yi Zheng, David A. Williams, and James C. Mulloy. "Rac GTPases Are Required for MLL-AF9-Induced Mixed Lineage Leukemia." Blood 112, no. 11 (November 16, 2008): 3368. http://dx.doi.org/10.1182/blood.v112.11.3368.3368.
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Abstract The t(9;11) translocation fusion gene MLL-AF9 (MA9) is commonly found in acute myeloid and lymphoid leukemia and is associated with intermediate to poor outcome. The specific signaling pathways downstream of MA9 are still poorly understood. It has recently been reported that MA9 leukemia cells express higher levels of the small GTPase protein Rac and CDC42 when compared to in vitro MA9 immortal cells in a murine model. To determine the importance of Rac GTPase signaling in MA9-induced transformation, we used an MA9 leukemia model we recently established involving MA9 fusion gene expression in human CD34+ cells. Treatment with the Rac specific inhibitor NSC23766, or transient knockdown of Rac expression by RNAi, induced rapid apoptosis in MA9 cells but not in normal cord blood or t(8;21) translocation fusion gene AML-ETO expressing cells. These data demonstrate that the Rac signaling pathway plays a critical role in the growth and survival of MA9 leukemia cells. To extend this work to an in vivo genetic model, we used mice deficient in Rac2 (Rac2-KO) or with floxed alleles of Rac1 in mice transgenic for Mx-Cre. Leukemia development was compared in mice transplanted with MA9-transduced wild type, Rac1 or Rac2 deficient low density bone marrow cells. Poly I:C injections were performed 2 weeks after transplantation to delete Rac1. Rac deletion was confirmed by PCR and western blot analysis. Mice that received either WT or Rac1−/ − MA9 expressing cells uniformly developed AML and died at 3 to 5 months. Mice transplanted with Rac2-KO cells expressing MA9 showed a decreased incidence and increased latency (6 to 10 months) of AML development despite the persistent engraftment of MA9-expressing cells. All three groups of mice maintained MA9 EGFP+ cells in peripheral blood over the entire experiment, and eventually gave rise to similar end stage AML with a Gr-1+/Mac-1+/Kit+/B220-/CD3- phenotype and myelomonocytic blast morphology. Combined with our observation in human CD34+ cord blood cells transduced with MA9, these in vitro and in vivo data indicate that MA9-mediated transformation and survival requires Rac and their downstream effectors. Rac2 signaling appears to be particularly important in the murine MA9 AML model. Therapeutic targeting of Rac could be a unique and important approach to treating MLL leukemia.
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Matsuoka, Shuji, Hiromichi Tsurui, Masaaki Abe, Kazuo Terashima, Kazuhiro Nakamura, Yoshitomo Hamano, Mareki Ohtsuji, et al. "A Monoclonal Antibody to the α2 Domain of Murine Major Histocompatibility Complex Class I that Specifically Kills Activated Lymphocytes and Blocks Liver Damage in the Concanavalin A Hepatitis Model." Journal of Experimental Medicine 198, no. 3 (July 28, 2003): 497–503. http://dx.doi.org/10.1084/jem.20021301.
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We earlier found that a rat monoclonal antibody (mAb) RE2 can induce rapid death of murine activated, but not resting, lymphocytes and lymphocyte cell lines, in a complement-independent manner, a cell death differing from typical apoptosis or necrosis. We here found that this cell death is independent of pathways involving Fas, caspase, and phosphoinositide-3 kinase. With the advantage of producing human B cell line transfectants with stable expression of human/mouse xeno-chimeric MHC class I genes, we found that RE2 epitope resides on the murine class I α2 domain. However, the α3 domain plays a key role in transducing the death signal, which mediates extensive aggregation of the MHC class I-integrin-actin filament system, giving rise to membrane blebs and pores. In mouse models with T/NKT cell activation-associated fulminant hepatitis, administration of mAb RE2 almost completely inhibited the development of liver cell injuries. Taken collectively, this form of cell death may be involved in homeostatic immune regulation, and induction of this form of cell death using the mAbs may be potentially therapeutic for subjects with immunological diseases mediated by activated lymphocytes.
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Shibata, T., V. Kindler, Y. Chicheportiche, P. Vassalli, and S. Izui. "Interleukin 3 perfusion prevents death due to acute anemia induced by monoclonal antierythrocyte autoantibody." Journal of Experimental Medicine 171, no. 5 (May 1, 1990): 1809–14. http://dx.doi.org/10.1084/jem.171.5.1809.
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We have evaluated the therapeutic activity of rIL-3, in comparison with recombinant granulocyte-macrophage CSF (rGM-CSF) and recombinant erythropoietin (rEpo), on a lethal form of acute anemia induced by a single injection of a monoclonal IgG1 anti-mouse RBC (MRBC) autoantibody. Continuous perfusion of rIL-3 before the administration of anti-MRBC mAb prevented animals from the death due to anemia with a rapid recovery in greater than 90% of the cases, while only partial protection (one third of the cases) was obtained by rEpo perfusion, and no protection by rGM-CSF. Since the anti-MRBC mAb induced a marked agglutination of RBC in spleens and livers, and subsequent hemodynamic failure may be an additional contributing factor to the animals' death, the activation of Fc gamma receptor-dependent phagocytosis by rIL-3, as well as the increased number of monocytes/macrophages resulting from rIL-3 perfusion, may also facilitate rapid elimination of these agglutinated RBC, resulting in the further amelioration of the animals' survival. Our results suggest that the therapeutic effect of rIL-3 on anti-MRBC autoantibody-induced anemia is achieved by: (a) its activity to promote the growth and differentiation of erythroid progenitors responsive to Epo and of monocyte/macrophage lineage; and (b) its activity to enhance the phagocytic activity of macrophages to efficiently eliminate agglutinated RBC in spleens and livers.
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Meng, Zhen, Zhengyi Wang, Bingshi Guo, Wei Cao, and Huaqiong Shen. "TJC4, a Differentiated Anti-CD47 Antibody with Novel Epitope and RBC Sparing Properties." Blood 134, Supplement_1 (November 13, 2019): 4063. http://dx.doi.org/10.1182/blood-2019-122793.
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Introduction Tumor cells overexpress CD47 which engages signal-regulatory protein (SIRPa) on macrophages to deliver a "do not eat" signal to avoid being phagocytosed. Blocking CD47 using SIRPa-Fc or anti-CD47 antibodies (Ab) has emerged as a promising strategy to neutralize CD47 and promote tumor eradication. However, targeting CD47 led to significant anemia and thrombocytopenia in both pre-clinical studies and phase I trials as CD47 is also expressed on normal red blood cells (RBCs) and platelets. I-Mab has developed a novel CD47 antibody, TJC4 also known as TJ011133, which was endowed with an RBC sparing property and unique binding epitope, may have better safety profile based on the pre-clinical data. Methods A naïve human single chain variable fragment (ScFv) library was subjected for the binders to human CD47-extracellular domain (ECD). All the binders with unique sequences were converted to full antibodies and screened against human RBCs and tumor cells, leading to the discovery of TJC4. A series of head to head experiments have been performed with other CD47 antibodies to compare the in vitro RBC binding and hemagglutination, ability to block the CD47-SIRPa interaction and enhance the macrophage mediated phagocytosis of tumor cells. Different in vivo tumor models were employed to evaluate the anti-tumor efficacy of TJC4 either by mono or combination treatment. In addition, a comprehensive analysis of the hematological parameters was assessed in cynomolgus monkeys receiving a single intravenous infusion or weekly repeated injections. To explore the underlined mechanism of the RBC sparing properties of TJC4, the binding pose and epitope were identified by X-ray crystallography and the influence of CD47 glycosylation in RBCs were further examined. Results TJC4 is a fully human anti-CD47 IgG4 antibody that shares a similar binding affinity to human and cynomolgus monkey CD47. Like other anti-CD47 antibodies, TJC4 blocks the interaction of CD47 and SIRPa, leading to the enhanced macrophage phagocytosis of various CD47+ tumor cell lines and primary AML cells. Mono-treatment of TJC4 completely eradicated tumor cells in a Raji cell xenograft model and significantly extended the overall survival of treated mice in an AML model. When combined with Rituximab, TJC4 showed a superior efficacy in a DLBCL model over the mono-treatment group. TJC4 has unique RBC sparing properties as evident by the negligible binding to healthy human RBCs and platelets respectively. Single dose or repeat dose treatment of TJC4 minimally and transiently impacts RBCs in cynomolgus monkeys and no other safety findings were observed up to the highest dose (100 mg/kg). No impact was observed in platelets. The unique functional properties of TJC4 can be explained in part by its structure when in complex with CD47, which reveals an almost straight head-to-head binding and a novel conformational epitope that is distinct from other CD47 antibodies. Upon the structural analysis of the binding epitope, we identified a potential N-linked glycosylation site located nearby the two critical epitopes on the CD47 protein. Due to the nature of the high glycosylation degree of membrane proteins by RBCs, the N-linked glycan is hypothesized to function as a "shield" to block the exposure of the epitopes and prevent the TJC4 binding to human RBCs. This hypothesis is validated by the restoration of TJC4 binding to the deglycosylated RBCs after the PNGase treatment. Conclusion In summary, TJC4 is a next generation therapeutic anti-CD47 antibody that is devoid of the hematological liabilities while maintaining anti-tumor efficacy. These attributes of TJC4 differentiate it from other CD47 targeting agents currently in clinical evaluation. Disclosures Meng: I-Mab Biopharma: Employment, Equity Ownership. Wang:I-Mab Biopharma: Employment, Equity Ownership. Guo:I-Mab Biopharma: Employment, Equity Ownership. Cao:I-Mab Biopharma: Employment, Equity Ownership. Shen:I-Mab Biopharma: Employment, Equity Ownership.
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Haagen, I. A., A. J. Geerars, M. R. Clark, and J. G. van de Winkel. "Interaction of human monocyte Fc gamma receptors with rat IgG2b. A new indicator for the Fc gamma RIIa (R-H131) polymorphism." Journal of Immunology 154, no. 4 (February 15, 1995): 1852–60. http://dx.doi.org/10.4049/jimmunol.154.4.1852.
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Abstract Rat mAbs receive considerable interest for immunologic intervention in man. The rat IgG2b isotype has previously been found to be optimally active both in vivo and in vitro. We found that both a rat IgG2b CD3 mAb and a monovalent hybrid rat IgG2b-mouse IgG1 bispecific Ab triggered T cell activation in PBMC. Inhibition analyses with mAb blocking different human IgG Fc receptors (Fc gamma R) showed a dimorphic pattern. In donors expressing an Fc gamma RIIa-R/R131 allotype (previously defined on the basis of interaction with mouse (m) IgG1 as "high responder") anti-Fc gamma RI mAb 197 inhibited rat IgG2b induced T cell mitogenesis almost completely. In Fc gamma RIIa-H/H131 ("low responder" allotype) donors, however, both anti-Fc gamma RI mAb 197 and anti-Fc gamma RII mAb IV.3 were essential for optimal inhibition of mitogenesis. T cell proliferation experiments performed with the use of Fc gamma R-transfected fibroblasts as accessory cells showed the high affinity Fc gamma RIa (CD64) to interact with both rat IgG2b and rat IgG2b-mlgG1 hybrid CD3 mAb. The use of the two types of Fc gamma RIIa (CD32)-transfectants instead showed rat IgG2b CD3 mAb to interact solely with the IIa-H/H131 allotype. Interestingly, rat IgG2b-mlgG1 hybrid mAb did not interact effectively with this low affinity Fc gamma R. This suggests a requirement for only one rat IgG2b H chain for Fc gamma RIa-mediated binding, whereas two identical H chains seem to be necessary for proper interaction with Fc gamma RIIa. Ab-sensitized RBC-rosette experiments performed with the use of a rat IgG2b anti-NIP mAb confirmed the interaction pattern observed with rat CD3 mAb, supporting the phenomena to be isotype-, and not mAb-, dependent. These analyses point to a unique reactivity pattern for rat IgG2b Abs, interacting both with the high affinity Fc gamma RIa in all donors and Fc gamma RIIa of individuals expressing the IIa-H131 allotype.
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Mizukawa, Benjamin, Junping Wei, Mark Wunderlich, Fu-Sheng Chou, Yi Zheng, David A. Williams, and James C. Mulloy. "Bcl-XL Is a Critical Mediator of Rac Signaling in MLL-AF9-Induced Acute Myeloid Leukemia." Blood 114, no. 22 (November 20, 2009): 1971. http://dx.doi.org/10.1182/blood.v114.22.1971.1971.
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Abstract Abstract 1971 Poster Board I-994 Rac GTPases have been found to play an important role in MLL-AF9 (MA9) acute myeloid leukemia. Rac activity is increased in MA9 leukemia cells, and inhibition of Rac by shRNA or the Rac specific inhibitor NSC23766 induces rapid apoptosis in MA9 cells but not in normal cord blood cells or cells expressing the t(8;21) fusion protein AML1-ETO (Wei et al, Cancer Cell 2008). More recently, we demonstrated that MA9-mediated leukemogenesis is significantly delayed in Rac2 knockout (Rac2 KO) low-density bone marrow (LDBM) cells in contrast to the rapid development of leukemia seen after transplantation of wild-type (WT) murine LDBM expressing MA9. Loss of Rac2 has previously been shown to affect Bcl-2 family expression, specifically resulting in decreased levels of the pro-survival protein Bcl-xL and increased levels of the pro-apoptotic protein Bad (Yang et al, Immunity 2000). Here we investigate the role of Bcl-xL in mediating the effects of Rac signaling in MA9 leukemia. Interestingly, treatment with NSC23766 inhibits Bcl-xL expression in MA9 cells. This effect was also seen in Bcl-xL-transduced MA9 cells, implicating a role for Rac in post-transcriptional regulation of Bcl-xL expression in addition to the transcriptional effect that has been previously reported. Significantly fewer Annexin V+ cells were found in NSC23766-treated MA9 cells expressing ectopic Bcl-xL protein when compared to control MA9-pBabe cells, demonstrating that excess Bcl-xL expression can partially rescue the phenotype associated with Rac inhibition. To target the Bcl-2 survival signals in MA9 cells, we used the small-molecule pan-Bcl-2 inhibitor GX15-070 and found a significant inhibition of survival and growth of treated MA9 cells at much lower doses than required for control cells not expressing MA9. Similarly, shRNA knockdown of Bcl-xL in MA9 cells inhibited growth. To determine whether ectopic expression of Bcl-xL could compensate for loss of Rac2 in vivo and restore short latency MA9-mediated leukemogenesis, LDBM was harvested from Rac2 KO mice and transduced with a retroviral vector expressing MA9 and a second retrovirus to express either Bcl-xL or empty vector control. Unsorted cells were transplanted into congenic CD45.1 mice to also establish a competitive assay between singly transduced cells expressing MA9 alone and doubly transduced cells co-expressing both MA9 and Bcl-xL. All recipients of Rac2 KO cells from the MA9+empty vector transduction group remain alive at 300 days from transplant. All recipients of Rac2 KO cells from the MA9+Bcl-xL transduction group died of leukemia with a mean latency of 184 days (range 102 — 280 days), and analyzable tumors showed co-expression of MA9 and Bcl-xL. Two mice that died later in this cohort (day 224 and day 280) also showed a second population of cells expressing MA9 without co-expression of Bcl-xL. Experiments are underway to determine which population has leukemogenic ability upon secondary transplant. Mice that received WT LDBM transduced with MA9+Bcl-xL died of leukemia with the expected mean latency of 190 days for this model (range 130 — 250 days). In this group, the number of WT MA9 tumors co-expressing Bcl-xL was in equal proportion to tumors co-expressing empty vector, suggesting the advantage bestowed by Bcl-xL overexpression is more pronounced in the context of Rac2 deficiency. Together, these findings suggest a critical role for Rac signaling through the Bcl-xL pathway in MA9 leukemia and support therapeutic approaches targeting this pathway in MLL leukemia. Disclosures: No relevant conflicts of interest to declare.
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Oosterwijk-Wakka, J. C., P. M. M. Van Hauten, Eijk S. Lafferty-Van, R. J. A. Maas, J. Cany, H. Dolstra, P. F. A. Mulders, and E. Oosterwijk. "Natural killer cells in combination with mAb cG250: a potential new therapy approach for RCC." European Urology Supplements 18, no. 8 (October 2019): e3138. http://dx.doi.org/10.1016/s1569-9056(19)33380-9.
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Mizukawa, Benjamin, Mahesh Shrestha, Mark Wunderlich, Fu-Sheng Chou, Andrea Griesinger, Ashish Kumar, Yi Zheng, David A. Williams, and James C. Mulloy. "Rac GTPase Survival Signaling Through Anti-Apoptotic Bcl-2 Proteins Reveals a Target for Combination Therapy in MLL-AF9 Acute Myeloid Leukemia." Blood 118, no. 21 (November 18, 2011): 1536. http://dx.doi.org/10.1182/blood.v118.21.1536.1536.
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Abstract Abstract 1536 The Rac family of small Rho GTPases has attracted interest as a therapeutic target in hematologic malignancies due to their central role in coordinating diverse cellular processes such as adhesion, cytoskeletal organization, proliferation, and survival. Rac activity is increased in MLL-AF9 (MA9) acute myeloid leukemia, and Rac inhibition using the small-molecule NSC23766 induces apoptosis in MA9 cells. We recently found that loss of Rac2 delays development of MA9 leukemia in a murine genetic model. Furthermore, latency of disease can be rescued by ectopic expression of Bcl-xL in the Rac2 knockout cells implicating Rac2 regulation of pro-survival Bcl-2 proteins in MA9 leukemogenesis. Whether Rac survival signaling through Bcl-2 proteins can be exploited therapeutically in the developed MA9 leukemia is untested. We hypothesized that Rac2 signaling is critical for MA9 leukemia cell survival, and that inhibition of Rac or downstream Bcl-2 proteins could be an effective therapeutic strategy alone or in combination. We tested our hypothesis in MA9 cells derived from human CD34+ umbilical cord blood (UCB) cells expressing MA9 by retroviral transduction. Lentiviral knockdown was used to determine the specific contribution of Rac2 to MA9 cell survival and maintenance. Decreased levels of Bcl-xL and Bcl-2 were seen in MA9 cells expressing Rac2-targeting shRNA, compared to non-targeting control. Rac2 knockdown induced apoptosis and impaired growth of MA9 cells in culture. Furthermore, Rac2 deficiency reduced in vitro colony-forming ability indicating impairment of the clonogenic MA9 cell. MA9 cells expressing two different Rac2 shRNA vs. non-targeting control were injected into NOD/LtSz-scid-SGM3 (NSS) mice to determine whether Rac2 deficiency impairs engraftment and progression of the MA9 leukemia stem cell in a xenotransplantation assay. Flow cytometric analysis of bone marrow aspirates showed markedly reduced MA9 engraftment in the Rac2 knockdown groups. Whereas all mice in the control group eventually died of MA9 leukemia (N=5), only one death from MA9 cells expressing Rac2 shRNA was seen in the knockdown groups (N=10). We next evaluated the effects of direct inhibition of Bcl-2 proteins downstream from Rac using the BH3-mimetic ABT-737. Three different MA9 cell lines, as well as the THP-1 cell line bearing an MLL-AF9 fusion, were highly sensitive (IC50 ∼30 nM) to ABT-737, with no toxicity seen in control UCB cells in the dose range tested. To determine in vivo efficacy, ABT-737 was administered to NSS mice engrafted with human MA9 cells. End of treatment aspirates showed a marked decrease in leukemia engraftment in the ABT-737 treatment group compared to vehicle control (<2% vs. 43%, respectively; p < 0.002). To determine whether combined inhibition of Rac and Bcl-2 proteins produces an additive effect, MA9 cell lines were treated with serial doses of ABT-737 in the presence or absence of a sub-therapeutic dose of NSC23766 (20 μM). Normal UCB cells, as well as HL-60 and two cell lines expressing the t(8;21) fusion protein AML1-ETO, were tested as controls. MA9 cell lines were exquisitely sensitive to the combined inhibition of Rac and Bcl-2 proteins, with a 2.5- to 9-fold reduction in the IC50 of ABT-737 in the presence of 20 μM NSC23766. In contrast, little to no effect was seen in any of the control cells. Our findings demonstrate for the first time that specific inhibition of Rac2 induces apoptosis and impairs the clonogenic MA9 cell in vitro and in vivo, in association with decreased expression of downstream pro-survival Bcl-2 proteins, and identify a pathway that can be exploited therapeutically. We conclude that survival signaling through Rac and Bcl-2 family proteins can be effectively targeted with small-molecule inhibitors alone and in combination in MLL-AF9 acute myeloid leukemia. Disclosures: No relevant conflicts of interest to declare.
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Rottini, G., L. Roncelli, G. Narchi, P. Patriarca, G. Protani, S. Perissutti, and F. Tedesco. "Monoclonal antibodies as probes to investigate the molecular changes of C5 associated with the different stability of the molecule on sheep erythrocytes and Escherichia coli 0111:B4." Journal of Immunology 146, no. 2 (January 15, 1991): 643–47. http://dx.doi.org/10.4049/jimmunol.146.2.643.
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Abstract The fifth C component (C5) exhibits a different stability when bound to sheep E or Escherichia coli 0111:B4, being fairly stable on the bacterial intermediate sensitized E. coli 0111:B4 coated with C components up to C5 (BAC1-5) and extremely labile on the RBC intermediate sensitized sheep E coated with C components up to C5 (EAC1-5). We examined the possibility that molecular changes of membrane-bound C5 might be responsible for the different functional behavior of the two intermediates using mAb to C5 and sensitive immunoassays to detect bound C5. The decay of EAC1-5 over 30 min of incubation at 37 degrees C was associated with a significant drop in the reactivity of bound C5 with three of four mAb used. These results contrasted with those obtained with BAC1-5, which showed unchanged reactivity with all mAb tested over the same period of incubation. The effect of mAb on the activity of C5 was then investigated in an attempt to relate the change of the reactivity pattern of EAC1-5 with the functional modification of bound C5. MAb 1.5 and 1.6 were the only antibodies that interfered with the functional activity of C5, although through a different mechanism. In particular, mAb 1.5 was active both on fluid-phase and on membrane-bound C5 and is therefore likely to interact with the binding site for the late components on C5. Conversely, mAb 1.6 was only effective on fluid-phase C5 and acted by promoting a decay of BAC1-5 similar to the spontaneous decay of EAC1-5. We suggest that the bacterial outer membrane may protect C5 from functional decay and that mAb 1.6 interferes with the stabilizing effect of the bacteria in an as yet unclear manner.
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Stefanová, I., and V. Horejsí. "Association of the CD59 and CD55 cell surface glycoproteins with other membrane molecules." Journal of Immunology 147, no. 5 (September 1, 1991): 1587–92. http://dx.doi.org/10.4049/jimmunol.147.5.1587.
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Abstract mAb against human glycosyl-phosphatidylinositol-linked leucocyte surface Ag CD59 and CD55 immunoprecipitated from detergent lysates of HPB ALL cell line in addition to the respective Ag a common 80-kDa glycoprotein component and (glyco)lipids. The 80-kDa glycoprotein is different from otherwise similar CD44 Ag. The CD59 immunoprecipitate contained also a small amount of the CD55 glycoprotein and the CD55 immunoprecipitate minute amount of the CD59 Ag. These results are interpreted in terms of existence of noncovalent complexes resistant to dissociation by mild detergents and consisting of the 80-kDa glycoprotein, CD59 and CD55 glycoproteins, relatively tightly bound (glyco)lipids and possibly other so far unidentified components. These complexes contain probably also other glycosyl-phosphatidylinositol-linked Ag, as an anti-CD48 mAb immunoprecipitated also an apparently very similar complex. The complexes immunoprecipitated by mAb against the CD55, CD59, and CD48 Ag also contain a protein kinase activity. This type of complexes could not be demonstrated in several other cell types such as RBC, PBMC, and HeLa cells. However, a qualitatively very similar set of components was immunoprecipitated from the murine thymoma EL-4 cell line by an anti-Thy-1 mAb.
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Swanson, Rebecca L., Natalie J. Morey, Paul W. Doetsch, and Sue Jinks-Robertson. "Overlapping Specificities of Base Excision Repair, Nucleotide Excision Repair, Recombination, and Translesion Synthesis Pathways for DNA Base Damage in Saccharomyces cerevisiae." Molecular and Cellular Biology 19, no. 4 (April 1, 1999): 2929–35. http://dx.doi.org/10.1128/mcb.19.4.2929.
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ABSTRACT The removal of oxidative damage from Saccharomyces cerevisiae DNA is thought to be conducted primarily through the base excision repair pathway. The Escherichia coliendonuclease III homologs Ntg1p and Ntg2p are S. cerevisiae N-glycosylase-associated apurinic/apyrimidinic (AP) lyases that recognize a wide variety of damaged pyrimidines (H. J. You, R. L. Swanson, and P. W. Doetsch, Biochemistry 37:6033–6040, 1998). The biological relevance of theN-glycosylase-associated AP lyase activity in the repair of abasic sites is not well understood, and the majority of AP sites in vivo are thought to be processed by Apn1p, the major AP endonuclease in yeast. We have found that yeast cells simultaneously lacking Ntg1p, Ntg2p, and Apn1p are hyperrecombinogenic (hyper-rec) and exhibit a mutator phenotype but are not sensitive to the oxidizing agents H2O2 and menadione. The additional disruption of the RAD52 gene in the ntg1 ntg2 apn1 triple mutant confers a high degree of sensitivity to these agents. The hyper-rec and mutator phenotypes of the ntg1 ntg2 apn1 triple mutant are further enhanced by the elimination of the nucleotide excision repair pathway. In addition, removal of either the lesion bypass (Rev3p-dependent) or recombination (Rad52p-dependent) pathway specifically enhances the hyper-rec or mutator phenotype, respectively. These data suggest that multiple pathways with overlapping specificities are involved in the removal of, or tolerance to, spontaneous DNA damage in S. cerevisiae. In addition, the fact that these responses to induced and spontaneous damage depend upon the simultaneous loss of Ntg1p, Ntg2p, and Apn1p suggests a physiological role for the AP lyase activity of Ntg1p and Ntg2p in vivo.
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Li, Chao Ying, and Jin Qing Jiang. "Development of Monoclonal Antibody Based Heterologous Immunoassay for Ractopamine Residue." Advanced Materials Research 461 (February 2012): 58–61. http://dx.doi.org/10.4028/www.scientific.net/amr.461.58.
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This study aimed to develop a monoclonal antibody based icELISA method for Ractopamine (Rac) residue. For this purpose, mixed anhydride method was employed to synthesize the immunogen of Rac-BSA and 1, 4-butanediol diglycidyl ether was used to prepare the coating antigen of Rac-OVA, thus pursue the heterologous sensitivity. Through cell fusion technology, four Hybridoma named R1-B5, R2-B3, R2-C6, and R4-C8 were screened out, and the Kas of all mAbs were between 2.7 and 4.8×109 L/mol. Based on the R1-B5 mAb, a heterologous icELISA standard curve was developed. The working range was from 0.013 to 33.7 ng/mL, with LOD and IC50 value of 0.007 ng/mL and 0.67 ng/mL, respectively. Therefore, this icELISA can be used for detecting Rac residue in animal products.
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MacLeod, C. L., K. Finley, D. Kakuda, C. A. Kozak, and M. F. Wilkinson. "Activated T cells express a novel gene on chromosome 8 that is closely related to the murine ecotropic retroviral receptor." Molecular and Cellular Biology 10, no. 7 (July 1990): 3663–74. http://dx.doi.org/10.1128/mcb.10.7.3663.
Full textAbstract:
A novel cDNA clone (20.5) which is differentially expressed between two closely related T-lymphoma cell clones was isolated by subtraction-enriched differential screening. SL12.4 cells, from which the cDNA was isolated, have characteristics of thymocytes at an intermediate stage in development. A sister cell clone derived from the same tumor, SL12.3, does not express this mRNA, has a distinct phenotype, and expresses fewer genes required for mature T-cell function. The cDNA sequence predicts a highly hydrophobic protein (approximately 49.5 kilodaltons) which contains seven putative membrane spanning domains. The gene was expressed on concanavalin A-activated T lymphocytes and was designated Tea (T-cell early activation gene). The Tea gene mapped to chromosome 8 and appeared to be conserved among mammalian and avian species. The Tea gene is distinct from, but bears extensive amino acid and DNA sequence similarity with, the murine ecotropic retroviral receptor which is encoded by the Rec-1 gene. Neither gene product displayed significant homology with other known transmembrane-spanning proteins. Thus, the Tea and Rec-1 genes establish a new family encoding multiple membrane-spanning proteins.
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Conger, J. D., H. J. Sage, and R. B. Corley. "Diversity in the available repertoire of murine antibodies reactive with bromelain-treated isologous erythrocytes." Journal of Immunology 143, no. 12 (December 15, 1989): 4044–52. http://dx.doi.org/10.4049/jimmunol.143.12.4044.
Full textAbstract:
Abstract Antibodies specific for bromelain-treated mouse RBC (BrMRBC) are of interest as models of "natural autoantibodies" and because of their primary source is Ly-1+ (CD5+) B cells. In earlier work by others, anti-BrMRBC hybridomas prepared by using CBA or NZB "spontaneously activating" peritoneal B cells were all found to produce mAb with a single common H chain V region sequence, by using a novel gene (VH11p), and a single common L chain V region sequence, a member of the Vk9 group (VkBrMp). We prepared anti-BrMRBC hybridomas by using LPS-activated B10.A splenic B cells in order to reveal the maximum available diversity in this repertoire. Data based on binding studies, Northern blot analyses with V region-specific probes, and mRNA nucleotide sequence analysis indicated that there is combining-site diversity in the repertoire of anti-BrMRBC hybridomas. There was considerable variation in trimethylammonium (a constituent of phosphatidyl choline) binding efficiency, and one of the anti-BrMRBC mAb showed no detectable binding. Northern blot analyses indicated 6 of 11 mAb to be of the VH11p/VkBrMp type, including one dual reactive anti-[BrMRBC + SRBC] mAb. Sequence analyses of the H chain V regions of four of the non-VH11p mAb revealed utilization of four distinct VH, three of which are very similar to the VH expressed by Ly-1+ B cell clones or lymphomas, as reported by others. However, because the VH11p/VkBrMp-type mAb were all relatively efficient at lysing BrMRBC and binding trimethylammonium, we suggest that affinity considerations may determine the selective predominance of B cells with this V region configuration from an available repertoire of considerable diversity.
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Sung, S. S., and J. A. Walters. "Identification and characterization of a hamster monoclonal antibody anti-2.28 directed against a 70-kilodalton activation antigen on human monocytes." Journal of Immunology 142, no. 6 (March 15, 1989): 1903–8. http://dx.doi.org/10.4049/jimmunol.142.6.1903.
Full textAbstract:
Abstract Hamster mAb against activated human monocytes were examined for their reactivities against monocyte activation Ag. One mAb, anti-2.28, stained only monocytes activated with LPS plus IFN-gamma, but not unactivated peripheral blood monocytes, polymorphonuclear leukocytes, lymphocytes, RBC, and platelets. However, it stained peripheral blood T cells activated with PMA plus anti-CD3 and peripheral blood and tonsillar B cells activated with PMA plus anti-mu. Of the 35 cell lines of diverse origin examined for immunofluorescence staining by anti-2.28, only EBV-transformed cell lines showed strong staining by this mAb. One pre-B cell line, Nalm-12, could be induced by PMA to exhibit intermediate staining. Immunoprecipitation studies identified the 2.28 Ag as a 70- to 85-kDa monomer. Immunofluorescence staining, immunoprecipitation, and peptide mapping studies indicated that 2.28 was different from a number of monocyte and lymphocyte surface Ag including Mo3e, B-4 (CD19), B-5, CD39, and the G28-8 Ag Bgp 95. These studies suggest that 2.28 may be a novel hemopoietic non-lineage-specific activation Ag.
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Lin, S. L., D. Derr, and J. E. Hildreth. "A monoclonal antibody against a novel 20-kDa protein induces cell adhesion and cytoskeleton-dependent morphologic changes." Journal of Immunology 149, no. 8 (October 15, 1992): 2549–59. http://dx.doi.org/10.4049/jimmunol.149.8.2549.
Full textAbstract:
Abstract A new murine IgA mAb (JKT.M1), developed against Jurkat T cells chronically infected with HIV IIIB induces in vitro homotypic aggregation in several hemopoietic cell lines. The JKT.M1 Ag is expressed on a wide variety of cell types including human lymphocytes, monocytes, platelets, RBC, human umbilical vein endothelial cells, many T cell lines, myelomonocytic cell lines, and a primate kidney cell line. The JKT.M1 Ag shows differential expression on myelomonocytic cells; it is present on K562 and HL60 cell lines, which represent precursors of E and monocytes, respectively, but is not expressed on the surface of U937 and THP-1 cell lines, which appear to represent intermediate cell types of the monocytic cell lineage. However, the JKT.M1 Ag is expressed on mature peripheral blood monocytes and the MonoMac cell line. Immunoprecipitation from cell lysates (Jurkat, SupT1, PBMC, MonoMac) with the JKT.M1 mAb yields a 20-kDa Ag with few if any carbohydrate residues as determined by N-glycanase and neuraminidase treatments. The pI appears acidic by two-dimensional gel analysis, and the nonreduced form migrates more slowly than the reduced form when analyzed by SDS-PAGE suggesting the presence of intramolecular disulfide bridge(s). JKT.M1 mAb-induced cell adhesion is shown to be divalent cation- and temperature-dependent. The adhesion induced by JKT.M1 mAb is inhibited by 20 microM cytochalasin B and also by 2 mM 2-deoxyglucose plus 10 mM sodium azide suggesting that cytoskeletal changes and metabolic energy are required. Aggregation induced by JKT.M1 appears to be independent of CD43, CD44, and VLA4 (CD29/CD49d), mAb against which have also been shown to induce homotypic cell adhesion. Anti-CD18 mAb have been shown to inhibit homotypic aggregation in other studies but failed to do so in the present study. Thus JKT.M1-induced adhesion also appears to be independent of CD18, the beta-chain of leukocyte integrins. However, like mAb against LFA-1, immobilized JKT.M1 stimulates a T cell line to undergo dramatic morphologic changes which could be enhanced by the addition of phorbol ester. These data suggest that the novel 20-kDa molecule recognized by the JKT.M1 mAb may trigger cell adhesion through a previously undescribed mechanism.
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MacLeod, C. L., K. Finley, D. Kakuda, C. A. Kozak, and M. F. Wilkinson. "Activated T cells express a novel gene on chromosome 8 that is closely related to the murine ecotropic retroviral receptor." Molecular and Cellular Biology 10, no. 7 (July 1990): 3663–74. http://dx.doi.org/10.1128/mcb.10.7.3663-3674.1990.
Full textAbstract:
A novel cDNA clone (20.5) which is differentially expressed between two closely related T-lymphoma cell clones was isolated by subtraction-enriched differential screening. SL12.4 cells, from which the cDNA was isolated, have characteristics of thymocytes at an intermediate stage in development. A sister cell clone derived from the same tumor, SL12.3, does not express this mRNA, has a distinct phenotype, and expresses fewer genes required for mature T-cell function. The cDNA sequence predicts a highly hydrophobic protein (approximately 49.5 kilodaltons) which contains seven putative membrane spanning domains. The gene was expressed on concanavalin A-activated T lymphocytes and was designated Tea (T-cell early activation gene). The Tea gene mapped to chromosome 8 and appeared to be conserved among mammalian and avian species. The Tea gene is distinct from, but bears extensive amino acid and DNA sequence similarity with, the murine ecotropic retroviral receptor which is encoded by the Rec-1 gene. Neither gene product displayed significant homology with other known transmembrane-spanning proteins. Thus, the Tea and Rec-1 genes establish a new family encoding multiple membrane-spanning proteins.
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Embury, Stephen H., Christine E. Baran, Colleen A. Hefner, Christi K. Seto, and Neil M. Matsui. "The Nature of the P-Selectin Ligands on Sickle Cells." Blood 104, no. 11 (November 16, 2004): 363. http://dx.doi.org/10.1182/blood.v104.11.363.363.
Full textAbstract:
Abstract Elucidation of the adhesive interactions that effect microvascular occlusion in sickle cell disease both increases our understanding of the pathophysiology of vasoocclusion and identifies molecular targets for the development of therapeutic interventions. Work from our laboratory has established that sickle RBC adhere to P-selectin on thrombin-activated endothelial cells and to immobilized, recombinant P-selectin in vitro (Matsui et al. Blood 98:1955, 2001) and that this adhesion can be inhibited by agents that block P-selectin (Matsui et al. Blood 100:3790, 2002). Based on these findings, we established that sickle RBC adherence to endothelial P-selectin has a substantial influence on microvascular blood flow in vivo and that blocking P-selectin enhances microvascular flow (Embury et al. Blood In Press). We reasoned that characterization of the cognate ligands for P-selectin ligand on sickle RBC could identify additional targets for therapeutic intervention. We had determined that that sickle RBC did not express the P-selectin ligand, P-selectin glycoprotein-1, but that membrane sialic acid is required for sickle RBC binding to P-selectin. Here we describe further characterization of the P-selectin binding determinants on sickle RBC membranes. We assessed the expression of sialyl Lewis X (sLeX) on sickle RBC using flow cytometry and the importance of sLeX expression to the rolling adhesion of sickle RBC to P-selectin in vitro. Using the monoclonal antibodies (mAb) HECA-452 and CSLEX-1 in flow cytometry we detected significant expression of sLeX on sickle RBC (p < 0.003 and p < 0.02, respectively) but not on non-sickle RBC (p < 0.07 and p < 0.3, respectively). Treatment of sickle RBC with sialidase caused a partial, dose dependant reduction of the level of detectable sLeX and of rolling adhesion to immobilized P-selectin (approximately 40% and 85%, respectively), which correlated positively. To assess the possible selective contribution of reticulocytes as a subset of higher sLeX expressing sickle RBC we employed dual label flow cytometry to determine whether sLeX and the transferrin receptor (CD71) are co-expressed. Using mAb YDJ1.2.2 for the transferrin receptor as a reticulocyte marker and CSLEX-1 showed that sLeX was expressed both on sickle reticulocytes and on older sickle RBC. Treatment of sickle RBC with O-sialoglycoprotein endopeptidase, which cleaves sialylated O-glycans, also reduced both their sLeX expression and rolling adhesion on P-selectin (approximately 30% and 65%, respectively). Treatment of sickle RBC with N-glycosidase F did not reduce sLeX or adhesion levels, trypsin treatment produced inconsistent effects, and phosphatidylinositol-specific phospholipase C caused a significant decrease in adhesion but not a significant reduction in sLex expression. These findings suggest that sickle RBC possess more than one type of glycoprotein as a ligand for P-selectin. We also used a solid-phase binding assay to detect a significant level of P-selectin binding to membrane lipids extracted from sickle RBC. Thus, the P-selectin binding determinants on sickle RBC include sialic acid, sLeX, O-linked glycans, PI-linked glycoproteins, and glycolipids. Each of these P-selectin ligands represents a potential target of new adhesion blocking drugs for the treatment of sickle cell disease.
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Engel, P., Y. Nojima, D. Rothstein, L. J. Zhou, G. L. Wilson, J. H. Kehrl, and T. F. Tedder. "The same epitope on CD22 of B lymphocytes mediates the adhesion of erythrocytes, T and B lymphocytes, neutrophils, and monocytes." Journal of Immunology 150, no. 11 (June 1, 1993): 4719–32. http://dx.doi.org/10.4049/jimmunol.150.11.4719.
Full textAbstract:
Abstract CD22 is a B lineage-restricted member of the Ig superfamily that serves as an adhesion receptor expressed by mature B lymphocytes. In this study, the ability of different cell types to attach to COS cells transiently transfected with a full-length CD22 cDNA (COS-CD22) was examined to determine the cellular distribution of the ligand for CD22. T and B lymphocytes, monocytes, erythrocytes, and neutrophils formed specific rosettes with COS-CD22 cells at 4 degrees C. A panel of 33 new mAb directed against CD22 were developed to examine the regions of CD22 that mediate adhesion. Four of these mAb, HB22-7, -22, -23, and -33 (at 1 to 5 micrograms/ml) specifically blocked adhesion (75 to 95%) of all cell types to COS-CD22 cells. Each of these mAb cross-blocked each other's binding, suggesting that ligand binding occurs through a single region of CD22. These mAb also identify a region of CD22 distinct from those defined by previously described CD22 mAb. CD22-mediated adhesion of cell lines to COS-CD22 cells was independent of CD45RO and CDw75 expression, and it was not inhibited by mAb against known integrins. Although alpha-2,6-linked sialic acid expressed on the surface of COS cells did not serve as a ligand for CD22, the CD22 ligand may contain a critical sialic acid determinant, as neuraminidase treatment of all target cells eliminated CD22-mediated adhesion. CD22-mediated adhesion was Ca2+/Mg2+ independent, again suggesting that integrins were not involved. An inhibitory substance for CD22-mediated adhesion was found to be present in FCS and some ascites fluid. Analysis of CD22 mRNA and protein revealed that although multiple mRNA splice variants of CD22 mRNA can be detected, only a single protein isoform was detected on the cell surface. Therefore, although the identity of the CD22 ligands remains incompletely characterized, it is possible that a single major ligand is expressed by RBC and leukocytes, which binds to a single region of CD22.
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Pincus, S. H., K. Wehrly, and B. Chesebro. "Treatment of HIV tissue culture infection with monoclonal antibody-ricin A chain conjugates." Journal of Immunology 142, no. 9 (May 1, 1989): 3070–75. http://dx.doi.org/10.4049/jimmunol.142.9.3070.
Full textAbstract:
Abstract mAb 907 is directed against the envelope protein of the HIV. The epitope recognized by this antibody is expressed in moderate density on the surface of tissue culture cells infected with the LAV/HTLV-IIIB strain of HIV. We have coupled antibody 907 to ricin A chain (RAC). The antibody-RAC conjugate inhibited protein synthesis and cell growth in HIV-infected cells. An irrelevant antibody conjugated to RAC had no effect. Most important, treatment of infected cells with the conjugate markedly inhibited the production of infectious virus, as measured by the production of viral foci on susceptible monolayer cells. Exposure of HIV-infected target cells to the conjugate for as short a period as 1 h resulted in cell death. Serum of AIDS patients inhibited, but did not completely suppress, the toxicity of the 907-RAC conjugate. A second antibody, designated BM-1, which recognizes a carbohydrate Ag on the surface of virally infected cells, was conjugated to RAC. The BM-1-RAC conjugate did not kill HIV-infected cells, highlighting the importance of the target Ag. Immunotoxins produced with antibodies that recognize Ag on the surface of HIV-infected cells may have utility in the therapy of AIDS.
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Ko, Myung S., Kate K. Xu, and Marilyn J. Telen. "Mechanism of Soluble Laminin-Mediated Enhancement of Sickle Red Cell Adhesion to Endothelial Cells." Blood 108, no. 11 (November 16, 2006): 1237. http://dx.doi.org/10.1182/blood.v108.11.1237.1237.
Full textAbstract:
Abstract We have previously shown that erythrocytes from patients homozygous for hemoglobin S (SS RBC) adhere to immobilized laminins −10,11 (α5 LAM) with high affinity through the B-CAM/LU receptor. SS RBC also avidly bind soluble LAM via B-CAM/LU. In addition, the interaction between endothelial cell (EC) integrin αVβ3 and erythrocyte ICAM4 is a significant contributor to the adhesion of SS RBC to EC. We have recently shown that soluble LAM, either purified or in plasma, enhances SS RBC adhesion to EC. We have now studied the mechanism by which soluble LAM enhances RBC adhesion to EC and the possible relationship between LAM-enhanced and ICAM-4 mediated adhesion of SS RBC. We tested the adhesion of SS RBC to EC under various potentially adhesion-blocking conditions to determine possible modes of interaction between SS RBC, LAM, SS RBC pre-incubated in LAM (SS RBC+LAM), and EC. We first studied whether blocking RBC-bound LAM with an anti-LAM antibody (Ab) could reduce the adhesion-enhancing effects of LAM. SS RBC were pre-incubated in 10 μg/mL soluble LAM for 1 hr at 37°C. After LAM incubation, SS RBC+LAM were washed and re-incubated for 1 hr in 4C7 mAb, an anti-LAM Ab, followed by washing. Adhesion of SS RBC+LAM incubated in 4C7 was compared to adhesion of SS RBC+LAM incubated with an irrelevant Ab and SS RBC not first incubated in LAM. SS RBC+LAM showed on average more than a two-fold increase in adhesion compared to SS RBC not incubated with LAM, at shear stresses of both 2 and 5 dynes/cm2 (n=3). In contrast, SS RBC+LAM incubated in Ab 4C7 showed < a 50% increase in adhesion compared to SS RBC not pre-incubated in LAM. However, due to variability in adhesion among patient samples, these differences were not significant. In order to determine the EC structure to which SS RBC+LAM might bind, we then incubated EC for 1 hr in an IgM Ab to dystroglycan, a putative LAM receptor on EC. SS RBC+LAM were tested for adhesion to untreated EC, EC treated with anti-dystroglycan, and EC treated with a control IgM. Treatment of EC with anti-dystroglycan decreased the adhesion of SS RBC+LAM by 39.1% and 50.1% at shear stresses of 2 and 5 dynes/cm2, respectively, while EC treated with control IgM showed insignificant decreases in adhesion of 3.3% and 6.6% at shear stresses of 2 and 5 dynes/cm2, respectively (n=3; p=.0013 at 2 dynes/cm2; p=.0175 at 5 dynes/cm2). These results suggest that enhancement of SS RBC adhesion in the presence of LAM is due to LAM interaction with an EC receptor, possibly dystroglycan. We then explored whether the adhesion enhancing effects of LAM could still be observed when the interaction between erythrocyte ICAM4 and EC αVβ3 was blocked. EC were pre-treated for 1 hr with 25 μg/mL of soluble recombinant (sr) ICAM4, in order to block αvβ3 binding sites. We found that pre-incubation of SS RBC in 10 μg/mL LAM failed to enhance RBC adhesion to sr-ICAM4 treated EC (p=.0276 at 2 dynes/cm2, n=3). Similar results were obtained with the 7E3 mAb directed against αVβ3. This suggests that blocking the interaction between ICAM4 and αVβ3 abolishes adhesion despite the presence of RBC-bound LAM. Therefore, we conclude that soluble LAM enhances SS RBC adhesion to EC, probably by providing secondary adhesive interactions that can be abrogated by anti-LAM as well as anti-dystroglycan antibodies. However, the contribution of LAM to SS RBC adhesion was negligible in the absence of interaction between ICAM4 and αVβ3. Thus, the mechanism by which LAM enhances SS RBC adhesion to EC appears to be a complex process, dependent on both the binding of RBC-bound LAM to EC as well as ICAM4 interaction with EC αVβ3.