An Agonist Murine Monoclonal Antibody to the Human c-Mpl Receptor Stimulates Megakaryocytopoiesis

Thrombopoietin (TPO) is a hematopoietic growth factor that stimulates megakaryocytopoiesis and platelet production in vivo and promotes the development of identifiable megakaryocytes in vitro. We have developed a murine monoclonal antibody, BAH-1, raised against human megakaryocytic cells, which specifically recognizes the c-Mpl receptor and shows agonist activity by stimulating megakaryocytopoiesis in vitro. BAH-1 antibody specifically binds to platelets and to recombinant c-Mpl with high affinity. Similar to TPO, BAH-1 alone supported the formation of colony-forming unit-megakaryocyte (CFU-MK) colonies. The combination of BAH-1 plus interleukin-3 or of BAH-1 plus human TPO significantly increased the number of human CFU-MK colonies. In addition, BAH-1 monoclonal antibody stimulated the proliferation and maturation of primary bone marrow megakaryocytes in a dynamic heterogeneous liquid culture system. Individual large megakaryocytes as well as small megakaryocytic cells were observed in cultures of CD34+ CD41+cells in the presence of BAH-1 antibodies. Similar to TPO, BAH-1 antibody induced a significant response of murine immature megakaryocytes as observed by an increase in the detectable numbers of acetylcholinesterase-positive megakaryocytes. No effects of BAH-1 antibody were observed on colony-forming unit–granulocyte-macrophage, burst-forming unit-erythroid, or colony-forming unit-erythroid colonies. In vivo studies showed that BAH-1, alone or in combination with TPO, expands the numbers of megakaryocytic progenitor cells in myelosuppressed mice. This antibody should prove useful in understanding the structure-function aspects of the c-Mpl receptor as well as in evaluating the effects of the sustained activation of this receptor in preclinical models of severe thrombocytopenia. © 1998 by The American Society of Hematology.

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An Agonist Murine Monoclonal Antibody to the Human c-Mpl Receptor Stimulates Megakaryocytopoiesis

Blood ◽

10.1182/blood.v92.6.1981 ◽

1998 ◽

Vol 92 (6) ◽

pp. 1981-1988 ◽

Cited By ~ 29

Author(s):

Bijia Deng ◽

Naheed Banu ◽

Beth Malloy ◽

Philip Hass ◽

Jian Feng Wang ◽

...

Keyword(s):

Monoclonal Antibody ◽

Murine Monoclonal Antibody ◽

In Vivo Studies ◽

Severe Thrombocytopenia ◽

Colony Forming Unit ◽

Liquid Culture System ◽

Primary Bone ◽

American Society

Abstract Thrombopoietin (TPO) is a hematopoietic growth factor that stimulates megakaryocytopoiesis and platelet production in vivo and promotes the development of identifiable megakaryocytes in vitro. We have developed a murine monoclonal antibody, BAH-1, raised against human megakaryocytic cells, which specifically recognizes the c-Mpl receptor and shows agonist activity by stimulating megakaryocytopoiesis in vitro. BAH-1 antibody specifically binds to platelets and to recombinant c-Mpl with high affinity. Similar to TPO, BAH-1 alone supported the formation of colony-forming unit-megakaryocyte (CFU-MK) colonies. The combination of BAH-1 plus interleukin-3 or of BAH-1 plus human TPO significantly increased the number of human CFU-MK colonies. In addition, BAH-1 monoclonal antibody stimulated the proliferation and maturation of primary bone marrow megakaryocytes in a dynamic heterogeneous liquid culture system. Individual large megakaryocytes as well as small megakaryocytic cells were observed in cultures of CD34+ CD41+cells in the presence of BAH-1 antibodies. Similar to TPO, BAH-1 antibody induced a significant response of murine immature megakaryocytes as observed by an increase in the detectable numbers of acetylcholinesterase-positive megakaryocytes. No effects of BAH-1 antibody were observed on colony-forming unit–granulocyte-macrophage, burst-forming unit-erythroid, or colony-forming unit-erythroid colonies. In vivo studies showed that BAH-1, alone or in combination with TPO, expands the numbers of megakaryocytic progenitor cells in myelosuppressed mice. This antibody should prove useful in understanding the structure-function aspects of the c-Mpl receptor as well as in evaluating the effects of the sustained activation of this receptor in preclinical models of severe thrombocytopenia. © 1998 by The American Society of Hematology.

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Evaluation of immunotoxins containing single-chain ribosome-inactivating proteins and an anti-CD22 monoclonal antibody (OM124): in vitro and in vivo studies

British Journal of Haematology ◽

10.1046/j.1365-2141.1998.00665.x ◽

1998 ◽

Vol 101 (1) ◽

pp. 179-188 ◽

Cited By ~ 29

Author(s):

Andrea Bolognesi ◽

Pier Luigi Tazzari ◽

Fabiola Olivieri ◽

Letizia Polito ◽

Roberto Lemoli ◽

...

Keyword(s):

Monoclonal Antibody ◽

In Vivo Studies ◽

Single Chain ◽

Ribosome Inactivating Proteins

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Ectopic expression of murine TPO receptor (c-mpl) in mice is pathogenic and induces erythroblastic proliferation

Blood ◽

10.1182/blood.v88.5.1656.bloodjournal8851656 ◽

1996 ◽

Vol 88 (5) ◽

pp. 1656-1665 ◽

Cited By ~ 1

Author(s):

L Cocault ◽

D Bouscary ◽

C Le Bousse Kerdiles ◽

D Clay ◽

F Picard ◽

...

Keyword(s):

Progenitor Cell ◽

Leukemia Virus ◽

Ectopic Expression ◽

Hematopoietic Progenitor Cell ◽

Severe Thrombocytopenia ◽

Colony Forming Unit ◽

Adult Mice ◽

Cell Assays

c-mpl, the cellular homologue of the v-mpl oncogene transduced in the myeloproliferative leukemia virus (MPLV), encodes the receptor for thrombopoietin, a cytokine involved in the proliferation and differentiation of cells of the megakaryocytic lineage. Here, we show that a retrovirus containing murine c-mpl cDNA (HSFmmpl) is pathogenic in vivo when inoculated in adult mice. All mice developed hepatosplenomegaly and died within 9 to 12 weeks after infection. Histological analysis showed that spleen, liver, and peripheral blood were invaded by erythroblasts at every stage of differentiation. In contrast to the myeloproliferative syndrome induced by MPLV, we did not observe an infiltration of these organs with cells from the granulocytic lineage nor a thrombocytosis. In fact, the platelet count of HSFmmpl mice progressively decreased and a severe thrombocytopenia was observed late in the course of the disease. Further characterization of the target progenitor of HSFmmpl virus in the spleen and bone marrow of diseased animals was accomplished using in vitro clonogenic progenitor cell assays. This analysis indicated that both late and early erythroid compartment (colony-forming unit- erythroid and burst-forming unit-erythroid) were largely increased in the spleens. The colony-forming unit-granulocyte-macrophage compartment was also increased but to a lesser extent. This study shows for the first time that ectopic expression of a member of the cytokine receptor superfamily promotes hematopoietic progenitor cell proliferation and could play a role in leukemogenesis.

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Mechanism of Thrombocytopenia Induced by Anti-CD40 Ligand Immune Complexes and the Prevalence of CD40 Ligand Autoantibodies in Patients with Thrombotic Autoimmune Disorders

Blood ◽

10.1182/blood.v112.11.2857.2857 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2857-2857

Author(s):

Ali Amirkhosravi ◽

Todd V Meyer ◽

Liza Robles-Carillo ◽

Monica Davila ◽

Florian Langer ◽

...

Keyword(s):

Platelet Activation ◽

Lupus Erythematosus ◽

Immune Complexes ◽

Cd40 Ligand ◽

In Vivo Studies ◽

Severe Thrombocytopenia ◽

Thrombotic Risk ◽

Control Subjects

Abstract Anti-CD40 ligand (anti-CD40L) immunotherapy in patients with systemic lupus erythematosus (SLE, a chronic inflammatory autoimmune disease) resulted in unexpected thromboembolic fatalities. In our laboratory, previous in vitro mechanistic (flow cytometry, aggregation, and dense granule release) studies have shown that monoclonal anti-CD40L immune complexes potently activate platelets via the IgG receptor (FcγRIIa). The data suggested this activity was also dependent on the CD40L receptor (CD40), which is constitutively expressed on resting and activated platelets. This raised the possibility that autoantibodies against CD40L maybe present in patients with thrombotic autoimmune diseases such as SLE and anti-phospholipid syndrome (APS) and possibly contribute to the pathogenesis of thrombosis in such patients. We hypothesized that monoclonal anti-CD40L immune complexes (anti-CD40L IC) should exhibit prothrombotic effects in animals via IC-induced platelet activation, and CD40 ligand autoantibodies may be prevalent in patients with thrombotic auto-immune disorders. Mouse platelets, however, do not carry FcγRIIa. Therefore, to study anti-CD40L IC-induced platelet activation in vivo, we used mice transgenic for human FcγRIIa (“hFcR” mice). Immune complexes consisting of the anti-CD40L monoclonal antibody, M90, plus recombinant soluble CD40L (M90+sCD40L), or control reagents were injected intravenously (tail vein) into wild type (WT) or hFcR mice. Platelets were counted from 10–60 minutes thereafter. Additionally, plasma samples from patients with SLE (n=54), APS, (n=8), idiopathic thrombosis (n=34), and control subjects (n=86) were tested for the presence of IgG-type anti-CD40L autoantibodies using a highly optimized in-house ELISA. The injection of M90+CD40L IC (100–500 nM) produced symptoms consistent with thrombotic shock and induced severe thrombocytopenia (10–30% of basal platelet count) in hFcR (n=10–20) but not WT (n=5) mice—indicating that IC-induced thrombocytopenia was mediated via platelet FcγRIIa, as was found in vitro. Platelet priming by subaggregatory amounts of ADP greatly increased the sensitivity of hFcR mice to anti-CD40L IC (≥ eight-fold—as low as 12.5 nM). Furthermore, sequential injections of sCD40L followed by M90 in hFcR mice caused similar effects, indicating that ICs can also form while circulating. Injections of M90 or sCD40L alone were inactive in all animals. The prevalence of CD40L autoantibodies was notably higher in patients with SLE or APS compared to control subjects [13/54 (24%) or 3/12 (25%) vs. 5/86 (6%), P=0.002 and P=0.09 respectively]. Although CD40L autoantibodies were also more prevalent in patients with SLE and APS than in those with idiopathic thrombosis [2/34 (6%)], this difference was not statistically significant (P=0.058 and 0.2 respectively). Our findings demonstrate that the platelet activation caused by of anti-CD40L IC can be reproduced in mice, but only in those transgenic for the human IgG receptor (Fcγ RIIa). These in vivo findings may shed light on the thromboembolic complications associated with CD40L immunotherapy. Furthermore, our hFcR mouse model is a promising approach for assessing the hemostatic safety of CD40L—and possibly other—therapeutic antibodies. Our results also show that autoantibodies to CD40L occur at relatively high frequency in patients with SLE and APS. While a causal relationship between such antibodies and thrombotic risk remains unidentified, our in vivo studies suggest further investigation is warranted.

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Humanized Monoclonal Antibody That Passively Protects Mice against Systemic and Intranasal Ricin Toxin Challenge

Clinical and Vaccine Immunology ◽

10.1128/cvi.00088-16 ◽

2016 ◽

Vol 23 (9) ◽

pp. 795-799 ◽

Cited By ~ 17

Author(s):

Greta Van Slyke ◽

Erin K. Sully ◽

Natasha Bohorova ◽

Ognian Bohorov ◽

Do Kim ◽

...

Keyword(s):

Monoclonal Antibody ◽

Lethal Dose ◽

Murine Monoclonal Antibody ◽

Serum Concentrations ◽

Immunodominant Epitope ◽

Ricin Toxin ◽

Humanized Monoclonal Antibody

ABSTRACTPB10 is a murine monoclonal antibody against an immunodominant epitope on ricin toxin's enzymatic subunit. Here, we characterize a fully humanized version of PB10 IgG1 (hPB10) and demonstrate that it has potentin vitroandin vivotoxin-neutralizing activities. We also report the minimum serum concentrations of hPB10 required to protect mice against 10 times the 50% lethal dose of ricin when delivered by injection and inhalation.

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A rat monoclonal antibody reacting specifically with the tyrosylated form of alpha-tubulin. I. Biochemical characterization, effects on microtubule polymerization in vitro, and microtubule polymerization and organization in vivo.

The Journal of Cell Biology ◽

10.1083/jcb.97.5.1467 ◽

1983 ◽

Vol 97 (5) ◽

pp. 1467-1475 ◽

Cited By ~ 97

Author(s):

J Wehland ◽

M C Willingham ◽

I V Sandoval

Keyword(s):

Monoclonal Antibody ◽

Biochemical Characterization ◽

Mammalian Species ◽

Antigenic Site ◽

In Vivo Studies ◽

Alpha Tubulin ◽

3T3 Fibroblasts ◽

Microtubule Polymerization

The antigenic site recognized by a rat monoclonal antibody (clone YL 1/2) reacting with alpha-tubulin (Kilmartin, J.V., B. Wright, and C. Milstein, 1982, J. Cell Biol., 93:576-582) has been determined and partially characterized. YL 1/2 reacts specifically with the tyrosylated form of brain alpha-tubulin from different mammalian species. YL 1/2 reacts with the synthetic peptide Gly-(Glu)3-Gly-(Glu)2-Tyr, corresponding to the carboxyterminal amino acid sequence of tyrosylated alpha-tubulin, but does not react with Gly-(Glu)3-Gly-(Glu)2, the constituent peptide of detyrosylated alpha-tubulin. Electron microscopy as well as direct and indirect immunofluorescence microscopy shows that YL 1/2 binds to the surface of microtubules polymerized in vitro and in vivo. Further in vitro studies show that the antibody has no effect on the rate and extent of microtubule polymerization, the stability of microtubules, and the incorporation of the microtubule-associated proteins (MAP2) and tau into microtubules. In vivo studies using Swiss 3T3 fibroblasts injected with YL 1/2 show that; when injected at low concentration (2 mg IgG/ml in the injection solution), the antibody binds to microtubules without changing their distribution in the cytoplasm. Injection of larger concentration of YL 1/2 (6 mg IgG/ml) induces the formation of microtubule bundles, and still higher concentrations cause the aggregation of microtubule bundles around the nucleus (greater than 12 mg IgG/ml).

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Ectopic expression of murine TPO receptor (c-mpl) in mice is pathogenic and induces erythroblastic proliferation

Blood ◽

10.1182/blood.v88.5.1656.1656 ◽

1996 ◽

Vol 88 (5) ◽

pp. 1656-1665 ◽

Cited By ~ 37

Author(s):

L Cocault ◽

D Bouscary ◽

C Le Bousse Kerdiles ◽

D Clay ◽

F Picard ◽

...

Keyword(s):

Progenitor Cell ◽

Leukemia Virus ◽

Ectopic Expression ◽

Hematopoietic Progenitor Cell ◽

Severe Thrombocytopenia ◽

Colony Forming Unit ◽

Adult Mice ◽

Cell Assays

Abstract c-mpl, the cellular homologue of the v-mpl oncogene transduced in the myeloproliferative leukemia virus (MPLV), encodes the receptor for thrombopoietin, a cytokine involved in the proliferation and differentiation of cells of the megakaryocytic lineage. Here, we show that a retrovirus containing murine c-mpl cDNA (HSFmmpl) is pathogenic in vivo when inoculated in adult mice. All mice developed hepatosplenomegaly and died within 9 to 12 weeks after infection. Histological analysis showed that spleen, liver, and peripheral blood were invaded by erythroblasts at every stage of differentiation. In contrast to the myeloproliferative syndrome induced by MPLV, we did not observe an infiltration of these organs with cells from the granulocytic lineage nor a thrombocytosis. In fact, the platelet count of HSFmmpl mice progressively decreased and a severe thrombocytopenia was observed late in the course of the disease. Further characterization of the target progenitor of HSFmmpl virus in the spleen and bone marrow of diseased animals was accomplished using in vitro clonogenic progenitor cell assays. This analysis indicated that both late and early erythroid compartment (colony-forming unit- erythroid and burst-forming unit-erythroid) were largely increased in the spleens. The colony-forming unit-granulocyte-macrophage compartment was also increased but to a lesser extent. This study shows for the first time that ectopic expression of a member of the cytokine receptor superfamily promotes hematopoietic progenitor cell proliferation and could play a role in leukemogenesis.

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