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1.
Curr Drug Discov Technol ; 11(1): 65-73, 2014 Mar.
Article in English | MEDLINE | ID: mdl-23978038

ABSTRACT

Since Kohler and Milstein developed the process of generating hybridomas by fusing antibody secreting B cells with an immortal myeloma cell line, the techniques used to develop monoclonal antibodies for use as human therapeutics have progressed significantly. Here, we will briefly review hybridoma technology and the evolution of therapeutic antibodies for the treatment of human disease. We will focus on the evolution of humanized mouse models for the generation of therapeutic human antibodies, comparing the early models, such as severe combined immunodeficient (SCID) mice which do not engraft human leukocytes well due to residual innate immunity, to the more recently developed models such as non-obese diabetic (NOD)/SCID IL-2Rγ-deficient mice in which numerous human hematopoietic lineages can be cultivated. Building on the identification of suitable host strains for the reconstitution of human immune cells, focus has now shifted onto humanizing the murine microenvironment in order to support human immune cell function. Although several recent studies have shown that the provision of human soluble factors can support maturation and function of human immune cells, particularly within the myeloid compartment, this does not appear to impact antibody production significantly. Moreover, models in which grafting of human tissues is performed to provide human microenvironments which support human leukocyte maturation do show improved humoral immune function, but require several surgical manipulations for generation of the model. Ultimately the most desirable scenario is to generate transgenic models that can be bred efficiently and express a sufficient number of human molecules to support functional human immune cells and several groups have made progress in making this idea a reality. These studies in the context of the generation of human antibodies will be discussed.


Subject(s)
Antibodies, Monoclonal , Animals , Drug Discovery , Hematopoietic Stem Cell Transplantation , Humans , Hybridomas , Mice
2.
Fibrogenesis Tissue Repair ; 4(1): 23, 2011 Oct 19.
Article in English | MEDLINE | ID: mdl-22011363

ABSTRACT

BACKGROUND: Fibrocytes are a population of circulating bone-marrow-derived cells that express surface markers for leukocytes and mesenchymal cells, and are capable of differentiating into myofibroblasts. They have been observed at sites of active fibrosis and increased circulating numbers correlate with mortality in idiopathic pulmonary fibrosis (IPF). Inhibition of chemokine (C-C motif) receptor 2 (CCR2) during experimental models of lung fibrosis reduces lung collagen deposition, as well as reducing lung fibrocyte accumulation. The aim of the present study was to determine whether human and mouse fibrocytes express functional CCR2. RESULTS: Following optimized and identical human and murine fibrocyte isolation, both cell sources were shown to be positive for CCR2 by flow cytometry and this expression colocalized with collagen I and CD45. Human blood fibrocytes stimulated with the CCR2 ligand chemokine (C-C motif) ligand 2 (CCL2), demonstrated increased proliferation (P < 0.005) and differentiation into myofibroblasts (P < 0.001), as well as a chemotactic response (P < 0.05). Murine fibrocytes also responded to CCR2 stimulation, with CCL12 being more potent than CCL2. CONCLUSIONS: This study directly compares the functional responses of human and murine fibrocytes to CCR2 ligands, and following comparable isolation techniques. We have shown comparable biological effects, strengthening the translatability of the murine models to human disease with respect to targeting the CCR2 axis to ameliorate disease in IPF patients.

3.
Hybridoma (Larchmt) ; 30(2): 153-62, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21529288

ABSTRACT

ST2L is a transmembrane receptor that belongs to the IL-1 receptor family. The receptor is expressed on various cell types including Th2 cells, mast cells, basophils, growth-activated fibroblasts, and vascular endothelial cells. ST2L activation by its ligand IL-33 has been implicated in Th2-mediated immunity, inflammation, and allergic responses in vivo. Inhibition of ST2L activity can attenuate Th2-dominated immune responses such as lung eosinophilia, airway hyper-responsiveness, and arthritis in animal models. Here we report the generation and in vitro characterization of a panel of rat anti-mouse ST2L monoclonal antibodies. We demonstrate that the antibodies specifically bind to recombinant receptor protein and that a subset of the binders inhibits mouse ST2L activity in multiple in vitro assays. Four of the identified anti-mouse ST2L antibodies were shown to prevent IL-33 from binding to ST2L, down-regulate IL-33-induced NF-κB signaling, and neutralize the ability of IL-33 to stimulate mouse Th2 cell proliferation. The characterized monoclonal antibodies are important tools that will be used to study mouse ST2L receptor functionality in vivo.


Subject(s)
Antibodies, Monoclonal , Interleukins/immunology , Receptors, Interleukin-1/immunology , Recombinant Proteins/immunology , Signal Transduction/drug effects , Th2 Cells/drug effects , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/isolation & purification , Antibodies, Monoclonal/pharmacology , Antibody Specificity , Biotin/chemistry , Biotin/metabolism , Cell Proliferation/drug effects , Down-Regulation , HEK293 Cells , Humans , Hybridomas/immunology , Hybridomas/metabolism , Hypersensitivity/drug therapy , Hypersensitivity/immunology , Immunoconjugates/chemistry , Immunoconjugates/metabolism , Inflammation/drug therapy , Inflammation/immunology , Interleukin-1/immunology , Interleukin-33 , Interleukins/metabolism , Mice , Mice, Inbred BALB C , NF-kappa B/metabolism , Plasmids/genetics , Plasmids/metabolism , Rats , Receptors, Interleukin-1/genetics , Receptors, Interleukin-1/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Signal Transduction/immunology , Th2 Cells/immunology , Transfection
4.
Endocrinology ; 150(4): 1597-606, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19036881

ABSTRACT

Implicated in the pathogenesis of breast cancer, prolactin (PRL) mediates its function in part through the prolactin receptor (PRLr)-associated Janus kinase 2 (Jak2)/signal transducer and activator of transcription 5 (Stat5) signaling complex. To delineate the mechanisms of Stat5a regulation in breast cancer, transcription factor-transcription factor (TF-TF) array analysis was employed to identify associated transcriptional regulators. These analyses revealed a PRL-inducible association of Stat5a with the transcription factor and protooncogene c-Myb. Confirmatory co-immunoprecipitation studies using lysates from both T47D and MCF7 breast cancer cells revealed a PRL-inducible association between these transcription factors. Ectopic expression of c-Myb enhanced the PRL-induced expression from both composite and synthetic Stat5a-responsive luciferase reporters. Chromatin immunoprecipitation assays also revealed a PRL-inducible association between c-Myb and endogenous Stat5a-responsive CISH promoter, which was associated with an enhanced expression of CISH gene product at the RNA and protein levels. Small interfering RNA-mediated c-Myb knockdown impaired the PRL-induced mRNA expression of five Stat5-responsive genes. DNA binding-defective mutants of c-Myb, incapable of activating expression from a c-Myb-responsive reporter, maintained their ability to enhance a Stat5a-responsive reporter. At a cellular level, ectopic expression of c-Myb resulted in an increase in T47D proliferation. Taken together, these results indicate that c-Myb potentiates Stat5a-driven gene expression, possibly functioning as a Stat5a coactivator, in human breast cancer.


Subject(s)
Prolactin/pharmacology , Proto-Oncogene Proteins c-myb/metabolism , Proto-Oncogene Proteins c-myb/physiology , STAT5 Transcription Factor/metabolism , Tumor Suppressor Proteins/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Chromatin Immunoprecipitation , Gene Expression Regulation, Neoplastic/drug effects , Humans , Immunoprecipitation , Promoter Regions, Genetic/genetics , Protein Binding/drug effects , Proto-Oncogene Proteins c-myb/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/physiology , Reverse Transcriptase Polymerase Chain Reaction , STAT5 Transcription Factor/genetics , Signal Transduction/drug effects , Signal Transduction/genetics , Suppressor of Cytokine Signaling Proteins/genetics , Suppressor of Cytokine Signaling Proteins/metabolism , Tumor Suppressor Proteins/genetics
5.
Hybridoma (Larchmt) ; 27(1): 25-30, 2008 Feb.
Article in English | MEDLINE | ID: mdl-18294073

ABSTRACT

ABSTRACT Herein we describe the use of an agonistic anti-murine CD40 MAb as a B cell proliferative agent to enhance the generation of monoclonal antibodies (MAbs) in Balb/c mice. While hybridoma technology has been validated repeatedly over the decades, little work has been described to improve upon the overall numbers of in vivo B cells and specific antibodies obtained from a fusion. To begin to address this situation, strategies to boost B lymphocyte yields for hybridoma production were employed. Anti-CD40 agonist antibodies have been reported to activate and amplify human resting B lymphocytes in vitro, resulting in increased cell numbers available for the generation of human hybridomas or B cell clones. An agonistic anti-murine CD40 MAb was administered to immunized mice 3 days prior to splenic harvest, and B lymphocyte yields were found to be approximately 2-fold higher in treated animals when compared to untreated animals. Moreover, the resulting hybridoma fusions using lymphocytes from treated animals yielded 5- to 10-fold more antigen reactive hybrids when compared to untreated animals. This novel addition to conventional approaches utilizes the proliferative effects of agonistic anti-CD40 MAbs to markedly enhance monoclonal antibody generation.


Subject(s)
Antibodies, Monoclonal/immunology , CD40 Antigens/agonists , Hybridomas/immunology , Receptors, Antigen, B-Cell/immunology , Animals , B-Lymphocytes/cytology , B-Lymphocytes/immunology , CD40 Antigens/immunology , Cell Fusion , Female , Humans , Hybridomas/cytology , Immunization , Mice , Mice, Inbred BALB C , Spleen/cytology , Spleen/immunology
6.
Hum Antibodies ; 15(3): 61-9, 2006.
Article in English | MEDLINE | ID: mdl-17065737

ABSTRACT

The generation of anti-variable region monoclonal antibodies (mAbs) against therapeutic antibodies is essential in the pharmacokinetic/pharmacodynamic (PK/PD) assessments of the drugs in clinical study samples. Sandwich EIA and other methods are typically employed to achieve sensitivity and selectivity for the PK/PD analyses. These assays usually require generation of mAb reagents that bind specifically to the drug in non-competing pair combinations. Thus, large panels of anti-variable region mAbs must be generated in an expeditious manner to increase the probability of success. Herein we describe a novel immunization method that utilizes type 1 interferons (IFNs) as immunomodulators coupled with an agonistic anti-CD40 mAb as a B cell proliferative agent to drive immune responses. This novel protocol allows for rapid and robust mAb reagent generation without the use of conventional protein-denaturing adjuvants. The use of IFNs allowed for the generation of comparable and in some cases, increased numbers of anti-variable region mAbs in a dramatically shorter timeframe. This IFN based, immunostimulatory approach utilizing a non-denaturing adjuvant, likely presents conformational epitopes and may optimize the humoral response for the rapid generation of anti-variable region reagents to therapeutic mAb candidates.


Subject(s)
Adjuvants, Immunologic/pharmacology , Antibodies, Monoclonal/biosynthesis , Interferons/immunology , Animals , Antibodies, Monoclonal/immunology , B-Lymphocytes/immunology , Enzyme-Linked Immunosorbent Assay , Female , Mice , Mice, Inbred BALB C , Sensitivity and Specificity , Spleen/immunology
7.
Hum Antibodies ; 15(4): 155-62, 2006.
Article in English | MEDLINE | ID: mdl-17522437

ABSTRACT

The generation of anti-variable region monoclonal antibodies (mAbs) against therapeutic antibodies is essential in the pharmacokinetic/pharmacodynamic (PK/PD) assessments of the drugs in clinical study samples. Sandwich EIA and other methods are typically employed to achieve sensitivity and selectivity for the PK/PD analyses. These assays usually require generation of mAb reagents that bind specifically to the therapeutic mAb candidate in non-competing pair combinations. Thus, large panels of anti-variable region mAbs must be generated in an expeditious manner to increase the probability of success. Previously, we described a novel immunization method using type 1 interferons (IFNs) coupled with an agonistic anti-CD40 mAb to drive immune responses (Staquet et al., Human Antibodies 15 (2006), 61-69). This protocol allows for rapid and robust generation of large panels of anti-variable region mAbs. In order to quickly characterize and efficiently identify optimal anti-variable region antibody pairs early in the hybridoma process using crude supernatants, an inexpensive, high-throughput ELISA method was developed. The ability to rapidly identify appropriate mAb pairs will save resources by eliminating the time-consuming and laborious process of subcloning irrelevant hybridomas.


Subject(s)
Antibodies, Monoclonal/biosynthesis , CD40 Antigens/immunology , Enzyme-Linked Immunosorbent Assay/methods , Immunoglobulin Variable Region/immunology , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/pharmacokinetics , Antibodies, Monoclonal/therapeutic use , B-Lymphocytes , Biotechnology/methods , Female , Humans , Immunization , Immunoglobulin G/administration & dosage , Immunoglobulin G/immunology , Interferon Type I , Mice , Mice, Inbred BALB C , Time Factors
8.
Infect Immun ; 72(12): 7202-11, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15557645

ABSTRACT

Unmethlylated CpG dinucleotides induce a strong T-helper-1-like inflammatory response, presumably mediated by Toll-like receptor 9 (TLR9). However, the nature and cellular localization of TLR9 in primary human cells remain controversial. Here we demonstrate, using flow cytometry and immunofluorescence microscopy techniques, that TLR9 can be expressed at the cell surface. The primary human cell subsets that were positive for TLR9 expression were distinct depending on the tissues analyzed. Specifically, in human peripheral blood mononuclear cells (PBMC) the majority of cell surface TLR9(+) cells were confined to the major histocompatibility complex (MHC) class II(+) CD19(-) populations that express CD11c and/or CD14, whereas in tonsils the same gated population contained primarily MHC class II(+) CD19(+) cells. Cells positive for surface expression represented a minor fraction of the total cell populations examined, varying between 2 and 10%. In addition, we found that TLR9 expression at the surface of PBMC was up-regulated approximately fourfold following stimulation with the gram-negative bacterial cell wall component lipopolysaccharide, suggesting a potential modulatory role of TLR4 agonists on TLR9 expression. Taken together, these data validate human TLR9 expression at the surface of primary cells, in addition to the previously described intracellular localization. Further, our results suggest that human antigen-presenting cells comprise the major cell populations expressing cell surface TLR9.


Subject(s)
DNA-Binding Proteins/analysis , Leukocytes, Mononuclear/chemistry , Palatine Tonsil/chemistry , Receptors, Cell Surface/analysis , Amino Acid Sequence , Antigens, CD19/analysis , CD11c Antigen/analysis , Flow Cytometry , Fluorescent Antibody Technique , Humans , Interleukin-3 Receptor alpha Subunit , Lipopolysaccharides/pharmacology , Molecular Sequence Data , Receptors, Interleukin-3/analysis , Staining and Labeling , Toll-Like Receptor 9
9.
Endocrine ; 20(1-2): 83-90, 2003.
Article in English | MEDLINE | ID: mdl-12668872

ABSTRACT

Precedent data have revealed that peptidyl isomerases can modulate the function of cell-surface receptors, but no such interactions have been previously shown for the members of the cytokine receptor superfamily. We demonstrate here that a functional interaction occurs between the prolactin receptor (PRLR) and peptidyl prolyl cis/trans isomerase cyclophilin A (CypA). CypA was co-immunoprecipitated with the PRLR in vivo from the breast epithelial cell line T47D and Chinese hamster ovary transfectants overexpressing transfected human PRLR. In addition, in vitro binding assays demonstrated a direct interaction of CypA with the PRLR, in the presence or absence of cyclosporine. Co-immunoprecipitation studies also showed an association of CypA with Jak2. Functional analysis revealed that overexpression of CypA inhibited PRL-induced Rac activation, while simultaneously prolonging Jak2 phosphorylation. These proximal actions had profound downstream effects: CypA overexpression significantly enhanced the basal and PRL-stimulated expression from a beta-casein reporter construct. Hence, the interaction between CypA and the PRLR plays a differential regulatory role in the various signaling pathways leading from the PRLR.


Subject(s)
Cyclophilin A/metabolism , Proto-Oncogene Proteins , Receptors, Prolactin/metabolism , Animals , Breast/cytology , CHO Cells , Caseins/metabolism , Cricetinae , Cyclophilin A/pharmacology , Down-Regulation , Epithelial Cells/cytology , Humans , Janus Kinase 2 , Phosphorylation/drug effects , Protein-Tyrosine Kinases/metabolism , Receptors, Prolactin/genetics , Signal Transduction/physiology , Transfection , rac1 GTP-Binding Protein/metabolism
10.
Mol Endocrinol ; 16(10): 2310-22, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12351696

ABSTRACT

Prolactin (PRL)-dependent signaling occurs as the result of ligand-induced homodimerization of the PRL receptor (PRLr). To date, short, intermediate, and long human PRLr isoforms have been characterized. To investigate the expression of other possible human PRLr isoforms, RT-PCR was performed on mRNA isolated from the breast carcinoma cell line T47D. A 1.5-kb PCR fragment was isolated, subcloned, and sequenced. The PCR product exhibited a nucleotide sequence 100% homologous to the human long isoform except bp 71-373 were deleted, which code for the S1 motif of the extracellular domain. Therefore, this isoform was designated the deltaS1 PRLr. Northern analysis revealed variable deltaS1 PRLr mRNA expression in a variety of tissues. Transfection of Chinese hamster ovary cells with deltaS1 cDNA showed the isoform is expressed at the protein level on the cell surface with a molecular mass of approximately 70 kDa. Kinetic studies indicated the deltaS1 isoform bound ligand at a lower affinity than wild-type receptor. The deltaS1 PRLr was also shown to activate the proximal signaling molecule Jak2 upon addition of ligand to transfected cells, and, unlike the long PRLr, high concentrations of ligand did not function as a self-antagonist to signaling during intervals of PRL serum elevation, i.e. stress and pregnancy. Given its apparent widespread expression, this PRLr isoform may contribute to PRL action. Furthermore, the functionality of this receptor raises interesting questions regarding the minimal extracellular domain necessary for ligand-induced receptor signaling.


Subject(s)
Proto-Oncogene Proteins , Receptors, Prolactin/genetics , Receptors, Prolactin/metabolism , Animals , Base Sequence , Breast Neoplasms/genetics , CHO Cells , Cloning, Molecular , Cricetinae , Extracellular Matrix/metabolism , Fibronectins/metabolism , Hormones/metabolism , Humans , Janus Kinase 2 , Molecular Sequence Data , Prolactin/metabolism , Protein Isoforms , Protein Structure, Tertiary , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Reference Values , Signal Transduction , Tumor Cells, Cultured
11.
Proc Natl Acad Sci U S A ; 99(10): 6790-5, 2002 May 14.
Article in English | MEDLINE | ID: mdl-11997457

ABSTRACT

The nuclear translocation of peptide hormones, such as the somatolactogenic hormone prolactin, after receptor internalization has been widely reported. Prolactin has been demonstrated to interact with cyclophilin B, a member of the immunophilin family of proteins. Cyclophilin B interaction with prolactin potentiated prolactin-induced proliferation, cell growth, and the nuclear retrotransport of prolactin. These effects could be abrogated by the removal of the peptidyl-prolyl isomerase activity of cyclophilin B. Our findings indicate that the intranuclear prolactin/cyclophilin B complex acts as a transcriptional inducer by interacting directly with Stat5, resulting in the removal of the Stat-repressor protein inhibitor of activated Stat 3 (PIAS3), thereby enhancing Stat5 DNA-binding activity and prolactin-induced, Stat5-mediated gene expression.


Subject(s)
Cyclophilins/metabolism , DNA-Binding Proteins/metabolism , Milk Proteins , Prolactin/metabolism , Trans-Activators/metabolism , Transcription, Genetic , Animals , Carrier Proteins/metabolism , Caseins/genetics , Cell Division , Cell Nucleus/metabolism , Cyclophilins/genetics , Peptidylprolyl Isomerase , Rats , STAT5 Transcription Factor , Tumor Cells, Cultured
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