Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 12 de 12
Filter
Add more filters










Publication year range
1.
MAbs ; 6(5): 1265-73, 2014.
Article in English | MEDLINE | ID: mdl-25517311

ABSTRACT

We report a chimeric monoclonal antibody (mAb) directed to a neo-epitope that is exposed in the IgG lower hinge following proteolytic cleavage. The mAb, designated 2095-2, displays specificity for IdeS-generated F(ab')2 fragments, but not for full-length IgG or for closely-related F(ab')2 fragments generated with other proteases. A critical component of the specificity is provided by the C-terminal amino acid of the epitope corresponding to gly-236 in the IgG1 (also IgG4) hinge. By its ability to bind to IdeS-cleaved anti-CD20 mAb, mAb 2095-2 fully restored antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against WIL2-S cells to the otherwise inactive anti-CD20 IgG1 F(ab')2 fragment. Similarly, 2095-2 reinstated ADCC against MDA-MB-231 cells to an anti-CD142 IgG1 F(ab')2 fragment. mAb 2095-2 was also capable of eliciting both CDC and ADCC to IgG4 F(ab')2 fragments, an IgG subclass that has weaker ADCC and CDC when intact relative to intact IgG1. The in vitro cell-based efficacy of 2095-2 was extended to the in vivo setting using platelets as a cell clearance surrogate. In a canine model, the co-administration of 2095-2 together with IdeS-generated, platelet-targeting anti-CD41/61 F(ab')2 fragment not only restored platelet clearance, but did so at a rate and extent of clearance that exceeded that of intact anti-CD41/61 IgG at comparable concentrations. To further explore this unexpected amplification effect, we conducted a rat study in which 2095-2 was administered at a series of doses in combination with a fixed dose of anti-CD41/61 F(ab')2 fragments. Again, the combination, at ratios as low as 1:10 (w/w) 2095-2 to F(ab')2, proved more effective than the anti-CD41/61 IgG1 alone. These findings suggest a novel mechanism for enhancing antibody-mediated cell-killing effector functions with potential applications in pathologic settings such as tumors and acute infections where protease activity is abundant.


Subject(s)
Antibodies, Monoclonal/immunology , Immunoglobulin Fab Fragments/immunology , Immunoglobulin G/immunology , Animals , Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal, Murine-Derived/immunology , Antibodies, Monoclonal, Murine-Derived/metabolism , Antibodies, Monoclonal, Murine-Derived/pharmacology , Antibody-Dependent Cell Cytotoxicity/drug effects , Antibody-Dependent Cell Cytotoxicity/immunology , Bacterial Proteins/metabolism , Blood Platelets/immunology , Blood Platelets/metabolism , Cell Line , Cell Line, Tumor , Cysteine Endopeptidases/metabolism , Dogs , Enzyme-Linked Immunosorbent Assay , Epitopes/immunology , Epitopes/metabolism , Humans , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin Fab Fragments/pharmacology , Immunoglobulin G/metabolism , Matrix Metalloproteinase 3/metabolism , Platelet Count , Proteolysis , Rats , Rituximab
2.
Cytokine ; 65(2): 167-74, 2014 Feb.
Article in English | MEDLINE | ID: mdl-24345576

ABSTRACT

Interleukin-17A (IL-17A) is the prototype of IL-17 family and has been implicated in the pathogenesis of a variety of autoimmune diseases. Therefore its structural and functional properties are of great medical interest. During our research on a recombinant human IL-17A (rhIL-17A) variant, four isoforms were obtained when it was refolded. While isoforms 1 and 2 represented non-covalent dimers, isoforms 3 and 4 were determined to be covalent dimers. All four isoforms were structurally similar by Circular Dichroism and fluorescence spectroscopy studies, but differential scanning calorimetry demonstrated thermal stability in the order of isoform 1=isoform 2

Subject(s)
Disulfides/metabolism , Interleukin-17/metabolism , Recombinant Proteins/metabolism , Amino Acid Sequence , Calorimetry, Differential Scanning , Circular Dichroism , Electrophoresis, Polyacrylamide Gel , Humans , Interleukin-17/chemistry , Mass Spectrometry , Molecular Sequence Data , Peptides/chemistry , Peptides/metabolism , Protein Isoforms/metabolism , Protein Multimerization , Protein Refolding , Solutions , Spectrometry, Fluorescence
3.
Mol Immunol ; 47(14): 2422-6, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20554002

ABSTRACT

A chimeric antibody was constructed from two unrelated antibodies by combining their heavy and light chains. The "double chimera" consists of the mouse variable regions of different specificity (IL-13 and EMMPRIN) and the constant regions of different origin (mouse and human). The Fab fragment of this chimeric antibody was expressed in mammalian cells, and the crystal structure was determined at 1.6A resolution. Despite a large number of amino acid substitutions in the double chimera with respect to the parent antibodies, the heavy and light chains associate into a stable molecule. Comparison to the structure of one of the parent antibodies reveals that the variable domain interface, as well as the conformation of antigen-binding loops, is preserved without major rearrangements due to conservation of amino acids in key positions. Comparison to the structures of the all-human and all-mouse constant domains indicates a remarkable plasticity of the inter-chain interface that can tolerate residue relocations of up to 6A.


Subject(s)
Antibodies/chemistry , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/immunology , Amino Acid Sequence , Animals , Antibodies/genetics , Base Sequence , Complementarity Determining Regions , Crystallography, X-Ray , DNA Primers/genetics , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Heavy Chains/chemistry , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/chemistry , Immunoglobulin Light Chains/genetics , Mice , Models, Molecular , Molecular Sequence Data , Protein Conformation , Protein Engineering , Protein Structure, Tertiary , Recombinant Fusion Proteins/genetics , Sequence Homology, Amino Acid
4.
Protein Expr Purif ; 67(2): 182-9, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19442740

ABSTRACT

Fab (fragment that having the antigen binding site) of a monoclonal antibody (mAb) is widely required in biopharmaceutical research and development. At Centocor, two routes of Fab production and purification were used to enable a variety of research and development efforts, particularly, crystallographic studies of antibody-antigen interactions. One route utilizes papain digestion of an intact monoclonal antibody for Fab fragment production. After digestion, separation of the Fab fragment from the Fc (fragment that crystallizes) and residual intact antibody was achieved using protein A affinity chromatography. In another route, His-tagged Fab fragments were obtained by transient expression of an appropriate construct in mammalian cells, and typical yields are 1-20mg of Fab fragment per liter of cell culture. The His-tagged Fab fragments were first captured using immobilized metal affinity chromatography (IMAC). To provide high quality protein sample for crystallization, Fabs from either proteolytic digestion or from direct expression were further purified using size-exclusion chromatography (SEC) and/or ion-exchange chromatography (IEC). The purified Fab fragments were characterized by mass spectrometry, SDS-PAGE, dynamic light scattering, and circular dichroism. Crystallization experiments demonstrated that the Fab fragments are of high quality to produce diffraction quality crystals suitable for X-ray crystallographic analysis.


Subject(s)
Antibodies, Monoclonal/metabolism , Immunoglobulin Fab Fragments/biosynthesis , Immunoglobulin Fab Fragments/isolation & purification , Papain/metabolism , Antibodies, Monoclonal/chemistry , Cell Line , Circular Dichroism , Histidine/genetics , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/genetics , Light , Oligopeptides/genetics , Papain/chemistry , Recombinant Fusion Proteins/biosynthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Scattering, Radiation , X-Ray Diffraction
5.
Biochim Biophys Acta ; 1791(8): 746-56, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19362163

ABSTRACT

Serine palmitoyltransferase (SPT) has been localized to the endoplasmic reticulum (ER) by subcellular fractionation and enzymatic assays, and fluorescence microscopy of epitope-tagged SPT; however, our studies have suggested that SPT subunit 1 might be present also in focal adhesions and the nucleus. These additional locations have been confirmed by confocal microscopy using HEK293 and HeLa cells, and for focal adhesions by the demonstration that SPT1 co-immunoprecipitates with vinculin, a focal adhesion marker protein. The focal adhesion localization of SPT1 is associated with cell morphology, and possibly cell migration, because it is seen in most cells before they reach confluence but disappears when they become confluent, and is restored by a standard scratch-wound healing assay. Conversely, elimination of SPT1 using SPTLC1 siRNA causes cell rounding. Thus, in addition to its "traditional" localization in the ER for de novo sphingolipid biosynthesis, SPT1 is present in other cellular compartments, including focal adhesions where it is associated with cell morphology.


Subject(s)
Cell Nucleus/enzymology , Cell Shape , Endoplasmic Reticulum/enzymology , Focal Adhesions/enzymology , Protein Subunits/metabolism , Serine C-Palmitoyltransferase/metabolism , ATP Binding Cassette Transporter 1 , ATP-Binding Cassette Transporters/metabolism , Cell Adhesion , Cell Line , Cell Membrane/enzymology , Gene Silencing , Humans , Immunoprecipitation , Protein Transport , RNA, Small Interfering/metabolism , Reproducibility of Results , Sphingolipids/metabolism , Subcellular Fractions/enzymology , Vinculin/metabolism
6.
Viral Immunol ; 21(2): 173-88, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18570589

ABSTRACT

The functional role of IL-12 and IL-23 in host defense and disease following viral infection of the CNS was determined. Instillation of mouse hepatitis virus (MHV, a positive-strand RNA virus) into the CNS of mice results in acute encephalitis followed by a chronic immune-mediated demyelinating disease. Antibody-mediated blocking of either IL-23 (anti-IL-23p19) or IL-12 and IL-23 (anti-IL-12/23p40) signaling did not mute T-cell trafficking into the CNS or antiviral effector responses and mice were able to control viral replication within the brain. Therapeutic administration of either anti-IL-23p19 or anti-IL-12/23p40 to mice with viral-induced demyelination did not attenuate T-cell or macrophage infiltration into the CNS nor improve clinical disease or diminish white matter damage. In contrast, treatment of mice with anti-IL-12/23p40 or anti-IL-23p19 resulted in inhibition of the autoimmune model of demyelination, experimental autoimmune encephalomyelitis (EAE). These data indicate that (1) IL-12 and IL-23 signaling are dispensable in generating a protective T-cell response following CNS infection with MHV, and (2) IL-12 and IL-23 do not contribute to demyelination in a model independent of autoimmune T-cell-mediated pathology. Therefore, therapeutic targeting of IL-12 and/or IL-23 for the treatment of autoimmune diseases may offer unique advantages by reducing disease severity without muting protective responses following viral infection.


Subject(s)
Central Nervous System/immunology , Coronavirus Infections/immunology , Interleukin-12/immunology , Interleukin-23/immunology , Murine hepatitis virus/immunology , T-Lymphocytes/immunology , Animals , Autoimmune Diseases , Central Nervous System/pathology , Coronavirus Infections/pathology , Demyelinating Diseases/immunology , Encephalomyelitis/immunology , Female , Mice
7.
Cell Immunol ; 248(2): 103-14, 2007 Aug.
Article in English | MEDLINE | ID: mdl-18048020

ABSTRACT

Toll-like receptors are a family of pattern-recognition receptors that contribute to the innate immune response. Toll-like receptor 3 (TLR3) signals in response to foreign, endogenous and synthetic ligands including viral dsRNA, bacterial RNA, mitochondrial RNA, endogenous necrotic cell mRNA and the synthetic dsRNA analog, poly(I:C). We have generated a monoclonal antibody (mAb CNTO2424) that recognizes the extracellular domain (ECD) of human TLR3 in a conformation-dependent manner. CNTO2424 down-regulates poly(I:C)-induced production of IL-6, IL-8, MCP-1, RANTES, and IP-10 in human lung epithelial cells. In addition, mAb CNTO2424 was able to interfere with the known TLR3-dependent signaling pathways, namely NF-kappaB, IRF-3/ISRE, and p38 MAPK. The generation of this neutralizing anti-TLR3 mAb provides a unique tool to better understand TLR3 signaling and potential cross-talk between TLR3 and other molecules.


Subject(s)
Antibodies, Monoclonal , Toll-Like Receptor 3/antagonists & inhibitors , Toll-Like Receptor 3/immunology , Animals , Antibodies, Blocking/metabolism , Antibodies, Monoclonal/metabolism , Binding Sites, Antibody , Cell Line , Cell Line, Transformed , Female , Humans , Mice , Mice, Inbred BALB C , Pilot Projects , Toll-Like Receptor 3/metabolism
8.
Protein Expr Purif ; 55(2): 279-86, 2007 Oct.
Article in English | MEDLINE | ID: mdl-17646110

ABSTRACT

While well established in bacterial hosts, the effect of coding sequence variation on protein expression in mammalian systems is poorly characterized outside of viral proteins or proteins from distant phylogenetic families. The potential impact is substantial given the extensive use of mammalian expression systems in research and manufacturing of protein biotherapeutics. We are studying the effect of codon engineering on expression of recombinant antibodies with an emphasis on developing manufacturing cell lines. CNTO 888, a human mAb specific for human MCP-1, was obtained by antibody phage display in collaboration with MorphoSys AG. The isolated DNA sequence of the antibody was biased towards bacterial codons, reflecting the engineering of the Fab library for phage display expression in Escherichia coli. We compared the expression of CNTO 888 containing the parental V-region sequences with two engineered coding variants. In the native codon exchanged (NCE) variant, the V-region codons were replaced with those used in naturally derived human antibody genes. In the human codon optimized (HCO) variant the V-region codons were those used at the highest frequency based on a human codon usage table. The antibody expression levels from stable transfections in mammalian host cells were measured. The HCO codon variant of CNTO 888 yielded the highest expressing cell lines and the highest average expression for the screened populations. This had a significant positive effect on the process to generate a CNTO 888 production cell line and indicates the potential to improve antibody expression in mammalian expression systems by codon engineering.


Subject(s)
Antibodies, Monoclonal/genetics , Codon , Genetic Engineering , Amino Acid Sequence , Antibodies, Monoclonal/chemistry , Base Sequence , Cell Line , DNA, Recombinant , Humans , Molecular Sequence Data , Plasmids
9.
Int Immunopharmacol ; 6(6): 880-91, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16644473

ABSTRACT

Suppression of T cell response is the key to enhance graft survival and control autoimmune diseases. A mitogenic anti-CD3 monoclonal antibody (mAb), OKT3, has been used for decades to control acute rejection in organ transplantation. Although effective, the clinical use was limited by its side effects, such as cytokine release mediated by T cell activation. A low mitogenic humanized OKT3 with reduced FcR-binding (hgammaOKT3 Ala-Ala) was generated and tested in several clinical studies. Although hgammaOKT3 Ala-Ala demonstrated maintained efficacy and better safety it still activated T cells. To investigate if a non-mitogenic anti-CD3 mAb can be equally effective in immune suppression, a chimeric non-FcR-binding anti-mouse CD3 mAb (anti-CD3 IgG2a Ala-Ala) was generated. Unlike the hgammaOKT3 Ala-Ala, the mouse IgG2a Ala-Ala anti-CD3 mAb did not induce T cell activation as measured by proliferation, cytokine production and apoptosis. Nevertheless, the IgG2a Ala-Ala anti-CD3 mAb was equally effective in the inhibition of antigen-specific CD4+ T cell activation in vitro to that of the mitogenic anti-CD3 mAb (Anti-CD3 IgG2a). In vivo, the IgG2a Ala-Ala anti-CD3 mAb only induced transient reduction of peripheral and spleen T cells and did not trigger detectable cytokine release. Nonetheless, this non-mitogenic anti-CD3 mAb significantly prolonged islet graft survival as effectively as the mitogenic anti-CD3 mAb in an allogenic islet transplantation model. These results demonstrated that a non-mitogenic anti-CD3 mAb could be used as an effective immune modulator. It may also indicate that a true non-mitogenic version of OKT3 could further improve its safety profile for clinical use.


Subject(s)
Antibodies, Monoclonal/pharmacology , CD3 Complex/immunology , Immunosuppressive Agents/pharmacology , Lymphocyte Activation/drug effects , T-Lymphocytes/drug effects , Animals , Antibodies, Monoclonal/metabolism , Apoptosis/drug effects , Apoptosis/immunology , Cell Proliferation/drug effects , Cytokines/blood , Cytokines/metabolism , Diabetes Mellitus, Experimental/immunology , Diabetes Mellitus, Experimental/surgery , Graft Survival/drug effects , Graft Survival/immunology , Humans , Immunoglobulin G/genetics , Immunoglobulin G/metabolism , Immunoglobulin G/pharmacology , Islets of Langerhans Transplantation/immunology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Lymphocyte Count , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Muromonab-CD3/pharmacology , Protein Binding , Receptors, Fc/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/immunology , U937 Cells , fas Receptor/metabolism
10.
Biochem J ; 387(Pt 3): 727-35, 2005 May 01.
Article in English | MEDLINE | ID: mdl-15579134

ABSTRACT

Gas6 (growth-arrest-specific gene 6) is a vitamin K-dependent protein known to activate the Axl family of receptor tyrosine kinases. It is an important regulator of thrombosis and many other biological functions. The C-terminus of Gas6 binds to receptors and consists of two laminin-like globular domains LG1 and LG2. It has been reported that a Ca2+-binding site at the junction of LG1 and LG2 domains and a hydrophobic patch at the LG2 domain are important for receptor binding [Sasaki, Knyazev, Cheburkin, Gohring, Tisi, Ullrich, Timpl and Hohenester (2002) J. Biol. Chem. 277, 44164-44170]. In the present study, we developed a neutralizing human monoclonal antibody, named CNTO300, for Gas6. The antibody was generated by immunization of human IgG-expressing transgenic mice with recombinant human Gas6 protein and the anti-Gas6 IgG sequences were rescued from an unstable hybridoma clone. Binding of Gas6 to its receptors was partially inhibited by the CNTO300 antibody in a dose-dependent manner. To characterize further the interaction between Gas6 and this antibody, the binding kinetics of CNTO300 for recombinant Gas6 were compared with independently expressed LG1 and LG2. The CNTO300 antibody showed comparable binding affinity, yet different dependence on Ca2+, to Gas6 and LG1. No binding to LG2 was detected. In the presence of EDTA, binding of the antibody to Gas6 was disrupted, but no significant effect of EDTA on LG1 binding was evident. Further epitope mapping identified a Gas6 peptide sequence recognized by the CNTO300 antibody. This peptide sequence was found to be located at the LG1 domain distant from the Ca2+-binding site and the hydrophobic patch. Co-interaction of Gas6 with its receptor and CNTO300 antibody was detected by BIAcore analysis, suggesting a second receptor-binding site on the LG1 domain. This hypothesis was further supported by direct binding of Gas6 receptors to an independently expressed LG1 domain. Our results revealed, for the first time, a second binding site for Gas6-receptor interaction.


Subject(s)
Antibodies, Monoclonal/chemistry , Intercellular Signaling Peptides and Proteins/chemistry , Amino Acid Sequence , Binding Sites , Calcium/chemistry , Epitope Mapping , Humans , Kinetics , Molecular Sequence Data , Protein Binding , Protein Structure, Tertiary
11.
J Histochem Cytochem ; 51(6): 715-26, 2003 Jun.
Article in English | MEDLINE | ID: mdl-12754283

ABSTRACT

Metastatic processes, including cell invasion, extracellular matrix degradation, and tissue remodeling, require cellular reorganization and proliferation. The cell signaling molecules required and the proteins involved in cell restructuring have not been completely elucidated. We have been studying the role of sphingolipids in normal cell activity and in several pathophysiological states. In this study we used immunohistochemistry to observe the presence of the two known subunits of serine palmitoyltransferase (SPT) in proliferating cells, in an in vitro model of wound repair, and in human malignant tissue. We report increased expression of the two subunits, SPT1 and SPT2, in the proliferating cells in these models. We also demonstrate a change in subcellular localization of the SPT subunits from predominantly cytosolic in quiescent cells to nuclear in proliferating cells. In addition, we observed SPT1 and SPT2 immunoreactivity in reactive stromal fibroblasts surrounding the carcinoma cells of some of the tumors. This enhanced SPT expression was absent in the stromal fibroblasts surrounding normal epithelial cells. Our results suggest a potential role for overexpression of SPT in the processes of cell metastasis.


Subject(s)
Acyltransferases/metabolism , Fibroblasts/metabolism , Neoplasms/metabolism , Cell Division , Cell Line, Transformed , Humans , Immunohistochemistry/methods , Infant, Newborn , Protein Subunits , Serine C-Palmitoyltransferase
12.
J Histochem Cytochem ; 51(5): 687-96, 2003 May.
Article in English | MEDLINE | ID: mdl-12704216

ABSTRACT

Sphingolipids serve as structural elements of cells and as lipid second messengers. They regulate cellular homeostasis, mitogenesis, and apoptosis. Sphingolipid signaling may also be important in various pathophysiologies such as vascular injury, inflammation, and cancer. Serine palmitoyltransferase (SPT) catalyzes the condensation of serine with palmitoyl-CoA, the first, rate-limiting step in de novo sphingolipid biosynthesis. This integral microsomal membrane protein consists of at least two subunits, SPT1 and SPT2. In this study we analyzed the expression of SPT1 and SPT2 in normal human tissues. Strong SPT1 and SPT2 expression was observed in pyramidal neurons in the brain, in colon epithelium, and in mucosal macrophages. However, SPT2 expression was more prominent than SPT1 in the colon mucosal macrophages, the adrenomedullary chromaffin cells and endothelium, and in the uterine endothelium. SPT2 was localized in both nuclei and cytoplasm of the adrenomedullary chromaffin cells, whereas SPT1 was primarily cytoplasmic. These observations link enhanced SPT expression to proliferating cells, such as the lung, stomach, intestinal epithelium, and renal proximal tubular epithelium, and to potentially activated cells such as neurons, chromaffin cells, and mucosal macrophages. A baseline expression of SPT, established by this study, may serve as a measure for aberrant expression in various disease states.


Subject(s)
Acyltransferases/metabolism , Acyltransferases/immunology , Antibody Specificity , Humans , Immunohistochemistry , Organ Specificity , Protein Subunits , Serine C-Palmitoyltransferase
SELECTION OF CITATIONS
SEARCH DETAIL
...