(1/471) Specific lysis of melanoma cells by receptor grafted T cells is enhanced by anti-idiotypic monoclonal antibodies directed to the scFv domain of the receptor.
Malignant transformation of melanocytes is frequently associated with abnormalities in antigen processing and in human leukocyte antigen class I antigen expression. Here, we evaluated a human leukocyte antigen class I antigen-independent approach to target cytotoxic T lymphocytes to melanoma cells by grafting cytotoxic T lymphocytes with a chimeric receptor that consists of both a domain binding to high molecular weight-melanoma associated antigen and a cellular activation domain. The binding domain is a single-chain antibody fragment (scFv) derived from the monoclonal anti-high molecular weight-melanoma associated antigen antibody 763.74 by phage display techniques. The cellular activation domain is the signaling unit of the FcepsilonRI receptor gamma chain. Both domains constitute the chimeric receptor scFv763.74-gammaR. Cytotoxic MD45 T cells grafted with the scFv763.74-gammaR receptor bind specifically to high molecular weight-melanoma associated antigen-positive melanoma cells and lyse melanoma cells in a human leukocyte antigen class I independent fashion. Pre-incubation of receptor grafted T cells with immobilized anti-idiotypic (id) monoclonal antibody MK2-23 binding to the scFv domain of the receptor enhanced the lysis of melanoma cells indicating that the specific cytolytic activity of receptor grafted T cells can be increased by costimulation with cross-linked anti-idiotypic monoclonal antibodies that recognize the antigen binding domain of the chimeric receptor. (+info)
(2/471) ErbB2 and ErbB3 receptors mediate inhibition of calcium-dependent chloride secretion in colonic epithelial cells.
We have previously demonstrated that epidermal growth factor (EGF) inhibits calcium-dependent chloride secretion via a mechanism involving stimulation of phosphatidylinositol 3-kinase (PI3-K). The muscarinic agonist of chloride secretion, carbachol (CCh), also stimulates an antisecretory pathway that involves transactivation of the EGF receptor (EGFR) but does not involve PI3-K. Here, we have examined if ErbB receptors, other than the EGFR, have a role in regulation of colonic secretion and if differential effects on ErbB receptor activation may explain the ability of the EGFR to propagate diverse signaling pathways in response to EGF versus CCh. Basolateral, but not apical, addition of the ErbB3/ErbB4 ligand alpha-heregulin (HRG; 1-100 ng/ml) inhibited secretory responses to CCh (100 microM) across voltage-clamped T(84) epithelial cells. Immunoprecipitation/Western blot studies revealed that HRG (100 ng/ml) stimulated tyrosine phosphorylation and dimerization of ErbB3 and ErbB2, but had no effect on phosphorylation of the EGFR. HRG also stimulated recruitment of the p85 subunit of PI3-K to ErbB3/ErbB2 receptor dimers, while the PI3-K inhibitor, wortmannin (50 nM), completely reversed the inhibitory effect of HRG on CCh-stimulated secretion. Further studies revealed that, while both EGF (100 ng/ml) and CCh (100 microM) stimulated phosphorylation of the EGFR, only EGF stimulated phosphorylation of ErbB2, and neither stimulated ErbB3 phosphorylation. EGF, but not CCh, stimulated the formation of EGFR/ErbB2 receptor dimers and the recruitment of p85 to ErbB2. We conclude that ErbB2 and ErbB3 are expressed in T(84) cells and are functionally coupled to inhibition of calcium-dependent chloride secretion. Differential dimerization with other ErbB family members may underlie the ability of the EGFR to propagate diverse inhibitory signals in response to activation by EGF or transactivation by CCh. (+info)
(3/471) Pharmacology and biological efficacy of a recombinant, humanized, single-chain antibody C5 complement inhibitor in patients undergoing coronary artery bypass graft surgery with cardiopulmonary bypass.
BACKGROUND: Cardiopulmonary bypass (CPB) induces a systemic inflammatory response that causes substantial clinical morbidity. Activation of complement during CPB contributes significantly to this inflammatory process. We examined the capability of a novel therapeutic complement inhibitor to prevent pathological complement activation and tissue injury in patients undergoing CPB. METHODS AND RESULTS: A humanized, recombinant, single-chain antibody specific for human C5, h5G1.1-scFv, was intravenously administered in 1 of 4 doses ranging from 0.2 to 2.0 mg/kg before CPB. h5G1.1-scFv was found to be safe and well tolerated. Pharmacokinetic analysis revealed a sustained half-life from 7.0 to 14.5 hours. Pharmacodynamic analysis demonstrated significant dose-dependent inhibition of complement hemolytic activity for up to 14 hours at 2 mg/kg. The generation of proinflammatory complement byproducts (sC5b-9) was effectively inhibited in a dose-dependent fashion. Leukocyte activation, as measured by surface expression of CD11b, was reduced (P<0.05) in patients who received 1 and 2 mg/kg. There was a 40% reduction in myocardial injury (creatine kinase-MB release, P=0.05) in patients who received 2 mg/kg. Sequential Mini-Mental State Examinations (MMSE) demonstrated an 80% reduction in new cognitive deficits (P<0.05) in patients treated with 2 mg/kg. Finally, there was a 1-U reduction in postoperative blood loss (P<0. 05) in patients who received 1 or 2 mg/kg. CONCLUSIONS: A single-chain antibody specific for human C5 is a safe and effective inhibitor of pathological complement activation in patients undergoing CPB. In addition to significantly reducing sC5b-9 formation and leukocyte CD11b expression, C5 inhibition significantly attenuates postoperative myocardial injury, cognitive deficits, and blood loss. These data suggest that C5 inhibition may represent a novel therapeutic strategy for preventing complement-mediated inflammation and tissue injury. (+info)
(4/471) Structural dynamics of green fluorescent protein alone and fused with a single chain Fv protein.
Structural information on intracellular fusions of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria with endogenous proteins is required as they are increasingly used in cell biology and biochemistry. We have investigated the dynamic properties of GFP alone and fused to a single chain antibody raised against lipopolysaccharide of the outer cell wall of gram-negative bacteria (abbreviated as scFv-GFP). The scFv moiety was functional as was proven in binding assays, which involved the use of both fluorescence correlation spectroscopy observing the binding of scFv-GFP to gram-negative bacteria and a surface plasmon resonance cell containing adsorbed lipopolysaccharide antigen. The rotational motion of scFv-GFP has been investigated with time-resolved fluorescence anisotropy. However, the rotational correlation time of scFv-GFP is too short to account for globular rotation of the whole protein. This result can only be explained by assuming a fast hinge motion between the two fused proteins. A modeled structure of scFv-GFP supports this observation. (+info)
(5/471) Green fluorescent antibodies: novel in vitro tools.
We produced a fluorescent antibody as a single recombinant protein in Escherichia coli by fusing a red-shifted mutant of green fluorescent protein (EGFP) to a single-chain antibody variable fragment (scFv) specific for hepatitis B surface antigen (HepBsAg). GFP is a cytoplasmic protein and it was not previously known whether it would fold correctly to form a fluorescent protein in the periplasmic space of E.COLI: In this study we showed that EGFP alone or fused to the N'- and C'-termini of the scFv resulted in fusion proteins that were in fact highly fluorescent in the periplasmic space of E.COLI: cells. Further characterization revealed that the periplasmic N'-terminal EGFP-scFv fusion was the most stable form which retained the fluorescent properties of EGFP and the antigen binding properties of the native scFv; thus representing a fully functional chimeric molecule. We also demonstrated the utility of EGFP-scFv in immunofluorescence studies. The results showed positive staining of COS-7 cells transfected with HepBsAg, with comparable sensitivity to a monoclonal antibody or the scFv alone, probed with conventional fluorescein-labelled second antibodies. In this study, we developed a simple technique to produce fluorescent antibodies which can potentially be applied to any scFv. We demonstrated the utility of an EGFP-scFv fusion protein for immunofluorescence studies, but there are many biological systems to which this technology may be applied. (+info)
(6/471) Molecular analysis of human anti-factor VIII antibodies by V gene phage display identifies a new epitope in the acidic region following the A2 domain.
One of the major binding sites for factor VIII inhibitors is located within the A2 domain. In this study, phage display technology was used to isolate 2 human monoclonal antibodies, termed VK34 and VK41, directed toward the heavy chain of factor VIII. The V(H) domain of a single-chain variable domain antibody fragment (scFv) VK34 is encoded by germline gene segment DP-10. Epitope-mapping studies revealed that scFv VK34 is directed against amino acid residues Arg(484)-Ile(508), a previously identified binding site for factor VIII inhibitors in the A2 domain. ScFv VK34 inhibited factor VIII activity with a titer of 280 BU/mg. The V(H) domain of VK41 was encoded by germline gene segment DP-47. A phage corresponding to VK41 competed with a monoclonal antibody for binding to amino acid residues Asp(712)-Ala(736) in the acidic region adjacent to the A2 domain. Reactivity of VK41 with a factor VIII variant in which we replaced amino acid residues Asp(712)-Ala(736) for the corresponding region of heparin cofactor II was strongly reduced. In addition, substitution of Tyr(718719723) for Phe abrogated binding of VK41 to factor VIII. ScFv VK41 did not inhibit factor VIII activity. This study not only defines the primary structure of human anti-factor VIII antibodies reactive with the A2 domain, it also describes an antibody with an epitope not previously identified in the antibody repertoire of hemophilia patients with an inhibitor. (Blood. 2000;96:540-545) (+info)
(7/471) Generation of a murine single chain Fv (scFv) antibody specific for cucumber mosaic virus (CMV) using a phage display library.
With the long-term goal of generating CMV-resistant transgenic plants using antibody genes, a single-chain variable fragment (scFv) antibody that binds to the cucumber mosaic virus was isolated from a scFv phage display library by four rounds of affinity selection with CMV-Mf as an antigen. The scFv has the identical binding specificity to CMV as a monoclonal antibody that is generated by the hybridoma fusion technique, and recognized purified preparations of CMV isolates belonging to either subgroup I or II in immunoblotting. The nucleotide sequences of the recombinant antibody showed that a heavy chain variable region (V(H)) gene belonged to the VH3 subgroup and the kappa light chain variable region (V kappa) came from the Vkappa4 subgroup. Our results demonstrate that the scFv phage display library, an alternative approach to the traditional hybridoma fusion technique, has a potential applicability in the study of plant virus and plant pathology. (+info)
(8/471) RASA, a recombinant single-chain variable fragment (scFv) antibody directed against the human sperm surface: implications for novel contraceptives.
BACKGROUND: A recombinant single-chain variable fragment (scFv) antibody was engineered to a tissue-specific carbohydrate epitope located on human sperm agglutination antigen-1 (SAGA-1), a sperm glycoform of CD52. METHODS AND RESULTS: cDNAs encoding the variable regions of the S19 [IgG(1)kappa] monoclonal antibody (mAb) were identified, linked, and cloned into the pCANTAB 5E vector. The recombinant anti-sperm antibody (RASA) was expressed in E. coli HB2151 cells as a 29 kDa monomer and, remarkably, also formed multimers of approximately 60 and 90 kDa. RASA reacted with the endogenous SAGA-1 antigen by Western blot analysis, labelled the entire human sperm surface by indirect immunofluorescence, and aggregated human spermatozoa in a tangled (head-to-head, head-to-tail, tail-to-tail) pattern of agglutination, as was also observed with the native S19 mAb. CONCLUSIONS: These results demonstrate that active recombinant antibodies can be produced to a tissue-specific carbohydrate epitope on the human sperm surface, thereby opening opportunities for novel contraceptive agents. (+info)