RESUMEN
OKT3, a mouse anti-human CD3 monoclonal antibody (mAb), is a potent immunosuppressive agent used in clinical transplantation to treat allograft rejection. Two major drawbacks of this therapy are the systemic release of several cytokines due to cross-linking mediated by the mAb between T cells and FcgammaR-bearing cells and the human anti-mouse antibody (HAMA) response. To overcome these side effects, three chimeric OKT3 single chain variable fragment (scFv) IgM antibodies, scOKT3-gamma DeltaIgM wt, scOKT3-gamma DeltaIgM C575S and scOKT3-gamma DeltaIgM VAEVD, were generated. They consist of the light and heavy variable binding domains of OKT3 mAb as well as the CH3 and CH4 domains of different human IgM variants linked with a human IgG3 hinge region to provide more flexibility and stability. Like the native IgM, scOKT3-gamma DeltaIgM antibodies are able to form polymeric structures, which lead to an increase in binding affinity and immunosuppressive potential compared with the parental OKT3 mAb. However, independently of their polymerization, all scOKT3-gamma DeltaIgM constructs do not induce any significant T cell proliferation or cytokine release (IL-2, TNF-alpha and IFN-gamma) in in vitro assays, while their CD3-modulating properties are retained. These results suggest that the use of scOKT3-gamma DeltaIgM antibodies may offer significant advantages over the OKT3 mAb in improving clinical immunosuppressive treatment.
Asunto(s)
Fragmentos de Inmunoglobulinas/farmacología , Inmunoglobulina M/farmacología , Inmunosupresores/farmacología , Activación de Linfocitos/efectos de los fármacos , Muromonab-CD3/farmacología , Proteínas Recombinantes de Fusión/farmacología , Linfocitos T/efectos de los fármacos , Adulto , Secuencia de Aminoácidos , Línea Celular , Activación de Complemento/efectos de los fármacos , Complemento C1q/metabolismo , Estabilidad de Medicamentos , Femenino , Humanos , Fragmentos de Inmunoglobulinas/inmunología , Inmunoglobulina M/inmunología , Datos de Secuencia Molecular , Muromonab-CD3/inmunología , Linfocitos T/inmunologíaRESUMEN
We have shown recently that cell surface-bound, single-chain Fv antibodies (scFv) are a powerful tool for the improvement of cellular tumor vaccines. To simplify this approach and to develop a general tool for the generation and improvement of cellular tumor vaccines, we chose an scFv against a peptide from the human proto-oncogene c-myc that could anchor any c-myc-tagged protein to the cell surface. The retroviral vector p50-Mx-neo (pMESV) was used to express scFv on the surface of the human melanoma line SkMel63. The cell-bound anti-c-myc scFv bound specifically to a soluble purified anti-CD28 scFv carrying a c-myc peptide-tag at its C terminus. Proof of principle was determined by incubating human peripheral blood lymphocytes with a mixture of (a) anti-c-myc-transfected SkMel63 cells binding the anti-CD28 scFv and (b) SkMel63 cells transfected with an anti-CD3 scFv. A clear synergistic effect on T-cell activation was observed that was comparable with that obtained in previous studies using SkMel63 cells transfected with the gene for the anti-CD28 scFv. As the cell surface-displayed anti-c-myc scFv can bind any c-myc-tagged protein of interest, this technique facilitates the genetic engineering of cellular vaccines for the therapy of virtually all human neoplasias.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos de Superficie/inmunología , Vacunas contra el Cáncer/uso terapéutico , Genes myc/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Activación de Linfocitos/inmunología , Linfocitos T/inmunología , Sitios de Unión , División Celular/efectos de los fármacos , Cartilla de ADN/química , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Expresión Génica , Genes myc/genética , Vectores Genéticos , Humanos , Inmunogenética , Interferón gamma/análisis , Interleucina-4/análisis , Reacción en Cadena de la Polimerasa , Proto-Oncogenes Mas , Células Tumorales Cultivadas/efectos de los fármacosRESUMEN
CD28 is a key regulatory molecule in T cell responses. Ag-TCR/CD3 interactions without costimulatory signals provided by the binding of B7 ligands to the CD28R appear to be inadequate for an effective T cell activation. Indeed, the absence of B7 on the tumor cell surface is probably one of the factors contributing to the escape of tumors from immunological control and destruction. Therefore, to increase the immunogenicity of tumor cell vaccines, we have expressed anti-CD3 and anti-CD28 single-chain Abs (scFv) separately on the surface of a human melanoma SkMel63 cell line (HLA-A*0201). A mixture of cells expressing anti-CD3 with cells expressing anti-CD28 resulted in a marked activation of allogeneic human PBL in vitro. The apparent induction of a Th1 differentiation pathway was accompanied by the proliferation of MHC-independent NK cells and MHC-dependent CD8+ T cells. PBL that had been cultured together with transfected SkMel63 tumor cells were able to specifically induce apoptosis in untransfected SkMel63 cells. In contrast, three other tumor cell lines expressing HLA-A*0201, including two melanoma cell lines, showed no significant apoptosis. These results provide valuable information for both adoptive immunotherapy and the generation of autologous tumor vaccines.
Asunto(s)
Especificidad de Anticuerpos , Apoptosis/inmunología , Región Variable de Inmunoglobulina/fisiología , Melanoma/inmunología , Melanoma/patología , Receptores de Antígenos de Linfocitos B/metabolismo , Animales , Especificidad de Anticuerpos/genética , Apoptosis/genética , Antígenos CD28/inmunología , Citocinas/biosíntesis , Citocinas/genética , Pruebas Inmunológicas de Citotoxicidad , Técnica del Anticuerpo Fluorescente Indirecta , Vectores Genéticos/inmunología , Vectores Genéticos/metabolismo , Humanos , Región Variable de Inmunoglobulina/biosíntesis , Región Variable de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/metabolismo , Activación de Linfocitos/inmunología , Melanoma/genética , Melanoma/metabolismo , Ratones , Muromonab-CD3/metabolismo , Muromonab-CD3/farmacología , Receptores de Antígenos de Linfocitos B/biosíntesis , Receptores de Antígenos de Linfocitos B/genética , Linfocitos T Citotóxicos/inmunología , Células TH1/metabolismo , Transfección/inmunología , Células Tumorales CultivadasRESUMEN
Isolated microtubule proteins from the Atlantic cod (Gadus morhua) assemble at temperatures between 8 and 30 degrees C. The cold-adaptation is an intrinsic property of the tubulin molecules, but the reason for it is unknown. To increase our knowledge of tubulin diversity and its role in cold-adaptation we have further characterized cod tubulins using alpha- and beta-tubulin site-directed antibodies and antibodies towards posttranslationally modified tubulin. In addition, one cod brain beta-tubulin isotype has been sequenced. In mammals there are five beta-tubulins (betaI, betaII, betaIII, betaIVa and betaIVb) expressed in brain. A cod betaIII-tubulin was identified by its electrophoretic mobility after reduction and carboxymethylation. The betaIII-like tubulin accounted for more than 30% of total brain beta-tubulins, the highest yield yet observed in any animal. This tubulin corresponds most probably with an additional band, designated beta(x), which was found between alpha- and beta-tubulins on SDS-polyacrylamide gels. It was found to be phosphorylated and neurospecific, and constituted about 30% of total cod beta-tubulin isoforms. The sequenced cod tubulin was identified as a betaIV-tubulin, and a betaIV-isotype was stained by a C-terminal specific antibody. The amount of staining indicates that this isotype, as in mammals, only accounts for a minor part of the total brain beta-tubulin. Based on the estimated amounts of betaIII- and betaIV-tubulins in cod brain, our results indicate that cod has at least one additional beta-tubulin isotype and that beta-tubulin diversity evolved early during fish evolution. The sequenced cod betaIV-tubulin had four unique amino acid substitutions when compared to beta-tubulin sequences from other animals, while one substitution was in common with Antarctic rockcod beta-tubulin. Residues 221, Thr to Ser, and 283, Ala to Ser, correspond in the bovine tubulin dimer structure to loops that most probably interact with other tubulin molecules within the microtubule, and might contribute to cold-adaptation of microtubules.
Asunto(s)
Peces/metabolismo , Microtúbulos/metabolismo , Tubulina (Proteína)/química , Adaptación Fisiológica , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Anticuerpos Monoclonales , Secuencia de Bases , Western Blotting , Encéfalo/metabolismo , Bovinos , Clonación Molecular , Frío , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Evolución Molecular , Biblioteca de Genes , Hígado/metabolismo , Datos de Secuencia Molecular , Miocardio/metabolismo , Óvulo/metabolismo , Paclitaxel/farmacología , Fosforilación , Procesamiento Proteico-Postraduccional , Análisis de Secuencia de ADN , Tubulina (Proteína)/metabolismoRESUMEN
The interaction between tubulin subunits and microtubule-associated proteins (MAPs) such as tau is fundamental for microtubule structure and function. Previous work has suggested that the "microtubule binding domain" of tau (composed of three or four imperfect 18-amino acid repeats, separated by 13- or 14-amino acid inter-repeat regions) can bind to the C-terminal ends of both alpha and beta tubulin monomers. Here, using covalent cross-linking strategies, we demonstrate that there are two distinct tau cross-linking sites (designated as "C-terminal" and "internal") on each alpha and beta tubulin monomer. The C-terminal tau cross-linking site is located within the 12 C-terminal amino acids of both alpha and beta tubulin, while the internal tau cross-linking site is located within the C-terminal one-third of alpha and beta tubulin but not within the last 12 amino acids. In addition, we show that tau cross-links to the C-terminal site via its repeat 1 and/or the R1-R2 inter-repeat. The cross-linking of tau to the internal site is mediated by some subset of its other repeat units. Integrating these and earlier data with the 3.7 A resolution model of the alphabeta tubulin dimer recently presented by E. Nogales et al. [(1998), Nature 391, 199-203], we propose a new model for the tau-microtubule interaction.
Asunto(s)
Reactivos de Enlaces Cruzados/metabolismo , Tubulina (Proteína)/metabolismo , Proteínas tau/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Etildimetilaminopropil Carbodiimida/metabolismo , Humanos , Microtúbulos/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Estructura Terciaria de Proteína , Ratas , Tubulina (Proteína)/química , Proteínas tau/químicaRESUMEN
To provide an efficient means of coupling proteins, peptides and other suitable moieties to cells, we have constructed a retroviral expression vector for cell surface display of a single-chain antibody (scFv) against the hapten 4-ethoxymethylene-2-phenyl-oxazo-line-5-one (phOx). The hapten phOx can be easily conjugated to primary amino and sulfhydryl groups, thus providing points of attachment for the cell surface-bound anti-phOx scFv. This universal cell coupling system could prove to be particularly useful for anchoring monoclonal antibodies for tumor targeting and to present co-stimulatory molecules and other ligands (even mixtures) at the cell surface for gene therapy.
Asunto(s)
Reacciones Antígeno-Anticuerpo , Fragmentos de Inmunoglobulinas/genética , Animales , Células Cultivadas , Citometría de Flujo , Haptenos/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Retroviridae/genética , TransfecciónRESUMEN
CI 980 (NSC 613862; [S-(-)]) and NSC 613863 [R-(+)] are the two chiral isomers of ethyl 5-amino 1,2-dihydro-2-methyl-3-phenylpyrido[3,4-b]pyrazin-7-ylcar bamate (NSC 370147), which is a mitotic inhibitor with in vivo and in vitro activity against murine multidrug-resistant sublines. We have characterized the inhibition of in vitro microtubule assembly by the S (CI 980) and R (NSC 613863) enantiomers, their actions on the cytoplasmic microtubule network of epithelial-like PtK2 cells, and on the cell cycle of different human and murine leukemias and PtK2 cells. Assembly of purified tubulin, or tubulin plus microtubule-associated proteins, into microtubules was substoichiometrically inhibited by both compounds, which also induced a slow depolymerization of preassembled microtubules. Half inhibitory concentrations were 0.4-0.7 microM and 1.6-2.1 microM for the S and R isomers, respectively. Excess of both drugs induced polymerization of liganded tubulin into abnormal polymers similar to colchicine. The cytoplasmic microtubules of PtK2 cells were disrupted by both compounds in a concentration- and time-dependent manner, which was observed by indirect immunofluorescence microscopy and quantified by an enzyme-linked immunoassay of cytoskeletal tubulin. Half inhibitory concentrations were 6 nM S isomer, 100 nM R isomer, and 1 microM colchicine. Twenty nM S isomer or 500-700 nM R isomer gave nearly maximal effect. At these concentrations, half maximal microtubule depolymerization took place after 2 h of treatment. After drug removal, slow microtubule assembly and nearly complete reorganization of the cytoplasmic microtubules of PtK2 cells were observed (24 h). One nM S enantiomer or 25 nM R enantiomer induced mitotic arrest in 8 h in U937, HL60, and EL4 leukemias. PtK2 cells also stopped in mitosis after a 24-h incubation with 50 nM R isomer or 5 nM S isomer. The inhibition of cell division was irreversible in the leukemic cells, while PtK2 cells partially resumed growth. Although the interactions of CI 980 with microtubules in vitro are not very different from other drugs, it is a most potent cellular microtubule and mitotic inhibitor.
Asunto(s)
Antineoplásicos/farmacología , Carbamatos/farmacología , Ciclo Celular/efectos de los fármacos , Proteínas de Microtúbulos/efectos de los fármacos , Microtúbulos/efectos de los fármacos , Pirazinas/farmacología , Piridinas/farmacología , Animales , Línea Celular , Humanos , Leucemia Mieloide/patología , Leucemia Promielocítica Aguda/patología , Ratones , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Células Tumorales CultivadasRESUMEN
The effects of taxoids (taxol and Taxotere) were followed on two human cancerous cell lines (bladder carcinoma J82 cells and epidermoid carcinoma KB 3-1 cells). Three cellular parameters were studied, viz., the qualitative effect on cellular microtubules, the quantitation of tubulin, and the antimitotic action, using two-parametric flow-cytometric analyses in treated cells. In both of the cell lines the tubulin content increased after taxoid treatment before the accumulation of cells in the G2/M phase. The effects of taxoids on tubulin appeared at about a 10-fold lower concentration on KB cells than on J82 cells. After drug exposure, the microtubule network showed a striking difference between the two cell lines: microtubule bundles were predominant in the J82 cell line, whereas multiple asters were prevalent in the KB cell line. The formation of these structures was dose- and time-dependent. Asters were observed in mitotic cells and bundles were seen in interphase cells. The reversibility of these structures in both cell lines varied with the duration of exposure to drug. Some differences were shown between taxol and Taxotere: the effects of Taxotere as compared with taxol appeared at a 2-fold lower concentration and their reversibility was slower.