RESUMEN
Synthetic vaccines that are based on peptides representing immunogenic epitopes require a carrier molecule as well as an adjuvant in order to be effective. The choice of carriers or adjuvants approved for use in humans is very limited, and a considerable effort is devoted to develop new and efficient delivery systems. One of these vehicles utilizes preparations of outer membranes of meningococci, that form hydrophobic interactions, denoted proteosomes. Immunogenic proteins and peptides can be anchored to these proteosomes vesicles, which may serve as both carrier and adjuvant functions. In the present study we examined the ability of proteosomes to present epitopes of influenza, to elicit specific anti-influenza responses and to protect mice against viral challenge after intranasal immunization. Three influenza peptides were used--one corresponding to amino acid residues 91-108 of the haemagglutinin surface glycoprotein of H3 subtype, which comprises a B-cell epitope, and two from the internal nucleoprotein--a T-helper cell (Th) epitope (residues 55-69) and a cytotoxic T-lymphocyte (CTL) epitope (147-158). Mice were immunized intranasally (i.n.) with preparations containing each of the above epitopes, or various combinations thereof. The results obtained with this system demonstrate that influenza epitopes represented by synthetic peptides anchored to a proteosome carrier elicit both humoral and cellular specific immune responses, that can lead to partial protection of the mice from viral challenge. The importance of immunizing with vaccines containing both B- and T-cell peptide epitopes was emphasized by the demonstration that such vaccines elicited longer lasting immunity and led to more effective protection against influenza viral challenge.
Asunto(s)
Virus de la Influenza A/inmunología , Vacunas contra la Influenza/farmacología , Infecciones por Orthomyxoviridae/prevención & control , Péptidos/inmunología , Péptidos/farmacología , Vacunas Sintéticas/farmacología , Proteínas Virales/inmunología , Administración Intranasal , Secuencia de Aminoácidos , Animales , Linfocitos B/inmunología , Embrión de Pollo , Epítopos/inmunología , Vacunas contra la Influenza/inmunología , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Infecciones por Orthomyxoviridae/inmunología , Linfocitos T/inmunología , Vacunas Sintéticas/inmunología , Proteínas Virales/farmacologíaRESUMEN
Normal strains of mice are rendered sensitive to small amounts (3-10 micrograms) of staphylococcal enterotoxin B (SEB) by transplanting bone marrow cells of SCID donor mice to lethally irradiated recipients. Four to 12 weeks post-transplantation, SEB induces 56-100% lethality. Transplantation of normal mouse bone marrow cells, either alone or with the SCID mouse selected bone marrow cells, does not confer SEB sensitivity. These data imply that either irradiation ablates certain cell population(s), that confer resistance to SEB in normal mice (populations that are absent in the SCID donor mice) or that the donor cells selectively repopulate recipients with SEB-sensitive cells. This model will help elucidate the cells, cytokines and the SEB peptide fragments responsible for SEB toxicity and will be useful in identifying promising vaccine candidates and in developing preventive medicines to protect against this potent toxin.
Asunto(s)
Trasplante de Médula Ósea/efectos adversos , Enterotoxinas/toxicidad , Quimera por Radiación/inmunología , Traumatismos Experimentales por Radiación/inmunología , Staphylococcus aureus/inmunología , Irradiación Corporal Total/efectos adversos , Animales , Relación Dosis-Respuesta Inmunológica , Inmunidad Innata , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones SCID , Datos de Secuencia Molecular , Traumatismos Experimentales por Radiación/patología , Traumatismos Experimentales por Radiación/prevención & controlRESUMEN
Cis-diamminedichloroplatinum (II) (cis-Pt) complexed to a carboxymethyl dextran-avidin conjugate was targeted to biotin-monoclonal antibody 108 (b-MAb 108). This MAb recognizes the extracellular domain of the epidermal growth factor receptor (EGF-R) on human epidermoid carcinoma (KB) cells over-expressing EGF-R. Cis-Pt-carboxymethyl-dextran-avidin (Pt-dex-Av) containing 60-90 M cis-Pt/M avidin was administered 24 hr following b-MAb108 containing 3-5 M biotin/M MAb. This treatment was potentially more effective in suppressing the growth of established KB tumor xenografts, or in inhibiting the development of lung metastases in nude mice, than free MAb 108, free drug or MAb 108 followed by drug. Replacing b-MAb 108 by unbiotinylated antibody or by b-MAb of a different specificity also yielded lower suppressive effects. The sequential administration of Pt-dex-Av following b-MAb was more effective than introduction of the Pt-dex-Av when already complexed to b-MAb 108. The results presented in this preliminary investigation suggest that Pt-dex-Av is specifically removed from the circulation by b-MAb 108 concentrated at the tumor site.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Antineoplásicos/administración & dosificación , Cisplatino/administración & dosificación , Células KB/efectos de los fármacos , Animales , Avidina , Biotina , Terapia Combinada , Dextranos , Esquema de Medicación , Portadores de Fármacos , Humanos , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Ratones , Ratones Desnudos , Trasplante de NeoplasiasRESUMEN
Monoclonal antibodies that recognize the extracellular domain of the epidermal growth factor receptor (mAb108) were conjugated with doxorubicin through a dextran bridge. Several antibody-drug conjugates, containing different amounts of doxorubicin, retained binding capacity to human epidermoid carcinoma (KB) cells overexpressing epidermal growth factor receptors. Slight decrease in drug cytotoxicity was seen in in vitro tests, as determined either by inhibition of thymidine incorporation into cells or by reduction in number and size of KB-cell colonies. Yet, when tested in vivo against KB tumor xenografted into nude mice, the anti-epidermal growth factor-receptor drug conjugates with high drug-substitution levels were significantly more effective than free doxorubicin, antibody alone, mixture of dextran-doxorubicin and antibody, or drug conjugated with irrelevant antibody. When the labile covalent bonds linking antibody to dextran bridge were stabilized by reduction, the therapeutic efficacy of the conjugate was markedly decreased. These results show that antibodies against the extracellular domain of the epidermal growth factor can deliver doxorubicin specifically and efficiently to tumor sites that express high receptor levels exerting a specific antitumor effect.
Asunto(s)
Doxorrubicina/administración & dosificación , Receptores ErbB/inmunología , Inmunotoxinas/administración & dosificación , Neoplasias Experimentales/terapia , Animales , Anticuerpos Monoclonales/administración & dosificación , Carcinoma de Células Escamosas/terapia , División Celular , Humanos , Inmunoterapia , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Células Tumorales CultivadasRESUMEN
Conjugates were constructed between daunorubicin or vindesin and a monoclonal antibody to human milk fat globule membrane associated antigen. This antibody recognizes a high molecular weight glycoprotein present at the cell surface of human normal and tumour epithelial cells; after specific binding to plasma membrane of cultured MCF-7 human breast carcinoma cells, it is endocytosed and gains access to lysosomes, wherein it is broken down (Aboud-Pirak et al., Cancer Res 48: 3188-3196, 1988). Covalent linkage of daunorubicin (through a succinylated tetrapeptide arm) or of vindesin (through a hemisuccinate arm) yields conjugates with maximal molar ratios (drug molecule/specific IgG under monomeric form, i.e. unaggregated) or 2.0 and 4.5 respectively. The conjugate with daunorubicin inhibits the binding of the 3H labelled antibody to MCF-7 cells as efficiently as the native unconjugated antibody, whereas the conjugate with vindesin inhibits it only by 56%. Both conjugates are entirely stable in plasma and serum; after 24 hr incubation at pH 4.8 in the presence of rat liver lysosomal enzymes, 60 and 33% of daunorubicin and vindesin respectively are released from the conjugates. Adherent non-confluent cultures of cells recognized (MCF-7) or not (Hep-G2, human hepatocarcinoma cells) by the antibody were incubated from 1 hr to 6 days with different concentrations of daunorubicin or vindesin, free or conjugated to the specific or to a control monoclonal antibody. LD50, defined as the drug concentration required to reach 50% of the amount of cell associated protein obtained in the absence of drug were determined at the end of 6 days continuous incubation or after shorter incubation followed by reincubation in drug free medium up to 6 days. Both cell lines are almost equally susceptible to the free drugs. The conjugate between daunorubicin and the antibody appears inactive, even at saturating concentrations of antibody. This could result from the extrusion out of the cells of daunorubicin molecules released from the conjugate, impairing the drug to reach the intracellular concentration required for cytotoxicity. In contrast, conjugation of vindesin to the specific but not to a control antibody restricts the activity of the drug to cells selectively recognized by the specific antibody. However, even after corrections for the loss of immunoreactivity and for the incomplete release of vindesin from the conjugate, cytotoxicity is achieved at higher concentrations or requires longer exposure to the conjugated than to the free drug.(ABSTRACT TRUNCATED AT 400 WORDS)
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Daunorrubicina/administración & dosificación , Vindesina/administración & dosificación , Reacciones Antígeno-Anticuerpo , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Hidrólisis , Glicoproteínas de Membrana/inmunología , Mucina-1 , Células Tumorales CultivadasRESUMEN
Iodine-125-labeled monoclonal antibody 108.4 (108.4 mAb), raised against the extracellular domain of the epidermal growth factor (EGF) receptor, was shown to visualize sc xenografts of human oral epidermoid carcinoma (KB) cells in nude mice. In vitro, although EGF caused an increase in the number of KB cell colonies (150% at a concentration of 160 mM), the anti-EGF receptor antibodies reduced clone formation. At a concentration at which EGF caused a 50% increase in colony number, the addition of a 100-fold molar excess of 108.4 mAb resulted in a decrease in the number of cell colonies to 20% of the original value. Therefore, the effect of antibody on the KB tumor was studied in vivo in three different modes of tumor transplantation. Antitumor activity was demonstrated first by retardation (versus controls) of the growth of tumor cells as sc xenografts (P greater than .017), then by prolongation of the life span of animals with the ip form of the tumor (P less than .001), and finally on an experimental lung metastasis by a reduction in the number and size of tumors (P less than .05). When the anti-EGF receptor antibodies were added together with cisplatin, the antitumor effect was greatly enhanced, suggesting that the toxic activity of these agents is synergistic (P less than .007). The antitumor effect persisted when animals were treated with the F(ab)'2 fragment of the antibody, although it was less efficient. The Fab fragment of the antibody, whose ability to bind to the cell-associated receptor was completely conserved, did not affect the growth of the tumor. The activity manifested by the F(ab)'2 fragment of the anti-EGF receptor antibodies suggested that the antitumor effect was not due to immune mechanisms requiring the Fc portion of the antibody.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Receptores ErbB/inmunología , Neoplasias Experimentales/terapia , Animales , Fragmentos Fab de Inmunoglobulinas , Células KB , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Trasplante HeterólogoRESUMEN
The aim of this study was to analyze whether a monoclonal antibody to human milk fat globule membrane-associated antigens, recognized specifically and homogeneously by human breast carcinoma cells but also by normal epithelial cells active in secretion, could be used to restrict the access of antitumoral drugs to cells exposing the epitope. The drug-antibody conjugate to be used is constructed by means of a covalent peptidic linkage stable in extracellular medium but hydrolyzed by lysomal enzymes after endocytosis of the drug-carrier conjugate. This monoclonal antibody specifically immunoprecipitates radioactive material from MCF-7 cells biosynthetically radiolabeled with galactose, glucosamine, palmitic acid, or acetic acid but not with mannose, leucine, or methionine. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol, the label migrates as two bands with apparent molecular weights of about 350,000 and 400,000. These bands disappear, or their molecular weight is affected, after treatment of the cells with cycloheximide or of cell lysates with trypsin, Pronase, or neuraminidase but not treatment of the immunoprecipitate with endoglycosidase F. This suggests that these antigens are glycoproteins with O-linked oligosaccharides containing sialic acid in the epitope. By analogy, they should be similar, if not identical, to those recognized by the monoclonal antibodies designated HMFG1 (H. Burchell, H. Durbin, and J. Taylor-Papadimitriou, J. Immunol., 131:508-513, 1983) and DF3 (H. Sekine, T. Ohno, and D.W. Kufe, J. Immunol., 135:3610-3615, 1985). Binding at 4 degrees C of the 3H-labeled antibody by MCF-7 cells indicates the specific attachment of about 1.2 X 10(6) IgG molecules per cells with a Kd of about 14 nM. At 37 degrees C, cells take up the 3H-labeled antibody in amounts much higher than the binding capacity. In addition to cell-associated material, labeled digestion products are released into the culture medium. Cell fractionation by differential centrifugation and isopycnic equilibration on sucrose gradient indicates that the bulk of cell-associated antibody is distributed like the marker enzyme of lysosomes. Although the total uptake of the antibody by the cells is unaffected by either 50 microM chloroquine or 3 micrograms/ml cycloheximide, the release of digestion products is completely inhibited by chloroquine. Antigen-antibody dissociation is pH dependent, since, respectively, 50 and 84% of membrane-bound antibody are released during washing at pH 4.6 and 4.1.(ABSTRACT TRUNCATED AT 400 WORDS)