IgG antibody response to polyethylene glycol-modified adenosine deaminase in patients with adenosine deaminase deficiency.

Polyethylene glycol (PEG)-modified bovine adenosine deaminase (ADA) is used for replacement therapy of severe combined immunodeficiency disease due to inherited ADA deficiency. We monitored IgG anti-ADA antibody in 17 patients treated by intramuscular injections of PEG-ADA for 1 to greater than 5.5 yr. ELISA-detectable anti-ADA IgG appeared in 10 patients, usually between the third and eighth months of treatment. Anti-ADA levels did not correlate with trough plasma ADA activity, which averaged 1.8-5 times normal blood (erythrocyte) ADA activity, depending on dose (15-60 U/kg per wk). ELISA-detectable anti-ADA antibodies were directed primarily at bovine-specific peptide (rather than PEG-containing) epitopes. Enhanced enzyme clearance, mediated by antibody that directly inhibited native and PEG-modified bovine ADA, and native, but not PEG-modified human ADA, occurred in two patients. In one, tolerance was induced; in the second, twice weekly injections of PEG-ADA compensated for accelerated clearance. We speculate that inhibitory antibodies recognize conserved, relatively PEG-free epitope(s) encompassing the active site, and that in human, but not bovine, ADA a PEG-attachment site "shields" the active site from immune recognition. We conclude that PEG-modification largely prevents the development of high affinity, or high levels of clearing antibodies to bovine ADA, and that PEG-modified human ADA should be further investigated as a possible treatment for ADA deficiency.

Effects of a hybrid recombinant human alpha interferon (A/D) on in vitro cytotoxicity and in vivo localization of monoclonal antibody L6-cytosine deaminase conjugate in a colon cancer model.

L6 is an IgG2a murine monoclonal antibody which we have demonstrated binds well to HT29 human colon carcinoma cells by flow cytometry, whole cell ELISA, and mixed hemadsorption. In vitro cytotoxicity studies revealed that the monoclonal antibody L6-cytosine deaminase (L6-CD) immunoconjugate plus the nontoxic prodrug, 5-fluorocytosine (5-FC), is equivalent to 5-fluorouracil (5-FU) in its ability to kill HT29 cells. Human alpha-interferon (A/D) was able to enhance this cytotoxic effect. The I.C.50's revealed that very small amounts of L6-CD are needed for this cytotoxic effect (approximately, 5 pg/ml resulted in 50% viability). The limiting factor was the amount of 5-FC employed with L6-CD (3 microM yielded 50% cell viability). alpha-Interferon (A/D) lowered the requirement of 5-FC to 1 microM to achieve 50% cell lethality. In vivo biodistribution experiments indicated that 1 microgram of L6-CD is nonspecifically taken up by the liver and spleen and cleared rapidly from the blood. Significant localization of L6-CD to HT29 tumors occurred only when 99 micrograms of unlabeled L6-CD was added to 1 microgram of 125I-labeled immunoconjugate injected intravenously. Further augmentation of tumor/blood ratios without reduction in percent injected dose per gram of tumor was possible with the intravenous injection of 100 micrograms of anti-idiotypic monoclonal antibody 13B, 24 hours after L6-CD, which bound unreacted L6-CD and cleared it from the blood. The addition of 100,000 U of alpha-interferon (A/D) given intraperitoneally every day increased the clearance of L6-CD by the liver and spleen, but impaired tumor localization (percent injected dose per gram). These studies demonstrated that in vivo localization of the L6-CD conjugate to HT29 tumors could be optimized by injecting excess L6-CD followed by an equal amount of L6 anti-idiotype mAb 13B, 24 hours after L6-CD.

Immobilization of adenosine deaminase onto agarose and casein.

In the present study adenosine deaminase (ADA) was immobilized onto two different polymeric materials, agarose and casein. The factors affecting the amount of enzyme attachment onto the polymeric supports such as incubation time were investigated. The maximum amount of enzyme immobilized onto different polymeric supports occurred at incubation pH value 7.5 and ADA concentration 42 units/g and the incubation time needed for the maximum amount of enzyme attachment to the polymeric supports was found to be 8 h. Some phsicochemical properties of the free and immobilized ADA such as operational stability, optimum temperature and thermal stability, pH optimum and stability, storage stability, and the effect of gamma-radiation were studied. The operational stability of the free and immobilized enzyme showed that the enzyme immobilized by a cross-linking technique using gultaric dialdehyde showed poor durability and the relative activity decreased sharply due to the leakage after repeated washing, while the enzymes immobilized by covalent bonds to the carriers showed a slight decrease in most cases in the relative activity (around 20%) after being used 10 times. Storage for 4-6 months, showed that the free enzyme lost its activity, while the immobilized enzyme showed the opposite behavior. Subjecting the immobilized enzyme to a dose of gamma radiation of 0.5-10 Mrad showed complete loss in the activity of the free enzyme at a dose of 5 Mrad, while the immobilized enzymes showed relatively high resistance to gamma radiation up to a dose of 5 Mrad.

Adenosindeaminasa como método diagnóstico en tuberculosis pleural

Estudiamos 128 pacientes con derrame pleural, a los cuales se les realizó un diagnóstico histológico o bacteriológico definitivo. La medición del ADA en líquido pleural separó claramente los grupos de tuberculosis y neoplasia con un punto de corte de 36U. Teniendo una sensibilidad de 97.59 por ciento y un valor predictivo negativo de un 94.8 por ciento.