Immunological and pharmacological characterization of poly-DL-alanyl-modified Erwinia carotovora L-asparaginase.

The covalent attachment of poly-DL-alanine peptides to lysyl residues on the surface of Erwinia carotovora L-asparaginase has produced a modified enzyme which is much less immunogenic in mice and demonstrates 100-fold longer plasma half-life in the rhesus monkey. Immunogenic responses towards both the immunoglobulin G (IgG) and immunoglobulin E (IgE) antibody subclasses were evaluated in C57BL x DBA/2 F1 mice exposed to 250 rads of whole-body irradiation 4 hr prior to immunization with 5-diazo-4-oxynorvaline-inactivated native and modified L-asparaginase in complete Freund's adjuvant. Under these immunologically stressful conditions, the native enzyme evoked an IgE and IgG response which could be further amplified by a secondary immunization, whereas the modified enzyme evoked no IgE or IgG response even after a tertiary immunization. In experiments mimicking an intensive therapeutic schedule, whereby two groups of mice were given weekly injections of 5 to 10 units of either native or modified asparaginase for up to 14 weeks, neither enzyme form evoked a significant IgE response, and only the mice given injections of the native enzyme produced an IgG response. In a preliminary patient study, skin testing of a child who had shown an allergic reaction to the native enzyme resulted in a negative response after an intradermal injection of the modified enzyme, whereas a wheal and flare reaction was observed to both the native enzyme and a histamine control. All of these results suggest that the modified enzyme should show a definite reduction in immunological reactions associated with L-asparaginase treatment of childhood leukemia.

Cloning, purification, and characterization of beta-cystathionase from Escherichia coli.

The Clarke-Carbon clone bank of hybrid plasmid Escherichia coli DNA has been screened for plasmids able to complement an E. coli strain deficient for the production of beta-cystathionase. Clone 4-14 had the ability to complement a deletion mutation at this locus and expressed higher levels of beta-cystathionase than the wild-type strain. The transfer of the plasmid carried by this clone to a strain that constitutively expresses all the enzymes of the methionine biosynthetic pathway results in 100-fold overproduction of beta-cystathionase as compared to wild-type levels. With use of this strain, an efficient three-step purification scheme is described that gives 90% pure enzyme in 54% yield with a specific activity of 215 IU/mg. This enzyme is characterized as to molecular weight (280 000), number of subunits (six), pyridoxal phosphate binding (5.7 mol of pyridoxal phosphate bound/mol of protein, Km of 0.005 mM), amino acid composition, substrate specificity, and kinetic properties.

Pharmacology of L-asparaginase and the effects of host and enzyme modification.

Pharmacology is the study of the properties of a drug with respect to its interaction with the host, toxicology, and with the disease, therapeutics. Since both the toxicology and therapeutic activity of L-asparaginase are presented by others at this symposium, I will give only a brief overview of the pharmacology of L-asparaginase with particular reference to how it differs from other chemotherapeutic agents. Primarily, I would like to discuss how it may be possible to modify some of the pharmacological constraints on the therapeutic responses to L-asparaginase by combining the enzyme with inhibitors of asparagine synthetase and by using polymer-modified forms of the enzyme.

Improvement in the therapeutic, immunological, and clearance properties of Escherichia coli and Erwinia carotovora L-asparaginases by attachment of poly-DL-alanyl peptides.

The chemical modification of both Escherichia coli and Erwinia carotovora asparaginases by a DL-alanine-N-carboxyanhydride polymerization technique produced modified enzymes which had greater protease stability, retained most of their catalytic activity, and demonstrated a 7- to 10-fold prolongation in plasma clearance properties in normal mice and rats. Concomitantly, plasma substrate depletion was also extended 5 to 13 days longer for the modified as compared with the native enzymes. For preparations of modified enzymes with plasma half-lives longer than 24 hr, the therapeutic activity was superior to that of the native enzymes. In addition, the modified E. coli preparations were less immunogenic in mice than was the native enzyme, and they cross-reacted with antibodies developed to the native enzyme to a 300-fold lesser degree, such that the modified enzyme still showed prolonged clearance in an animal which had been immunized previously to the native enzyme. The native enzyme was immediately cleared from the plasma of such immune animals, although hyperimmune animals would rapidly clear both the native and modified enzymes. Similarly, the modified E. carotovora enzyme would cross-react to a 500-fold lesser degree with antibodies developed against the native E. carotovora enzyme.

L-cyst(e)ine requirements of malignant cells and progress toward depletion therapy.

The L-cyst(e)ine requirements of normal and malignant cells are reviewed and expanded within the context of establishing whether the measurement of gamma-cystathionase levels constitutes a predictive test for tumor sensitivity to L-cyst(e)ine depletion. The ability of both purified L-cysteine desulfhydrase and gamma-cystathionase to inhibit the growth of the L-cystine-dependent L1210 leukemia in culture is presented, as well as approaches to circumvent the limitations of these enzymes for in vivo therapy. The ability of proparagylglycine to inhibit L-cysteine biosynthesis in vivo is reviewed for its possible use in combination therapy. In addition, the ability of poly D,L-alanine modification of Escherichia coli L-asparaginase to increase the plasma half-life in mice tenfold as well as to decrease the immunogenicity of the enzyme is presented.