Cancer therapy with chemically modified enzymes. II. The therapeutic effectiveness of arginase, and arginase modified by the covalent attachment of polyethylene glycol, on the taper liver tumor and the L5178Y murine leukemia.

Monomethoxypolyethylene glycol (PEG) was attached covalently to arginase. PEG-arginase was effective in prolonging the survival times of mice injected with the Taper liver tumor, whereas unmodified arginase was ineffective. PEG-arginase was more effective than arginase in the in vitro destruction of L5178Y mouse leukemia. However, neither PEG-arginase nor arginase inhibited the in vivo growth of this tumor.

Induction of tolerance in mice by uricase and monomethoxypolyethylene glycol-modified uricase.

The ability to induce tolerance to uricase by the administration of native uricase, and uricase modified by the covalent attachment of monomethoxypolyethylene glycol (PEG) was examined. Uricase, and uricase with PEG attached to 35% (PEG-uricase 35%) and 70% (PEG-uricase 70%) of available amino groups were found to induce tolerance in mice not previously sensitized to uricase. There was a dampening of the IgG, IgE and IgM antibody response to uricase which persisted even after a second sensitizing dose of uricase was administered to these animals. PEG-uricases were found to have little or no immunogenicity when injected into mice and a reduced immunogenicity and antigenicity when tested in rabbits. Native uricase, however, was found to be immunogenic and antigenic in mice and rabbits. Mice sensitized to native uricase were injected with uricase, PEG-uricase 35% or 70% to induce tolerance. After a second sensitizing injection of uricase, circulating levels of IgE, IgG and IgM were measured. All three enzymes induced tolerance in the IgE class of antibody but there was no significant change in the hemagglutinating antibody levels of the mice. Mice injected with 1 mg of uricase died from anaphylaxis. PEG-uricase 35% was found to induce the most effective tolerance in both unsensitized and sensitized mice.

Properties of two urate oxidases modified by the covalent attachment of poly(ethylene glycol).

Poly(ethylene glycol) of 5 000 daltons has been attached covalently to preparations of urate oxidase (urate: oxygen oxidoreductase, EC 1.7.3.3) from hog liver and Candida utilis. Attachment of sufficient poly(ethylene glycol) to either urate oxidase renders the enzyme incapable of eliciting antibody production in mice, or of reacting with antibodies to the unmodified enzyme. The poly(ethylene glycol) : urate oxidase conjugates exhibit higher Km and lower V values than the unmodified urate oxidases. Optimal pH values are increased for the poly(ethylene glycol) : urate oxidases, and optimal temperatures are decreased. The blood circulating lives of the modified urate oxidases following intravenous injection are much longer than those of the unmodified urate oxidases: repetitive injections over a period of 90 days dd not alter the blood circulating lives of the poly(ethylene glycol) : urate oxidases. The unmodified enzymes, on the other hand, were cleared from the blood with extreme rapidity after a few intravenous injections.

Spectral model of leishmaniasis in congenic strains of mice.

Nineteen congenic, resistant strains of mice on C57BL/10ScSn genetic background were infected with Leishmania donovani and the course of infection quantitated. Early in the infection, parasite burdens in the liver were similar for all strains, indicating that the parasite was able to establish, grow, and reproduce in the liver macrophages of each strain with equal facility. Differences in acquired resistance, indicated by decreases in parasite burden, among the strains were first noted at day 21 and became distinct by day 35 postinfection. The extremes were represented by B10.129(10M) mice in which the parasite burden continued to increase at day 35, and B10.LP-H-3b in which only 10% of the peak parasite population remained at this time. The other strains formed a complete continuum between the two extremes. Differences in hepatic pathology were noted among strains, but the severity was not related directly to the strength of the immune response as indicated by reduction in parasite burden; instead, it was more correlated with spleen-to-body weight ratios. Because of the range of responses observed, congenic strains of mice may be of use not only for immunization and chemotherapy studies of leishmaniasis, but also may yield fundamental information on spectral diseases in general.

Genetic control of acquired resistance to visceral leishmaniasis in mice.

A series of H-2 and non-H-2 congenic resistant (CR) strains on a C57BL/10Sn background were infected with 10(7) amastigotes of Leishmania donovani. Non-H-2 congenic strains B10.LP-H-3b and B10.CE(30NX) and (B10.LP-H-3b x B10)F1 hybrids showed a very rapid decrease in liver-parasite burdens beyond day 21. Parasite counts for these strains at day 35 were significantly lower than for all other strains tested. The rapid decrease in parasite numbers, massive lymphocellular infiltration into the liver and strong delayed hypersensitivity reactions to parasite antigens in strains congenic for a portion of chromosome 2 indicated that acquired immunity to L. donovani was controlled by a dominant gene at or near the Ir-2 locus. In addition, B10.129(10M) mice, which differ from C57BL/10Sn at the H-11 locus, showed highly significant increases in parasite numbers at day 35. Other observations supporting the absence of acquired immunity in B10.129(10M) included negative delayed hypersensitivity tests to parasite antigens and the absence of lymphocellular infiltrate into the liver. Although the differences were not as pronounced, H-2 CR strains with H-2b, H-2a, and H-2k haplotypes also showed significantly greater decreases in parasite numbers by day 35 as compared to other H-2 CR strains.

Some properties of polyethylene glycol:phenylalanine ammonia-lyase adducts.

Methoxypolyethylene glycol of 5000 daltons (PEG) was attached covalently to phenylalanine ammonia-lyase from Rhodotorula glutinis. Attachment of sufficient quantities of PEG to phenylalanine ammonia-lyase substantially reduces immunological recognition and clearance of the conjugated enzyme in mice. The modified enzyme demonstrates altered catalytic properties such as shifts in the pH and temperature optima, an increase in the Michaelis-Menten constant, and a lowered Vmax in comparison with the native enzyme. PEG-phenylalanine ammonia-lyase has increased resistance to proteolytic digestion, particularly when in the presence of cinnamate, a competitive inhibitor, while the native enzyme is rapidly inactivated. In the ultracentrifuge PEG-phenylalanine ammonia-lyase exhibits a lower sedimentation rate than the unmodified enzyme, despite the fact that it is much larger. The electrophoretic mobility of PEG-phenylalanine ammonia-lyase is greatly decreased in comparison to the unmodified enzyme. PEG-phenylalanine ammonia-lyase had a much longer blood-circulating life in mice, both initially and after a number of injections, than did the native enzyme. PEG-phenylalanine ammonia-lyase was a good immunogen but a poor antigen in mice and rabbits, that is, it readily induced antibody formation, but reacted poorly in vitro with the antibodies that were formed against it.

Treatment of L5178Y tumor-bearing BDF1 mice with a nonimmunogenic L-glutaminase-L-asparaginase.

An L-glutaminase-L-asparaginase from Achromobacter has been rendered nonimmunogenic by the covalent attachment of polyethylene glycol (PEG) to nonessential amine groups of the enzyme. PEG-L-glutaminase-L-asparaginase exhibits a greatly enhanced half-life in the bloodstream compared to the unmodified enzyme in normal mice, and is effective in prolonging the survival of BDF1 mice inoculated ip with L5178Y cells. PEG-L-glutaminase-L-asparaginase appears rapidly in the blood following ip injection.

Preparation of a non-immunogenic arginase by the covalent attachment of polyethylene glycol.

Methoxypolyethylene glycol of 5000 daltons (PEG) was attached covalently to bovine liver arginase using 2,4,6-trichloro-s-triazine as the coupling agent. The conjugate (PEG-arginase), with PEG attached to 53% of the amino groups, retained 65% of its original enzymatic activity. Mice were injected intravenously with arginase or PEG-arginase for periods of one to three months. The blood-circulating life of PEG-arginase was greatly extended over that of arginase. The half-life of injected arginase at day 30 was less than 1 h, whereas that of the PEG-enzyme was 12 h. Antisera from mice injected with native arginase reacted against arginase but not against PEG-arginase when tested by immunodiffusion. Antisera from animals injected with PEG-arginase reacted neither with native arginase nor PEG-arginase. The data indicate that arginase modified by PEG has been rendered both non-immunogenic and non-antigenic when tested in mice. The injection of PEG-arginase into mice did not induce tolerance toward the native enzyme. Injected PEG-arginase, in the presence of precipitating antibody directed against native arginase, circulated at the same level as in virgin animals. The attachment of PEG to arginase altered its kinetic properties.

Alteration of immunological properties of bovine serum albumin by covalent attachment of polyethylene glycol.

Methoxypolyethylene glycols of 1900 and 5000 daltons have been attached covalently to bovine serum albumin using cyanuric chloride as the coupling agent. When sufficient polymer is attached, the modified bovine serum albumin appears to lose its immunogenicity in the rabbit and, on intramuscular or intravenous injection, elicits antibodies neither to itself nor to native bovine serum albumin. It does not react with antibodies raised against native bovine serum albumin. Bovine serum albumin to which methoxypolyethylene glycol has been attached exhibits a blood circulating life in the rabbit rather similar to native bovine serum albumin, except that it is not removed from circulation by the eventual development of antibodies. Modified bovine serum albumins which had been iodinated with 125I, or prepared with [14C]cyanuric chloride, were injected intravenously in rabbits. Both labels appeared almost quantitatively in the urine after 30 days. The modified bovine serum albumins showed substantial changes in properties, such as solubility, electrophoretic mobility in acrylamide gel, ion exchange chromatography, and sedimentation, as compared with the unmodified protein.

Effect of covalent attachment of polyethylene glycol on immunogenicity and circulating life of bovine liver catalase.

Methoxypolyethylene glycols of 1900 daltons (PEG-1900) or 5000 daltons (PEG-5000) were covalently attached to bovine liver catalase using 2,4,6-trichloro-s-triazine as the coupling agent. Rabbits were immunized by the intravenous and intramuscular routes with catalase modified by covalent attachment of PEG-1900 to 43% of the amino groups (PEG-1900-catalase). The intravenous antiserum did not yield detectable antibodies against PEG-1900-catalase or native catalase, as determined by Ouchterlony and complement fixation methods, whereas the intramuscular antiserum contained antibodies to both PEG-1900-catalase and catalase. PEG-1900 did not react with either antiserum. Catalase was prepared in which PEG-5000 was attached to 40% of the amino groups (PEG-5000-catalase). This catalase preparation did not react with either antiserum. PEG-1900-catalase retained 93% of its enzymatic activity; PEG-5000-catalase retained 95%. PEG-5000-catalase resisted digestion by trypsin, chymotrypsin, and a protease from Streptomyces griseus. PEG-1900-catalase and PEG-5000-catalase exhibited enhanced circulating lives in the blood of acatalasemic mice during repetitive intravenous injections. No evidence was seen of an immune response to injections of the modified enzymes. Mice injected repetitively with PEG-5000-catalase remained immune competent for unmodieied catalase, and no evidence of tissue or organ damage was seen.

Stimulation of resistance in mice to sporozoite-induced Plasmodium berghei malaria by injections of avian exoerythrocytic forms.

Mice received a series of injections of formalin-killed merozoites (FKM) of exoerythrocytic stages of Plasmodium fallax prior to challenge with sporozoites of P. berghei. In one study 4 of 16 FKM-immunized mice never exhibited parasitized erythrocytes after 2 challenges of 10(4) P. berghei sporozoites each, while all control animals died with high parasitemias. FKM-immunized mice were as susceptible as control mice to infections initiated with parasitized erythrocytes. In a second study, 14 of 16 mice immunized via the intravenous (i.v.) or combined intramuscular (i.m.) and i.v. routes were immune to an initial challenge with 10(4) sporozoites, but were susceptible to a second challenge. Three injections of FKM via the i.m. or intraperitoneal routes did not elicit a protective response against sporozoite challenge. Sera harvested from FKM-immunized and control mice prior to challenge produced no visible CSP reaction with P. berghei sporozoites, nor was infectivity of sporozoites altered after incubation in sera, showing that SNA was absent. In additional experiments results were less encouraging. An attempt to repeat the result of the second experiment failed. Each of 5 mice which received the same number of FKM by a similar schedule became infected after sporozoite challenge. In an additional study the immunization schedule was increased from 3 to 7 injections of FKM and 40% of FKM-immunized mice resisted challenge. However, mice which had received FKM prior to sporozoite challenge consistently displayed an increased prepatent period compared with control animals. A department from methods of the more successful studies was necessitated in these later studies in which FKM were harvested from cell cultures maintained for longer periods of time.

Comparison of two geographic strains of Leishmania donovani by resistance of mice to superinfection.

Groups of mice were injected with Leishmania donovani strains Et (Burma) or IS (Sudan) and the courses of infection were followed. Between days 20 and 30 after infection by treatment with pentostam. Et- and 1S-recovered mice were challenged by intravenous injection of amastigotes of each strain. Although resistance to superinfection was apparent the L. donovani strains could not be differentiated by this method.