Utilization of Wolinella succinogenes asparaginase to treat diseases associated with asparagine dependence

Abstract

Described herein are methods for producing recombinant forms of asparaginase derived from Wollinella succinogenes. In addition, methods for covalent modification of proteins, including asparaginases, by acylation are also provided. Certain embodiments provide for epitopic-labeling of the amino terminus of W. succinogenes asparaginase. Additional embodiments concern methods for the therapeutic utilization of the native, homotetrameric form of W. succinogenes asparaginase, as well as the use of epitopically-labeled or non-epitopically-labeled recombinant W. succinogenes asparaginase (or a covalently modified analog thereof) in the therapeutic treatment of malignant and non-malignant hematological disease and other diseases where asparagine depletion or deprivation would be efficacious or which respond to asparagine depletion or deprivation, as well as their potential utilization in the therapeutic treatment of autoimmune diseases such as rheumatoid arthritis, AIDS, and SLE.

Description

[0001] This application claims priority to U.S. provisional patent application 60 / 049,085, filed Jun. 9, 1997, which is hereby incorporated in its entirety.[0002] The present invention relates to methodologies for the production of microbial enzymes, particularly native and recombinant Wollinella succinogenes asparaginase and its analogs, which possesses potent in vitro and in vivo activity against diseases correlated with asparagine dependence. In addition, the present invention also relates to methods for the utilization of recombinant microbial enzymes in the treatment of diseases which respond to asparagine depletion, including various hematologic, autoimmune, and infectious diseases.BACKGROUND OF INVENTION[0003] The references cited below are not admitted to be prior art to the inventions described herein.[0004] Asparaginases are enzymes which catalyze the deamidation of L-asparagine (asparaginase activity) and L-glutamine (glutaminase activity). See Cantor, P. S. & Schimmell, ...

AI Technical Summary

Problems solved by technology

While these asparaginases possess potent anti-leukemic activity, clinical utilization of the aforementioned microbial asparaginases resulted in a wide range of host toxicity (e.g., hepatic, renal, splenic, pancreatic dysfunction and blood coagulation) and pronounced immunosuppression (see Ohno, R.

Although some investigators have reported that low dosages of E. coli asparaginase result in limited hepatotoxic complications, sensitive indicators of hepatic function in some patients receiving low dosages, however, still reveals significant hepatic disease which may result in life-threatening coagulopathy (see Crowther, D., Asparaginase and human malignant disease, 229 Nature 168 (1971)).

However, the significance of these in vitro studies is somewhat limited because the in vivo fates of asparaginases and the homeostatic control of asparagine and glutamine may result in a modification of the immunosuppressive effects of anti-neoplastic asparaginases.

Another significant problem associated with the use of microbial asparaginases is that patients treated with E. coli and E. carotovora asparaginases frequently develop neutralizing antibodies of the IgG and IgM immunoglobulin class (see, e.g, Cheung, N.

The development of antibodies directed against E. coli (EC) asparaginase and the modified PEG-asparaginase in patients is associated with neutralization of the enzymatic activity of both the EC and PEG-asparaginases in vivo, thus potentially resulting in an adverse clinical prognosis.

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