Modified Forms of Pseudomonas Exotoxin A

Abstract

Pseudomonas exotoxin A or “PE” is a 66 kD, highly potent, cytotoxic protein secreted by the bacterium Pseudomonas aeruginosa. Various forms of PE have been coupled to other proteins, such as antibodies, to generate therapeutically useful cytotoxin conjugates that selectively target cells of a desired phenotype (such as tumor cells). In the present invention, peptides spanning the sequence of an approximately 38 kD form of Pseudomonas exotoxin A protein were analyzed for the presence of immunogenic CD4+ T cell epitopes. Six immunogenic T cell epitopes were identified. Residues were identified within each epitope for introduction of targeted amino acid substitutions to reduce or prevent immunogenic T-cell responses in PE molecules which may be administered to a heterologous host.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit of U.S. Provisional Appl. No. 61 / 531,576, filed Sep. 6, 2011 which is herein incorporated by reference in its entirety.REFERENCE TO RELATED APPLICATION[0002]This application claims benefit of and priority based on U.S. Provisional Patent Application Ser. No. 61 / 531,576, filed Sep. 6, 2011, the contents of which are herein incorporated by reference in their entirety.NAMES OF THE PARTIES IN A JOINT RESEARCH AGREEMENT[0003]The claimed invention was made pursuant to a joint research agreement, as defined in 35 U.S.C. §103 (c)(3), that was in effect on or before the date the claimed invention was made, and as a result of activities undertaken within the scope of die joint research agreement, by or on behalf of the Intrexon Corp. (Foster City, Calif., U.S.A.) and Antitope Ltd. (Cambridge, UK).REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY[0004]A Sequence Listing is submitted electronically via EFS-Web...

AI Technical Summary

Benefits of technology

[0037]Peptides spanning the sequence of an approximately 38 kD (predicted molecular weight) form of Pseudomonas exotoxin A protein (SEQ ID NO:1) were analyzed for the presence of immunogenic CD4+ T cell epitopes. A total of 120 overlapping 15 mer peptides spanning this sequence (SEQ ID NO:1), but also including an amino terminal (Glyx5-Ser)x2 linker sequence (SEQ ID NO:3) to produce a 359 amino acid Gly-Set-PE38 polypeptide sequence (SEQ ID NO:2), were tested against a cohort of healthy human donors. CD4+ T cell responses against individual peptides were measured via proliferation assays. Assay data was used to compile a T cell epitope map of the PE38 sequence. Six immunogenic T cell epitopes were identified. Residues were then identified, within each of these epitopes for use in targeted amino acid substitutions to reduce or prevent PE38-induced immunogenicity. Reduction or prevention of PE immunogenicity should allow for multiple therapeutic administrations of cytotoxic PE for use, for example, in the targeted destruction of cancer cells in vivo (such as when administered as an immunoconjugate or cell-surface targeted fusion protein).

Problems solved by technology

These responses include those that elicit a weak clinical effect and those that limit efficacy which can occasionally result in morbidity or even mortality in patients.

In particular, serious complications can arise with the production of neutralizing antibodies, especially when they target recombinant self proteins and therefore have the potential to cross react with the patient's own endogenous protein (Lim, 2005).

PE-A typically produces death by causing liver failure.

A significant disadvantage in using PE-A for treatment of disease, however, is that

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