Mutated Pseudomonas exotoxins with reduced antigenicity
Inventors
Pastan, Ira H. • Onda, Masanori • Nagata, Satoshi • FitzGerald, David • Kreitman, Robert • Lee, Byungkook
Assignees
US Department of Health and Human Services
Publication Number
US-8907060-B2
Publication Date
2014-12-09
Expiration Date
2026-07-25
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Abstract
The invention provides mutated Pseudomonas exotoxins (PE) that have reduced immunogenicity compared to PEs containing the native sequence. The PEs of the invention have one or more individual mutations of positions of the native sequence of PE that reduce antibody binding to one or more PE epitopes. Nucleic acids encoding the mutated PEs, chimeric molecules comprising them, compositions comprising the chimeric molecules and methods of using them, are also provided.
Core Innovation
The invention provides mutated Pseudomonas exotoxins (PE) that have reduced immunogenicity compared to PEs containing the native sequence. The PEs of the invention have one or more individual mutations of positions of the native sequence of PE that reduce antibody binding to one or more PE epitopes. Nucleic acids encoding the mutated PEs, chimeric molecules comprising them, compositions comprising the chimeric molecules and methods of using them, are also provided.
Immunotoxins using bacterial toxins known as Pseudomonas exotoxin A (PE) have been studied for treating malignancies, especially hematological cancers. While current PE-based immunotoxins have clinical promise, their high immunogenicity limits repeated use in patients with solid tumors, as these patients rapidly develop neutralizing antibodies that render the immunotoxins ineffective after a single administration. Thus, it is desirable to develop less antigenic forms of PE to reduce the immune response and improve therapeutic utility.
The invention discloses that PE has seven major epitopes further divided into thirteen subepitopes and that mutating a single amino acid residue in ten of these subepitopes reduces or eliminates antibody binding to that epitope, thereby reducing overall antigenicity. Mutations can be combined in PE variants to reduce antigenicity while retaining cytotoxic activity. The mutated PEs can be used in immunotoxins with targeting moieties such as antibodies or cytokines. These mutated PEs are expected to provoke lower titers of neutralizing antibodies in vivo, extending the efficacy and usability of PE-based immunotoxins for solid tumor treatment.
Claims Coverage
The claims comprise multiple independent claims directed to mutated Pseudomonas exotoxin A polypeptides, chimeric molecules comprising them, compositions including these chimeras, and methods of inhibiting cell growth using these chimeras. There are several inventive features related to specific amino acid substitutions at defined residues of PE that reduce antigenicity while preserving cytotoxicity and enable use in targeted therapies.
Mutated Pseudomonas exotoxins with specific amino acid substitutions at epitope-related residues
Isolated Pseudomonas exotoxin A comprising substitutions at one or more amino acid residues selected from N314, D324, Q332, D403, R412, R427, E431, R432, R458, D461, R467, R505, R538, E548, R576, K590, and L597 of SEQ ID NO:1, with defined substitutions such as alanine, glycine, serine, glutamine, valine, leucine, or isoleucine. Specific rules apply to substitution of residues Q332, K590, and L597. Optionally, further substitution at residue R490 with various amino acids is included.
Chimeric molecules comprising targeting moieties fused or conjugated to mutated PEs
Chimeric molecules comprising targeting moieties (antibodies including scFv, dsFv, Fab, F(ab')2, or cytokines) conjugated or fused to mutated PEs having one or more substitutions at epitope-related residues as specified in the mutated PEs. The chimeras optionally include multiple substitutions including at residues R490, R313, R432, R513, E548, among others, to reduce immunogenicity while retaining cytotoxicity.
Pharmaceutical compositions comprising the chimeric molecules
Pharmaceutical compositions comprising the chimeric molecules described above along with pharmaceutically acceptable carriers, formulated for therapeutic administration.
Methods of inhibiting growth of target cells using mutated PE-containing chimeric molecules
Methods for inhibiting growth of cells bearing a target molecule by contacting the cells with chimeric molecules comprising a targeting moiety binding the target and a mutated PE as specified that reduces antigenicity while maintaining cytotoxic activity. The targeting moiety can be antibodies against cancer antigens or cytokines targeting cytokine receptors such as IL-13 receptor.
The claims cover mutated Pseudomonas exotoxins with specific amino acid substitutions that reduce antigenicity, their incorporation into chimeric immunotoxins with defined targeting moieties, pharmaceutical compositions thereof, and methods to inhibit target cell growth using such chimeras. The invention focuses on defined substitutions at specified PE residues and combinations thereof to optimize reduced immunogenicity with maintained cytotoxicity.
Stated Advantages
PE-based immunotoxins with reduced antigenicity provoke a lower immune response in patients, reducing the development of neutralizing antibodies.
Mutated PEs retain cytotoxic activity comparable to or greater than wild type, enabling effective targeting and killing of cancer cells.
Combining multiple epitope-disrupting mutations in PE reduces overall antigenicity without loss of therapeutic efficacy.
The mutations can be engineered into existing PE variants, improving the therapeutic utility of current immunotoxin clinical candidates.
Documented Applications
Treatment of hematological malignancies and solid tumors using immunotoxins containing mutated Pseudomonas exotoxin A linked to targeting moieties such as antibodies or cytokines.
Use of immunotoxins with mutated PEs to reduce tumor burden, eliminate metastases, and alleviate symptoms associated with cancers.
Targeting cells expressing cytokine receptors such as IL-13 receptor for treating cancers including gliomas and other diseases like asthma and allergic rhinitis.
In vitro or ex vivo elimination of targeted cells, for example purging malignant cells from cell cultures using immunotoxins comprising mutated PEs.
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