Pseudomonas exotoxin a with reduced immunogenicity
Inventors
Pastan, Ira H. • Beers, Richard • Onda, Masanori
Assignees
National Institutes of Health NIH • US Department of Health and Human Services
Publication Number
US-8936792-B2
Publication Date
2015-01-20
Expiration Date
2030-09-10
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Abstract
The present invention provides improved Pseudomonas Exotoxin A (PE) molecules with high cytotoxicity and reduced immunogenicity, compositions containing the improved (PE), and methods of use.
Core Innovation
The invention provides improved Pseudomonas exotoxin A (PE) molecules with high cytotoxicity and reduced immunogenicity, compositions containing the improved PE, and methods of use. The improved PE has Domain I removed, most of Domain II removed, and substitutions within Domain III amino acid residue positions D406, R432, R467, R490, R513, E548, K590 and Q592 with glycine, alanine or serine. Functionally, these improved PE molecules retain high cytotoxic activity while removal of B cell epitopes reduces immunogenicity.
The problem addressed is that current PE-based immunotoxins, while therapeutically promising, are highly immunogenic. This immunogenicity limits their repeated administration, especially in patients with solid tumors who develop neutralizing antibodies within weeks. This limits immunotoxin treatment to usually one administration, reducing their overall efficacy. The invention aims to create less antigenic forms of PE-based immunotoxins to reduce patients' immunogenic responses and extend treatment usability.
The invention discovers that mutating specific amino acid residues within PE's epitopes, particularly eight selected residues including D406 and Q592, reduces recognition by neutralizing antibodies while retaining cytotoxic function. The invention further removes Domain I and most of Domain II to reduce non-specific toxicity. The resulting PE variants, exemplified by LR-8M, show reduced immunogenicity in animal models with preserved or enhanced cytotoxicity, providing valuable immunotoxins with improved therapeutic profiles.
Claims Coverage
The patent contains multiple independent claims directed to isolated modified PE molecules, chimeric molecules containing modified PE, compositions containing these chimeric molecules, and methods of inhibiting cell growth using chimeric molecules with modified PE. The main inventive features concern the specific amino acid deletions and substitutions in PE, the fusion or conjugation to targeting moieties, and their functional use.
Isolated modified Pseudomonas exotoxin A with specific deletions and substitutions
An isolated Pseudomonas exotoxin A protein having residues 1-273 and 285-394 removed and substitutions of alanine, glycine or serine at amino acid residues D406, R432, R467, R490, R513, E548, K590 and Q592, corresponding to SEQ ID NO:1.
Expanded substitutions including additional amino acid residues
A PE molecule further containing substitutions of alanine, glycine or serine at one or more amino acid residues selected from D403, R412, R427, E431, R458, D461, R505, E522, R538, R551, R576 and L597.
Chimeric molecules with modified PE
Chimeric molecules comprising a targeting moiety conjugated or fused to a modified PE with the above deletions and substitutions.
Chimeric molecules with targeting moieties being antibodies
Chimeric molecules wherein the targeting moiety is an antibody including scFv, dsFv, Fab, single domain antibody, or F(ab')2, directed against cell surface antigens such as CD19, CD21, CD22, CD25, CD30, CD33, CD79b, transferrin receptor, EGF receptor, mesothelin, cadherin and Lewis Y.
Compositions comprising chimeric molecules and pharmaceutically acceptable carriers
Pharmaceutical compositions containing the chimeric molecules described, combined with pharmaceutically acceptable carriers.
Methods of inhibiting cell growth using chimeric molecules
Methods of inhibiting growth of cells bearing a target molecule by contacting the cells with chimeric molecules comprising a targeting moiety and the modified PE as specified, wherein the contacting inhibits cell growth.
The claims cover isolated PEs with precise domain deletions and multiple alanine, glycine or serine substitutions at defined amino acid residues; chimeric molecules combining these PEs with targeting moieties including antibodies; compositions of such chimeric molecules with carriers; and methods of inhibiting target cell growth using these chimeric molecules.
Stated Advantages
The improved PE molecules have reduced immunogenicity, enabling multiple administrations without eliciting neutralizing antibodies.
Retained or enhanced cytotoxic activity against target cells despite mutations reducing antigenicity.
Increased therapeutic utility of PE-based immunoconjugates, especially for treatments of solid tumors where immunogenicity limits efficacy.
Documented Applications
Use of improved PE molecules in immunotoxins for treatment of various cancers including chronic lymphatic leukemia (CLL), non-Hodgkin lymphoma, and solid tumors.
Treatment methods involving inhibiting growth, hyperproliferation or survival of cells bearing target cell surface markers by administering chimeric molecules with modified PE.
Inducing apoptosis in cells expressing specific cell surface markers using chimeric molecules containing the modified PE.
Diagnostic and therapeutic monitoring of tumor burden and treatment efficacy using compositions containing improved PE-based immunotoxins.
Ex vivo or in vitro purging of targeted cells from biological samples using chimeric molecules with modified PE.
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