Deletions in domain II of pseudomonas exotoxin a that remove immunogenic epitopes
Inventors
Pastan, Ira H. • Weldon, John • FitzGerald, David
Assignees
US Department of Health and Human Services
Publication Number
US-8871906-B2
Publication Date
2014-10-28
Expiration Date
2028-09-04
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Abstract
The invention provides mutated, cytotoxic forms of Pseudomonas exotoxin A (PE) comprising a furin cleavage sequence conjugated or fused directly to residues 395-613 of PE or variants of that sequence. These minimal forms of PE are smaller than previous cytotoxic forms of PE, reduce non-specific toxicity, and reduce immunogenicity due to domain II or domain Ib of PE. The invention further provides nucleic acids encoding said PEs, chimeric molecules containing them, and methods of use thereof.
Core Innovation
The invention provides mutated, cytotoxic forms of Pseudomonas exotoxin A (PE) comprising a furin cleavage sequence conjugated or fused directly to residues 395-613 of PE or variants of that sequence. These minimal forms of PE are smaller than previous cytotoxic forms of PE, reduce non-specific toxicity, and reduce immunogenicity due to domain II or domain Ib of PE. The invention further provides nucleic acids encoding said PEs, chimeric molecules containing them, and methods of use thereof.
Current PE-based immunotoxins have dose-limiting toxicities and high immunogenicity, limiting their therapeutic effectiveness, especially for solid tumors where patients develop neutralizing antibodies after one administration. Previously developed forms like PE38 retain some toxicity and immunogenic epitopes in domains II and Ib, which contribute to these limitations. Despite efforts, a need remains for improved PEs that reduce non-specific toxicity and immunogenicity while maintaining cytotoxic activity.
To address these problems, the invention deletes all of domain II except for the furin cleavage site as well as deletes domain Ib residues, generating smaller PEs that retain functional domain III (residues 395-613) and can be fused to ligands. These forms significantly reduce immunogenic epitopes and non-specific toxicity, allowing higher dosing and enhanced anti-tumor effects. The minimal forms retain cytotoxic activity in vitro and in vivo, show resistance to lysosomal degradation, and show improved therapeutic indexes relative to prior PEs like PE38.
Claims Coverage
The patent contains multiple independent claims defining isolated mutated Pseudomonas exotoxin A (PE) compositions, chimeric molecules containing such PEs fused to ligands, and methods of inhibiting target cell growth by contacting cells with these chimeric molecules.
Isolated mutated PE with domain II deletion and furin cleavage sequence
The PE lacks all of domain II except for a furin cleavage sequence and comprises an amino acid sequence with formula R1n-FCS-R2n-R3n-PE functional domain III (residues 395-613), optionally including specified substitutions to increase cytotoxicity or reduce immunogenicity.
Furin cleavage sequence with specific amino acid features
The furin cleavage sequence (FCS) can be minimal (P4-P3-P2-P1) or extended with defined residues at P6-P5 and P1'-P2' positions, with P1 being arginine or lysine, P2' tryptophan, and P4 as arginine, valine or lysine, provided certain basic residue requirements to ensure cleavage by furin.
Chimeric molecule comprising a ligand fused to the mutated PE
A ligand (not transforming growth factor α), such as an antibody or fragment retaining antigen recognition, conjugated or fused to the described mutated PE having the domain II deletion and furin cleavage sequence as defined above.
Method of inhibiting growth of target cells by contacting with the chimeric molecule
Contacting target cells expressing a specific antigen or receptor with the chimeric molecule comprising the ligand fused to the mutated PE to inhibit cell growth, where the PE lacks domain II except for the furin cleavage site.
The claims cover novel mutated PEs with substantial domain II deletion retaining a furin cleavage site, their fusion to targeting ligands (excluding TGFα), and methods of using these chimeric molecules to inhibit target cell growth. The features focus on structural modifications that reduce immunogenicity and toxicity while preserving cytotoxic function.
Stated Advantages
Reduced non-specific toxicity compared to prior PEs, allowing higher dosing and greater therapeutic window.
Reduced immunogenicity by eliminating epitopes in domains II and Ib of PE.
Smaller molecular size facilitating deeper tumor penetration, beneficial for treatment of solid tumors.
Retention of potent cytotoxic activity both in vitro and in vivo against target cells.
Resistance to lysosomal degradation enhancing intracellular stability and therapeutic effectiveness.
Ability to engineer functional chimeric molecules with ligand fused upstream of the furin cleavage site allowing appropriate intracellular processing.
Documented Applications
Use of the mutated PE as the toxic moiety in immunotoxins for treatment of CD22-expressing hematological malignancies such as chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), acute lymphoblastic leukemia (ALL), and lymphomas.
Use of mutated PE in immunotoxins targeting mesothelin-expressing solid tumors, including mesothelioma and ovarian cancer.
Use of chimeric molecules including antibodies or fragments thereof fused to the mutated PE to inhibit the growth of target cells expressing the relevant antigen or receptor.
In vitro purging of target malignant cells from biological samples or cultures using immunotoxins containing the mutated PE.
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