Pseudomonas exotoxin A with less immunogenic T cell and/or B cell epitopes
Inventors
Pastan, Ira H. • Mazor, Ronit • Onda, Masanori • Vassall, Aaron • Beers, Richard • Eberle, Jaime • Liu, Wenhai
Assignees
US Department of Health and Human Services
Publication Number
US-10428119-B2
Publication Date
2019-10-01
Expiration Date
2032-06-07
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Abstract
The invention provides a Pseudomonas exotoxin A (PE) comprising an amino acid sequence having a substitution of one or more B-cell and/or T-cell epitopes. The invention further provides related chimeric molecules, as well as related nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions. Methods of treating or preventing cancer in a mammal, methods of inhibiting the growth of a target cell, methods of producing the PE, and methods of producing the chimeric molecule are further provided by the invention.
Core Innovation
The invention provides a Pseudomonas exotoxin A (PE) comprising an amino acid sequence having a substitution of one or more B-cell and/or T-cell epitopes, specifically targeting substitutions at various amino acid residues as defined by reference to SEQ ID NO: 1. These substitutions include residues L294, L297, Y298, L299, and R302, with particular provisos regarding combinations involving alanine substitution. The inventive PEs may include further substitutions within B-cell epitopes and T-cell epitopes to reduce immunogenicity.
The problem being solved is that native Pseudomonas exotoxin A, though cytotoxic and potentially effective in treating diseases like cancer, is highly immunogenic. Administration of PE can stimulate immune responses that neutralize its cytotoxic activity, limiting the dosage that can be administered and thereby reducing therapeutic effectiveness. There is a need for improved PE variants with reduced immunogenicity that maintain cytotoxic function.
The invention also provides chimeric molecules comprising the modified PEs conjugated or fused to targeting moieties such as antibodies, as well as related nucleic acids, recombinant expression vectors, host cells, and pharmaceutical compositions. Methods of treating or preventing cancer through administration of these modified PEs or chimeric molecules and methods of producing them are further included. The modifications aim to retain or improve cytotoxic activity while reducing immunogenicity by removing or altering T-cell and B-cell epitopes.
Claims Coverage
The patent includes one independent claim directed to a Pseudomonas exotoxin A with defined amino acid sequence substitutions and related pharmaceutical and chimeric molecule claims. The claims emphasize specific structural features and substitutions that reduce immunogenic epitopes while maintaining functionality.
Pseudomonas exotoxin A amino acid sequence with defined substitutions
A PE comprising the amino acid sequence of Formula I, which includes a furin cleavage sequence (FCS), regions R1, R2, R3, and PE functional domain III, wherein R2 comprises a sequence with defined variable amino acids and excludes the sequence LVALYLAARLSW. Substitutions include amino acid residue D463 and optional substitutions within B-cell and/or T-cell epitopes defined relative to SEQ ID NO: 1.
Pharmaceutical composition comprising the modified Pseudomonas exotoxin A
A pharmaceutical composition comprising the inventive PE and a pharmaceutically acceptable carrier.
Chimeric molecule comprising a targeting moiety and modified PE
A chimeric molecule where a targeting moiety is conjugated or fused to the inventive PE. The targeting moiety may be a monoclonal antibody or antigen binding portion thereof, specific to defined cell surface markers such as CD19, CD22, mesothelin, and others, including the antigen binding portion of HA22.
Method of inhibiting growth of target cells using the modified PE
A method of inhibiting growth of a target cell by contacting the cell with the inventive PE in an effective amount, where the target cell is optionally a cancer cell.
Methods of producing the inventive PE and chimeric molecule
Methods comprising recombinant expression and purification of the inventive PE or chimeric molecule.
The claims focus on a Pseudomonas exotoxin A having specific amino acid substitutions that reduce immunogenicity by altering B-cell and T-cell epitopes, compositions containing such PEs, chimeric molecules conjugated to targeting moieties, methods of treating cancer by targeting specific cells, and methods of producing these molecules.
Stated Advantages
The inventive PEs may advantageously be less immunogenic than unsubstituted wild-type PE by removing or altering T-cell and/or B-cell epitopes, thereby reducing neutralizing immune responses.
The PEs retain at least about 20% and up to 100% or more of the native PE cytotoxic activity, with some substitutions increasing cytotoxicity while reducing immunogenicity.
Chimeric molecules utilizing the modified PEs can specifically target cell-surface markers, enhancing selective cytotoxicity to target cells such as cancer cells, minimizing off-target effects.
The invention provides variants and pharmaceutical compositions that can be used to treat or prevent cancer more effectively due to reduced immunogenicity and improved targeting properties.
Documented Applications
The inventive Pseudomonas exotoxin A and related chimeric molecules are explicitly described for use in methods of treating or preventing cancer in a mammal by administering an effective amount to treat or prevent cancer.
Methods of inhibiting the growth of target cells, including cancer cells expressing specific cell surface markers, by contacting the cells with the inventive PE, chimeric molecules, or pharmaceutical compositions are described.
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