Pseudomonas exotoxin A with less immunogenic B cell epitopes
Inventors
Pastan, Ira H. • Onda, Masanori • Liu, Wenhai
Assignees
US Department of Health and Human Services
Publication Number
US-10111927-B2
Publication Date
2018-10-30
Expiration Date
2032-09-13
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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 of amino acid residues E420, D463, Y481, L516, R563, D581, D589, and K606, wherein the amino acid residues are defined by reference to SEQ ID NO: 1. 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) with substitutions at one or more of the amino acid residues E420, D463, Y481, L516, R563, D581, D589, and K606, defined by reference to SEQ ID NO: 1. These substitutions are aimed at amino acid residues located within one or more B-cell epitopes of PE, which can reduce the immunogenicity of the toxin. The PE may also have additional substitutions within T-cell epitopes, deletions of continuous amino acid residues within certain domains, or combinations thereof.
The problem being solved addresses the high immunogenicity of Pseudomonas exotoxin A, which causes anti-PE immune responses, including production of antibodies and immune cells that neutralize the toxin's cytotoxic activity. This immunogenicity limits the dosage and effectiveness of PE in treatments such as cancer therapy. Therefore, the invention seeks to reduce PE immunogenicity while maintaining or enhancing cytotoxicity.
The invention further includes chimeric molecules where the modified PE is conjugated or fused to targeting moieties such as antibodies specific to cell surface markers. These chimeric molecules allow targeted delivery of the toxin to undesired cells, e.g., cancer cells, thereby improving therapeutic efficacy and reducing off-target effects. Related nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions comprising the modified PE or chimeric molecules are also part of the invention.
Claims Coverage
The patent includes multiple independent claims covering modified Pseudomonas exotoxin A (PE) with specific amino acid substitutions, chimeric molecules comprising the PE coupled to targeting moieties, pharmaceutical compositions, and methods of production and therapeutic use. The main inventive features relate to particular substitutions to reduce immunogenicity, optional substitutions within B-cell and T-cell epitopes, structural deletions, and the incorporation of targeting moieties.
Pseudomonas exotoxin A with specific amino acid substitutions to reduce immunogenicity
PE comprising substitutions at one or more amino acid residues selected from E420, D463, Y481, L516, R563, D581, and D589 as defined by SEQ ID NO: 1, with the condition that if position 516 is substituted with alanine, at least one other specified residue is also substituted. Further substitutions within one or more B-cell epitopes are included, characterized by substitutions at residues such as E285, P290, R313, and others. Optional further substitutions within T-cell epitopes and deletions of amino acid residues 1-273 and 285-394 are also covered.
Substitution types and combinations for reduced immunogenicity
Independent substitutions of alanine, glycine, serine, or glutamine in place of the specified amino acid residues. Specific embodiments target substitutions of residues D463, Y481, L516, or E420, Y481, R563, D581, D589 with various amino acids to modulate immunogenicity.
Substitutions within T-cell epitopes
Further substitutions of one or more amino acid residues within one or more T-cell epitopes, including residues such as L294, L297, Y298, L299, R302, and residues at positions 464-480, 482-515, and 517-519 of SEQ ID NO: 1, substituting alanine, glycine, serine, or glutamine to reduce T-cell mediated immunogenicity.
Modified PE with substitution of valine, leucine, or isoleucine at R490
PE comprising the specified substitutions plus a further substitution of valine, leucine, or isoleucine in place of residue R490 as defined by SEQ ID NO: 1, optionally with further T-cell epitope substitutions and deletions in specified regions.
Chimeric molecules comprising the modified PE and targeting moieties
Chimeric molecules that include a targeting moiety conjugated or fused to the inventive PE, where the targeting moiety is preferably a monoclonal antibody specific for cell surface markers such as CD19, CD22, mesothelin, Lewis Y, and others.
Pharmaceutical compositions and therapeutic methods
Pharmaceutical compositions comprising the modified PE and a pharmaceutically acceptable carrier. Methods of inhibiting growth of target cells, including cancer cells expressing specific cell surface markers, by contacting the cells with the modified PE. Methods of producing the modified PE and chimeric molecules by recombinant expression and purification, as well as linking targeting moieties to purified PE.
The claims broadly cover recombinant modified PE molecules with defined amino acid substitutions in B-cell and T-cell epitopes to reduce immunogenicity while maintaining function. They also encompass chimeric molecules with targeting moieties for cell-specific delivery, pharmaceutical compositions, and methods of production and therapeutic use in cell growth inhibition, particularly cancer treatment.
Stated Advantages
Reduced immunogenicity of Pseudomonas exotoxin A by eliminating B-cell and T-cell epitopes through specific amino acid substitutions.
Retention or enhancement of cytotoxic activity against target cells despite reduced immunogenicity.
Improved therapeutic efficacy by enabling higher or repeated dosing due to decreased immune neutralization.
Targeted delivery of cytotoxic activity to cancer cells via conjugation or fusion with targeting moieties such as monoclonal antibodies.
Versatility in production and formulation including recombinant expression, chemical synthesis, and pharmaceutical compositions suitable for various administration routes.
Documented Applications
Use of the modified PE and chimeric molecules in methods of treating or preventing cancer in mammals by administering an effective amount.
Inhibiting the growth of target cells, particularly cancer cells, by contacting them with the modified PE or chimeric molecules.
Construction of nucleic acids, recombinant expression vectors, and host cells for producing the modified PE or chimeric molecules.
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