Pseudomonas exotoxin A with less immunogenic B cell epitopes
Inventors
Pastan, Ira H. • Onda, Masanori • Liu, Wenhai
Assignees
United States, Helath And Human Services, Secretary of, Department of • US Department of Health and Human Services
Publication Number
US-9206240-B2
Publication Date
2015-12-08
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) comprising an amino acid sequence with substitutions at one or more of the amino acid residues E420, D463, Y481, L516, R563, D581, D589, and K606, as defined by reference to SEQ ID NO: 1. The PE may also optionally include further substitutions within one or more T-cell epitopes, further substitutions within one or more B-cell epitopes, and/or deletions of continuous amino acid residues 1-273 and 285-394 as defined by SEQ ID NO: 1. The invention also provides related chimeric molecules, nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions.
The invention addresses the problem that native Pseudomonas exotoxin A is highly immunogenic, stimulating an anti-PE immune response including production of anti-PE antibodies and immune cells that neutralize the cytotoxic activity of PE. This immunogenicity reduces the effective therapeutic dose that can be administered to patients, compromising treatment effectiveness against diseases such as cancer. Thus, there is a need for improved PEs that have reduced immunogenicity but retain cytotoxic activity.
Claims Coverage
The patent claims define multiple inventive features centered on specific substitutions to Pseudomonas exotoxin A (PE) to reduce immunogenicity while retaining functionality, including further modifications and applications.
Substitutions of specific amino acid residues to reduce immunogenic B-cell epitopes
A PE amino acid sequence with substitutions at one or more residues selected from E420, D463, Y481, L516, R563, D581, and D589, with the proviso regarding alanine substitution at position 516. Further substitutions may occur within B-cell epitopes, with specific residues listed for such substitutions.
Substitutions with selected amino acids to reduce immunogenicity
Substitutions of one or more of the residues E420, D463, Y481, L516, R563, D581, and D589 to alanine, glycine, serine, or glutamine.
Substitution of subsets of residues for further immunogenic reduction
Substitution restricted to residues D463, Y481, and L516 or alternatively to residues E420, Y481, R563, D581, and D589 or to residues E420, D463, Y481, L516, R563, and D581.
Further substitutions within selected B-cell epitope residues
Further substitutions to alanine, glycine, serine, or glutamine at one or more of a specified list of residues within B-cell epitopes, including notably residues R427, R458, R467, R490, R505, and R538.
Specific combination of substitutions for decreased immunogenicity
A substitution of alanine for D463 and further alanine substitutions for residues R427, R458, R467, R490, R505, and R538.
Optional further substitutions within T-cell epitopes and deletions
PE optionally has further substitutions within one or more T-cell epitopes and/or deletions of continuous amino acid residues 1-273 and 285-394 as defined by SEQ ID NO: 1.
Pharmaceutical compositions and chimeric molecules
Pharmaceutical compositions comprising the inventive PE and chimeric molecules comprising a targeting agent conjugated or fused to the inventive PE, wherein the targeting agent can be a monoclonal antibody.
Method of inhibiting growth of target cells
A method of inhibiting growth of a target cell by contacting the cell with the inventive PE in an amount effective to inhibit growth.
The claims cover Pseudomonas exotoxin A molecules with specific amino acid substitutions that reduce immunogenic B-cell epitopes, optionally combined with further substitutions and/or deletions, as well as compositions, chimeric molecules with targeting moieties, and methods for inhibiting target cell growth. These features collectively target the problem of immunogenicity reduction while preserving cytotoxic activity.
Stated Advantages
The inventive PEs may be less immunogenic than unsubstituted PE due to removal of B-cell epitopes through specific amino acid substitutions.
Further substitutions within T-cell epitopes and B-cell epitopes may further reduce immunogenicity by removing additional epitopes.
The PEs retain cytotoxic activity effective for destroying or inhibiting growth of target cells, including cancer cells.
Combination of substitutions can lead to reduced immunogenicity and increased cytotoxic activity simultaneously.
The chimeric molecules facilitate targeted delivery of PE cytotoxicity to cells expressing specific surface markers, reducing off-target effects.
Documented Applications
Treatment or prevention of cancer in a mammal by administering the inventive PE or chimeric molecules in an effective amount.
Inhibition of growth of target cells, including cancer cells, by contacting the cells with the inventive PE or related pharmaceutical compositions.
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