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-9657066-B2
Publication Date
2017-05-23
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 SEQ ID NO: 1. The substitutions may further include changes within one or more T-cell and/or B-cell epitopes, and optionally deletions of continuous amino acid residues from residues 1-273 and 285-394 of SEQ ID NO: 1. The PE may also include a furin cleavage sequence and portions of domains Ib and II and functional domain III, whereby domain III mediates cytotoxic ADP ribosylation activity.
The inventive PEs are designed to be less immunogenic by reducing or eliminating B-cell epitopes through targeted amino acid substitutions. This reduction in immunogenicity addresses the problem where administration of PE induces an antibody response that neutralizes the toxin's cytotoxicity, limiting the effective dose and reducing treatment efficacy for diseases such as cancer.
The invention further provides related chimeric molecules combining the inventive PE with targeting moieties such as antibodies, associated nucleic acids, recombinant vectors, expression hosts, and pharmaceutical compositions. Methods for producing these components and methods for treating or preventing cancer or inhibiting target cell growth by administration of the inventive PE or chimeric molecules are described.
Claims Coverage
The patent includes multiple independent claims focusing on a Pseudomonas exotoxin A (PE) with specified amino acid sequence features and chimeric molecules containing such PEs. The claims emphasize inventive substitutions reducing immunogenic B-cell epitopes and optional substitutions in T-cell epitopes, as well as related compositions and methods.
Pseudomonas exotoxin A with specific amino acid substitutions and epitope modifications
A PE comprises an amino acid sequence including a furin cleavage sequence (FCS), optional segments R1, R2, and R3 from SEQ ID NO: 1, and a functional domain III (residues 395-613) with substitutions at one or more residues selected from E420, D463, Y481, L516, R563, and D581. When L516 is substituted with alanine, at least one other specified residue is also substituted. The PE further has substitutions in one or more B-cell epitopes among a specified group of residues and optionally has substitutions within T-cell epitopes.
Substitution types for reduced immunogenicity
The substitutions at amino acid residues E420, D463, Y481, L516, R563, and D581 can be alanine, glycine, serine, or glutamine. Specific combinations include substitutions of D463, Y481, and L516 or of E420, Y481, R563, and D581.
Furin cleavage sequence with further B-cell epitope substitution
The FCS comprises residues 274-284 of SEQ ID NO: 1. Further substitution includes alanine, glycine, serine, or glutamine for residue E282 within B-cell epitopes.
Additional substitutions in B-cell epitopes for immunogenicity reduction and cytotoxicity enhancement
Further substitutions in one or more B-cell epitopes include alanine, glycine, serine, or glutamine at residues D403, D406, R412, R427, E431, R432, R458, D461, R467, R490, R505, R513, E522, R538, E548, R551, R576, K590, Q592, and L597, and substitution of valine, leucine, or isoleucine for R490 to improve cytotoxicity.
Specific preferred substitution set with FCS and B-cell epitope changes
A PE with m, n, p all zero, FCS residues 274-284, substitution of alanine for D463, and further alanine substitutions for R427, R458, R467, R490, R505, and R538 within B-cell epitopes.
Chimeric molecule comprising the inventive PE
A chimeric molecule comprising a targeting moiety conjugated or fused to the PE described above.
Targeting moiety as monoclonal antibody against specified cell surface markers
The targeting moiety is a monoclonal antibody specifically binding to a cell surface marker selected from CD19, CD21, CD22, CD25, CD30, CD33, CD79b, transferrin receptor, EGF receptor, mesothelin, cadherin, and Lewis Y.
Specific targeting moieties enumerated
The targeting moiety is selected from B3, RFB4, SS, SS1, MN, MB, HN1, HN2, HB21, MORAb-009, their antigen-binding portions, or the antigen binding portion of HA22.
Pharmaceutical composition comprising the inventive PE
A pharmaceutical composition comprising the inventive PE and a pharmaceutically acceptable carrier.
Methods of inhibiting target cell growth using the inventive PE
A method of inhibiting growth of target cells by contacting them with an effective amount of the inventive PE.
Target cell specificity in growth inhibition methods
The target cell is a cancer cell expressing a cell surface marker selected from CD19, CD21, CD22, CD25, CD30, CD33, CD79b, transferrin receptor, EGF receptor, mesothelin, cadherin, and Lewis Y.
Methods of producing the inventive PE and chimeric molecules
Methods comprising recombinantly expressing and purifying the inventive PE or chimeric molecules. Also, producing a chimeric molecule by expressing and purifying the PE and covalently linking a targeting moiety.
The claims cover Pseudomonas exotoxin A compositions with specific amino acid substitutions reducing B-cell epitope immunogenicity and optionally T-cell epitope substitutions, chimeric molecules with targeting moieties, pharmaceutical compositions, and methods of production and therapeutic use. The inventive features focus on detailed substitution patterns within defined PE domains and epitope regions to reduce immunogenic responses while retaining cytotoxic efficacy.
Stated Advantages
The inventive PEs may be less immunogenic than unsubstituted wild-type PE by removing B-cell epitopes through amino acid substitutions.
The immunotoxins can retain or increase cytotoxic activity simultaneously with decreased immunogenicity.
The inventive modification allows administration of PE-based therapeutics with reduced neutralization by anti-PE antibodies, enhancing effective dosing for cancer treatment.
Documented Applications
Use of the inventive Pseudomonas exotoxin A and chimeric molecules for treating or preventing cancer in mammals.
Use of the inventive PEs and chimeric molecules to inhibit growth of target cells, including cancer cells expressing specified cell surface markers.
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