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-9346859-B2
Publication Date
2016-05-24
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. 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.
Pseudomonas exotoxin A (PE) is a bacterial toxin with cytotoxic activity that may be effective for destroying or inhibiting the growth of undesirable cells, e.g., cancer cells. Accordingly, PE may be useful for treating or preventing diseases such as cancer. However, PE may be highly immunogenic. PE administration may stimulate an anti-PE immune response including, for example, the production of anti-PE antibodies and/or T-cells, that undesirably neutralizes the cytotoxic activity of PE. Such immunogenicity may reduce the amount of PE that can be given to the patient which may, in turn, reduce the effectiveness of the PE for treating the disease.
Claims Coverage
The patent contains multiple independent claims covering Pseudomonas exotoxin A variants with specific amino acid substitutions, pharmaceutical compositions, chimeric molecules, and methods of treatment and production.
Substitutions in Pseudomonas exotoxin A reducing immunogenicity
A PE comprising substitutions of one or more amino acid residues at positions 421, 422, 423, 425, 427, 429, 439, 440, 443, 444, 446, 447, 450, 463-479, 481-514, 516-519, 551, 552, 554, 555, 556, and 558 of SEQ ID NO: 1; with provisos on substitutions at positions 485, 516, 427, 467, 490, 505, 513, or 551; optionally with further substitutions within one or more B-cell epitopes of SEQ ID NO: 1.
Substitutions replacing certain amino acids with alanine, glycine, serine, or glutamine
PE with substitution of alanine, glycine, serine, or glutamine at selected positions including 493, 494, 495, 496, 498, 499, 500, 501, and 502 within positions 421-558 of SEQ ID NO: 1; optionally with further B-cell epitope substitutions.
Use of specific PE forms
PEs including PE4E, PE40, PE38, PE25, PE38QQR, PE38KDEL, PE-LR, or PE35 comprising the inventive substitutions.
Substitution of amino acid residues within one or more B-cell epitopes
Substitution of one or more amino acids within B-cell epitopes at positions 282, 285, 290, 313, 314, 319, 324, 327, 331, 332, 403, 406, 412, 431, 432, 458, 461, 522, 548, 576, 590, 592, and 597 of SEQ ID NO: 1 with alanine, glycine, serine or glutamine.
Pharmaceutical compositions containing the substituted PE
Pharmaceutical compositions comprising the PE variants with the substituted amino acids as described and a pharmaceutically acceptable carrier.
Chimeric molecules comprising PE and targeting agents
Chimeric molecules comprising a targeting agent conjugated or fused to the PE with the amino acid substitutions.
Use of monoclonal antibodies as targeting agents
Chimeric molecules where the targeting agent is a monoclonal antibody specifically binding to certain cell surface markers including CD19, CD21, CD22, CD25, CD30, CD33, CD79b, transferrin receptor, EGF receptor, mesothelin, cadherin, and Lewis Y.
Methods of inhibiting target cell growth or producing PEs and chimeric molecules
Methods of inhibiting growth of target cells by contacting with the substituted PE; and methods of producing the PE and chimeric molecules by recombinant expression and purification.
The claims cover PEs with specified amino acid substitutions to reduce immunogenicity while retaining cytotoxicity, chimeric molecules with targeting moieties including monoclonal antibodies, pharmaceutical compositions, and methods for treatment and production.
Stated Advantages
The inventive PEs may be less immunogenic than unsubstituted PE by removal of T-cell and B-cell epitopes.
Reduced immunogenicity enables administration of higher or more effective doses for treating diseases, such as cancer.
Some substitutions may increase cytotoxic activity.
Chimeric molecules allow targeted delivery of cytotoxic PE to cells expressing relevant surface markers, minimizing off-target effects.
Documented Applications
Methods of treating or preventing cancer in a mammal by administering the inventive PE, chimeric molecules, nucleic acids, recombinant vectors, host cells, populations of cells, or pharmaceutical compositions.
Methods of inhibiting growth of target cells using the inventive PE, chimeric molecules, nucleic acids, recombinant vectors, host cells, populations, or pharmaceutical compositions.
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