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-9765123-B2
Publication Date
2017-09-19
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 substitutions in one or more B-cell and/or T-cell epitopes, thereby reducing immunogenicity. The PE can have substitutions at specific amino acid residues defined by reference to SEQ ID NO: 1, optionally combined with deletions of domains or amino acid sequences. The PE may be incorporated into chimeric molecules with targeting moieties, provided as nucleic acids, expressed from recombinant vectors, and formulated in pharmaceutical compositions for therapeutic use.
The background identifies that while PE is a bacterial toxin effective for destroying or inhibiting undesirable cells such as cancer cells, it is highly immunogenic. Immunogenicity leads to an immune response generating anti-PE antibodies and/or T-cells that neutralize PE's cytotoxic activity. This response limits the dose that can be safely administered and reduces therapeutic effectiveness, demonstrating a need for PE with reduced immunogenicity.
The inventive PEs are designed with substitutions at key residues involved in B-cell and T-cell epitopes, such as L294, L297, Y298, L299, R302, D463, Y481, L516, and a host of other epitopes within domain II and domain III of PE. These substitutions can be alanine, glycine, serine, or glutamine, aiming to remove or reduce immunodominant epitopes. The invention also encompasses functional chimeric molecules that target specific cell surface markers using targeting moieties including monoclonal antibodies, allowing selective delivery of cytotoxic PE to cancer cells while minimizing immunogenicity.
Claims Coverage
The patent includes one independent claim focusing on the Pseudomonas exotoxin A with specific amino acid substitutions designed to reduce immunogenicity while retaining cytotoxic function.
Substitution of amino acid residues D463, Y481, and L516 with provisos
The PE comprises substitutions of one or more amino acid residues selected from D463, Y481, and L516 as defined by SEQ ID NO: 1, with the condition that if L516 is substituted with alanine, at least one of D463 or Y481 must also be substituted. The PE may also include further substitutions within B-cell and/or T-cell epitopes or deletions of amino acid stretches (residues 1-273 and 285-394).
Further substitutions within B-cell epitopes
The PE may have additional substitutions within one or more B-cell epitopes comprising amino acid residues such as E282, E285, P290, R313, N314, P319, D324, E327, E331, Q332, D403, D406, R412, R427, E431, R432, R458, D461, R467, R490, R505, R513, E522, R538, E548, R551, R576, K590, Q592, and L597 as per SEQ ID NO: 1.
Preferred amino acid substitutions lowering immunogenicity
Substitutions independently include alanine, glycine, serine, or glutamine in place of the specified residues D463, Y481, L516, and residues within B-cell epitopes such as R427, R458, R467, R490, R505, and R538.
Substitutions reducing T-cell epitopes
Substitutions of alanine, glycine, serine, or glutamine in place of one or more amino acid residues selected from I493, R494, N495, G496, L498, L499, R500, V501, and Y502 as defined in SEQ ID NO: 1 reduce T-cell immunogenicity.
Chimeric molecules with targeting moieties
The invention covers chimeric molecules comprising targeting moieties conjugated or fused to the substituted PE. The targeting moiety may be a monoclonal antibody specific for cell surface markers including CD19, CD21, CD22, CD25, CD30, CD33, CD79b, transferrin receptor, EGF receptor, mesothelin, cadherin, and Lewis Y, or antigen binding portions thereof such as HA22.
Pharmaceutical compositions and therapeutic methods
Pharmaceutical compositions comprising the inventive PE and pharmaceutically acceptable carriers are claimed. Methods of inhibiting growth of target cells, including cancer cells, by contacting cells with the PE are claimed.
The claims cover Pseudomonas exotoxin A proteins with specific substitutions reducing B-cell and T-cell epitopes to lower immunogenicity, optionally combined with deletions and further substitutions, alone or in chimeric molecules with targeting moieties. The claims also cover pharmaceutical compositions of these PEs and their use in methods of inhibiting target cell growth, including for cancer treatment.
Stated Advantages
Substitutions reduce or eliminate immunogenic T-cell and/or B-cell epitopes, thereby diminishing immune responses such as production of neutralizing antibodies and T cell activation.
Reduced immunogenicity enables higher dosing of PE, potentially improving effectiveness for treating diseases like cancer.
The modified PEs retain cytotoxicity and can target specific cell populations when combined with targeting moieties.
Certain substitutions can simultaneously increase cytotoxicity while decreasing immunogenicity.
Documented Applications
Treating or preventing cancer in a mammal by administration of the modified PE, chimeric molecules, nucleic acids, recombinant vectors, host cells, populations of cells, or pharmaceutical compositions thereof.
Inhibiting growth of target cells, especially cancer cells, in vitro or in vivo by contacting the cells with the modified PE materials.
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