Modified Pseudomonas exotoxin A
Inventors
Pastan, Ira H. • Mazor, Ronit • Onda, Masanori • Lee, Byungkook • Niederfellner, Gerhard • Imhof-Jung, Sabine • Brinkmann, Ulrich • Georges, Guy
Assignees
Hoffmann La Roche Inc • US Department of Health and Human Services
Publication Number
US-9388222-B2
Publication Date
2016-07-12
Expiration Date
2034-10-03
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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 modified Pseudomonas exotoxin A (PE) proteins comprising amino acid substitutions that disrupt one or more B-cell and/or T-cell epitopes to reduce immunogenicity while retaining cytotoxic activity. These modified PE can be formulated into related chimeric molecules fused to targeting moieties such as humanized antibody fragments, as well as related nucleic acids, recombinant expression vectors, host cells, populations of cells, and pharmaceutical compositions. Methods for producing these modified PEs and chimeric molecules and methods of treating or preventing cancer and inhibiting target cell growth using these molecules are also provided.
The problem being addressed is the high immunogenicity of PE, which stimulates anti-PE immune responses, including anti-PE antibodies and T-cells, that neutralize PE's cytotoxic activity. This immunogenicity limits the administered dose, reducing therapeutic effectiveness in diseases like cancer. Prior deimmunized PE variants incompletely removed B-cell epitopes, leading to residual immunogenicity, and also suffered from reduced cytotoxic potency. Further, some deimmunized PE constructs such as PE24 had a reduced serum half-life due to smaller size, and prior targeting moieties (e.g., dsFv fragments) and linkers contained T-cell epitopes and caused poor developability due to stability issues. Prior immunotoxins also relied on mouse-derived targeting fragments, increasing immunogenicity. Therefore, there is a need for improved PE variants with lower immunogenicity, improved cytotoxic potency, enhanced serum half-life, and improved stability.
The innovative solution presented includes PE with specific substitutions at amino acid residues F443, R456, L477, R494, and L552, optionally combined with further substitutions at residues within one or more B-cell epitopes and/or T-cell epitopes, or deletions of continuous amino acid stretches corresponding to domains Ia and II, particularly generating combinations that eliminate all B-cell epitopes without substantially reducing cytotoxicity. These modified PEs can be fused to humanized antibody Fab fragments through optimized elongated linkers that reduce T-cell epitopes in the linker while improving thermal stability. The PEs retain cytotoxic domain III functional activity and have improved pharmacokinetic properties such as enhanced serum half-life and reduced off-target toxicity compared to prior constructs. Methods for the recombinant expression, purification, and formulation of these modified PE constructs and chimeric molecules are also provided. The targeted nature of these chimeric molecules allows specific delivery to cells expressing chosen cell-surface markers to inhibit growth or treat cancers.
Claims Coverage
The patent includes three independent claims covering modified Pseudomonas exotoxin A proteins with specific amino acid substitutions and chimeric molecules comprising these PEs fused to targeting moieties.
Modified pseudomonas exotoxin A with specific substitutions and optional further modifications
A Pseudomonas exotoxin A comprising substitutions of one or more amino acid residues F443, R456, L477, R494, and L552 (reference to SEQ ID NO: 1), optionally combined with (i) further substitutions in B-cell epitopes at specified residues, (ii) further substitutions in T-cell epitopes, (iii) deletions of residues 1-273 and 285-394, or (iv) combinations thereof.
Isolated mutated PE with formula R1n-FCS-R2n-R3n-PE functional domain III and specific substitutions
An isolated mutated PE with the formula R1n-FCS-R2n-R3n-PE functional domain III, where n is 0 or 1 per each R1, R2, R3, R1 and R2 are 1-10 amino acid residues, FCS is a furin cleavage sequence cleavable by furin, and PE functional domain III corresponds to residues 395-613 of SEQ ID NO: 1. One or more of residues F443, R456, L477, R494, and L552 are substituted, optionally with additional substitutions within B-cell and/or T-cell epitopes.
Chimeric molecules comprising the modified PE fused to targeting moieties
Chimeric molecule comprising a targeting moiety conjugated or fused to any of the inventive PEs described, where the targeting moiety can be a monoclonal antibody or its antigen binding fragment specific for a cell surface marker.
The patent claims cover modified Pseudomonas exotoxin A molecules with defined amino acid substitutions disrupting immunogenic epitopes, embodiments with specific domain deletions and furin cleavage sequences, and chimeric molecules comprising such PEs fused to targeting moieties for selective delivery, providing constructs with reduced immunogenicity and retained cytotoxicity.
Stated Advantages
Reduced immunogenicity by elimination of B-cell and T-cell epitopes, resulting in diminished anti-PE antibody and T-cell responses.
Retention or improvement of cytotoxic potency towards tumor cells despite deimmunization.
Increased serum half-life and improved pharmacokinetic properties due to optimized chimeric constructs and elongated linkers.
Reduced off-target toxicity, such as hepatotoxicity and vascular leak syndrome, compared to previous PE immunotoxins.
Improved thermal stability and developability of chimeric molecules with elongated linkers that remove T-cell epitopes in linker regions.
Documented Applications
Treatment or prevention of cancer in mammals by administration of the modified PE or chimeric molecules.
Inhibition of growth of target cells, particularly cancer cells expressing specific cell surface markers.
Use of nucleic acids, recombinant expression vectors, and host cells encoding the modified PE or chimeric molecules for production.
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