Multimeric protein toxins to target cells having multiple identifying characteristics
Inventors
Leppla, Stephen H. • Liu, Shi-Hui • Bugge, Thomas H.
Assignees
US Department of Health and Human Services
Publication Number
US-7947289-B2
Publication Date
2011-05-24
Expiration Date
2025-02-09
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Abstract
The present invention provides compositions comprising modified bacterial toxins and methods for using the modified bacterial toxins for targeting particular cell populations and for treating diseases.
Core Innovation
The problem solved by the invention lies in increasing the specificity of bacterial toxin targeting to particular cell populations. Prior homo-oligomeric modified bacterial toxins targeted cells based on a single characteristic, leading sometimes to undesired targeting of normal cells sharing that characteristic. There was a need for additional modified bacterial toxins with greater specificity targeting cells based on multiple identifying characteristics, thereby reducing non-specific toxicity to non-target cells.
Claims Coverage
The claims encompass compositions of modified bacterial toxin first effector components forming heterooligomers of modified monomers and related polypeptides, characterized by multiple specific modifications. There are 29 claims covering different aspects including monomer modifications, multimeric composition, second effector components, and pharmaceutical formulations.
Composition of a first effector component forming heterooligomers of modified monomers
A composition comprising a first effector component of a multimeric bacterial protein toxin, including at least two different modified monomers that form a heterooligomer. Each modified monomer is generated by modification of the unmodified monomer meeting at least two criteria: substitution of native cell-recognition domain for a non-native domain; substitution of native proteolytic activation site for a non-native site; mutation generating monomers that only form heterooligomers with specific partners; or mutation such that a second effector component binds only at a site formed by interaction of different monomers.
Substitution of native domains and sites with specific examples
Modification (a) comprises substituting native cell-recognition domain with a non-native domain including antibody, cytokine, or cell surface receptor ligand. Modification (b) comprises substituting native furin cleavage site with cleavage sites for various proteases such as metalloproteinases, cysteine proteases, aspartic acid proteases, plasminogen activators, kallikreins, type 1 or 2 transmembrane serine proteases, or GPI anchored serine proteases. Modification (c) includes at least two point mutations that create binding sites for heterooligomerization with different monomer types.
Multimeric first effector component composition
The first effector component forms a multimeric bacterial protein toxin comprising at least five, six, or seven modified monomers. Each additional monomer, beyond the first two, is either the same as the first or second modified monomer or different, and generated by modification meeting at least two of criteria (a) to (d).
Second effector component selection
The second effector component is selected from anthrax lethal factor (LF), anthrax edema factor (EF), amino acid residues 1-254 of anthrax lethal factor (LFn), or LFn fused to a heterologous polypeptide, including FP59 (LFn fused to an ADP-ribosylation domain of Pseudomonas exotoxin A).
Selected monomer modification combinations
Each first and second modified monomer is generated by modification meeting specific pairwise combinations of criteria: (a) and (b); (b) and (c); (c) and (d); (a) and (c); (a) and (d); or (b) and (d).
Inclusion of a third modified monomer
The composition may further comprise a third modified monomer different from the first two, generated by modification meeting at least two of the criteria (a), (b), (c), and (d).
Specific bacterial protein toxins covered
The bacterial protein toxins include anthrax toxin, cholera toxin, Shiga toxin, staphylococcus toxin α, and pertussis toxin, with anthrax toxin preferred.
Modification specifics of anthrax protective antigen monomers
First and second modified monomers are generated from unmodified anthrax protective antigen monomers, differing from each other. Modifications include substituting the native furin cleavage site with cleavage sites for specified proteases, mutating oligomerization sites so the monomers bind to each other only, and mutating lethal factor binding sites such that both monomers are required to bind the lethal factor.
Specific protease cleavage sites and mutations in anthrax protective antigen monomers
Protease cleavage sites include metalloproteinases (such as MMP-1, -2, -9, -13, -14, MT2-MMP), cysteine proteases, aspartic acid proteases, plasminogen activators (urokinase and tissue type), kallikreins (KLK2, KLK3/PSA), type 1 and 2 transmembrane serine proteases (including hepsin and matriptase), and GPI anchored serine proteases. Mutations for lethal factor binding involve specific residue substitutions (e.g., Arg178Ala, Lys197Ala, Arg200Ala, Ile207Ala, Ile210Ala, Lys214Ala).
Specific substitutions of native furin cleavage sites
Substitutions include replacing the native furin cleavage site of one anthrax protective antigen monomer with a cleavage site for a plasminogen activator and of the other monomer with a cleavage site for a metalloproteinase or matrix metalloproteinase.
Substitution of native cell recognition domains with cytokines
The native cell-recognition domain can be substituted with cytokines such as IL-2 or GM-CSF.
Pharmaceutical composition
A pharmaceutical composition comprising the above modified bacterial toxin composition together with a pharmaceutically acceptable carrier.
Isolated polypeptide monomers
Isolated polypeptide monomers which form heterooligomers, each generated by modification meeting at least two of the criteria (a) to (d).
Isolated polypeptides with specific sequences
Isolated polypeptides comprising amino acid sequences set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 16, or 20.
The claims define bacterial toxin compositions with hetero-oligomeric first effector components made of monomers modified by combinations of substitutions and mutations to increase targeting specificity. They cover specific modifications of anthrax protective antigen monomers, their protease cleavage sites, oligomerization and effector binding sites, and methods of producing pharmaceutical compositions containing such modified proteins.
Stated Advantages
Increased specificity for target cells expressing multiple identifying characteristics, reducing nonspecific toxicity to non-target cells.
Reduced toxicity in vivo compared to unmodified bacterial toxins.
Potent tumoricidal activity only when modified monomers complement each other, enhancing selectivity.
Documented Applications
Treatment of diseases including various cancers such as carcinoma, sarcoma, lymphoma, leukemia, melanoma, and many specific types like colon, breast, bladder, thyroid, lung, ovarian, pancreatic, and brain cancers.
Treatment of viral infections including HIV, CMV, HPV, HBV, HCV, HSV, and HZV.
Treatment of autoimmune diseases including rheumatoid arthritis, diabetes mellitus, myasthenia gravis, systemic lupus erythematosus, Grave's disease, and Addison's disease.
Targeting and killing specific cell populations that express multiple proteolytic enzymes or cell surface molecules.
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