Chlorotoxin polypeptides and conjugates and uses thereof
Inventors
Sentissi, Abdellah • Jacoby, Douglas B.
Assignees
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Publication Number
US-9018347-B2
Publication Date
2015-04-28
Expiration Date
Abstract
Reduced lysine chlorotoxin polypeptides that may be used to generate single species conjugates of chlorotoxin. Conjugates comprising such chlorotoxin polypeptides and pharmaceutical compositions thereof. Methods of using such compositions and/or conjugates.
Core Innovation
The patent addresses the problem that conjugation of wild-type chlorotoxin can yield mixed species, which is attributed to multiple lysine conjugation sites on the chlorotoxin polypeptide. The patent therefore provides reduced-lysine chlorotoxin polypeptides in which lysine residues are modified so that chemical conjugation reactions do not occur at most lysine sites.
The patent defines mono-lysine and no-lysine forms and specifies that the modified lysine residues are blocked from participating in a chemical conjugation reaction by a modification to the lysine residue. The design retains a lysine residue corresponding to position 27 in SEQ ID NO: 1, while lysine residues other than the position 27 lysine are blocked, supporting formation of single-species chlorotoxin conjugates.
The patent further describes chlorotoxin conjugates formed by covalent association of the reduced-lysine chlorotoxin polypeptides with a therapeutic agent and/or detection agent. The covalent association can be direct or mediated by a linker, and the disclosure expands the conjugate payload options to multiple therapeutic and diagnostic agent categories and applies these conjugates to cancer and aberrant angiogenesis, including ocular neovascularization.
Claims Coverage
The independent claims include a chlorotoxin polypeptide defined by sequence identity to SEQ ID NO: 1 and by lysine blocking behavior that limits chemical conjugation to a controlled lysine site, position 27. The claim set includes additional inventive features regarding the type of lysine modification and how therapeutic or detection agents are associated, including through a linker.
Sequence identity with controlled lysine modification pattern
A chlorotoxin polypeptide has at least 90% sequence identity with SEQ ID NO: 1, and each lysine residue present in the chlorotoxin polypeptide, except for the lysine residue corresponding to position 27 of SEQ ID NO: 1, is blocked from participating in a chemical conjugation reaction by a modification to the lysine residue.
Covalent or non-covalent lysine blocking
The lysine residue blocking modification is either a covalent modification or a non-covalent modification.
Covalent lysine modifications as pegylation or methylation
The covalent modification of the blocked lysine residue is pegylation or methylation.
Epsilon NH2 lysine functional group modification
The blocked lysine residue is modified at the epsilon NH2 group of the lysine residue.
Covalent association through a linker
The chlorotoxin polypeptide and the therapeutic agent or the detection agent are covalently associated through a linker.
Enumerated therapeutic agent types for the conjugate
The therapeutic agent is selected from the group consisting of radioisotopes, prodrug activating enzymes, radiosensitizers, interfering ribonucleic acids, superantigens, anti-angiogenic agents, alkylating agents, purine antagonists, pyrimidine antagonists, plant alkaloids, intercalating antibiotics, aromatase inhibitors, anti-metabolites, mitotic inhibitors, growth factor inhibitors, cell cycle inhibitors, enzymes, topoisomerase inhibitors, biological response modifiers, anti-hormones and anti-androgens.
Overall, the claim coverage centers on reduced-lysine chlorotoxin polypeptides defined by at least 90% identity to SEQ ID NO: 1 and lysine residues that are blocked from participating in chemical conjugation, except for the lysine residue corresponding to position 27. Dependent claim refinements specify the categories of lysine blocking modifications and further define conjugates in which therapeutic or detection agents are covalently associated, optionally through a linker.
Stated Advantages
Enables single-species chlorotoxin conjugates by reducing mixed species arising from multiple lysine conjugation sites.
Documented Applications
Cancer therapy and diagnostic use of chlorotoxin conjugates, including examples involving paclitaxel-chlorotoxin conjugates and gemcitabine-chlorotoxin conjugates, and assays including binding, cytotoxicity, uptake/imaging, and invasion.
Aberrant angiogenesis, including ocular neovascularization such as macular degeneration, using chlorotoxin conjugates.
Therapeutic and diagnostic imaging approaches using chlorotoxin conjugates, including radiolabeled imaging mention (SPECT/PET) and radiosensitizers/radioisotopes.
In vivo breast cancer model using paclitaxel-chlorotoxin conjugates.
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