Modified anthrax toxin protective antigen
Inventors
Liu, Shi-Hui • Leppla, Stephen H. • Bugge, Thomas H. • Wein, Alexander N. • Peters, Diane E. • Liu, Jie • Chen, Kuang-Hua
Assignees
US Department of Health and Human Services
Publication Number
US-10835593-B2
Publication Date
2020-11-17
Expiration Date
2036-08-25
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Abstract
Disclosed is a Bacillus anthracis protective antigen (PA) comprising a PA amino acid sequence, wherein one or more of amino acid residues I207, I210, E654, I656, R659, M662, Y681, and L687, as defined by reference to SEQ ID NO: 1, are, independently, substituted, with the proviso that amino acid residue I207 is not substituted with alanine and amino acid residue I210 is not substituted with alanine. Related compositions, nucleic acids, recombinant expression vectors, host cells, populations of cells, methods of treating or preventing cancer in a mammal, and methods of inhibiting the growth of a target cell are also disclosed.
Core Innovation
The invention provides modified Bacillus anthracis protective antigen (PA) proteins comprising specific amino acid substitutions at one or more of the residues I207, I210, E654, I656, R659, M662, Y681, and L687, with the proviso that I207 is not substituted with alanine and I210 is not substituted with alanine. These modified PAs can form functional, lethal-factor-binding hetero-oligomers through intermolecular complementation when combined appropriately, allowing targeted cytotoxic activity against tumor cells.
The problem being solved is the toxic nature of Bacillus anthracis anthrax toxins or their components to normal cells despite their potential as agents to inhibit or destroy undesirable cells such as cancer cells. Specifically, prior PA variants (e.g., PA-U2-R200A, PA-L1-I210A) exhibit non-specific toxicity toward normal tissues, limiting their clinical utility. Thus, there is a need for improved anthrax toxin compositions with reduced non-specific toxicity and enhanced tumor targeting.
The invention addresses this need by providing PA variants with substitutions at critical residues, notably I207 substituted with residues other than alanine (preferably arginine), which reduces non-specific toxicity while maintaining potent anti-tumor activity. Additionally, substitutions at residues I656, Y681, L687 enhance selectivity for the tumor endothelial cell receptor CMG2, and substitutions at E654, R659, M662 modulate selectivity for the TEM8 receptor, which may be useful research tools or anti-tumor agents for TEM8-expressing tumors. Compositions combining pairs of such modified PAs that individually cannot form LF-binding heptamers but together form functional complexes offer specificity in targeting tumor cells through selective receptor binding and activation by tumor-associated proteases.
Claims Coverage
The patent contains five claims incorporating inventive features related to compositions of Bacillus anthracis protective antigen variants and combinations with specific substitutions and cleavage site modifications.
Improved protective antigen hetero-oligomer composition
A composition comprising a first PA with amino acid residue I207 substituted with arginine, and its furin cleavage site replaced with an MMP sequence (SEQ ID NO: 6), combined with a second PA having amino acid residue R200 substituted with alanine and furin cleavage site replaced with a uPA sequence (SEQ ID NO: 9), where the first and second PA amino acid sequences differ.
Substitution at amino acid residue I656 in protective antigens
The composition of PA variants wherein amino acid residue I656 of the first PA, the second PA, or both is substituted, contributing to modified receptor selectivity and function.
Preferred amino acid substitutions at I656
A composition wherein I656 of the first PA, second PA, or both is substituted specifically with glutamine, valine, alanine, cysteine, or glutamic acid.
Specific glutamine substitution at I656 in both PAs
A composition wherein I656 of the first and second PA amino acid sequences is substituted with glutamine.
Inclusion of effector components for biological activity
The composition further comprising one or both Bacillus anthracis lethal factor (LF) and edema factor (EF), which bind to hetero-oligomeric PA complexes to deliver cytotoxic effects.
The claims cover compositions of modified Bacillus anthracis protective antigens with specific amino acid substitutions and cleavage site modifications that form hetero-oligomers capable of binding lethal and edema factors, with particular emphasis on AA residue substitutions at I207, I656, and R200 and their combinations, enhancing tumor specificity and reducing non-specific toxicity.
Stated Advantages
Reduced non-specific toxicity to normal tissues while maintaining potent anti-tumor activity.
Improved selectivity for tumor endothelial cell receptors CMG2 (via substitutions at I656, Y681, L687).
Potential to target TEM8-expressing tumors through substitutions at E654, R659, M662.
Formation of functional lethal-factor-binding hetero-oligomeric PA complexes dependent on intermolecular complementation, increasing tumor-targeting specificity.
Combination therapy with immunosuppressive agents such as pentostatin and cyclophosphamide reduces anti-toxin immune responses, allowing repeated administration and enhancing anti-solid tumor efficacy.
Selective toxicity toward proliferating tumor endothelial cells with sparing of normal endothelial cells, reducing systemic toxicity.
Documented Applications
Treatment or prevention of cancer, including solid tumors such as oral squamous carcinoma, melanoma, lung tumors, and ovarian tumors, by administration of modified PA compositions with or without effector components.
Methods of inhibiting growth of target (cancer) cells in vitro or in vivo using the modified PA variants.
Combination therapies including administration of pentostatin and cyclophosphamide for cancer treatment, either alone or with PA compositions, to reduce immune responses and improve efficacy.
Use of modified PAs as research tools for studying receptor binding and toxin specificity (e.g., CMG2 and TEM8 interactions).
Treatment of oral melanoma in veterinary subjects such as cats and dogs by intratumoral administration of combined modified PA variants plus LF.
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