Methods for DNA-dependent targeting of a cell permeant antibody
Inventors
Weisbart, Richard H. • Nishimura, Robert N.
Assignees
US Department of Veterans Affairs
Publication Number
US-10383945-B2
Publication Date
2019-08-20
Expiration Date
2036-02-18
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Abstract
The invention provides methods for selective targeting of live cells, which have undergone or are undergoing radiation or chemotherapy, at a site of interest with a cell-penetrating polypeptide. In one embodiment of the invention, the method comprises contacting the live cells with a cell-penetrating polypeptide comprising cell-penetrating determinants so that the cell-penetrating polypeptide binds extracellular DNA near or around the live cells so as to form a complex or association therewith such that the complex or associated polypeptide-DNA so bound bind the live cells and penetrates the live cells thereby selectively targeting live cells at a site of interest with a cell-penetrating polypeptide.
Core Innovation
The invention provides methods for selective targeting of live cells, which have undergone or are undergoing radiation or chemotherapy, at a site of interest with a cell-penetrating polypeptide. The method involves contacting live cells with a cell-penetrating polypeptide comprising cell-penetrating determinants so the polypeptide binds extracellular DNA near or around the live cells, forming a complex that binds and penetrates the live cells, thereby selectively targeting live cells at the site of interest.
Moreover, the invention includes methods for selective targeting of live cells at or near cellular injury sites with cell-penetrating polypeptides that may be joined to therapeutic agents. The polypeptides form complexes with extracellular DNA released from cellular injury, which then bind and penetrate live cells, thus selectively targeting and potentially treating injured cells at the site.
The problem addressed by the invention arises from the need to better understand and harness the mechanism by which certain anti-DNA antibodies, such as the lupus autoantibody 3E10, penetrate living cells. While 3E10 has been developed as a molecular delivery vehicle and has therapeutic potential ranging from cancer to ischemic conditions, the detailed mechanism enabling its cell and nuclear penetration and selective targeting to damaged or diseased tissues was previously unclear. The invention discovers that the cell penetrating ability of such antibodies depends on their binding to extracellular DNA, which facilitates their selective uptake by live cells near sites of cellular turnover or injury.
Claims Coverage
The patent includes 19 independent features focusing on methods and compositions for selectively targeting live cells using cell-penetrating polypeptides and extracellular DNA.
Method for selective targeting of live cells using cell-penetrating polypeptide and DNA
A method comprising introducing isolated DNA near or around live cells at a site of interest; administering a cell-penetrating polypeptide with cell-penetrating determinants; binding of the polypeptide to the DNA or its degradation product to form a complex; contacting the complex with live cells to bind and penetrate them; and permitting additional complexes to form and target more live cells. The method includes administration of DNA by injection, microinjection, microprojectile, implantation or implantation of DNA-containing objects releasing DNA over time; the polypeptide is a 3E10 antibody or its single chain Fv fragment.
Method for inhibiting tumors associated with ischemia or cellular death
A method inhibiting tumors linked to ischemia, necrosis, or apoptosis by selectively targeting live cells at a site of interest using the method involving cell-penetrating polypeptides and DNA.
Cell penetration dependence on nucleoside salvage pathway
Cell penetration by the cell-penetrating polypeptide is dependent on a salvage pathway, in particular a nucleoside salvage pathway mediated by equilibrative nucleoside transporters (ENTs) or the SLC29 family, specifically ENT2.
3E10 antibody identity and modifications
The cell-penetrating polypeptide is a 3E10 antibody or fragment thereof with binding specificity matching an antibody produced by the hybridoma ATCC PTA 2439; the antibody may be humanized; may have an amino acid sequence modification such as the D31N substitution enhancing cell penetration; and optionally linked to amino acid sequence AGIH at its amino terminus.
Diversity of DNA used for targeting
The isolated DNA introduced may be single-, double-, triple-, or four-stranded DNA or combinations thereof, with phosphodiester, phosphorothioate or methylphosphonate bonds or artificial DNA containing a pseudopeptide backbone with various chemical linkers.
Inclusion of therapeutic, cytotoxic, or cytoprotective agents and imaging markers
The cell-penetrating polypeptide may be linked or combined with therapeutic agents including chemotherapeutics, toxins, radioactive isotopes, or cytoprotective agents such as heat shock proteins, stress proteins, or chaperones, and may be further linked to imaging or detectable markers such as radioisotopes, fluorophores, enzymes, luminescent or chemiluminescent compounds, heavy metals, ferromagnetic agents or contrast agents.
Targeting of injury sites
The site of interest for targeting may be an injury site including intracranial, brain, myocardial infarction, skin, liver, gastrointestinal, lung, eye, kidney, pancreas, peritoneal, bone, nasopharyngeal, uterine, cervical, breast, organ, tissue, burn or radiation injury.
The claims collectively encompass methods for selectively targeting live cells at various sites by exploiting the formation of complexes between cell-penetrating polypeptides (notably 3E10 antibody and derivatives) and extracellular DNA or its degradation products, using nucleoside salvage pathways for cell penetration, optionally combined with therapeutic or imaging agents, with applications to tumor inhibition and injured tissue targeting.
Stated Advantages
Selective targeting of live cells at sites of injury or tumors reduces potential systemic toxicity of therapeutics.
Enhanced uptake of cell-penetrating polypeptides in target tissues enriched with extracellular DNA.
Capability to deliver therapeutic agents intracellularly, including to cell nuclei, via antibody-DNA complexes.
Use of extracellular DNA as a targeting ligand allows for increased efficacy and specificity in treatments.
Demonstrated stability and sustained presence of cell-penetrating polypeptides in targeted tumor tissues in vivo.
Documented Applications
Selective targeting and treatment of live cells undergoing or having undergone radiation or chemotherapy at tumor sites.
Inhibition of tumors associated with ischemia, tissue necrosis or apoptosis.
Therapeutic targeting of cellular injury sites such as brain injury, myocardial infarction, skin injury, liver injury, and other tissue injuries.
Delivery of therapeutic agents including cytotoxic drugs or cytoprotective proteins to cells at injury or cancerous sites.
Use in cancer immunotherapy including targeting tumors with modifications such as 3E10 scFv linked to tumor suppressors or chaperones.
Diagnosis or identification of sites of cell or tissue injury, ischemia, tumor sites, or cellular turnover by detecting intracellular presence of cell-penetrating polypeptides.
Enhancement of chemotherapy or radiation therapy by targeted delivery through extracellular DNA-mediated uptake.
Protection of cells from reactive oxygen species toxicity and treatment of acute injuries such as stroke or myocardial infarction via selective targeting.
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