Antibody-mediated transduction of heat shock proteins into living cells
Inventors
Nishimura, Robert N. • Weisbart, Richard H. • Hansen, James
Assignees
US Department of Veterans Affairs
Publication Number
US-9732146-B2
Publication Date
2017-08-15
Expiration Date
2033-03-15
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Abstract
The invention provides for a fusion protein comprising a 3E10 Fv joined to a Hsp-70, Hsp-27, Hsp-90 or GRP-78 or portion thereof, and optionally, the 3E10 Fv comprising an amino acid sequence AGIH at its amino terminus.
Core Innovation
The invention provides fusion proteins comprising a 3E10 Fv joined or attached to heat shock proteins including Hsp-70, Hsp-27, Hsp-90, or glucose regulated protein 78 kDa (GRP78), or portions thereof. These fusion proteins may optionally include an amino acid sequence AGIH at the amino terminus of the 3E10 Fv. The 3E10 Fv is a single-chain variable fragment of the monoclonal antibody 3E10, which utilizes the hENT2 nucleoside salvage pathway to penetrate living cells. The invention also contemplates joining the 3E10 Fv to localizing signals or enzyme cleavage sites to direct or release the fusion proteins intracellularly.
The problem addressed by the invention is that current therapies are limited to small molecules since cells are impermeable to large molecules such as proteins. The invention solves this by providing a method to transport proteins into cells by molecular fusion with the cell-penetrating antibody fragment 3E10 Fv. This method allows delivery of functional heat shock proteins into living cells, which was not feasible with previous technologies.
The invention demonstrates feasibility of transporting large molecules like heat shock proteins into cells by creating fusion proteins with 3E10 Fv. These fusion proteins can be recombinantly produced, purified, and administered pharmaceutically for therapeutic or diagnostic purposes. The fusion proteins retain the cell-penetrating function of 3E10 and the biological activity of the attached heat shock or glucose regulated proteins, enabling potential protection against oxidative or cellular stress.
Claims Coverage
The claims include one independent claim detailing a fusion protein and further dependent claims elaborating on its specific features and embodiments. The main inventive features involve the structure and composition of the fusion protein and its functional components.
Fusion protein comprising 3E10 Fv joined to Hsp-70 with a specific linker and optional AGIH sequence
A fusion protein comprising a 3E10 Fv joined to a Hsp-70, connected via a peptide linker including an immunoglobulin heavy chain constant domain CH1 or portion thereof, and a swivel sequence that permits the 3E10 Fv and Hsp-70 to swivel. The 3E10 Fv optionally contains an amino acid sequence AGIH at its amino terminus. The swivel sequence is specifically LESSGS positioned between the 3E10 Fv and Hsp-70.
Arrangement of functional peptide sequence in the fusion protein
The fusion protein has a defined amino- to carboxyl-terminus sequence arrangement: AGIH, 3E10 Fv, CH1 domain, swivel sequence, and Hsp-70.
3E10 Fv derivatives from monoclonal antibody 3E10
The 3E10 Fv is a derivative of monoclonal antibody 3E10 from 3E10 hybridoma (ATCC PTA 2439) comprising light chain CDR1, CDR2, CDR3 and heavy chain CDR1, CDR2, CDR3 regions of 3E10 antibody and competes with the original antibody, preserving cell penetration and epitope recognition.
Swivel sequence and CH1 domain specifications
The portion of the immunoglobulin heavy chain constant domain CH1 comprises specific amino acid sequences as positions 361-374 or 361-373 of SEQ ID NO:6 or NO:30 with the swivel sequence attached at the C-terminus of CH1, consisting of positions 375-380 of SEQ ID NO:6.
Fusion protein conjugated to therapeutic or diagnostic agents
The fusion protein can be joined to therapeutic agents, including cytotoxic agents such as ricin, doxorubicin, taxol, and others, or diagnostic agents like enzymes, biotin, fluorophores, chromophores, heavy metals, paramagnetic isotopes, or radioisotopes.
Fv sequence variations and recombinant formats
The Fv sequence can be recombinant, chimeric, humanized, or fully human sequences, encoded by specified nucleic acids corresponding to 3E10 antibody CDRs shown in the sequence listing, preserving antibody specificity and function.
The claims cover a fusion protein combining a 3E10 Fv with Hsp-70 via a defined peptide linker with a swivel sequence, optionally containing an AGIH motif, and include derivatives preserving binding and cell penetration, arrangements of the fusion components, and conjugation possibilities with therapeutic or diagnostic agents. Variations in the antibody fragment sequence and format are also claimed.
Stated Advantages
Enables transport of large proteins such as heat shock proteins into living cells, overcoming cell impermeability to large molecules.
Utilizes a unique cell penetration mechanism via the hENT2 nucleoside salvage pathway, distinguishing it from other cell-penetrating peptides or domains.
Fusion proteins can be produced recombinantly and formulated pharmaceutically for therapeutic uses targeting diseases associated with oxidative stress or reactive oxygen species toxicity.
The fusion protein format allows modular attachment of therapeutic or diagnostic agents, expanding potential applications.
Documented Applications
Use for protecting cells from oxidative injury induced by hydrogen peroxide or reactive oxygen species (ROS).
Treatment or inhibition of diseases or disorders associated with hydrogen peroxide or ROS toxicity including brain injury (brain trauma, spinal cord injury, peripheral nerve injury, stroke), heart injury (myocardial infarction), skin injury (wound, burn, decubitus ulcer), radiation injury (burn or poison), acute renal failure, acute organ failure, liver injury, bowel infarction, peripheral vascular disease, pulmonary failure, and cancer.
Pharmaceutical compositions for administration via mucosal membranes including nasal administration, inhalation, sublingual, intravenous, intramuscular, or intratumor routes.
Combination therapies with chemotherapeutic agents, immune modulators, or cocktails of different Fvs targeting different epitopes for synergistic therapeutic effects.
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