Antibody-mediated transduction of heat shock proteins into living cells
Inventors
Weisbart, Richard H. • Nishimura, Robert N. • Hansen, James E.
Assignees
US Department of Veterans Affairs
Publication Number
US-10703807-B2
Publication Date
2020-07-07
Expiration Date
2033-03-15
Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
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 single-chain variable fragment (Fv) joined or attached to various heat shock proteins (Hsps) or portions thereof, including Hsp-70, Hsp-27, Hsp-90, and glucose regulated protein 78 kDa (GRP78). The 3E10 Fv may optionally include an amino acid sequence AGIH at its amino terminus. These fusion proteins enable the transport of large proteins into living cells, exploiting the unique cell-penetrating property of the 3E10 antibody fragment through the hENT2 nucleoside salvage pathway.
The background problem addressed by the invention is that current therapies are limited to small molecules because cells are impervious to large molecules such as proteins. Existing cell-penetrating peptides (CPPs) and protein transduction domains (PTDs) do not utilize the unique hENT2 nucleoside salvage pathway for cell entry. The invention solves this problem by providing a method to transport proteins into cells as molecular fusion proteins comprising fragments of the 3E10 antibody, enabling intracellular delivery and therapeutic applications of heat shock proteins.
The invention further encompasses pharmaceutical compositions comprising these fusion proteins and methods of use thereof. The 3E10 Fv portion can be modified or replaced with derivatives or variants that maintain the binding specificity and cell-penetrating properties. Additionally, the fusion proteins may be joined to localizing signals to target intracellular compartments and include cleavage sites for separation within the cell. The invention demonstrates feasibility and utility in promoting cytoprotection against oxidative and reactive oxygen species (ROS) toxicity.
Claims Coverage
The patent discloses a set of independent claims focusing on therapeutic methods employing fusion proteins comprising 3E10 Fv linked to Hsp-70 with a specific peptide linker including a swivel sequence. The inventive features focus on the structural characteristics of the fusion protein and its use in inhibiting diseases associated with hydrogen peroxide or ROS cytotoxicity.
Fusion protein structure comprising 3E10 Fv linked to Hsp-70 with CH1 peptide linker and swivel sequence
The fusion protein comprises a 3E10 single-chain variable fragment (Fv) joined to Hsp-70 via a peptide linker including a portion of an immunoglobulin heavy chain constant domain CH1 and a swivel sequence. The swivel sequence, consisting of the peptide LESSGS (positions 375-380 in SEQ ID NO:2), is located between the 3E10 Fv and Hsp-70 and permits swiveling of these domains. The 3E10 Fv optionally includes the amino acid sequence AGIH at its amino terminus.
Therapeutic method of inhibiting diseases by administering the fusion protein composition
Methods are claimed for inhibiting or treating diseases or disorders by promoting hydrogen peroxide or reactive oxygen species cytoprotection through administering a pharmaceutical composition comprising the specific fusion protein with the defined peptide linker and swivel sequence, and pharmaceutically acceptable carriers. The administration treats diseases including acute renal failure, organ failure, liver injury, brain injury, cancer, and related conditions.
Inclusion of derivatives and variants of 3E10 Fv maintaining specificity and cell-penetration
The 3E10 Fv may be a derivative of monoclonal antibody 3E10 from hybridoma ATCC PTA 2439 or antibodies competing with it, comprising one or more complementarity determining regions (CDRs). The derivatives retain ENT2-dependent cell penetration and epitope recognition, and may be identified by phage display screening of sequences corresponding to light and heavy chain CDRs.
Specific amino acid sequences defining linker and swivel regions within fusion protein
The peptide linker includes sequences from positions 361 to 374 or 373 of SEQ ID NO:6 or SEQ ID NO:30, respectively, corresponding to the heavy chain constant domain CH1. The swivel sequence is attached at the C-terminus of the CH1 and includes positions 375 to 380 of SEQ ID NO:6.
The claims focus on compositions and methods employing fusion proteins comprising a 3E10 Fv linked to Hsp-70 by a specific immunoglobulin heavy chain constant domain CH1 peptide linker and a swivel sequence allowing spatial flexibility. The invention covers the structure-function relationship of these fusion proteins and their therapeutic use for treating diseases linked to oxidative stress and reactive oxygen species.
Stated Advantages
The invention overcomes the limitation that cells are impervious to large molecules such as proteins by enabling direct intracellular delivery through fusion with 3E10 Fv.
The use of 3E10 antibody fragments that enter cells via the hENT2 nucleoside salvage pathway provides a unique and effective method for cellular transduction of proteins.
Fusion proteins with heat shock proteins promote cytoprotection against hydrogen peroxide or reactive oxygen species toxicity, potentially protecting against acute injuries and various diseases.
The fusion proteins can be targeted to intracellular compartments and designed to release active components inside cells through engineered cleavage sites.
Pharmaceutical formulations including nasal delivery increase accessibility for emergency situations and enable self-administration outside medical settings.
Documented Applications
Inhibition and treatment of diseases and disorders associated with hydrogen peroxide toxicity or reactive oxygen species (ROS) toxicity, including brain injury, heart injury, skin injury, radiation injury, acute renal failure, acute organ failure, liver injury, bowel infarction, peripheral vascular disease, pulmonary failure, and cancer.
Treatment of brain injuries such as brain trauma, spinal cord injury, peripheral nerve injury, and stroke.
Treatment of heart injury, including myocardial infarction.
Treatment of skin injuries, including wounds, burns, and decubitus ulcers.
Treatment of radiation injuries such as burns or poison exposure.
Use in pharmaceutical compositions administered via intravenous, intramuscular, intradermal, intranasal (mucosal membrane), or inhalation routes.
Interested in licensing this patent?