Polymeric forms of H-NOX proteins
Inventors
KAPP, Gregory • SERWER, LAURA • LE MOAN, NATACHA • Cary, Stephen P. L.
Assignees
Publication Number
US-11117952-B2
Publication Date
2021-09-14
Expiration Date
2033-01-07
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Abstract
The invention provides polymeric H-NOX proteins for the delivery of oxygen with longer circulation half-lives compared to monomeric H-NOX proteins. Polymeric H-NOX proteins extravasate into and preferentially accumulate in tumor tissue for sustained delivery of oxygen. The invention also provides the use of H-NOX proteins as radiosensitizers for the treatment of brain cancers.
Core Innovation
The invention provides polymeric H-NOX proteins comprising two or more H-NOX domains, including homologous or heterologous H-NOX domains that are covalently linked. These polymeric forms of H-NOX proteins have longer circulation half-lives compared to monomeric H-NOX proteins, enabling sustained delivery of oxygen. The polymeric H-NOX proteins preferentially extravasate into and accumulate in tumor tissue, allowing oxygen transport through normoxic regions and release deep within hypoxic tumor zones. This innovation represents a significant advance in oxygen carrier therapeutics, particularly in modifying hypoxic tumor niches to enhance radiotherapy, chemotherapy and other treatments dependent on tumor oxygenation.
The background describes that while H-NOX proteins are desirable as oxygen carriers due to their low nitric oxide (NO) reactivity and high NO stability, their small size leads to rapid renal clearance and short circulation half-life. Existing hemoglobin-based oxygen carriers have adverse side effects such as hypertensive and renal toxicity due to NO scavenging, which H-NOX proteins avoid. However, their use is limited unless circulation half-life is extended. The problem solved is thus the need for H-NOX proteins with longer circulation half-lives that can effectively bind and deliver oxygen and/or NO to distal tissues, especially tumors, over sufficient periods for clinical efficacy.
Claims Coverage
The claims cover a recombinant nucleic acid and methods related to a specific fusion H-NOX protein with key features, described in 25 inventive features.
Fusion H-NOX protein comprising a mutated T. tengcongensis H-NOX domain and a trimerization domain
A recombinant nucleic acid encoding a fusion H-NOX protein comprising a Thermoanaerobacter tengcongensis H-NOX domain that has an L144F substitution and a trimerization domain which is the foldon domain of bacteriophage T4 fibritin.
C-terminal covalent linkage of H-NOX domain to trimerization domain
The encoded fusion protein has the C-terminus of the H-NOX domain covalently linked to the trimerization domain.
Use of an amino acid linker between H-NOX and foldon domain
The H-NOX domain is fused to the foldon domain of T4 bacteriophage fibritin via an amino acid linker in the encoded protein, which may be of length 3 to 10 amino acids, preferably three amino acids such as Gly-Ser-Gly.
Specific sequence features of the H-NOX domain
The T. tengcongensis H-NOX domain comprises the amino acid sequence of SEQ ID NO:2 except for an L144F amino acid substitution; the fusion protein comprises the amino acid sequence of SEQ ID NO:8; and a nucleic acid sequence of SEQ ID NO:7 encodes the fusion protein.
Vectors and host cells for expression
Vectors comprising the recombinant nucleic acid and host cells (including bacterial cells) containing the vectors are claimed.
Methods of production of trimeric H-NOX protein
Methods comprising culturing host cells carrying the recombinant nucleic acid, allowing expression and association of the fusion H-NOX protein to form a trimeric H-NOX protein, optionally including purification steps.
The claims collectively cover recombinant nucleic acids encoding a fusion protein of T. tengcongensis H-NOX domain with an L144F mutation linked to a T4 bacteriophage foldon trimerization domain, host cells carrying such nucleic acids, vectors, and methods for producing and purifying the trimeric H-NOX protein.
Stated Advantages
Polymeric H-NOX proteins have longer circulation half-lives compared to monomeric H-NOX proteins, allowing for sustained oxygen delivery.
They preferentially extravasate and accumulate in tumor tissues, improving oxygen delivery to hypoxic tumor zones.
H-NOX proteins have low nitric oxide reactivity, avoiding the hypertensive and renal side effects associated with hemoglobin-based oxygen carriers.
The polymeric H-NOX proteins can tune oxygen dissociation constants and nitric oxide reactivity to optimal ranges for therapeutic oxygen delivery.
The invention provides stable trimers with molecular weights large enough to avoid renal clearance but small enough to perfuse ischemic cerebral tissues.
Documented Applications
Delivery of oxygen to humans and animals for therapeutic purposes, including veterinary uses.
Delivery of oxygen to hypoxic tumor tissues, including brain tumors such as glioblastoma.
Use as radiosensitizers to enhance the efficacy of radiation therapy during cancer treatment.
Treatment of brain cancer and reduction of brain tumor growth when combined with radiation or chemotherapy.
Improving oxygenation in solid tumors to increase the effectiveness of radiotherapy and chemotherapy.
Use in various mammalian subjects including humans, pets, laboratory and farm animals.
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