Polymeric forms of H-NOX proteins
Inventors
KAPP, Gregory • SERWER, LAURA • LE MOAN, NATACHA • Cary, Stephen P. L.
Assignees
Publication Number
US-10385116-B2
Publication Date
2019-08-20
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 covalently or non-covalently linked. These polymeric forms demonstrate longer circulation half-lives compared to monomeric H-NOX proteins and preferentially accumulate in tumors, delivering sustained oxygen. The polymeric proteins can be dimers, trimers, tetramers, or pentamers and may include homologous or heterologous H-NOX domains, often fused with polymerization domains such as the foldon domain of bacteriophage T4 fibritin.
The problem addressed by the invention stems from the short circulation half-life of monomeric H-NOX proteins due to their relatively small size and potential filtration through the kidneys. There is a need for H-NOX proteins with longer circulation half-lives capable of binding and delivering oxygen to distal tissues for sufficient time. The invention also solves the limitation of existing hemoglobin-based oxygen carriers that scavenge nitric oxide causing hypertensive side effects, by providing H-NOX proteins that are nitric-oxide-neutral with low NO reactivity and high NO stability, making them suitable blood substitutes.
Further, the invention includes mutant H-NOX proteins with tailored oxygen and nitric oxide binding affinities through amino acid substitutions, such as L144F and W9F/L144F in T. tengcongensis H-NOX domains. The polymeric H-NOX proteins, particularly trimeric forms created by fusing H-NOX domains to trimerization sequences like the foldon domain, facilitate oxygen delivery deep within hypoxic tumor zones. These proteins preferentially extravasate and persist in tumor tissue, thereby enhancing efficacy of radiotherapy, chemotherapy, and other oxygen-dependent anti-cancer treatments.
Claims Coverage
The claims focus on the trimeric H-NOX protein comprising three monomers each with inventive features directed to the H-NOX domain, trimerization via foldon domain, and specific mutations affecting oxygen and nitric oxide binding properties.
Trimeric H-NOX protein composition
A trimeric H-NOX protein comprising three H-NOX monomers, each monomer including a T. tengcongensis H-NOX domain with a L144F amino acid substitution and a foldon domain from bacteriophage T4 fibritin as the trimerization domain.
Covalent linkage arrangement
Each H-NOX domain in the monomers is covalently linked at the C-terminus to the trimerization foldon domain.
Oxygen dissociation and nitric oxide reactivity characteristics
The trimeric H-NOX protein exhibits an oxygen dissociation constant within 2 orders of magnitude of wild type human hemoglobin and a nitric oxide reactivity at least 10-fold lower than that of wild type human hemoglobin.
Specific oxygen affinity range
The dissolved oxygen dissociation constant of the trimeric H-NOX protein ranges between 1 μM and 10 μM at 20° C.
Low nitric oxide reactivity threshold
Nitric oxide reactivity of the trimeric H-NOX protein is less than 700 s−1 at 20° C., supporting reduced side effects.
Highly reduced nitric oxide reactivity
Nitric oxide reactivity is at least 100-fold lower than that of wild type human hemoglobin.
Oxygen dissociation rate limits
The oxygen dissociation rate constant (koff) is less than or equal to 0.65 s−1 or between 1.35 s−1 and 2.9 s−1 at 20° C.
Autoxidation stability
The rate of heme autoxidation of the trimeric H-NOX protein is less than 1 h−1 at 37° C.
Molecular weight
The trimeric H-NOX protein has a molecular weight greater than 50 kilodaltons.
Preferential tissue accumulation
The polymeric H-NOX protein preferentially accumulates in one or more tissues after administration compared to a corresponding monomeric H-NOX protein with a single domain.
Fusion protein sequence feature
A fusion H-NOX protein comprising a T. tengcongensis H-NOX domain (SEQ ID NO:2) with the L144F substitution linked to the foldon trimerization domain from bacteriophage T4 fibritin.
PEGylation of trimeric monomers
Each of the three H-NOX monomers in the trimeric protein can be PEGylated to potentially enhance pharmacokinetics or reduce immunogenicity.
The claims cover trimeric H-NOX proteins comprising three T. tengcongensis H-NOX domains bearing a L144F mutation linked via foldon trimerization domains, exhibiting specific oxygen binding and nitric oxide reactivity properties, structural linkages, molecular weight, preferential tissue accumulation, pharmaceutical compositions comprising these proteins, kits, and methods of treating cancers such as glioblastoma with or without radiation or chemotherapy.
Stated Advantages
Polymeric H-NOX proteins exhibit longer circulation half-lives compared to their monomeric counterparts.
These polymeric proteins preferentially extravasate into and accumulate within tumor tissues for sustained oxygen delivery.
H-NOX proteins have low nitric oxide reactivity, avoiding hypertensive and renal side effects associated with existing hemoglobin-based oxygen carriers.
The polymeric H-NOX proteins provide sustained oxygenation of hypoxic tumor regions, enhancing the effectiveness of radiotherapy, chemotherapy, and other oxygen-dependent cancer treatments.
Trimerization using foldon domains confers structural stability and improved physiological persistence.
Documented Applications
Therapeutic oxygen delivery to hypoxic tumor tissues to enhance cancer treatment efficacy, particularly as radiosensitizers in brain cancers such as glioblastoma.
Treatment of brain cancer by administering polymeric H-NOX proteins in combination with radiation therapy or chemotherapy.
Reduction of tumor hypoxia in animal models of brain cancer and other cancers like lung, colorectal, or skin cancer.
Use as blood substitutes and oxygen carriers in situations such as trauma, hemorrhage, ischemia, and surgery requiring enhanced oxygen delivery.
Potential use in veterinary medicine for animals including pets, farm, and research animals.
Use as imaging agents due to distribution properties allowing for enhanced optical coherence tomography.
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