Stabilized single human CD4 domains and fusion proteins
Inventors
Dimitrov, Dimiter S. • Chen, Weizao • Ponraj, Prabakaran
Assignees
US Department of Health and Human Services
Publication Number
US-10647754-B2
Publication Date
2020-05-12
Expiration Date
2034-03-12
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Abstract
The invention provides a polypeptide comprising a single domain CD4, as well as a fusion protein comprising the single domain CD4 and one or more fusion partners. A nucleic acid encoding the polypeptide or fusion protein, as well as compositions or cells comprising the polypeptide, fusion protein, or nucleic acid also are provided.
Core Innovation
The invention provides polypeptides comprising single domain CD4 (such as SEQ ID NOs: 1 or 2), fusion proteins comprising the single domain CD4 and one or more fusion partners optionally joined via linkers, and multivalent constructs comprising these fusion proteins. Nucleic acids encoding these polypeptides or fusion proteins, as well as compositions, cells comprising them, and methods of inhibiting HIV infection by administering these components, are also provided.
The problem being solved addresses the limitations of prior CD4 domain designs, which were either unstable, had lower affinity, or improper folding, and/or had limited solubility and stability under physiological conditions. Specifically, prior single domain CD4 constructs were often only stable at low pH and exhibited lower affinity compared to full-length CD4. There remained a need for new single domain CD4s that are correctly folded, highly soluble and stable under physiological conditions, while preserving full binding activity, specificity, and functions such as inducing conformational changes in HIV-1 gp120.
The invention overcomes these challenges by providing improved single domain CD4 mutants with enhanced soluble expression, stability, specificity, and HIV-1 envelope glycoprotein binding capabilities. These single domain CD4 polypeptides exhibit superior biological properties such as improved binding kinetics, high solubility, stability, minimization of immunogenicity, and improved tissue penetration. Fusion proteins and multivalent constructs combining these polypeptides with fusion partners, including antibodies, Fc regions, and others, further enhance stability, half-life, and/or potency, enabling effective therapeutic and prophylactic HIV inhibition strategies.
Claims Coverage
The claims include two independent compositions related to multivalent fusion proteins composed of specific fusion proteins with defined covalent bonds and their respective constructs, nucleic acids, cells, conjugates, and methods of viral inhibition.
Multivalent fusion protein composition comprising specific fusion proteins with covalent bonding
A composition comprising two fusion proteins each consisting essentially of SEQ ID NO: 21 and two fusion proteins comprising or consisting essentially of SEQ ID NO: 20, wherein these fusion proteins are covalently bonded to each other in a defined arrangement with bonding especially at Fc and constant regions.
Pharmaceutical composition including the multivalent fusion protein
A composition comprising the above multivalent fusion proteins and a pharmaceutically acceptable carrier.
Nucleic acids encoding the fusion proteins
Nucleic acids encoding each fusion protein of the multivalent composition.
Cells comprising nucleic acids encoding fusion proteins
Isolated or purified cells comprising nucleic acids encoding each fusion protein of the multivalent compositions.
Conjugates of fusion protein composition and cytotoxic agent
Conjugates comprising the multivalent fusion protein composition and a cytotoxic agent.
Therapeutic method of inhibiting viral replication
A method of therapeutically inhibiting viral replication in a cell or host by administering the multivalent fusion protein compositions.
Disulfide bonding for covalent linkage in fusion proteins
The covalent bonds between the fusion proteins are identified as disulfide bonds, linking Fc regions and constant domains in the specific construct architectures.
Overall, the independent claims cover specific multivalent fusion protein compositions comprising two types of fusion proteins with precise covalent disulfide bond-mediated linkages, their pharmaceutical formulations, encoding nucleic acids and cells, conjugates with cytotoxic agents, and methods to therapeutically inhibit viral replication using these compositions.
Stated Advantages
Improved soluble expression, stability, and specificity of the single domain CD4 mutants compared to previous versions.
Maintains functional activity including binding specificity and ability to induce conformational changes in HIV-1 gp120.
Fusion proteins with enhanced half-life, potency, and reduced nonspecific binding to MHC-expressing cells.
Bispecific multivalent fusion proteins exhibit superior neutralization potency against diverse HIV-1 isolates, exceeding broadly neutralizing antibodies and existing inhibitors.
Multivalent fusion proteins show high solubility, desirable stability against aggregation and degradation in physiological conditions.
Reduced enhancement of HIV-1 infectivity at low concentrations compared to full-length constructs.
Documented Applications
Therapeutic or prophylactic inhibition of HIV-1 infection in cells or hosts by administering the single domain CD4 polypeptides, fusion proteins, or constructs.
Use in methods to inhibit replication of a broad range of viruses including various retroviruses (e.g., HTLV-1, HTLV-2, FIV, SIV), hepatitis viruses, herpes viruses, filoviruses (e.g., Ebola), SARS, influenza, and rubella viruses.
Conjugates comprising cytotoxic agents for targeted destruction of virus-infected cells, such as HIV-1 infected cells, by targeting viral envelope glycoproteins.
Combination use with other antiviral therapies including HAART, protease inhibitors, reverse transcriptase inhibitors, entry inhibitors, vaccines, and biological response modifiers.
Probiotic delivery via engineered host cells (e.g., lactobacilli) expressing the fusion proteins for localized administration.
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