High-affinity fully functional soluble single-domain human CD4, antibodies, and related fusion proteins
Inventors
Dimitrov, Dimiter S. • Chen, Weizao
Assignees
US Department of Health and Human Services
Publication Number
US-8911728-B2
Publication Date
2014-12-16
Expiration Date
2031-05-20
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Abstract
The invention provides engineered antibody domains (eAds), a polypeptide comprising a single domain CD4, as well as a fusion protein comprising the same. Nucleic acids encoding eAd and/or polypeptide or the fusion protein thereof, as well as compositions or cells comprising the eAd, polypeptide, fusion protein, or nucleic acid also are provided.
Core Innovation
The invention provides engineered antibody domains (eAds), a polypeptide comprising a single-domain CD4, as well as fusion proteins comprising one or more eAds and/or single-domain CD4. The eAds specifically comprise or consist essentially of SEQ ID NO: 139, with amino acid variations allowed at positions x1-x7, excluding the sequence of SEQ ID NO: 1. The single-domain CD4 comprises or consists essentially of SEQ ID NO: 11, with variable amino acids at positions x1-x14, excluding SEQ ID NO: 12. Fusion proteins comprise the eAd or single-domain CD4 and one or more fusion partners, optionally joined via linkers.
The invention addresses the problem of HIV-1 entry into cells, which is initiated by binding of viral envelope glycoprotein gp120 to cellular receptor CD4, leading to conformational changes needed for virus fusion and entry. Previous soluble CD4 (sCD4) containing domains or anti-gp120 antibodies can inhibit HIV-1 entry but are limited in effectiveness. There is a need for new and effective anti-HIV therapies with improved binding, potency, and biological properties.
The invention provides fusion proteins that enhance HIV-1 neutralization and binding characteristics compared to previous constructs. The single-domain CD4 has advantages such as improved binding kinetics, solubility, stability, specificity, minimized immunogenicity, and better tissue penetration. Fusion partners increase stability and half-life of these molecules in vivo. The invention also includes nucleic acids, vectors, cells, methods of inhibiting HIV infection by administration of these molecules, and compositions comprising them.
Claims Coverage
The claims cover isolated polypeptides comprising specific amino acid sequences of single-domain CD4, fusion proteins including these polypeptides and fusion partners, compositions containing them, and constructs comprising multiple fusion proteins. The independent claims focus on these aspects.
Isolated single-domain CD4 polypeptide excluding SEQ ID NO: 12
An isolated polypeptide having the amino acid sequence of SEQ ID NO: 11, provided that it does not comprise the amino acid sequence of SEQ ID NO: 12. This polypeptide may include variable amino acids at specified positions as defined in dependent claims.
Fusion protein comprising single-domain CD4 and fusion partners
A fusion protein comprising (i) the single-domain CD4 polypeptide defined above and (ii) one or more fusion partners. The fusion partners may be joined via linkers and include engineered antibody domains, HIV envelope glycoproteins, CD4 fragments or mimics, Fc regions or portions thereof, immunoglobulin heavy and light chain constant regions, or combinations thereof.
The independent claims principally cover isolated single-domain CD4 polypeptides that exclude a particular amino acid sequence, and fusion proteins comprising these polypeptides linked to fusion partners through linkers, along with compositions and constructs containing such fusion proteins.
Stated Advantages
Improved binding kinetics and specificity compared to full-length CD4.
Enhanced neutralization potency against multiple HIV-1 clades.
Increased stability and serum half-life when fused to fusion partners such as Fc regions.
Better solubility and expression in recombinant systems such as E. coli.
Minimized immunogenicity in animals.
Better penetration into tissues where HIV-1 replicates, such as lymphoid environments.
Documented Applications
Therapeutic and prophylactic inhibition of HIV-1 and HIV-2 infection in cells or mammals by administration of the eAds, single-domain CD4, fusion proteins, nucleic acids, vectors, cells, or compositions comprising them.
Use as vaccine immunogens by exposing conserved neutralizing epitopes on gp120 stabilized in the CD4-bound state.
Combination therapies with existing anti-HIV treatments including highly active antiretroviral therapies (HAART), cytokines, vaccines, and other antiviral agents.
Ex vivo or in vivo gene and cell therapies using cells expressing the fusion proteins.
Formulations for delivery via intravenous, subcutaneous, intranasal, oral, or topical routes.
Use of viable engineered host cells, including probiotic bacteria, as delivery vehicles for the fusion proteins to targeted body sites.
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