Bispecific multivalent fusion proteins
Inventors
Dimitrov, Dimiter S. • Chen, Weizao • Ying, Tianlei
Assignees
Fudan University • US Department of Health and Human Services
Publication Number
US-10472412-B2
Publication Date
2019-11-12
Expiration Date
2035-12-16
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Abstract
The invention provides a construct comprising two or more fusion proteins of Formulas (I)-(IV): A-(optional linker)-C-(optional linker)-B (Formula I), B-(optional linker)-D-(optional linker)-E-(optional linker)-B (Formula II), A-(optional linker)-C (Formula III), and B-(optional linker)-D-(optional linker)-E (Formula IV), wherein A denotes an antibody or antibody fragment, B denotes a single domain CD4, C denotes an immunoglobulin light chain constant region D denotes an immunoglobulin heavy chain constant region, and E denotes an Fc region or a portion thereof that is optionally defucosylated.
Core Innovation
The invention provides constructs comprising two or more fusion proteins of specific formulas combining an antibody or antibody fragment, a single domain CD4, immunoglobulin light and heavy chain constant regions, and an Fc region or a portion thereof that can be defucosylated. These constructs enable the assembly of multivalent (bivalent, tetravalent, or octavalent) molecules with enhanced binding and functional properties.
The constructs include various forms such as A-(optional linker)-C, B-(optional linker)-D-(optional linker)-E-(optional linker)-B, and combinations thereof, where the components are defined by specific immunoglobulin domains and single domain CD4. Linkers serve as flexible connectors, and defucosylation of Fc regions enhances particular biological functions. The invention also provides compositions, conjugates with cytotoxic agents, nucleic acids encoding the fusion proteins, vectors, host cells expressing the proteins, and methods to prophylactically or therapeutically inhibit viral infections, especially HIV.
The problem being solved arises from the need for highly effective compositions and methods to prophylactically or therapeutically inhibit viral infections such as HIV-1 infection. Existing antibodies and fusion proteins have limitations in binding affinity, stability, multimerization, pharmacokinetics, and effector functions. In particular, inefficient heterodimerization between the immunoglobulin heavy and light chain constant domains (CH1-CK) poses challenges in producing stable bispecific and multivalent fusion proteins.
Claims Coverage
The patent claims include one independent claim with multiple dependent claims defining the features of the inventive constructs and their compositions.
Construct comprising specific fusion proteins
The construct comprises two fusion proteins of A-(optional linker)-C (Formula III) and two fusion proteins of B-(optional linker)-D-(optional linker)-E-(optional linker)-B (Formula II), wherein A is an antibody or antibody fragment, B is a single domain CD4, C is a modified immunoglobulin light chain constant region (SEQ ID NOs: 29-34), D is a modified immunoglobulin heavy chain constant region (SEQ ID NOs: 22-27), and E is an Fc region or portion thereof.
Single domain CD4 composition
The single domain CD4 comprises SEQ ID NO: 1 or SEQ ID NO: 2.
Antibody fragment specificity
The antibody or antibody fragment binds to an HIV envelope glycoprotein and can be an engineered antibody domain (eAd) comprising amino acid sequences from SEQ ID NOs: 10-14.
Fc region characteristics
The Fc region or portion thereof comprises sequences from SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 51 and is defucosylated to enhance function.
Optional linker composition
The optional linker comprises one or more G4S motifs or a portion of the human IgG1 hinge, including amino acid sequences from SEQ ID NO: 7, 8, 9, 35, or 36.
Preferred modified constant regions
The modified immunoglobulin light chain constant region preferably comprises SEQ ID NO: 31 and the modified immunoglobulin heavy chain constant region preferably comprises SEQ ID NO: 24.
Exemplary fusion protein pairings
The construct comprises paired fusion proteins of Formula II and III with specific SEQ ID NOs selected from defined pairs, including combinations from SEQ ID NOs: 39-50 as outlined in the patent.
Compositions and conjugates
Compositions comprising the construct and a carrier, conjugates of the construct with cytotoxic agents such as toxins, and the compositions of these conjugates are claimed.
The claims cover constructs combining specific fusion proteins with defined sequences, including modified immunoglobulin constant regions and defucosylated Fc portions, linked by specified linkers, and forming multivalent bispecific molecules. The claims extend to compositions, conjugates with cytotoxic agents, and their pharmaceutical formulations.
Stated Advantages
Defucosylation of the Fc region increases FcγRIIIa binding and enhances antibody-dependent cell-mediated cytotoxicity (ADCC) without compromising HIV-1 Env binding or neutralization.
Improved CH1-CK heterodimerization via structure-guided mutations leads to more stable and efficient bispecific and multivalent fusion proteins.
Variants with modified linkers and stabilized constant regions show increased expression yields, enhanced pharmacokinetics, and lower aggregation propensity.
Fc engineering to enhance FcRn binding further improves half-life and pharmacokinetics of the constructs in vivo.
Defucosylated constructs exhibit superior efficacy in suppressing HIV-1 infection and eliminating infected cells through NK cell-mediated ADCC in humanized mouse models.
Documented Applications
Prophylactic and therapeutic inhibition of HIV infection in cells or hosts by administering the construct, conjugate, nucleic acid, vector, or cell expressing the fusion proteins.
Eradication of HIV-1-infected cells in subjects through administration of conjugates comprising the construct and cytotoxic agents.
Inhibition or treatment of a broad range of viral infections including various retroviruses, herpes viruses, filoviruses, SARS, influenza, and rubella viruses.
Use in pharmaceutical compositions for administration via multiple routes including parenteral, topical, oral, or inhalation to mammals including humans.
Combination therapy with existing HIV treatments such as HAART, antiviral drugs, vaccines, and immunotherapies.
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