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Antibody fusion proteins with modified fcrn binding sites

A technology of fusion protein and antibody, applied in the field of Fc-IFNβ fusion protein, which can solve problems such as reducing serum half-life

Inactive Publication Date: 2012-04-04
MERCK PATENT GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Variant IgG1 with His435 mutated to alanine resulted in selective loss of FcRn binding and significantly reduced serum half-life (Firan et al. 2001, International Immunology 13:993)

Method used

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  • Antibody fusion proteins with modified fcrn binding sites
  • Antibody fusion proteins with modified fcrn binding sites
  • Antibody fusion proteins with modified fcrn binding sites

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Example 1: Construction of DNA sequence for expressing huFcV4h-single-L-DI-IFNβ

[0114] The huFcγ4h-mono-L-DI-IFNβ (huFcγ4 hinge mutant-linker monomer deimmunized interferon-β) antibody fusion protein is a heterologous human Fcγ4h chain and human Fc-γ4h-linker-DI-IFNβ chain dimer. The protein was produced in mammalian cells by co-expressing human Fcγ4h chain and human Fc-γ4h-linker-DI-IFNβ chain with its transcription unit contained in a single plasmid or 2 separate plasmids.

[0115] DNA encoding huFcγ4h (huFcγ4 hinge mutant) was derived from human IgG4 genomic sequence and subsequently engineered to contain a modified γ1 hinge region in order to minimize half-molecule formation. The formation of IgG4 half-molecules that do not form covalent disulfide bonds at the hinge region has been previously reported (Angal et al. (1993) M0l. Immunol. 30:105).

[0116] Construction of DNA Sequence Encoding Fc Fragment of Human Immunoglobulin-γ4 (Fcγ4)

[0117] The genomic ...

Embodiment 2

[0141] Example 2: Construction for expression of huFcv4h-single - DNA sequence of the L-DI-IFNβ variant

[0142] The DNA sequence for expression of the huFcv4h-mono-L-DI-IFNβ variant containing the H435A substitution (Kabat numbering) in γ4 was constructed. This includes genes encoding the heterodimers huFcγ4h(H435A) / huFcγ4h-L-DI-IFNβ, huFcγ4h / huFcγ4h(H435A)-L-DI-IFNβ and huFcγ4h(H435A) / huFcγ4h(H435A)-L-DI-IFNβ DNA sequence. By overlapping PCR with mutagenic primers (Daugherty et al. (1991) Nucleic Acids Res.19:2471-2476), introducing a mutation from the CAC codon to GCG encoding the H435A substitution in either the naked huFcγ4h chain or the huFcγ4h-L-DI-IFNβ fusion protein chain using the forward primer 5′-GGCTCTGCACAAC GCG TACACGCAGAAGAG (SEQ ID NO: 12), wherein GCG Encoding alanine substitution, and reverse primer 5′-CTCTTCTGCGTGTA CGC GTTGTGCAGAGCC (SEQ ID NO: 13), wherein CGC is the anticodon for alanine substitution.

Embodiment 3

[0143] Embodiment 3: the expression of fusion protein

[0144] The huFcγ4h-mono-L-DI-IFNβ heterodimer was produced in mammalian cells by co-expressing the human Fcγ4h chain and the human Fc-γ4h-linker-DI-IFNβ chain, wherein the transcriptional unit of the chain is contained in 1 single plasmid or 2 separate plasmids. For rapid analysis of protein expression, plasmids pdCs-Fcγ4h and pdCs-Fc Fcγ4h-linker-DI-IFNβ or variants were introduced into human kidney 293T cells by transient transfection using lipofectamine (Invitrogen, Carlsbad, CA) (GenHunter Corporation, Nashville, TN).

[0145] Mouse myeloma NS / 0 and Chinese hamster ovary cells were used to obtain stably transfected clones expressing the huFcγ4h-mono-L-DI-IFNβ heterodimer. For high-level expression, plasmid pdCs containing human Fcγ4h chain and human Fcγ4h-linker-DI-IFNβ chain transcription unit were introduced into mouse myeloma NS / 0 cells by electroporation. NS / 0 cells were grown in Dalbecco's Modified Eagle's M...

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Abstract

Disclosed are antibody fusion proteins with a modified FcRn binding site and nucleic acid molecules encoding them. The antibody fusion protein include two polypeptide chains, wherein the first polypeptide chain includes a biologically, preferably therapeutically active molecule linked to at least a portion of an immunoglobulin constant region. The second polypeptide chain includes at least a portion of an immunoglobulin constant region. One of the polypeptide chains includes a mutation in the constant region that results in reduction or loss of FcRn binding but does not cause substantial reduction of serum half-life of said antibody fusion protein. In a preferred embodiment the invention relates to respectively constructed Fc-IFNss molecules. Also disclosed are methods of producing the fusion proteins and methods of using the fusion proteins for treating diseases and conditions alleviated by the administration of the fusion proteins.

Description

technical field [0001] The present invention relates to antibody fusion proteins with modified FcRn binding sites and nucleic acid molecules encoding the same. The antibody fusion protein comprises two polypeptide chains, wherein the first polypeptide chain comprises a biologically active, preferably therapeutically active molecule linked to at least part of an immunoglobulin constant region. The second polypeptide chain includes at least a portion of an immunoglobulin constant region. One of the two polypeptide chains includes a mutation in the constant region that results in a reduction or loss of FcRn binding without causing a substantial decrease in the serum half-life of the antibody fusion protein. In a preferred embodiment, the present invention relates to a correspondingly constructed Fc-IFNβ fusion protein. Background of the invention [0002] Antibody fusion proteins linking a protein of interest to an immunoglobulin constant region have the advantages associated...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/00
CPCC07K2317/53C07K2319/02C07K2316/52C07K2317/52C07K16/00C07K2319/30A61P25/00A61P31/12A61P35/00A61P5/00A61P5/10A61P7/06C07K19/00C12N15/62A61K39/395C07K14/565
Inventor K-M·劳P·A·斯泰因
Owner MERCK PATENT GMBH
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