Amino acid sequences which enhance peptide conjugate solubility

Inventors

Weisbart, Richard H.

Assignees

Wesdyne Sweden ABUS Department of Veterans Affairs

Publication Number

US-9155801-B2

Publication Date

2015-10-13

Expiration Date

2030-05-27

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Abstract

The present invention provides peptide conjugates having improved solubility as well as increased secretion during cell based production, as well as methods of utilizing such peptides. The peptide conjugates include a short peptide domain defined by the amino acid sequence AGIH (SEQ ID NO: 8) and may include a biologically active molecule useful in intracellular and intranuclear transport of the biologically active molecule to treat various disorders and diseases.

Core Innovation

The invention provides peptide conjugates having improved solubility and increased secretion during cell-based production. Central to the invention is a short peptide domain defined by the amino acid sequence AGIH (SEQ ID NO: 8), which when conjugated to biologically active molecules, enhances their solubility and secretion. The peptide conjugates include the AGIH sequence and may include a biologically active molecule useful in intracellular and intranuclear transport for treating various disorders and diseases.

The problem being solved is the difficulty in producing large amounts of peptides, such as the 3E10 Fv peptide used for intracellular and intranuclear transport, while maintaining their solubility. Existing delivery vehicles such as protein transduction domains and anti-DNA autoantibodies like mAb 3E10 have limitations including potential immunogenicity and poor solubility during production. The present invention addresses these issues by introducing a short peptide domain that markedly improves solubility and secretion of conjugated peptides.

Claims Coverage

The patent contains one independent claim directed to a peptide-antibody conjugate which includes key inventive features related to the peptide sequence, antibody specificity, conjugation site, and possible inclusion of additional biologically active molecules.

Peptide-antibody conjugate with the AGIH sequence conjugated to N-terminus of mAb 3E10 or functional fragment

A peptide comprising about 4 to 50 amino acids including the AGIH (SEQ ID NO: 8) sequence conjugated to the N-terminus of mAb 3E10 or a functional fragment thereof, where the antibody has the binding specificity of that produced by the hybridoma with ATCC accession number PTA 2439.

Functional fragment options

The functional antibody fragment can be selected from Fab, F(ab′)2, Fv, and single chain Fv (scFv) fragments, including scFv of mAb 3E10 or fragments comprising variable regions of heavy and kappa light chains.

Linker molecule and genetic fusion conjugation

The peptide may be joined to the antibody by a linker molecule or expressed as a genetic fusion, preserving biological activity.

Inclusion of additional biologically active molecule

The conjugate may further include an additional biologically active molecule, which may be a nucleic acid sequence or protein, including antibodies, antibody fragments, enzymes, transcription factors, siRNA molecules, DNA, RNA, and conjugates of siRNA with proteins, peptides, or antibodies.

Expanded types of biologically active molecules

The additional biologically active molecule may be selected from various antibody types such as chimeric, humanized, CDR-grafted, bifunctional, single chain antibodies, or antibody polypeptide dimers.

The claims cover peptide-antibody conjugates specifically including the AGIH sequence at the N-terminus of mAb 3E10 or its functional fragments, conjugated via linkers or genetic fusions, optionally including additional biologically active molecules with broad applicability. The inventive features focus on the peptide sequence, conjugation site, antibody specificity, and modular inclusion of active molecules.

Stated Advantages

The AGIH peptide markedly enhances secretion of peptide conjugates during production in host cells.

The presence of AGIH greatly improves solubility of the peptide conjugates, preventing aggregation.

The invention provides a delivery vehicle capable of transporting biologically active molecules into cells without the toxicity or immunogenicity issues of previous vectors like other foreign proteins.

Documented Applications

Delivery of biologically active molecules including proteins, nucleic acids (such as siRNA), enzymes, transcription factors, and antibodies into cells for therapeutic or diagnostic purposes.

Treatment of various disorders and diseases through intracellular and intranuclear transport of biologically active molecules conjugated to the peptide including AGIH.

Production of peptide conjugates with improved solubility and secretion for use in cell-based production systems.

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