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Toxin peptides with extended blood halflife

A toxin peptide, half-life technology, applied in the field of biochemistry, can solve the problems of expensive synthesis and refolding production process, short in vivo half-life and so on

Inactive Publication Date: 2008-07-30
AMGEN INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, Ziconotide TM The synthetic and refolding production process of ® is expensive and only results in small peptides with a very short in vivo half-life (approximately 4 hours)

Method used

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  • Toxin peptides with extended blood halflife
  • Toxin peptides with extended blood halflife
  • Toxin peptides with extended blood halflife

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0655] Mammalian expression of Fc-L10-ShK[1-35]

[0656] Fc-L10-ShK[1-35], also known as "Fc-2xL-ShK[1-35]", is an inhibitor of Kv1.3. The DNA sequence encoding the Fc region of human IgG1 was constructed as described below, which was fused in-frame with the linker sequence and the monomer of the Kv1.3 inhibitor peptide ShK[1-35]. Disclosed herein are methods for expressing and purifying peptide antibodies from mammalian cells (HEK 293 and Chinese hamster ovary cells).

[0657]The expression pcDNA3.1(+)CMVi was constructed and expressed by replacing the CMV promoter between MluI and HindIII in pcDNA3.1(+) with CMV promoter plus intron (Invitrogen) (Figure 13A). The expression vector pcDNA3 was prepared by cloning the PCR product of the HindIII-NotI digested human Fc-linker-ActivinRIIB fusion protein containing the 5'Kozak sequence, the signal peptide, and a large fragment of pcDNA3.1(+)CMVi digested with HindIII-NotI. 1(+)CMVi-hFc-ActivinRIIB (Figure 13B). The nucleotide and amin...

Embodiment 2

[0678] Mammalian expression of Fc-L-ShK[2-35]

[0679] The DNA sequence encoding the Fc region of human IgG1 fused in-frame with the monomer of the Kv1.3 inhibitor peptide ShK[2-35] was constructed using standard PCR techniques. The Fc-2xL-ShK[1-35] in the original pcDNA3.1(+)CMVi was used as a template (Example 1, Figure 14) to prepare ShK[2-35] and molecules of 5, 10 or 25 amino acid linker portion. All ShK constructs should have the following amino acid sequence

[0680] SCIDTIPKSRCTAFQCKHSMKYRLSFCRKICGIC

[0681] (SEQ ID NO: 92)

[0682] The first amino acid of the wild-type sequence is removed.

[0683] The sequence of the primer used to prepare Fc-L5-ShK[2-35], also called "Fc-1xL-ShK[2-35]" is as follows:

[0684] cat gga tcc agc tgc atc gac acc atc / / SEQ ID NO: 661;

[0685] cat gcg gcc gct cat tag c / / SEQ ID NO: 662;

[0686] The sequence of the primer used to prepare Fc-L10-ShK[2-35], also called "Fc-2xL-ShK[2-35]" is as follows:

[0687] cat gga tcc gga gga gga...

Embodiment 3

[0705] Bacterial expression of Fc-L-ShK[1-35]

[0706] Description of bacterial peptide antibody expression vector and procedures for cloning and expressing peptide antibody . The cloning vector used for bacterial expression (Examples 3-30) is based on pAMG21 (originally described in US Patent 2004 / 0044188). It has been modified to replace the kanamycin resistance component with ampicillin resistance. This is accomplished by the following steps: excise the DNA between the unique BstBI and NsiI sites of the vector and use the PCR primer CCA ACA CAC TTC GAAAGA CGT TGA TCG GCA C (SEQ ID NO: 667) and CAC CCA ACAATG CAT CCT TAA AAA AAT TAC GCC C (SEQ ID NO: 668), using pUC19 DNA as the β-lactamase conferring resistance to ampicillin The template source of the gene, replace the excised DNA with a suitably digested PCR fragment carrying the β-lactamase gene. The new version is called pAMG21ampR.

[0707]Description of the cloning vector pAMG21ampR-Fc-Pep used in Examples 3-30 (except E...

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Abstract

Disclosed is a composition of matter of the formula (X<1>)a-(F<1>)d-(X<2>)b-(F<2>)e-(X<3>)c and multimers thereof, in which F<1> and F<2> are half-life extending moieties, and d and e are each independently 0 or 1, provided that at least one of d and e is 1; X<1>, X<2>, and X<3> are each independently -(L)f-P-(L)g-, and f and g are each independently 0 or 1; P is a toxin peptide of no more than about 80 amino acid residues in length, comprising at least two intrapeptide disulfide bonds; L is an optional linker; and a, b, and c are each independently 0 or 1, provided that at least one of a, b and c is 1. Linkage to the half-life extending moiety or moieties increases the in vivo half-life of the toxin peptide, which otherwise would be quickly degraded. A pharmaceutical composition comprises the composition and a pharmaceutically acceptable carrier. Also disclosed are a DNA encoding the inventive composition of matter, an expression vector comprising the DNA, and a host cell comprising the expression vector. Methods of treating an autoimmune disorder, such as, but not limited to, multiple sclerosis, type 1 diabetes, psoriasis, inflammatory bowel disease, contact-mediated dermatitis, rheumatoid arthritis, psoriatic arthritis, asthma, allergy, restinosis, systemic sclerosis, fibrosis, scleroderma, glomerulonephritis, Sjogren syndrome, inflammatory bone resorption, transplant rejection, graft-versus-host disease, and lupus and of preventing or mitigating a relapse of a symptom of multiple sclerosis are also disclosed.

Description

[0001] This application claims the priority of a U.S. non-provisional application (serial number undetermined) filed on April 17, 2006. The non-provisional application claims the priority of U.S. Provisional Application No. 60 / 672,342 filed on April 22, 2005. These two applications are hereby incorporated for reference. [0002] This application introduces all the topics contained on the disk by reference. The disk file was established on April 17, 2006, the file name is A-1006.ST25.txt, and the size is 744KB. [0003] In this application, multiple published documents are cited in parentheses or square brackets. The disclosures of these publications are incorporated in full in this application as a reference in order to more fully describe the state of the art to which the present invention belongs. Background of the invention [0004] 1. Invention field [0005] The present invention relates to the field of biochemistry, in particular to therapeutic peptide...

Claims

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Application Information

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IPC IPC(8): A61K47/48C12N15/62A61K38/16A61P37/06C07K14/435A61K47/65A61K47/68
Inventor J·K·萨利文J·G·麦吉文L·P·米兰达H·Q·阮K·W·沃尔克S.S.－F.胡C·V·小格格S·I·麦多诺夫
Owner AMGEN INC
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