Conjugates of biologically active proteins having a modified in vivo half-life

A bioactive, bioactive polypeptide technology, applied in the field of changing the half-life of bioactive proteins, can solve the problems of increasing drug complexity, changing the biological activity or safety of bioactive proteins, and increasing costs

Inactive Publication Date: 2008-01-23
CELL THERAPUETICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods create other problems
Extending the half-life of biologically active proteins by physical means often adds complexity to the drug, adding cost

Method used

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  • Conjugates of biologically active proteins having a modified in vivo half-life
  • Conjugates of biologically active proteins having a modified in vivo half-life
  • Conjugates of biologically active proteins having a modified in vivo half-life

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0077] Cloning of chimeric DNA molecules

[0078] To facilitate downstream protein purification, it was decided to secrete the G-CSF-polymer protein into the cell culture medium. Previous experience with various cytokine-polymer constructs and bacterial (ST2) secretion signals has shown that they are secreted inefficiently in both prokaryotic and eukaryotic systems. However, the Schizosaccharomycen pombe secretion signal sequence using pho1 + acid phosphatase (PHO) is known to enable secretion of heterologous proteins (GFP and HPV 16 E7) into the medium. Therefore, this secretion signal was tested in the CHO cell expression system with the G-CSF-polymer construct described below. Synthetic vectors for production of G-CSF-polymer constructs and expression of G-CSF-polymer are also described below.

[0079] 1.A. Generation of PHO x pBSK constructs

[0080] PHO x PBSK construct

[0081] The first construct synthesized was simply the PHO leader cloned into the bacterial cloni...

Embodiment 2

[0152] Transformation of host cells and expression of chimeric DNA molecules

[0153] 2. A. Generation of PHO-G-CSF-(NNT)155 x pCE2 construct

[0154] Preparation of vector pCE2

[0155]Plasmid pCE2 was generated from plasmid pREP7b by the following operation. First, a 2000 bp EBNA coding region was deleted. Second, pBR322ori was replaced with pKS-ori. Then, the RSV promoter region was replaced with the CMV enhancer and elongation factor-1a (EF-1a) promoter and intron.

[0156] The CMV enhancer was derived from a 380 bp XbaI-Sph1 fragment generated from pCEP4 (Invitrogen, San Diego, CA) by polymerase chain reaction (PCR) with the following primers:

[0157] 5'-GGCTCTAGAT ATTAATAGTA ATCAATTAC-3'; and

[0158] 5'-CCTCACGCAT GCACCATGGT AATAGC-3'.

[0159] The EF-la promoter and intron (Uetsuki et al., J. Biol. Chem. 264:5791-5798 (1989)) were derived from a 1200 bp SphI-Asp718I fragment generated by PCR from human genomic DNA using the following primers:

[0160] 5'-GGTG...

Embodiment 3

[0192] Pharmacokinetic Evaluation of G-CSF-(NNT)155 Conjugates

[0193] The pharmacokinetic parameters of the G-CSF-(NNT)155 protein conjugate were evaluated. Three other compounds were included as positive controls. Controls were Neulasta (PEG-G-CSF), Neupogen (rh-G-CSF) and G-CSF compounds. The compound was tested by a single intravenous (i.v.) administration to mice and blood was collected 72 hours post-dose. Plasma was analyzed by ELISA.

[0194] 3.A. Analysis method

[0195]The assay employs a quantitative sandwich enzyme immunoassay technique. Microplates were pre-coated with G-CSF-specific monoclonal antibodies. Add standards and samples to each well. The immobilized antibody binds the G-CSF present. After washing away unbound material, a G-CSF-specific enzyme-linked polyclonal antibody was added to each well. After washing away unbound antibody-enzyme reagent, the substrate solution is added to each well, producing a color proportional to the amount of G-CSF bo...

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Abstract

Disclosed are biologically active protein conjugates that comprise a biologically active polypeptide coupled via a peptide bond to a polypeptide comprising from 2 to about 500 units of a repeating peptide motif, wherein the biologically active protein conjugate exhibits a modified plasma half-life compared to the intrinsic half-life of the unconjugated biologically active polypeptide or protein. Also disclosed are methods of making and using the conjugated proteins, as well as methods for determining whether a given conjugate exhibits a modified half life relative to the intrinsic half life of the unconjugated polypeptide.

Description

[0001] Cross References to Related Applications [0002] Pursuant to 35 U.S.C 119(e), this application claims priority to US Application Nos. 60 / 712,585, filed August 30, 2005, and 60 / 647,119, filed January 25, 2005, the contents of which are incorporated by reference. technical field [0003] The present invention relates generally to biologically active proteins, and more particularly to altering the half-life of biologically active proteins. Background of the invention [0004] When administered as human therapeutics, biologically active proteins often have suboptimal half-lives. Their inherent half-lives make dosing regimens and dosage regimens often suboptimal for therapeutic effect, creating compliance problems and inconvenience to the patient. [0005] In the production of bioactive proteins for human therapeutics, attempts have been made through physical methods (such as altering the route of administration, nanoparticle packaging, and liposomal encapsulation), chem...

Claims

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Application Information

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IPC IPC(8): C07K14/435
CPCY02A50/30
Inventor M·贝斯曼S·齐普曼D·里昂J·辛格
Owner CELL THERAPUETICS INC
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