Immunoglobulin fusion proteins

a technology of immunoglobulin and fusion proteins, which is applied in the direction of fusion polypeptides, peptide/protein ingredients, fungi, etc., can solve the problems of heterogeneous product mixtures, high protein-specific factors, and inability to improve the biological activity of a second cytokine, so as to achieve the effect of maximizing the bioactivity of the fused epo

Inactive Publication Date: 2010-11-11
BOLDER BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

By their very nature, such factors will be highly protein-specific and unpredictable.
In one case where biological activity of the fusion protein was significantly reduced, modifying the amino acids at the junction between the cytokine / growth factor and the IgG domain resulted in a fusion protein with improved biological activity (Chen et al., 1994) This same modification did not improve biological activity of a second cytokine fused to the same IgG domain.
Lot to lot variability can result in heterogeneous product mixtures with varying activities.
Because Bam HI has a specific DNA recognition sequence, GGATCC, which is not present at the junctions of the EPO or Ig-Fc, Ig-CH or light chain constant regions, this method is not useful for constructing EPO-Ig direct fusions.
The methods described by EP 0464533A are, therefore, not useful for creating Ig direct fusions or Ig fusions that do not contain AspProGlu or ProGlu linkers.
The methods described in U.S. Pat. No. 5,349,053 are not useful for creating cytokine / growth factor-Ig direct fusions, or cytokine / growth factor-Ig fusions that do not contain a Ser linker.

Method used

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example 1

Construction of Growth Factor / Cytokine-IgG Gene Fusions

A. General Strategy.

[0046]Growth factor / cytokine (GF)-IgG gene fusions were constructed as described below. The general strategy employed for these constructions is outlined here and the specifics of individual cloning steps are detailed below. Cloning of the IgG4-CH coding sequence involved additional variations to the general strategy and these variations are described below. The human growth factor genes (GH, EPO and G-CSF) were cloned as cDNAs from various RNA sources detailed below. PCR primers used in these clonings added an optimized Kozak sequence (GCCACC; Kozak, 1991) and a Hind III restriction site to the 5′ end of each these clones and a portion of a peptide linker (ser-gly-gly-ser) (SEQ.ID.NO.1) terminating in a Bam HI restriction site, to the 3′ end of each of these clones. The growth factor genes were cloned as Hind III-Bam HI fragments into the mammalian cell expression vector pCDNA3.1(+) (Invitrogen, Inc., San Di...

example 2

Expression and Purification of GF-IgG Fusion Proteins

A. Small Scale Transfection of COS Cells

[0060]Expression and bioactivity of the GF-IgG fusion proteins were assessed initially by small-scale transfection of COS cells. Endotoxin-free plasmid DNAs were prepared using an “Endo-Free Plasmid Purification Kit” (Qiagen, Inc.) according to the vendor protocol and used to transfect COS-1 cells (available from the American Type Culture Collection, Rockville, Md.). The COS-1 cells were grown in Delbecco's Modified Eagle's Media supplemented with 10% FBS, 50 units / ml penicillin, 50 μg / ml streptomycin and 2 mM glutamine (COS cell growth media). Initial transfection experiments were carried out in Costar 6 well tissue culture plates (VWR Scientific) using the following protocol. Briefly, 2-3×105 cells were seeded into each well in 2 ml of COS cell growth media and allowed to incubate overnight at 37° C. and 5% CO2 by which time the cells had reached 50-60% confluency. For each well, 0.8 μg of...

example 3

In Vitro Bioactivities of Purified GF-IgG Fusion Proteins

A. General Strategy

[0066]Cell proliferation assays were developed to measure in vitro bioactivities of the IgG fusion proteins. The assays measure uptake and bioreduction of the tetrazolium salt MTS [3-(4,5-dimethylthiazol-2-yl)-5-3-carboxyphenyl)-2-(4-sulphenyl)-2H-tetrazolium]. In the presence of an electron coupler such as phenazine methosulfate (PMS), MTS is converted to a formazan product that is soluble in tissue culture media and can be measured directly at 490 nm. Cell number is linear with absorbance values up to about 2. For EPO and G-CSF we were able to use existing cell lines to develop the bioassays. For GH, we needed to create a cell line that proliferates in response to GH. Such a cell line was created by stably transforming a murine leukemia cell line with a rabbit GH receptor.

[0067]In general, the bioassays were set up by washing the appropriate cells three times with media (no additives) and resuspending the ...

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Abstract

The present invention relates to novel methods for making fusion proteins comprising a cytokine or growth factor fused to an immunoglobulin domain. The growth factor / cytokine can be fused directly to an immunoglobulin domain or through a peptide linker. The purified growth factor / cytokine-IgG fusion proteins produced by the novel methods are biologically active and can be used to treat diseases for which the non-fused growth factor / cytokine are useful.

Description

FIELD OF THE INVENTION[0001]This invention relates generally to methods for constructing chimeric proteins and more specifically to methods for constructing recombinant immunoglobulin fusion proteins.BACKGROUND OF THE INVENTION[0002]Prolonging the circulating half-lives of protein pharmaceuticals is of interest to patients and healthcare providers. Long acting protein therapeutics should require less frequent injections and can be effective at lower doses than proteins with shorter circulating half-lives. It is known that increasing the effective size of a protein can increase its circulating half-life by preventing removal of the protein by the kidney (Knauf et al., 1988; Mahmood, 1998). One method that can be used to increase the effective size of a protein is to use recombinant DNA technology to covalently fuse the protein of interest to a second protein. The larger fusion protein often has a longer circulating half-life than the non-fused protein (Capon et al., 1989; Zeng et al....

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K39/395C07K16/18C07H21/00C12N5/10C12P21/00A61P37/02C12N15/09A61K38/00A61P7/00A61P7/04A61P25/00A61P31/12A61P35/00C07K14/475C07K14/505C07K14/52C07K14/535C07K14/54C07K14/56C07K14/565C07K14/61C07K19/00C12N1/15C12N1/19C12N1/21C12P21/02C12R1/91
CPCA61K38/00C07K14/475C07K14/505C07K14/521C07K14/524C07K2319/30C07K14/5431C07K14/56C07K14/565C07K14/61C07K2319/00C07K14/535A61P25/00A61P31/12A61P35/00A61P37/02A61P7/00A61P7/04
Inventor COX, III, GEORGE N.DOHERTY, DANIEL H.
Owner BOLDER BIOTECH
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