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  Pharmaceutical Patents  

 

Title:  Recombinant transferrins, transferrin half-molecules and mutants thereof
United States Patent: 
8,080,640
Issued: 
December 20, 2011

Inventors:
 Funk; Walter D. (Dallas, TX), Woodworth; Robert C. (Shelburne, VT), Mason; Anne B. (Charlotte, VT), MacGillivray; Ross T. A. (Vancouver, CA)
Assignee:
  University of Vermont (Burlington, VT), The University of British Columbia (Vancouver, CA)
Appl. No.:
 10/887,711
Filed:
 July 8, 2004


 

Patheon


Abstract

Recombinant transferrin, non-glycosylated recombinant transferrin, transferrin half-molecules and mutant transferrins having altered metal-binding or other properties are described. The recombinant transferrin molecules are expressed in functional form by stable eukaryotic cell lines such as baby hamster kidney cells transformed with an expression vector encoding the recombinant molecule. The recombinant transferrins can be used in metal chelation therapy to bind and clear excess toxic metals in patients suffering from metal overloads or as tissue culture medium supplements or replacements.

Description of the Invention

BACKGROUND OF THE INVENTION

The iron-binding pseudoglobulins collectively called transferrins or siderophilins comprise a class of proteins with strikingly similar features. X-ray crystallographic analyses of human lactoferrin (Anderson, B. F. et al. (1987) Proc. Natl. Acad. Sci. USA 84:1769-1773) and rabbit serum transferrin (Bailey, S. et al. (1988) Biochemistry 27:5804-5812) reveal that these proteins consist of two similar lobes connected by a short bridging peptide and that each lobe contains two domains defining a deep cleft containing the binding site for a metal ion and a synergistic anion.

The chicken ovotransferrin gene has been expressed in transgenic mice (McKnight, G. S. et al. (1983) Cell (Cambridge, Mass.) 34:335-341) and a fusion protein of part of rat transferrin with galactosidase has been expressed in E. coli (Aldred, A. et al. (1984) Biochem. Biophys. Res. Commun. 122:960-965). Except for this fusion protein, attempts to express transferrin or portions of the molecule in prokaryotic systems have been unsuccessful (Aldred, A. et al. (1984) Biochem. Biophys. Res. Commun. 122:960-965). The highly convoluted structure of the protein and large number of disulfide bridges in the molecule are probably the major impediments to expression in bacterial hosts. Attempts to mimic partially the natural protein folding environment by targeting the protein for bacterial membrane transport via an attached alkaline phosphatase signal sequence have been unsuccessful.

SUMMARY OF THE INVENTION

This invention pertains to recombinant transferrin, to recombinant transferrins that bind to the transferrin receptor, to recombinant transferrin half-molecules comprising at least the metal-binding domains of a single lobe (amino-terminal or carboxy-terminal) of transferrin and to stable cell culture system for expression of the transferrin. The recombinant transferrin can be expressed in stable, transformed eukaryotic cells, such as baby hamster kidney cells, to yield essentially homogeneous (monodisperse) preparations of the full or half-molecule forms. The invention also pertains to mutant transferrins, non-glycosylated transferrins and transferrin half-molecules which have metal-binding or other properties which are different from the natural (wild-type) form of the transferrin. These include mutant transferrins and transferrin half-molecules which bind iron or other metals more or less avidly than natural transferrin.

Transferrin half-molecules can be used in metal chelation therapy to treat individuals affected with abnormalities of metal regulation or with metal poisoning. For example, transferrin half-molecules, especially mutant forms which bind iron with a higher avidity than natural transferrin, can be administered to iron-overloaded individuals, e.g., thalassemics, in order to clear excess toxic iron from their bodies. In addition, half-molecules, or mutants thereof having altered metal ion selectivities, could be used to clear other toxic metals, e.g., lead, mercury, cadmium, copper and zinc from the body.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides for the production of recombinant transferrin, recombinant transferrin half-molecules and mutant forms of full-length transferrin and transferrin half-molecules which have altered properties, such as improved metal-binding capability, compared to the natural transferrin molecules. Recombinant transferrins can be produced in large quantities and in substantially homogeneous (monodisperse) form. For example, recombinant half-molecules of human serum transferrin can be produced as an essentially homogeneous preparation substantially free of other human serum proteins. In contrast, half-molecules prepared by proteolysis of the holo-protein are difficult to purify and, in fact, the carboxy-terminal half of human transferrin cannot be satisfactorily prepared by proteolytic means. Recombinant techniques also allow the application of mutagenesis to design and produce new forms of transferrin.

In general, a recombinant transferrin of this invention is produced by transfecting a suitable host cell with a nucleic acid construct encoding the transferrin, culturing the transfected host cell under conditions appropriate for expression and recovering the recombinant transferrin expressed by the cell. The amino acid sequences for eight transferrins have been reported (See S. S. Baldwin Comp. Biochem Physiol. 106b: 203-218 (1993)). The DNA sequence (SEQ ID NO: 1) and amino acid sequence (SEQ ID NO: 2) for human serum transferrin has been determined (Yang, F. et al. (1984) Proc. Natl. Acad. Sci. USA 81:2752-2756). Full-length DNA for production of recombinant transferrins or truncated DNA encoding either the amino-terminal or carboxy-terminal lobe of transferrin or a portion thereof can be obtained from available sources or can be synthesized according to the known sequences by standard procedures. In order to provide for secretion of the recombinant transferrin into cell culture medium, DNA encoding a transferrin signal sequence (or other signal sequence suitable for the expression system) is positioned upstream of the transferrin encoding DNA.

Through receptor-mediated endocytosis, cell-surface transferrin receptors deliver transferrin with its bound iron to peripheral endosomes where the iron is released into the cell and then the iron-free transferrin or apotransferrin is recycled to the extracellular fluid. Accordingly, another aspect of the invention is a homogenous preparation of human transferrin that is recognized by a transferrin receptor and is free of other human proteins.

Mutant forms of transferrin and transferrin half-molecules can be produced by standard techniques of site-directed mutagenesis. See Taylor et al. (1985) Nucleic Acids Res. 13; 8749-8764; Zoller, M. J. and Smith, M. (1983) Meth. Enzymol 100:458-500. In particular, mutagenesis can be used to produce mutant transferrins which have metal-binding properties that are different from natural transferrin. For example, mutants capable of binding iron more avidly than natural transferrin can be produced. To produce such mutants, metal-binding domains can be mutagenized to replace one or more amino acids involved in binding with different amino acids. In human serum transferrin, the amino acids which are ligands for metal chelation are shown below (the number beside the amino acid indicates the position of the amino acid residue in the primary sequence where the first valine of the mature protein is designated position 1)

In other types of transferrin, the numbering is different, but the ligands (amino acids) are the same.

Other regions of transferrin control binding and these too can be targeted for mutagenesis. These are usually positively charged amino acids such as lysine, histidine or arginine. For example, a mutant transferrin half-molecule which binds iron more avidly than natural transferrin can be produced by replacing the lysine residue at position 206 with glutamine (AAG.fwdarw.CAG) or by replacing the histidine residue at position 207 with glutamic acid (CAG.fwdarw.GAG).

Further, human serum transferrin contains two N-linked oligosaccharides at Asn-413 and Asn-611 corresponding to AAT and AAC, respectively. These glycosylation sites can be removed by changing the codons to GAT and GAC which correspond to aspartic acid using, for example, oligonucleotide-directed mutagenesis. Thus, a non-glycosylated transferrin can be produced recombinantly.

The transferrin-encoding DNA is cloned into a eukaryotic expression vector containing appropriate regulatory elements to direct expression of the DNA. A preferred eukaryotic expression vector is the plasmid pNUT described by Palmiter, R. D. et al. (1987) Cell 50:435-443. This plasmid contains the mouse metallothionein promoter which induces transcription of the transferrin encoding DNA in the presence of heavy metal and transcription termination signals of human growth hormone. In addition, pNUT contains dihydrofolate reductase gene under control of the SV40 early promoter with transcription termination signals from human hepatitis B virus to allow selection in cell culture. The gene encodes a mutant form of the enzyme which has a 270-fold lower affinity for the competitive inhibitor methotrexate. This allows for the immediate selection of transfected cells in very high concentrations (0.5 mM) of methotrexate and abrogates the need for a recipient cell line that is deficient in dihydrofolate reductase. pNUT also contains pUC18 derived sequences which allows it to be amplified in E. coli to provide sufficient amounts of the plasmid for transfection of recipient cells.

The expression vector containing the DNA encoding the transferrin is incorporated into an appropriate host cell. The preferred host cell is a eukaryotic cell which can be transformed with the vector to yield a stable cell line which expresses a functionally active transferrin construct. A particularly useful cell is the baby hamster kidney cell. Baby hamster kidney cells can be transfected with a vector carrying the DNA construct encoding a transferrin (such as the pNUT plasmid) to provide a stable cell culture system which expresses and secretes a functionally active transferrin (full or half-molecule). These cells are well-suited for economical, large scale growth and can be obtained from readily available sources.

Standard techniques, such as calcium phosphate coprecipitation or electroporation can be used to transfect the eukaryotic host cell with the vector. The cell is then cultured under conditions appropriate to induce expression of the transferrin. For example, baby hamster kidney cells transfected with the pNUT vector are stimulated to express the transferrin construct in the presence of heavy metals. Baby hamster kidney cells are preferably cultured in the medium Dulbecco's Modified Eagle's medium-Ham's F-12 nutrient mixture with the serum substitute ULTROSER-G.TM. (Gibco) (serum substitute) at about 1%.

After an appropriate culture period, the expressed and secreted transferrin can be recovered from the culture medium. Standard purification procedures can be employed to yield a substantially homogeneous preparation of the recombinant transferrin. In one embodiment, the transferrin in the culture medium is saturated with iron and then purified by anion exchange chromatography.

The recombinant transferrins of the invention can be used to chelate and clear iron or other toxic metals from the body. The customary approach to iron chelation in vivo has been to assess a wide variety of naturally-occurring siderophores of microbial origin and synthetic iron chelators for their physiological effects, primarily the ability to bind and clear iron from the body. Many such compounds have been studied with varying abilities to clear iron and often with unacceptable side effects (Pitt, C. G. et al. (1979) J. Pharm. Exp. Therap. 208:12-18). As a result, the only iron chelator used for clearing excess iron from humans remains deferoxamine, a cyclic peptide from Streptomyces pilosis.

A preferred transferrin for iron chelation therapy is a mutant transferrin half-molecule which binds iron more avidly than natural transferrin. The use of a mutant half-molecule allows for more efficient chelation and removal of the metal. A particularly preferred mutant half-molecule is K206Q, described in the Exemplification below, which contains a glutamine rather than a lysine at position 206.

A transferrin half-molecule is advantageous because unlike the holo-proteins, it passes through the glomeruli of the kidney and is excreted in the urine, so that metal is not only chelated but also cleared from the body. Moreover, the single half-molecules do not bind to transferrin receptors on the membrane of tissue cells and therefore do not deliver iron to these tissues. Further, half-molecules of human transferrin would probably be recognized as "self" by the human body and therefore would not elicit an immunological response.

In addition, mutant half-molecules can be designed to have altered metal ion selectivities. The chelators could be used to clear other toxic metals from the body, e.g., lead, mercury, cadmium, and copper.

For chelation therapy, the recombinant transferrin is administered to a patient in amounts sufficient to chelate the metal and reduce circulating levels below toxic levels. Generally, it is administered in a physiologically acceptable vehicle, such as saline, by a parenteral route (typically intravenously).

Recombinant full-length human transferrin can be used in nonserum supplements or replacements for cell culture media. Transferrin is required for iron uptake by growing cells. The use of recombinant transferrin avoids the risk of contamination with, e.g., HIV or hepatitis virus associated with transferrin purified from human serum or prions from fetal bovine serum.
 

Claim 1 of 24 Claims

1. A nonserum supplement for cell culture medium comprising a homogenous preparation of recombinant, eukaryotically expressed, metal-binding full length human serum transferrin, wherein the transferrin has the mature peptide amino acid sequence set forth in SEQ ID NO:2, wherein at least one of Asn413 and Asn61 of SEQ ID NO:2 are mutated to an amino acid which does not allow glycosylation.

 

 

 

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