WO2005005476A1

WO2005005476A1 - Modified epidermal growth factors

Info

Publication number: WO2005005476A1
Application number: PCT/CN2003/000566
Authority: WO
Inventors: Guihong Tu; Huangjin Li
Original assignee: SHENZHEN WATSIN GENETECH Ltd
Current assignee: SHENZHEN WATSIN GENETECH Ltd
Priority date: 2003-07-15
Filing date: 2003-07-15
Publication date: 2005-01-20
Anticipated expiration: 2006-01-15
Also published as: AU2003304317A1

Abstract

Biological activity of previously reported EGF peptide fragments is sometimes very low, and therefore a need remains for the synthesis of more bioactive forms. A modified epidermal growth factor having an N-terminal cap covalently bonded to an amino terminus of an epidermal growth factor having an N-terminal is devised in this invention. The resulting modified EGF is found to be able to delay inactivation of existing epidermal growth factor.

Description

Modified Epidermal Growth Factors

Field of the Invention This invention relates to modified epidermal growth factors.

Background of the Invention Epidermal growth factor is a single-chain polypeptide having a molecular weight of 6 Kd (53 amino acid residues). The EGF peptide is already known to be a powerful nitogenic agent for a variety of cells in culture. In particular, EGF has been shown to stimulate the growth of epithelial cell tissue in a variety of preparations. However, biological activity of previously reported EGF peptide fragments is sometimes very low, and therefore a need remains for the synthesis of more bioactive forms. Peptide analogs already tested in the art have been found to be of varying potencies, some having greater bioactivity than others. A peptide having sufficient bioactivity so as to be a therapeutically effective clinical pharmaceutical agent has yet to be synthesized. Therefore, despite the promises that l EGF would become an important wound healing therapeutic, there has been few successful formulations thereof, partly due to the fact that hEGF as a protein is very unstable, particularly in aqueous solutions and at temperatures 20 or near normal body temperature. For example, Journal of Surgery Research 43,333(1987) discloses that at room temperature, hEGF has less than 1 hour in half life which is far shorter than the time required to induce DNA synthesis of cells in the wound site. Furthermore, it is known that hEGF quickly losses its biological activity from attack by proteolytic enzymes which causes denaturation and decomposition in the wound site.

In order to overcome this short coming of hEGF, US Patent No. 4,944,948 discloses a liposome gel formulation for the delivery of hEGF to the wound site. EP0312208 further discloses pharmaceutical carriers for slow release formulations for hEGF. PCT Application No. W099/44631 is another patent application that attempts to extend the wound healing life of hEGF in order to effect useful treatment for cutaneous injury.

Despite the above-described attempts to improve the usefulness of hEGF, the stability of the enzyme remains a problem as long as it stays in an aqueous environment above 0 ° C.

Objects of the Invention Therefore, it is an object of this invention to resolve at least one or more of the problems as set forth in the prior art. As a minimum, it is an object of this invention to provide the public with a useful choice.

Summary of the Invention Accordingly, this invention provides a modified epidermal growth factor having an N-terminal cap covalently bonded to an amino terminus of an epidermal growth factor having an N-terminal to delay inactivation of said modified epidermal growth factor comparing to said epidermal growth factor.

Preferably, said epidermal growth factor is human epidermal growth factor. Further, said N-terminal cap has at least three amino acids. More preferably, said N-terminal cap is selected from the group consisting of Ala- Arg-Ile, Cys-Xaa-Xaa, Xaa-Cys-Xaa, Xaa-Xaa-Cys. Additionally, said N- terminal cap is covalently linked to the N-terminal of EGF according to the following manner: Ala-Arg-Ile(-N-terminal), Cys-Xaa-Xaa(-N-terminal), Xaa- Cys-Xaa(-N-terminal), or Xaa-Xaa-Cys(-N-terminal). Moreover, said N- terminal cap is preferred to be Ala-Arg-Ile. The N-terminal of EGF is preferred to be Asn.

This invention also provides a composition including the above modified epidermal growth factor.

It is another aspect of this invention to provide a nucleic acid molecule of SEQ ID NO:!.

It is yet another aspect of this invention to provide a method of manufacturing the above modified EGF including the steps of: generating a vector including the nucleic acid molecule of SEQ ID NO: 1 ; transferring said vector to an expression cell; incubating said expression cell to express said modified EGF.

Brief description of the drawings Preferred embodiments of the present invention will now be explained by way of example and with reference to the accompany drawings in which: Figure 1 shows an example of a vector used in manufacturing the modified EGF of this invention.

Detailed Description of the Preferred Embodiment Objects, features, and aspects of the present invention are disclosed in or are obvious from the following description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention, which broader aspects are embodied in the exemplary constructions. As used herein, the term "having substantially the biologically activity of EGF" refers to a factor that has the mitogenic activity and wound healing ability of the naturally occurring EGF and an amino acid sequence of at least 90% homolog.

It was found in this invention that the stability of EGF can be enhanced if a small peptide fragment is attached to the N-terminal of EGF. The N-terminal refers to the Asn end of the EGF peptide, although this amino acid may be replaced. The small peptide fragment, which may be called the N-cap, is a peptide fragment having at least three amino acids. In one preferred embodiment, the N-cap is selected from the group consisting of Ala-Arg-Ile, Cys-Xaa-Xaa, Xaa-Cys-Xaa, Xaa-Xaa-Cys. The N-cap may be covalently linked to the N-terminal of EGF according to the following manner: Ala-Arg- Ile(-N-terminal), Cys-Xaa-Xaa(-N-terminal), Xaa-Cys-Xaa(-N-terminal), Xaa- Xaa-Cys(-N-terminal), even though the N-cap may be connected to the N- terminal of the EGF reversely. The most preferred N-cap may be Ala-Arg-Ile.

The modified EGF may be manufactured using the recombinant vector as shown in Figure 1. The vector can then be transferred into E. coli to express the modified EGF of this invention. In one example, the modified EGF was found to be expressed in a maximum amount after induced four hours with IPTG. The cells are then harvested by centrifuge after eight hours of incubation, and the cell walls are then broken down using known methods. The modified EGF can then be purified by chromatography technique. A person skilled in the art would be familiar with the above incubation techniques and would appreciate that modification to the above procedures will be required under different circumstances (purity of reagents, for example). The EGF sequence in the vector may have the following sequence SEQ ID NO: l:

GCTAGAATTAATTCCGACTCTGAATGCCCGCTGTCTCACGACGGTTA CTGCCTACACGAT GGTGTTTGCATGTATATCGAAGCTCTGGACAAATACGCGTGCAACTG TGTTGTTGGTTAC

ATCGGTGAACGTTGCCAGTACCGTGACCTGAAATGGTGGGAACTGC GTTAA Other sequences of the vector in Fig.l are readily available to a person skilled in the art, for example, the ampicillin resistance gene. The various sequences can then be linked by convention techniques.

Examples The bioactivity of three batches, named 990701, 990702, and 990703, of a composition containing 2000IU/ml of the modified EGF of this invention having the N-cap Ala-Arg-Ile(-N-terminal) is tested. Each sample in test has a volume of 15.0ml of 20mM phosphate buffer containing 10% glycerol and 1 % mannitol. 100 samples from each batch were used for each of the tests. The samples were stored under 2-8°C and 25°C and the bioactivity in terms of lU/ml was tested when the test began, 6 months later, 12 months later, then every three months until 24 months later, and then every 6 months until 36 months later. Balb/c3T3 cells were used as the testing medium, and MTT chromatography was used to determine the bioactivity. The results are shown in the following table: Table 1 Bioactivity (IU/ml) of composition storing at 2-8°C over time

Table 2 Bioactivity (Iϋ/ml) of composition storing at 25°C over time

The above results show that the modified EGF of this invention can maintain 80-92% bioactivity after 18 months if stored at 2-8°C. If the modified EGF is stored at room temperature, this period may be reduced to 12 months.

While the preferred embodiment of the present invention has been described in detail by the examples, it is apparent that modifications and adaptations of the present invention will occur to those skilled in the art. Furthermore, the embodiments of the present invention shall not be interpreted to be restricted by the examples or figures only. It is to be expressly understood, however, that such modifications and adaptations are within the scope of the present invention, as set forth in the following claims. For instance, features illustrated or described as part of one embodiment can be used on another embodiment to yield a still further embodiment. Thus, it is intended that the present invention cover such modifications and variations as come within the scope of the claims and their equivalents.

Claims

1. A modified epidermal growth factor having an N-terminal cap covalently bonded to an amino terminus of an epidermal growth factor having an N- terminal to delay inactivation of said modified epidermal growth factor comparing to said epidermal growth factor.

2. The modified epidermal growth factor of Claim 1, wherein said epidermal growth factor is human epidermal growth factor.

3. The modified epidermal growth factor of Claim 2, wherein said N- terminal cap has at least three amino acids.

4. The modified epidermal growth factor of Claim 3, wherein said N- terminal cap is selected from the group consisting of Ala-Arg-Ile, Cys- Xaa-Xaa, Xaa-Cys-Xaa, Xaa-Xaa-Cys.

5. The modified epidermal growth factor of Claim 4, wherein said N- terminal cap is covalently linked to the N-terminal of EGF according to the following manner: Ala-Arg-Ile(-N-terminal), Cys-Xaa-Xaa(-N- terminal), Xaa-Cys-Xaa(-N-terminal), or Xaa-Xaa-Cys(-N-terminal).

6. The modified epidermal growth factor of Claim 4, wherein said N- terminal cap is Ala-Arg-Ile.

7. The modified epidermal growth factor of Claim 1, wherein the N- terminal of EGF is Asn.

8. A composition including a modified epidermal growth factor according to any one of Claims 1 to 7.

9. A nucleic acid molecule of SEQ ID NO: 1.

10. A method of manufacturing a modified EGF as claimed in Claim 1 including the steps of: generating a vector including the nucleic acid molecule of Claim 9; transferring said vector to an expression cell; incubating said expression cell to express said modified EGF.