HER2 extracellular domain

Details HER2 extracellular domain HER2 extracellular domain

1995-04-14

2001-12-25

Kunz, Gary L. (Department: 1647)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

C435S069100, C435S242000, C435S252300, C435S320100, C536S023500

Reexamination Certificate

active

06333169

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention is generally directed to the extracellular domain of p185
HER2
, a receptor-like protein which is encoded by the human homolog of the rat neu oncogene.
More specifically, the present invention is directed to a form of the extracellular domain which is essentially free of transmembrane and cytoplasmic domains, to the DNA encoding this form, and to a process for producing this form of the extracellular domain in a host cell.
2. Description of Background and Relevant Materials
Human epidermal growth factor receptor 2 (HER2, also known as NGL and human c-erbB-2, or ERBB2), is the human homolog of the rat proto-oncogene neu. HER2 encodes a 1,255 amino acid tyrosine kinase receptor-like glycoprotein with homology to the human epidermal growth factor receptor. Although no ligand binding to this probable growth factor receptor has yet been isolated, the HER2 gene product, p185
HER2
, has the structural and functional properties of subclass I growth factor receptors (Yarden et al.,
Ann. Rev. Biochem
., 57:443-478 (1988); Yarden et al.,
Biochem
., 27:3113-3119 (1988)).
The receptor tyrosine kinases all have the same general structural motif; an extracellular domain that binds ligand, and an intracellular tyrosine kinase domain that is necessary for signal transduction, or in aberrant cases, for transformation. These 2 domains are connected by a single stretch of approximately 20 mostly hydrophobic amino acids, called the transmembrane spanning sequence. This sequence is thought to play a role in transferring the signal generated by ligand binding from the outside of the cell to the inside. It has also been suggested to play a role in the proper positioning of the receptor in the plasma membrane.
Consistent with this general structure, the p185
HER2
glycoprotein, which is located on the cell surface, may be divided into three principle portions: an extracellular domain, or ECD (also known as XCD); a transmembrane spanning sequence; and a cytoplasmic, intracellular tyrosine kinase domain. While it is presumed that the extracellular domain is a ligand receptor, as stated above the corresponding ligand has not yet been identified.
The HER2 gene is of particular interest because its amplification has been correlated with certain types of cancer. Amplification of the HER2 gene has been found in human salivary gland and gastric tumor-derived cell lines, gastric and colon adenocarcinomas, and mammary gland adenocarcinomas. Semba et al.,
Proc. Natl. Acad. Sci. USA
, 82:6497-6501 (1985); Yokota et al.,
Oncogene
, 2:283-287 (1988); Zhou et al.,
Cancer Res
., 47:6123-6125 (1987); King et al.,
Science
, 229:974-976 (1985); Kraus et al.,
EMBO J
., 6:605-610 (1987); van de Vijver et al.,
Mol. Cell. Biol
., 7:2019-2023 (1987); Yamamoto et al.,
Nature
, 319:230-234 (1986).
Gene transfer experiments have shown that overexpression of HER2 will transform NIH 3T3 cells and also cause an increase in resistance to the toxic macrophage cytokine tumor necrosis factor. Hudziak et al., “Amplified Expression of the HER2/ERBB2 Oncogene Induces Resistance to Tumor Necrosis Factor Alpha in NIH 3T3 Cells”,
Proc. Natl. Acad. Sci. USA
85, 5102-5106 (1988).
Because amplification of the HER2 gene results in greatly increased numbers of the p185
HER2
protein residing on the surfaces of affected cells, there may be an interrelationship between increased amounts of p185
HER2
extracellular domain on the surfaces of affected cells and the resistance of these cells to treatment. It would therefore be highly desirable to be able to manipulate the p185
HER2
extracellular domain in order to investigate several possibilities for the treatment of conditions associated with amplification of the HER2 gene. These therapeutic modes relate not only to the extracellular domain, but also to the putative ligand, which it should be possible to isolate and characterize using the purified p185
HER2
extracellular domain.
SUMMARY OF THE INVENTION
The present invention is accordingly directed to an extracellular portion of the HER2 molecule containing at least 9 amino acids, and/or containing an immune epitope, which is essentially free of transmembrane and intracellular portions of the HER2 molecule. The extracellular portion may be substantially pure, or at least about 99% pure, and may extend to the entire extracellular portion of the HER2 molecule. Moreover, the extracellular portion may be antigenic in animals, and may be conjugated with a peptide having immunogenic properties; this peptide may contain an immune epitope.
In another embodiment, the present invention is directed to isolated DNA encoding the extracellular portion of the HER2 molecule. This isolated DNA terminates upstream of the DNA portion encoding the transmembrane domain of the HER2 molecule. The termination may occur at least 1 base pair upstream of the portion encoding the transmembrane domain of the HER2 molecule, and preferably occurs about 24 base pairs upstream of this portion.
The isolated DNA of the present invention encodes a sequence of at least 9 amino acids of the extracellular portion, and none of the transmembrane or intracellular portions of the HER2 molecule.
In a further embodiment, the present invention contemplates an expression vector, such as a plasmid or virus, containing the isolated DNA as described above, as well as a cell containing the expression vector. This cell may be eukaryotic or prokaryotic.
The present invention also extends to a process for producing an extracellular portion of the HER2 molecule, which includes the steps of ligating the isolated DNA as described above into an expression vector capable of expressing the isolated DNA in a suitable host; transforming the host with the expression vector; culturing the host under conditions suitable for expression of the isolated DNA and production of the extracellular portion; and isolating the extracellular portion from the host. The host cell may be a prokaryote, such as a bacterium, or a eukaryote.
In a yet further embodiment, the present invention extends to a vaccine comprising the extracellular portion of the HER2 molecule, which may be combined with suitable adjuvants.


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Drebin et al “Inhibition of Tumor Growth by a Monoclonal Antibody . . .”PNAS 83:9129-9133 (Dec. 1986).
Masuko et al “A Murine Monoclonal Antibody That Recognizes an Extracellular . . .”Jpn. J. Cancer Res. 80:10-14 (Jan. 1989).
Kraus et al “Overexpression of the EGF Receptor-Related Proto-Oncogene erbB-2 . . .”EMBO J. 6(3):605-610 (1986).
Hudziak et al., “Increased Expression of the Putative Growth Factor Receptor p185HER2. . . ”PNAS 84:7159-7163 (Oct. 1987).*
van de Vijver et al, “Amplification of the neu(c-erbB-2) Oncogene . . . ”Mol. Cell. Biol. 7(5):2019-2023 (May 1987).*
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