Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,... – Structurally-modified antibody – immunoglobulin – or fragment...
Reexamination Certificate
2001-10-12
2004-03-02
Mertz, Prema (Department: 1646)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
Structurally-modified antibody, immunoglobulin, or fragment...
C424S001110, C424S001490, C530S387100, C530S387300
Reexamination Certificate
active
06699473
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of molecular biology and monoclonal antibody technology. More specifically, the present invention relates to human single-chain antibodies that bind specifically to the epidermal growth factor receptor.
2. Description of the Related Art
The epidermal growth factor receptor (EGFR) is a 170 kDa transmembrane glycoprotein consisting of an extracellular ligand binding domain, a transmembrane region and an intracellular domain with tyrosine kinase activity. The binding of growth factors, EGF or TGF&agr;, to the epidermal growth factor receptor results in receptor dimerization, auto-phosphorylation and induction of a tyrosine kinase cascade, leading ultimately to DNA synthesis and cell division.
The epidermal growth factor receptor gene (c-erb-1), located on chromosome 7, is homologous to the avian erythroblastosis virus oncogene (v-erbB) that induces malignancies in chickens. The v-erbB gene encodes for a truncated protein product that lacks the extracellular ligand binding domain. The tyrosine kinase domain of the epidermal growth factor receptor has been found to have 97% homology to the v-erbB transforming protein.
The epidermal growth factor receptor is overexpressed in a number of malignant human tissues when compared to their normal tissue counterparts. The gene for the receptor is both amplified and overexpressed in a number of cancer cells. Overexpression of the epidermal growth factor receptor is often accompanied by the co-expression of the growth factors, EGF and TGF&agr;, suggesting that an autocrine pathway for control of growth may play a major part in the progression of tumors.
A high incidence of overexpression, amplification, deletion and structural rearrangement of the gene coding for the epidermal growth factor receptor has been found in biopsies of brain tumors. In fact, the amplification of the epidermal growth factor receptor gene in glioblastoma multiforme tumors is one of the most consistent genetic alterations known, with the EGFR being overexpressed in approximately 40% of malignant gliomas. In addition to glioblastomas, abnormal epidermal growth factor receptor expression has also been reported in a number of squamous epidermoid cancers and breast cancers. Many patients with tumors that overexpress the epidermal growth factor receptor have a poorer prognosis than those who do not. Consequently, therapeutic strategies which can potentially inhibit or reduce the aberrant expression of the EGFR are of great interest as potential anti-cancer agents.
Since the advent of hybridoma technology to produce murine monoclonal antibodies (mAbs) developed by Milstein and Köhler in 1975 (1), the therapeutic potential of antibodies is beginning to come to fruition for cancer therapy. There are many reports describing a few antibodies which inhibit cell proliferation of epidermal growth factor receptor-overexpressing cell lines (2-6). One such mouse antibody, mAb 225, was shown to inhibit cell proliferation and block ligand-induced epidermal growth factor receptor tyrosine kinase activity (2-3, 7). Further analysis showed mAb 225 induced a G
1
growth arrest and activated an apoptotic pathway after a 24 h exposure to increasing concentrations of antibody (8).
Other monoclonal antibodies which bind to the epidermal growth factor receptor and block ligand binding also show promise for cancer therapy. One group of rat monoclonal antibodies showed a dramatic antitumor effect in xenograft mouse models, with one antibody, ICR62 curing 4 out of 8 mice of the tumor (9). However, the problem with rat and mouse monoclonal antibodies or even the human-mouse chimeric antibody is the possibility of an immune or allergic response with prolonged treatment (10-13).
In order to avoid the human anti-murine antibody (HAMA) response in humans due to the repeated administration of murine mAbs, it is preferable to use human antibody in therapy or diagnostics. A 100% human monoclonal antibody against the epidermal growth factor receptor, E7.6.3, has been shown to completely eradicate human tumor xenografts in mice (4). This antibody is expected to elicit a minimal immune response in humans and shows promise for future cancer therapy. However due to the heterologous vascular structure around the tumor and the molecular size of the antibodies, monoclonal antibodies penetrate the tumor poorly and are unevenly distributed around the tumor.
In order to improve on the use of monoclonal antibodies, intact monoclonal antibodies have been reduced in size to antibody fragments or single-chain antibodies (scFvs). Therefore the development of human anti-EGFR scFvs will enhance its use as a diagnostic and/or therapeutic agent. One advantage of single-chain antibodies is their ability to penetrate deeper into the tumor (14). Thus, these molecules may potentially be more efficacious than intact antibodies for systemic administration. Also single-chain antibodies can be expressed intracellularly (intrabodies) and targeted to a subcellular compartment of the tumor cell or be secreted by the tumor cell and bind in an autocrine/paracrine fashion.
The prior art is deficient in the lack of a 100% human single-chain antibody that binds to the epidermal growth factor receptor. The present invention fulfills this longstanding need and desire in the art.
SUMMARY OF THE INVENTION
The present invention provides a 100% human single-chain antibody (scFv) which binds to the epidermal growth factor receptor. Two single-chain antibodies were isolated from a human IgM phage display library using purified epidermal growth factor receptor as antigen, and identified by ELISA as epidermal growth factor receptor-specific. Sequence analysis confirmed the two isolates as individual clones based on differences in their nucleotide and putative amino acid sequences. One single-chain antibody was shown to bind to the native full length epidermal growth factor receptor and the truncated and/or mutated epidermal growth factor receptor on human cells.
The present invention is directed to a human anti-epidermal growth factor receptor single-chain antibody having a sequence of SEQ ID No. 1 (clone 6) or SEQ ID No. 2 (clone 63), as well as DNA molecules and expression vectors that encode for the expression of the claimed human anti-epidermal growth factor receptor single-chain antibody.
The present invention is also drawn to a pharmaceutical composition comprising the disclosed human anti-epidermal growth factor receptor scFv and a therapeutic and/or diagnostic agent. Preferably, the therapeutic and/or diagnostic agent can be a toxin, a chemotherapeutic agent, a radioisotope, a transition metal or a gene therapy vector.
The present invention is also drawn to a method of treating or imaging a tumor, comprising the step of administering to a patient in need of such treatment or detection an effective amount of a radiolabeled anti-EGFR single-chain Fv of the present invention.
REFERENCES:
Beerli, et al. Inhibition of Signaling from Type-1 Receptor Tyrosine Kinases Via Intracellular Expression of Single-Chain Antibodies.Breast Cancer Research and Treatment, vol. 38, 1996, pp. 11-17.
Mokotoff, et al. Targeting Growth Factor Receptors With Bispecific Molecules.Current Medical Chemistry, vol. 3, 1996, pp. 87-100.
Bonner James Alan
Curiel David T.
Raisch Kevin Paul
Adler Benjamin Aaron
Mertz Prema
UAB Research Foundation
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