Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Patent
1995-11-17
1998-12-01
Scheiner, Toni R.
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
5303877, 53038822, 5303888, 53038885, 5303897, 53623553, 4241331, 4241351, 4241431, 435 6, 435 71, 435 72, 435 724, 4352523, 4353201, 435 691, C07K 1628, C07H 2104, A61K 39395, C12N 1563
Patent
active
058440933
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD OF THE INVENTION
This invention relates to new anti-EGFR antibodies and antibody fragments, preferrably single-chain Fvs (scFvs) which can be obtained from phage-antibody libraries constructed from cells of an immunized mammalian, preferably a mouse. The antibody fragments isolated from the phage-antibody libraries can be engineered to create partially humanized whole antibody molecules. These chimeric anti-EGFR antibodies contain constant regions of human immunoglobulins, and can be used as well as their fragments as agents for the diagnosis and therapy of human tumors.
Furthermore, the invention demonstrates that phage-antibody libraries are an alternative, and more versatile, method for isolating antibodies from immunized mammalians in comparison with the standard hybridoma technology.
The invention relates, moreover, to pharmaceutical compositions comprising said antibodies or fragments for the purposes of treating tumors like melanoma, glioma or carcinoma. The said antibodies or fragments can be used also for diagnostic applications regarding locating and assessing the said tumors in vitro or in vivo.
The specification relates to several technical terms which are herewith defined as follows:
"FRs" (framework regions) mean the four subregions of the light or heavy chain variable regions that support the three CDRs.
"CDRs" (complementarity determining regions) mean the three subregions of the light or heavy chain variable regions which have hypervariable sequences and form loop structures that are primarily responsible for making direct contact with antigen.
"Chimeric" or partially humanized antibodies mean antibodies comprising constant regions deriving from human sources and variable regions (CDRs included) deriving from non-human sources, e.g. from the mouse.
"Humanized" or fully humanized antibodies mean antibodies comprising constant regions and FRs deriving from human sources whereas the CDRs derive from non-human sources.
"EGF" and "EGFR" mean the epidermal growth factor ant its receptor.
"PCR" means the polymerase chain reaction.
"scFv" means single-chain Fv which is an antibody fragment.
"V.sub.L " means light chain variable region.
"V.sub.k " means kappa light chain variable region.
"V.sub.H " means heavy chain variable region.
PBS means phosphate buffered saline
FCS means fetal calf serum
HBSS means Hanks balanced salt solution
FITC means fluoresceineisothiocyanate
MTC means mixed cell culture
BACKROUND OF THE INVENTION
Epidermal growth factor (EGF) is a polypeptide hormone which is mitogenic for epidermal and epithelial cells. When EGF interacts with sensitive cells, it binds to membrane receptors (EGFR). The EGFR is a transmembrane glycoprotein of about 170 kD and is a gene product of the c-erb-B proto-oncogene.
MAb 425 is a murine monoclonal antibody raised against the well known human A431 carcinoma cell line (ATCC CRL 1555), binds to a polypeptide epitope of the external domain of the human EGFR, and inhibits the binding of EGF. MAb 425 (ATCC HB 9629) was found to mediate tumor cytotoxicity in vitro and to suppress tumor cell growth of epidermoid and colorectal carcinoma-derived cell lines in vitro (Rodeck et al., Cancer Res. 1987. 47: 3692). Humanized and chimeric versions of MAb 425 have been disclosed in WO 92/15683.
Over the last few years, methods have been described (Skerra and Pluckthun, Science 1988. 240: 1038; Better et al., Science 1988. 240: 1041) with which functional antibody fragments can be produced in eukaryotic host cells, such as E. coli. These include the Fv fragment and the Fab fragment, whereby the Fv fragment is of special interest. Single-chain Fvs (wherein the V.sub.L and the V.sub.H chain are linked together) have been also described (Bird et al., Science 1988. 242: 423; Huston et al., Proc. Natl. Acad. Sci. USA 1988. 85: 5879).
Phage-antibody libraries offer an alternative technology to hybridoma technology in the isolation of antibodies from immunized animals. Hybridoma technology works by immortalizing the cells that produce the antibodies.
REFERENCES:
patent: 5395750 (1995-03-01), Dillon et al.
Bender H. et al 1992 Cancer Res 52:121-126.
Masui, H. et al Cancer Res 49:3482-3488 1989.
McCafferty J. et al Noture 348:552-554 1990.
Chaudhary VK et al PNAS 87:1066-1070 1990.
Shin, S-U. Biotherapy 3:43-53 1991.
Adan Jaume
Ansell Keith H.
Bendig Mary M.
Blasco Francesc
Gussow Detlef
Johnson Nancy A.
Merck Patent Gesellschaft mit beschrankter Haftung
Scheiner Toni R.
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