Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Animal cell – per se – expressing immunoglobulin – antibody – or...
Patent
1994-12-15
1998-06-16
Feisee, Lila
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Animal cell, per se, expressing immunoglobulin, antibody, or...
435332, 4351722, 435 721, 5303882, 53038885, 5303913, C12N 512, C07K 1628, G01N 3353
Patent
active
057669465
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to monoclonal antibodies which have specificity for P-glycoprotein (Pgp). More particularly, the invention relates to monoclonal antibodies to an extracellular epitope of human Pgp and to their preparation and diagnostic and clinical uses.
The present description refers to various publications by reference numbers in brackets and these are listed towards the end of the text.
The development by Kohler and Milstein (1) of somatic cell fusion techniques for the production of hybrid cell lines which produce monoclonal antibodies has made it possible to prepare unlimited quantities of homogeneous antibody products which have-defined specificity for single antigenic determinants. The portion of a protein antigenic determinant that is recognised by and reacts with an antibody is known, and referred to herein, as the epitope and consists of one or more specific epitope-forming amino acid sequences.
Glycoprotein P is a plasma membrane protein which is over-expressed on the surfaces of cell lines or human tumour cells that exhibit an intrinsic or acquired multidrug-resistant (MDR) phenotype. The expression of this protein gives the cells the ability to multiply actively in the presence of concentrations of cytostatic drugs that are toxic for non-MDR parent cells. Transfection studies using cloned Pgp cDNAs have demonstrated the direct role of this protein in mediating the MDR phenotype (2). Pgp is thought to function as an energy-dependent drug efflux pump.
The amino acid sequence of Pgp has been deduced from the nucleotide sequences of the genes that confer the MDR phenotype in recipient cells (2, 3, 4). The secondary structure of Pgp has been predicted from analysis of its primary structure and is that of a transmembranal protein consisting of 12 hydrophobic transmembranal helices linked via 6 hydrophilic extracellular loops and 2 large cytoplasmic domains encoding 2 ATP-binding sequences.
A number of different monoclonal antibodies to Pgp have been isolated and characterised, and the specificities of some of these are reviewed by Cenciarelli et al. (5). More recently further monoclonals have been described which have specificity for the extracellular domain of Pgp (6, 7, 8). However monoclonal antibodies to Pgp for which the specific epitope-forming amino acid sequences have been characterised react either with the cytoplasmic domain of Pgp or with extracellular epitopes comprising discontinuous portions of the amino acid sequence of the protein. For example, Georges et al. (8) have mapped the epitope of the monoclonal antibody MRK-16, described by Hamada and Tsuruo (9), which comprises peptides present on 2 of the 6 extracellular peptide loops (the 1.sup.st and the 4.sup.th), predictable from the primary amino acid sequence.
Monoclonal antibodies to Pgp have been proposed for diagnostic and clinical uses, e.g. to monitor the level of Pgp expression on, and thereby the multi drug resistance (mdr) status of, human cells. Also such antibodies may find uses in the inhibition of Pgp-mediated MDR (7).
None of the known monoclonal antibodies to Pgp recognises a human-specific, extracellular epitope of glycoprotein P consisting of a continuous epitope-forming amino acid sequence. Moreover there is a continuing need for antibodies which are capable of recognising low levels of Pgp; for instance, when expressed on MDR cells at low levels or when the MDR cells are present as only a low proportion of a cell population. Monoclonal antibodies capable of recognising such human-specific epitopes and low Pgp levels would be useful for immunological testing.
Using the above mentioned somatic cell fusion techniques, a hybridoma secreting a monoclonal antibody (MM4.17) has been obtained which is capable of selective, high affinity binding to a previously unknown continuous extracellular epitope of human Pgp.
FIG. 1 is a graph showing FACS analysis results for human Pgp expression, according to the invention.
Accordingly the present invention provides a monoclonal antibody that recognises a structurally
REFERENCES:
patent: 5206352 (1993-04-01), Roninson
patent: 5369009 (1994-11-01), Arceci
patent: 5434075 (1995-07-01), Mechetner
Cianfroglia et al. Proceedings Am. Assoc. Canar Res. v. 34:317 Abstract #1889, Mar. 1993.
Chevallier et al. J. Cell Pharmacol 2:165-170, 1991.
Cenciarell;, Int.J. Cancer 47:533-547, 1991.
Hamada et al. NAR 18:1900, 1990.
Bruggemann, Biotechniques 10:202-209 1991.
Seaver, Gen. Eng. News, v.14 No. 114 pp. 10-21, Aug. 1994.
Chiatalas John L.
Feisee Lila
Instituto Superiore Di. Sanita'
Johnson Nancy A.
Lopez Gabriel
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