Chemistry: molecular biology and microbiology – Kit
Reexamination Certificate
1996-08-13
2001-05-15
Bui, Phuong T. (Department: 1638)
Chemistry: molecular biology and microbiology
Kit
C435S007100, C435S007230, C435S007240, C435S007900, C435S069100, C435S070210, C435S070300, C435S810000, C530S388100, C530S388220, C424S143100, C424S144100, C424S138100
Reexamination Certificate
active
06232123
ABSTRACT:
The invention relates to monoclonal antibodies against leucocyte-specific G protein-coupled receptors, processes for the production thereof and their use as well as kits containing the same. Furthermore, the invention concerns a process for the production of monoclonal antibodies against G protein-coupled receptors.
It is known that receptors play a decisive part in the transduction of signals in an organism. After the binding of the ligands to cell membrane-integrated receptors, the signal is transmitted intracellularly via various mechanisms and processed. There are various families of these receptors, which distinguish themselves by certain structural features. What is called G protein-coupled receptors, internationally abbreviated as GCR (G protein-coupled receptors), are comprised in one family. The G protein is a heterotrimeric GTP-binding protein complex which is composed of the three subunits G-alpha, G-beta and G-gamma. It regulates cellular activities by exchanging GDP by GTP at its alpha-subunit thus activating or inactivating a number of effectors such as adenylyl cyclases, phosphodiesterases, phospholipases and ion channels. Receptors for hormones and neurotransmitters are considered to be representatives of G protein-coupled receptors. They comprise adrenergic and muscarinergic receptors as well as receptors for dopamine, the substance P, thyreotropin, morphine and others.
In addition, there are more and more references to the effect that G protein-coupled receptors are also involved in the regulation of activation, inhibition, migration and cell—cell interaction of immunocompetent cells. It is known that the activation of leucocytes by inflammation mediators such as formyl-MLP, anaphylatoxin C5a, prostaglandins, interleukin-8, MIP-1&agr; and MIP-1&bgr; as well as RANTES, also takes place via G protein-coupled receptors. Blocking of these receptors would have an anti-inflammatory effect. However, means for this have not yet been found.
Therefore, it is the object of this invention to provide means by which the leucocyte-specific G protein-coupled receptors can be blocked.
According to the invention this is achieved by providing monoclonal antibodies against leucocyte-specific G protein-coupled receptors (L-GCR). These antibodies are obtainable by the following steps:
(a) introduction of an L-GCR-coding DNA into cells and expression of L-GCR,
(b) immunization of an animal with L-GCR-expressing cells of (a), and
(c) fusion of spleen cells from the immunized animal of (b) with myeloma cells and production of monoclonal L-GCR antibody-producing hybridoma cells.
The L-GCR-coding nucleic acid may be a DNA, particularly a genome DNA or cDNA. Furthermore, it may be an RNA. The nucleic acids may be provided by conventional methods known from the literature (cf) e.g. Maniatis et al., Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Laboratory (1982); Lipp et al., Eur. J. Immunol. 22 (1992), 2795-2799).
According to the invention, modifications such as additions, deletions and/or substitution of one or more bases, can be introduced into the L-GCR-coding nucleic acid. Additions comprise marker sequences which are fused e.g. to the 5′ end or 3′ end of L-GCR-coding nucleic acid. Such marker sequences are e.g. codons encoding a protein and protein fragment, respectively, against which a monoclonal antibody exists. The latter serves for detecting the expression of the protein and protein fragment, respectively, and thus also that of L-GCR (cf. von Zastrow and Kobilka, J. Biol. Chem. 267 (1992),
3530-14 3538
; von Zastrow et al., J. Biol. Chem. 268 (1993), 763-766; T. Emrich, M. Lipp, not published). Marker sequences can also be inserted in the nucleic acid encoding L-GCR, e.g. in the sequences encoding extracellular or intracellular domains. Further additions are sequences which guarantee a localization of L-GCR in the cell membrane of L-GCR-expressing cells. Such sequences may code for signal peptides, membrane proteins or fragments thereof. They are usually fused to the 5′ end of the L-GCR-coding nucleic acid.
According to the invention the L-GCR-coding nucleic acid may exist in an expression unit. It comprises the sequences necessary for the transcription and translation of the L-GCR-coding nucleic acid such as promoter, enhancer, ribosome binding, transcription start and stop as well as translation start and stop sequences. The expression unit may also be present in a vector which may also be a virus. A person skilled in the art knows which sequences of the expression unit are necessary in which arrangement to be able to express L-GCR in certain cells. In addition, the person skilled in the art knows which vectors are suitable for which cells.
According to the invention the L-GCR-coding nucleic acid is introduced into cells to express L-GCR. For this purpose, it is possible to use methods and conditions, known from the literature, for the transfection of cells with nucleic acid. Examples are the calcium phosphate precipitation, DEAE-dextran, electroporation and lipid vesicle methods. It is favorable to use the calcium phosphate precipitation method, about 1-2×10
6
cells being transfected with about 15 &mgr;g of L-GCR-coding nucleic acid. Eukaryotic and prokaryotic cells may be used as cells. Eukaryotic cells, particularly mammalian cells, e.g. CHO, COS and 293 cells, yeast and insect cells are preferred. The person skilled in the art is familiar with the cells, and they are generally available. The expression of L-GCR can be detected indirectly, e.g. by the detection of the expression of marker sequences, which are comprised by the L-GCR-coding nucleic acid, or by ligand binding. The detection can be made by means of conventional methods known from the literature, e.g. by immunofluorescence, immune enzymology, ELISA technique, flow cytometry and the ligand binding test.
According to the invention an animal is immunized with L-GCR-expressing cells. For this purpose, it is possible to chose conventional conditions known from the literature. It is favorable to give an animal about 3×10
7
cells twice, the interval of the administrations being about 28 days. Conventional experimental animals, particularly rats, hamsters, rabbits and mice, may be used as animals. Rats are particularly favorable.
According to the invention spleen cells are taken from the immunized animal. This can be done in usual and known manner. It is favorable to take from the animal spleen cells about 60 hours after the second administration of the antigen. They are then fused with myeloma cells by means of PEG. For this purpose, it is possible to use conventional conditions known from the literature. It is favorable to use as myeloma cells those of the mouse myeloma cell line X63-Ag8.653 (Kearney et al., J. Immunol. 123 (1979), 1548-50), and the ratio of spleen cells to myeloma cells is about 1:3. About 1 week after the fusion, the supernatants of the fused cells are tested for antibody production. For this purpose, it is possible to apply conventional methods known from the literature. It is favorable to carry out flow cytometry. It serves for carrying out comparative measurements of the hybridoma cell supernatants on cells which have been transfected with L-GCR-coding nucleic acid and not modified (untransfected), respectively. Antibody-producing hybridoma cells are then cloned by conventional methods known from the prior art, e.g. by the limiting dilution technique.
Such a hybridoma cell producing the antibody RF8B2 was deposited with the Deutsche Sammlung von Miroorganismen und Zellkulturen [German Type Collection of Microorganisms and Cell Cultures], Mascherodeweg 1b. 38124 Braunschweig, (DSM) under deposit number DSM ACC 2153 on Sep. 8, 1993.
The L-GCR antibodies according to the invention are suitable for diagnostic measures, particularly for determining a patient's immune status. For this purpose, they are labeled e.g. with fluorochromes or biotin and incubated together with other labeled, generally available T cell-specific antibodies and B c
Emrich Thomas
Förster Reinhold
Kremmer Elisabeth
Lipp Martin
Wolf Ingrid
Bui Phuong T.
Pennie & Edmonds LLP
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