EP1012190A1 - Recepteur d'eotaxine eosinophile - Google Patents

Recepteur d'eotaxine eosinophile

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Publication number
EP1012190A1
EP1012190A1 EP97925399A EP97925399A EP1012190A1 EP 1012190 A1 EP1012190 A1 EP 1012190A1 EP 97925399 A EP97925399 A EP 97925399A EP 97925399 A EP97925399 A EP 97925399A EP 1012190 A1 EP1012190 A1 EP 1012190A1
Authority
EP
European Patent Office
Prior art keywords
receptor
eosinophil eotaxin
eosinophil
nucleic acid
eotaxin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97925399A
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German (de)
English (en)
Other versions
EP1012190A4 (fr
Inventor
Bruce L. Daugherty
Julie A. Demartino
Martin S. Springer
Salvatore J. Siciliano
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Merck and Co Inc
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Merck and Co Inc
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Publication date
Priority claimed from GBGB9700894.0A external-priority patent/GB9700894D0/en
Application filed by Merck and Co Inc filed Critical Merck and Co Inc
Publication of EP1012190A1 publication Critical patent/EP1012190A1/fr
Publication of EP1012190A4 publication Critical patent/EP1012190A4/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates to an eosinophil eotaxin receptor ("CC CKR3"), in particular, the human eosinophil eotaxin receptor and nucleic acids encoding this receptor.
  • This invention further relates to assays which may be used to screen and identify compounds that bind to the eosinophil eotaxin receptor. Such compounds would be useful in the treatment and prevention of atopic conditions including allergic rhinitis, dermatitis, conjunctivitis, and particularly bronchial asthma.
  • Eosinophils play prominant roles in a variety of atopic conditions including allergic rhinitis, dermatitis, conjunctivitis, and particularly bronchial asthma (for a reviews see e.g. Gleich, G. J., et al., Eosinophils. J. I. Gallin, I. M. Goldstein, R. Snyderman, Eds., Inflammation: Basic Principles and Clinical Correlates (Raven Press, Ltd., New York, 1992) and Seminario, M. C, et al. (1994) Current Opinion in Immunology 6, 860-864).
  • a pivotal event in the process is the accumulation of eosinophils at the involved sites.
  • chemokines a family of 8-10 kDa proteins are more restricted in the leukocyte subtypes they target and are potential candidates for the recruitment of eosinophils in atopic diseases and asthma (Baggiolini, M., Dewald, B. and Moser, B. (1994) Advances in Immunology 55, 97-179).
  • ⁇ -chemokines particularly eotaxin and RANTES, the receptor which mediates these actions has not been identified.
  • the chemokines contain four conserved cysteines, and are divided into two sub-families based on the arrangement of the first cysteine pair (Baggiolini, M., Dewald, B. and Moser, B. (1994) Advances in Immunology 55, 97-179).
  • ⁇ -chemokine family which includes IL-8, MGS A, NAP-2 and IP- 10
  • these two cysteines are separated by a single amino acid
  • RANTES regulated on activation T expressed and secreted
  • MCP-1 monocyte chemotactic protein
  • MCP-2 monocyte chemotactic protein
  • MCP-3 macrophage inflammatory protein
  • MlP-l ⁇ macrophage inflammatory protein
  • eotaxin eotaxin
  • the ⁇ -chemokines act primarily on neutrophils, and the ⁇ -chemokines on monocytes, lymphocytes, basophils and eosinophils (Baggiolini, M., Dewald, B. and Moser, B. (1994) Advances in Immunology 55, 97-179).
  • RANTES, MCP-2, MCP-3, and MlP-l ⁇ have been shown to activate eosinophils in vitro (Dahinden, C. A., et al. (1994) Journal of Experimental Medicine 179, 751-756; Ebisawa, M., et al. (1994) Journal of Immunology 153, 2153-2160; Weber, M., et al.
  • Eosinophils are attracted by a number of ⁇ -chemokines, the most potent of which are eotaxin (Griffiths- Johnson, D. A., et al. (1993) Bichemical and Biophysical Research Communications 197, 1167- 1172; Jose, P. J., et al. (1994) Journal of Experimental Medicine 179, 881-887; Rothenberg, et al. (1995) Proceedings ofthe National Academy of Sciences 92, 8960-8964) and RANTES (Dahinden, C. A., et al. (1994) Journal of Experimental Medicine 179, 751-756; Ebisawa, M., et al. (1994) Journal of Immunology 153, 2153-2160; Weber, M., et al. (1995) Journal of Immunology 154, 4166-4172;
  • ⁇ -chemokines While elucidation of the actions of ⁇ -chemokines on eosinophils has contributed greatly to the understanding of eosinophil biology, information regarding the cell surface receptors which mediate these effects remain sparse. Furthermore, there are no reports describing binding studies of any of the ⁇ -chemokines to primary eosinophils.
  • the known ⁇ -chemokine receptors are members of the G protein-coupled receptor superfamily. Two of these receptors, CC CKR1 (12, 13) and CC CKR2 (MCP-1R) (Charo, I. F., et al. (1994) Proceecing of the National Academy of Sciences 91, 2752-2756; Myers, S. J., et al.
  • CC CKR1 binds RANTES with high affinity, but binds eotaxin poorly, and while the effects of eotaxin on CC CKR2 have not been studied this receptor has no avidity for RANTES (Myers, S. J., et al. (1995) Journal of Biological Chemistry 270, 5786-5792).
  • a human eotaxin receptor has been reported by Ponath, P.D., et al. J. Exp. Med. 183, 2437-2448 (June 1996) and Gerard, C.J., et al., PCT Publication No. WO 96/22371 (July 25, 1996).
  • the sequence disclosed in this publication possesses an error in the assignment of threonine rather than serine at position # 276 of the receptor.
  • functionality of the receptor was not fully demonstrated.
  • HIV-1 human immunodeficiency virus
  • LAV lymphoid virus
  • HTLV-III human immunodeficiency syndrome
  • ARV advanced immune deficiency syndrome
  • Entry of HIV-1 into a target cell requires cell- surface CD4 and additional host cell cofactors. Fusin has been identified as a cofactor required for infection with virus adapted for growth in transformed T-cells, however, fusin does not promote entry of macrophagetropic viruses which are believed to be the key pathogenic strains of HIV in vivo.
  • chemokine receptors CCR-5 and CXCR-4, as well as the primary receptor CD4
  • the principal cofactor for entry mediated by the envelope glycoproteins of primary macrophage-trophic strains of HIV-1 is CCR5, a receptor for the ⁇ -chemokines RANTES, MlP-l ⁇ and MIP- l ⁇ (Deng, et al., Nature. 381. 661-666 (1996)). HIV attaches to the CD4 molecule on cells through a region of its envelope protein, gpl20.
  • CD-4 binding site on the gpl20 of HIV interacts with the CD4 molecule on the cell surface, and undergoes conformational changes which allow it to bind to another cell-surface receptor, such as CCR5 and/or CXCR-4.
  • CCR5 and/or CXCR-4 another cell-surface receptor
  • ⁇ -chemokine ligands prevent HIV-1 from fusing with the cell (Dragic, et al., Nature. 381. 667-673 (1996)).
  • chemokine receptors may be used by some strains of HIV-1 or may be favored by non-sexual routes of transmission. Although most HIV-1 isolates studied to date utilize CCR-5 or fusin, some can use both as well as the related CCR-2B and CCR-3 as co-receptors (Nature Medicine. 2(11), 1240-1243 (1996)). The determination that chemokine receptors are critical co-receptors for the entry of HIV into cells was pronounced a "1996 Breakthrough of the Year" by Science Magazine (Science. 274. 1987-1991 (Dec. 20, 1996)).
  • orally-active agents which modulate the action of the eosinophil eotaxin receptor would be a significant advance in the treatment and prevention of atopic conditions including allergic rhinitis, dermatitis, conjunctivitis, and particularly bronchial asthma. Further, agents which could block the eosinophil eotaxin receptor in humans who possess normal chemokine receptors should prevent infection in healthy individuals and slow or halt viral progression in infected patients.
  • the present invention relates to a novel receptor which is the eosinophil eotaxin receptor.
  • This receptor is a human ⁇ -chemokine receptor and has been designated "CC CKR3".
  • One aspect of the present invention is directed to the human eosinophil eotaxin receptor, free from receptor-associated proteins.
  • a further aspect of this invention is the human eosinophil eotaxin receptor which is isolated or purified.
  • Another aspect of this invention are eosinophil eotaxin receptors which are encoded by substantially the same nucleic acid sequences, but which have undergone changes in splicing or other RNA processing-derived modifications or mutagenesis induced changes, so that the expressed protein has a homologous, but different amino acid sequence from the native forms.
  • These variant forms may have different and/or additional functions in human and animal physiology or in vitro in cell based assays.
  • the present invention further provides the eosinophil eotaxin receptor, CC CKR3, which is a ⁇ -chemokine receptor and which was cloned from primary eosinophils, and expressed in AML14.3D10 cells.
  • This receptor binds the potent eosinophil attractants, eotaxin, RANTES and MCP-3 with high affinity.
  • eotaxin and RANTES and to a lessor extent MCP-3, induce Ca 2+ -fluxes in cells expressing CC CKR3.
  • Correlation with the binding properties of primary eosinophils provide conclusive evidence that CC CKR3 is the primary endogenous receptor which mediates the effects of ⁇ - chemokines on eosinophils.
  • the present invention further relates to assays which employ a novel receptor which is the eosinophil eotaxin receptor.
  • This receptor is a human ⁇ -chemokine receptor and has been designated "CC CKR3".
  • One aspect of the present invention is directed to assays employing the the human eosinophil eotaxin receptor, free from receptor-associated proteins.
  • a further aspect of this invention is directed to assays which employ the human eosinophil eotaxin receptor which is isolated or purified.
  • the present invention provides assays in which the eosinophil eotaxin receptor is expressed in an AML14.3D10 cell line. DETAILED DESCRIP ⁇ ON OF THE INVENTION
  • the present invention is directed to an eosinophil eotaxin receptor "CC CKR3" which is a G protein-coupled receptor and has been cloned from human eosinophils and which when stably expressed in AML14.3D10 cells binds eotaxin, RANTES and MCP-3 with high affinity.
  • CC CKR3 eosinophil eotaxin receptor
  • Competition binding studies against 125 ⁇ _human eotaxin gives Kd values of 0.1, 2.7, and 3.1 nM, respectively for the three ⁇ - chemokines.
  • CC CKR3 also binds MCP-1 with lower affinity, but does not bind MlP-l ⁇ or MIP-l ⁇ .
  • Competition binding studies on primary eosinophils give binding affinities for the different chemokines which are indistinguishable from those measured with CC CKR3. Since CC CKR3 is prominently expressed in eosinophils it is concluded that CC CKR3 is the eosinophil eotaxin receptor. Eosinophils also express a much lower level of a second chemokine receptor, CC CKR1, which appears to be responsible for the effects of MlP-l ⁇ .
  • the eosinophil eotaxin receptor is a protein containing various functional domains, including one or more domains which anchor the receptor in the cell membrane, and at least one ligand binding domain. As with many receptor proteins, it is possible to modify many of the amino acids, particularly those which are not found in the ligand binding domain, and still retain at least a percentage of the biological activity of the original receptor. In accordance with this invention, it is suggested that certain portions of the eosinophil eotaxin receptor are not essential for its activation by ⁇ -chemokines. Thus this invention specifically includes modified functionally equivalent eosinophil eotaxin receptors which have deleted, truncated, or mutated portions.
  • This invention also specifically includes modified functionally equivalent eosinophil eotaxin receptors which contain modified and/or deletions in other domains, which are not accompanied by a loss of functional activity. Additionally, it is possible to modify other functional domains such as those that interact with second messenger effector systems, by altering binding specificity and/or selectivity. Such functionally equivalent mutant receptors are also within the scope of this invention.
  • a further aspect of this invention are nucleic acids which encode an eosinophil eotaxin receptor or a functional equivalent from human or other species. These nucleic acids may be free from associated nucleic acids, or they may be isolated or purified. For most cloning purposes, cDNA is a preferred nucleic acid, but this invention specifically includes other forms of DNA as well as RNAs which encode an eosinophil eotaxin receptor or a functional equivalent.
  • Yet another aspect of this invention relates to vectors which comprise nucleic acids encoding an eosinophil eotaxin receptor or a functional equivalent.
  • These vectors may be comprised of DNA or RNA; for most cloning purposes DNA vectors are preferred.
  • Typical vectors include plasmids, modified viruses, bacteriophage and cosmids, yeast artificial chromosomes and other forms of episomal or integrated DNA that can encode an eosinophil eotaxin receptor. It is well witnin the skill of the ordinary artisan to determine an appropriate vector for a particular gene transfer or other use.
  • a further aspect of this invention are host cells which are transformed with a gene which encodes an eosinophil eotaxin receptor or a functional equivalent.
  • the host cell may or may not naturally express an eosinophil eotaxin receptor on the cell membrane.
  • the host cells are able to express the eosinophil eotaxin receptor or a functional equivalent on the cell membrane.
  • the receptors of this invention were cloned from RNA isolated from eosinophils.
  • PCR was used with primers designed from both CCCKR1 and CCCKR2, and clones screened by expression in the AML14.3D10 cell line.
  • the cloning was made difficult by several factors. First, prior to this invention there was very little information available about the biochemical characteristics and intracellular signalling/effector pathways used by these receptors making screening procedures uncertain. Second, this receptor could not be expressed and/or functionally coupled in the cell lines normally used for clomng receptors such, as COS, CHO, HEK293. After repeated failures using standard lines, an obscure eosinophilic- like cell line, AML14.3D10, was tried and found to suitable for expression of the receptors described in this invention.
  • the present invention further relates to assays which employ a novel receptor which is the eosinophil eotaxin receptor.
  • This receptor is a human ⁇ -chemokine receptor and has been designated "CC CKR3".
  • One aspect of the present invention is directed to assays employing the the human eosinophil eotaxin receptor, free from receptor-associated proteins.
  • a further aspect of this invention is directed to assays which employ the human eosinophil eotaxin receptor which is isolated or purified.
  • the present invention provides assays in which the eosinophil eotaxin receptor is expressed in an AML14.3D10 cell line.
  • a particular embodiment of this invention is directed to an assay to determine the presence of a compound which binds to the eosinophil eotaxin receptor.
  • this invention also comprises a method to determine the presence of a compound which binds to an eosinophil eotaxin receptor comprising: (a) introducing a nucleic acid which encodes an eosinophil eotaxin receptor into a cell under conditions so that eosinophil eotaxin receptor is expressed;
  • the eosinophil eotaxin receptor is expressed in AML14.3D10 cells.
  • the binding of the compound suspected of binding to the eosinophil eotaxin receptor is compared to the binding or the influence of eotaxin, RANTES and MCP-3.
  • a further embodiment of this invention is directed to an assay to determine the presence of a compound which antagonizes the binding of a known ligand to the eosinophil eotaxin receptor.
  • this invention further comprises a method to determine the presence of a compound which antagonizes the eosinophil eotaxin receptor comprising:
  • the eosinophil eotaxin receptor is expressed in AML14.3D10 cells.
  • the known ligand of the eosinophil eotaxin receptor is eotaxin, RANTES and MCP-3.
  • One aspect of this invention is the development of a sensitive, robust, reliable and high-throughput screening assay which may be used to detect ligands which bind to the eosinophil eotaxin receptor, in particular, antagonists of the action of chemokines on eosinophils.
  • a typical protocol of such an assay is as follows. Assay buffer (50 mM Hepes, pH 7.2 w/ 0.5% BSA, 5 mM MgCl2, 1 mM CaCl2, 100 uM PMSF and 10 ug/ml phosphoramidon, leupeptin, aprotinin and chymostatin), test compound (or equivalent volume of solvent), 20 pM 125 ⁇ _ n uman eotaxin (2000 Ci/mmol), 25 ng unlabeled human eotaxin (non-specific binding wells only), and AML14.3D10 cells expressing eotaxin receptor cells, or eosinophils, are added sequentially in 96-well, round-bottom, polystyrene plates to a final volume of 250 uL.
  • Assay buffer 50 mM Hepes, pH 7.2 w/ 0.5% BSA, 5 mM MgCl2, 1 mM CaCl2, 100 uM PMSF and 10
  • Assay plates are then mixed and incubated for 60 minutes at 31°C. After incubation, assay plates are harvested onto Packard 96-well GF/C Unifilter plates treated with 0.33% polyethylenimine (PEI) using Packard Filtermate 196 cell harvester. Wells and filters are washed with 200 uL 50 mM Hepes, pH 7.2 with 0.5M NaCl and 0.02% NaN3- After filtration, GF/C plates are dried and sealed. 25 uL Packard Microscint-O scintillant are then added to each well and counted for 2 minutes on Packard Topcount (liquid 125j setting).
  • PKI polyethylenimine
  • Ligands detected using assays described herein may be used in the treatment and prevention of conditions which would be benefited by the modification of the activity of the eosinophil eotaxin receptor, such as in the treatment and prevention of atopic conditions including allergic rhinitis, dermatitis, conjunctivitis, and particularly bronchial asthma.
  • a further aspect of this invention is directed to novel ligands which are identified using the subject assays.
  • the eosinophil eotaxin receptor and fragments are immunogenic.
  • another aspect of this invention is antibodies and antibody fragments which can bind to eosinophil eotaxin receptor or an eosinophil eotaxin receptor fragment.
  • These antibodies may be monoclonal antibodies and produced using either hybridoma technology or recombinant methods. They may be used as part of assay systems or to deduce the function of an eosinophil eotaxin receptor present in a cell.
  • a further aspect of this invention are antisense oligonucleotides nucleotides which can bind to eosinophil eotaxin receptor nucleotides and modulate receptor function or expression.
  • a further aspect of this invention is a method of increasing the amount of eosinophil eotaxin receptor in a cell comprising, introducing into the cell a nucleic acid encoding an eosinophil eotaxin receptor, and allowing expression of the eosinophil eotaxin receptor.
  • the receptor protein Free from receptor-associated proteins—the receptor protein is not in a mixture or solution with other membrane receptor proteins.
  • nucleic acid Free from associated nucleic acids—the nucleic acid is not covalently linked to DNA which it is naturally covalently hnked in the organism's chromosome. Isolated receptor- the protein is not in a mixture or solution with any other proteins.
  • Isolated nucleic acid the nucleic acid is not in a mixture or solution with any other nucleic acid.
  • Functional equivalent a receptor which does not have the exact same amino acid sequence of a naturally occurring eosinophil eotaxin receptor, due to alternative splicing, deletions, mutations, or additions, but retains at least 1%, preferably 10%, and more preferably 25% of the biological activity of the naturally occurring receptor.
  • Such derivatives will have a significant homology with the natural eosinophil eotaxin receptor and can be detected by reduced stringency hybridization with a DNA sequence obtained from an eosinophil eotaxin receptor.
  • the nucleic acid encoding a functional equivalent has at least about 50% homology at the nucleotide level to a naturally occurring receptor nucleic acid.
  • Purified receptor the receptor is at least about 95% pure.
  • Purified nucleic acid the nucleic acid is at least about 95% pure.
  • the 5' genomic DNA flanking sequence encoding the human eosinophil eotaxin receptor further comprises the region beginning with nucleotide 1 and ending with nucleotide 3586 as depicted below (SEQ ID NO:3):
  • sequence for the cDNA encoding human eosinophil eotaxin receptor further comprises the terminator region beginning with nucleotide 4652 and ending with nucleotide 5099 as depicted below (SEQ ID NO:4):
  • this invention also includes alternative base sequences wherein a codon (or codons) are replaced with another codon such that the amino acid sequence translated by the DNA sequences remains unchanged.
  • a sequence bearing one or more such replaced codons will be defined as a degenerate variation.
  • mutations exchanges of individual amino acids which one skilled in the art would expect to have no effect on functionality, such as valine for leucine, arginine for lysine, and asparagine for glutamine.
  • CC CKR3 The amino acid sequence of CC CKR3 shares some sequence homology with CC CKR1 (Neote, K., et al. (1993) Cell 72, 415-425), CC CKR2B (Charo, I. F., et al. (1994) Proceecing ofthe National Academy of Sciences 91, 2752-2756), CC CKR4 (Power, C. A., et al. (1995) Journal of Biological Chemistry 270, 19495-19500) and V28 (Raport, C. J., et al. (1995) Gene 163, 295-299).
  • the sequence of this protein, designated CC CKR3 is comparable to that previously reported by Combadiere et al.
  • Genomic cloning provided confirmation of the subject sequence, including lysine at position 107.
  • the sequence discrepency which results from a substitution of G to T at the third position of the codon for residue 107, could represent a genetic polymorphism. This is highly unlikely, however, because all ⁇ - and ⁇ -chemokine receptors analyzed to date contain lysine in that position including the recently described basophilic ⁇ -chemokine receptor (Power, C. A., et al.
  • CC CKR3 An unusual feature of CC CKR3 is the cluster of negatively charged amino acids (ETEELFEET) distal to TMIV in the second extracellular loop. Expression of the Human CC CKR3 in AML14.3D10 Cells
  • the CC CKR3 transfected AML14.3D10 cell line has been placed on restricted deposit with American Type Culture Collection in Rockville, Maryland as ATCC No. CRL- 12079, on April 5, 1996.
  • Stable clones were selected for neomycin resistance, and a number were chosen for further analysis.
  • a western blot was performed using antisera generated against a peptide derived from the predicted C-terminus of CC CKR3. Immunoreactive bands migrating at approximately 45-55 kd are present in primary eosinophils and the 3.16 clone, indicating that CC CKR3 is indeed expressed in these cells. There was no immunoreactive bands present in neutrophils indicating that the antisera was indeed identifying an eosinophil-specific protein.
  • CC CKR3 is a high affinity receptor for eotaxin.
  • Kd 60 nM
  • MlP-l ⁇ , and MIP-l ⁇ failed to compete at all (See Table 1).
  • the ⁇ -chemokine IL-8 did not inhibit eotaxin binding.
  • CC CKR3 is functionally coupled in AML14.3D10 cells
  • CC CKR3 The selectivity of CC CKR3 for the various ⁇ -chemokines mirrors the effectiveness of these ligands as eosinophil chemoattractants suggesting that CC CKR3 is the primary mediator of chemokine induced eosinophil chemotaxis.
  • the affinites for RANTES and MCP-3 were also identical to those measured on CC CKR3, and as with CC CKR3, neither MlP-l ⁇ , or MIP-l ⁇ , showed any ability to compete with radiolabeled eotaxin (see Table 1).
  • the Kd's obtained by competition against 125 ⁇ _MCP-3 on eosinophils are effectively indistinguishable to those measured against cloned CC CKR3 (see Table 1).
  • MCP-1 binds with much lower affinity, while MlP-l ⁇ and MlP-l ⁇ fail to bind at all.
  • the selectivity of CC CKR3 mirrors most of the binding activity of primary eosinophils. In fact, when measured by competition against 125 ⁇ _ eotaxin, the binding affinities on eosinophils for all of these ⁇ - chemokines are indistinguishable from those obtained with cloned CC CKR3.
  • CC CKR3 was cloned from eosinophils, and as shown by Western blotting is heavily expressed in these cells.
  • the abilities of the different chemokines to activate CC CKR3 are consistent with the binding data as eotaxin, RANTES, and to a lessor extent MCP- 3 all stimulate Ca ⁇ + fluxes in clones which express the receptor, while MCP-1, MlP-l ⁇ and MIP-l ⁇ do not, even at concentrations as high as 1 ⁇ M.
  • CC CKR3 is the eosinophil eotaxin receptor.
  • the primers used for PCR corresponded to a consensus sequence encoded in transmembrane domains (TM) II and VII: 5 * -PCR primer (TMII) (SEQ ID NO:5):
  • the resultant PCR product of -700 bp was subcloned into plasmid pNoTA (Five Prime, Three Prime, Ine) and sequenced using Sequenase (USB).
  • the remaining 5' and 3' sequence encoding CC CKR3 was cloned by rapid amplification of cDNA ends (RACE) using both the 5'-RACE and 3'-RACE kits (Clontech) with the following primer sequences: (5 -RACE) (SEQ ID NO:7): 5'-TCTCGCTGTACAAGCCTGTGTG-3';
  • 5 -CCTTCTCTCTTCCTATCAATCC-3 * The resultant PCR products (5'-RACE, -450 bp; 3 * -RACE, -700 bp) were subcloned into pCRII (Invitrogen) and sequenced. Upon identification of the 5'-end of the cDNA containing the initiator ATG codon and the 3'-end containing the termination codon TAG, a new set of PCR primers were designed to reamplify the entire coding region from eosinophil total RNA for expression of CC CKR3. The primer sequences used for RT/PCR were: 5'-PCR primer (SEQ ID NO:9):
  • Plasmid pD5/Igh/Neo (Daugherty, B.L., et al. (1991) Nucleic Acids Research 19, 2471-2476) was digested with the restriction enzyme Sail, filled in with E. coli DNA polymerase I Klenow fragment to create a blunt end and subsequently digested with the restriction enzyme Notl.
  • CM VIE intron A fragment from plasmid p89-l 1 was digested with Clal, filled in to create a blunt end and subsequently digested with Hindill. These fragments were used in a three-way ligation with a Hindill and Notl fragment of the human C5a receptor cDNA.
  • the C5a receptor fragment was excised with Hindill and Notl and replaced with the eotaxin receptor cDNA of 1105 bp obtained by RT/PCR with oligonucleotides SEQ ID NO:9 and SEQ ID NO: 10 after digestion with Hindill and Notl.
  • Several clones were sequenced and one clone comprising the consensus sequence was chosen for expression of CC CKR3 in heterologous cells.
  • AML14.3D10 cells (Paul, C. C, et al. (1995) Blood 86, 3737-3744) were cultured in RPMI-1640, 10% FBS, 1 mM sodium pyruvate, 0.5 ⁇ M ⁇ -mercaptoethanol and 2 mM L-glutamine (complete medium). Cells were harvested at a density of 0.3 x 10°7mL, washed once in PBS, resuspended in RPMI at 10 7 /mL, and 25 ⁇ g of plasmid was added. Electroporation was carried out at 300 V, 960 ⁇ F using a Gene Pulser (BioRad).
  • eosinophils were isolated from granulophoresis preparations obtained from allergic and asthmatic donors (Bach, M. K., et al. (1990) Journal of Immunological Methods 130, 277-281). The leukocytes were mixed with equal volumes of HBSS and layered over LSM (Organon Teknika) as described (Rollins, T. E., et al. (1988) Journal of Biological Chemistry 263, 520-526). After lysis of erythrocytes with NH4CI, the granulocytes were subsequently treated with anti-CD 16 microbeads followed by MACS separation (Miltenyl Biotech) (Hansel, T. T., et al. (1991) Journal of Immunological Methods 145, 105-110). Typically the eosinophil preparations were >99% pure as determined using the LeukoStat staining kit (Fisher).
  • Polyclonal rabbit antisera was generated to CC CKR3 using the C-terminal decapeptide sequence TAEPELSIVF. Peptide synthesis, coupling to thyroglobulin and production of antisera was performed (Miller, D. K., et al. (1993) Journal of Biological Chemistry 268, 18062-18069). Whole cells were boiled and sonicated in Laemli sample buffer (Laemmli, U. K.
  • MCP-3, MCP-1, RANTES, murine and human eotaxin were obtained from Peprotech (Princeton, NJ).
  • 125 ⁇ _ MCP-3 and 125 ⁇ _MIP-l ⁇ were obtained from New England Nuclear (Boston, MA), and ⁇ i-human-eotaxin was obtained from Amersham.
  • Binding of 125 ⁇ _ ⁇ a beled ligands (typically a total of 2 x 104 cpm) in the presence of varying concentrations of unlabeled ligands to intact cells (typically 1.5 x 104 , 10 ⁇ , or 10 ⁇ for experiments with labeled eotaxin, MCP-3, or MlP-l ⁇ , respectively) were performed at 32°C (Van Riper, G., et al. (1993) Journal of Experimantal Medicine 111, 851-856).
  • CC CKR3 Binding Assay Assay buffer 50 mM Hepes, pH 7.2 w/ 0.5% BSA, 5 mM
  • AML14.3D10 expressing eotaxin receptor (CCCKR3) cells are incubated with test compound and stimulated with eotaxin, RANTES, or MCP-3, pelleted and lysed in 1 mL lysis buffer (1% Nonidet P-40, 100 mM NaCl, 20 mM Tris, pH 7.4, 10 mM iodoacetamide, 46 mM b- glyceraphosphate, 10 mM NaF, 1 mM PMSF, 1 ug/mL leupeptin, 1 ug/mL chymostatin, 1 ug/mL antipain, 1 ug/mL pepstatin A, and 1 mM sodium orthovanadate).
  • lysis buffer 1% Nonidet P-40, 100 mM NaCl, 20 mM Tris, pH 7.4, 10 mM iodoacetamide, 46 mM b- glyceraphosphate, 10 mM NaF
  • Ly sates are then pre-cleared for 1 hr with uncoupled protein A Affi-Gel beads. Immunoprecipitation is then performed with p85 polyclonal antiserum (1 ul/mL lysate; Upstate Biologies, New York, NY), coupled to protein A Affi-Gel beads (Bio- Rad) at 4°C for 2 hr.
  • Immunoprecipitates are washed and subjected to in vitro lipid kinase assays by using a lipid mixture, 100 ul 0.1 mg/ml Ptdlns and 0.1 mg/ml phosphatidylserine dispersed by sonication into solution in 20 mM HEPES, pH 7.0, and 1 mM EDTA.
  • the reaction is initiated by the addition of 100 mM ATP and 20 uCi [gamma- 32 P]ATP (3000 Ci/mmol) in 20 ul kinase buffer.
  • the reaction is then terminated after 15 min and the phosphoinositide lipids are separated by thin layer chromatography (TLC) and visualized by exposure to iodine vapor autoradiography.
  • TLC thin layer chromatography
  • AML14.3D10 expressing eotaxin receptor cells are isolated by centrifugation (van Riper, G., et al. (1994) J. Immunol. 152, 4055- 4061) for 15 min at 150 X g, washed and resuspended at 10' cells/ml in HBSS (pH 7.4) containing 1 mM CaCl2 and 1 mM MgCl2 (chemotaxis buffer). The chemotaxis experiments is then performed in Transwell dishes (6.5 mm, Costar, Cambridge, MA).
  • the lower chamber contains 0.6 ml of chemotaxis buffer and is separated from the upper chamber containing 10" cells by a 5-mm pore Nucleopore polycarbonate membrane (Nucleopore Co ⁇ oration, Pleasanton, CA). After a 15 min preincubation at 37°C, test compound and eotaxin, RANTES, or MCP-3 are added to the lower chamber to a final concentration of 300 nM. After 2 hrs at 37°C, the upper chamber inserts are removed, and the cells that migrate to the lower chamber are enumerated by a Coulter Counter (Coulter Electronics, Hialeah, FL).
  • Coulter Counter Coulter Electronics, Hialeah, FL.
  • AML14.3D10 expressing eotaxin receptor cells are labeled with [ 3 H] inositol (10 uCi/ml) for 24 hrs as described (Wu, D., et al. (1993) Science 261, 101-103).
  • Test compound and arious concentrations of eotaxin, MCP-3, or RANTES are then added to the cells for 30 min.
  • the cells are lysed in 10% perchloric acid, neutralized in 2 N KOH and centrifuged. The supernatant is transferred to columns containing 0.5 ml AG1-X8 anion exchange resin, washed with 6 ml borax buffer and eluted with 0.3 ml formic acid (0.1 M). The eluted samples are mixed with scintillation cocktail and counted.
  • AML14.3D10 expressing eotaxin receptor cells are subject to serum starvation for 16 hrs.
  • the cells are then mixed at a 3: 1 (v/v) ratio with low melting temperature agarose.
  • a 10 ul drop of the cell/agarose mixture is pipetted into a sterile Capsule Cup (Molecular Devices) at a cell density of approximately 200,000 cells/cup.
  • the cell/agarose drop forms a gel after about 5 min, and is assembled into the cup between two 3 um porosity polycarbonate membranes with running medium.
  • the assembled capsule cups is placed into the sensor chambers and then placed on the Cytosensor Microphysiometer (Molecular Devices) containing 1 ml of running medium.
  • the chambers are allowed to equilibrate for 1 hr at 37°C with a flow rate of 100 ul/min.
  • the experiment is initiated with an 8 min exposure of eotaxin, RANTES, MCP-3 and test compound at various concentrations and the acidification rate over baseline will be measured in the medium (McConnell, H.M., et al. (1992) Science 257, 1906-1912) until the cells return to the unstimulated level.
  • AML14.3D10 expressing eotaxin receptor cells are diluted in APA buffer (HBSS; 25mM Hepes; 0.2% BSA, pH7.2) at a concentration of 4 x lOfyml.
  • APA buffer HBSS; 25mM Hepes; 0.2% BSA, pH7.2
  • test compound and eotaxin, RANTES, or MCP-3 added into a 96-well plate and incubated at 37°C. 100 ml of cells are added to the plate and incubated for 20 sec to which 100 ml of APA cocktail (2 mis 8% formaldehyde; 460uLs 0.33uM Rhodamine- phalloidin; 1.85 mg 200ug/ml lysolecithin; 7.25 mis HBSS) is added.
  • characterization data observed may vary slightly according to and depending upon the particular assay or characterization method employed, and such expected variations or differences in the results are contemplated in accordance with the objects and practices of the present invention. It is intended, therefore, that the invention be defined by the scope of the claims which follow and that such claims be inte ⁇ reted as broadly as is reasonable.
  • CTGCTCTTCC TCGTCACCCT TCCATTCTGG ATCCACTATG TCAGGGGGCA TAACTGGGTT 300
  • ATCTTTTTCA TAATCCTGCT GACAATCGAC AGGTACCTGG CCATTGTCCA TGCTGTGTTT 420
  • GAGGGCTCTC CATTCCAGCC CAAGGAAGAC TAAGAATGAA TACCTCATGA GTATATTAGC 360
  • CTTCTTTCTT ATTGTTCTTA CTTATTTACG ATTACCCTAT CGTTTTCCCA AAATGTAAAA 960
  • TATATTGTTA TCATTATCTA GCCTGTTTTT TCCTGTTGTG TATTTCTTCC TTTAAATGCT 3300

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Abstract

Le récepteur d'éotaxine éosinophile a été isolé, cloné et séquencé. Ledit récepteur est un récepteur β-chémokine humain et a été baptisé 'CC CKR3'. Le récepteur d'éotaxine éosinophile peut être utilisé pour rechercher et identifier des composés qui se lient avec le récepteur d'éotaxine éosinophile. De tels composés seraient utiles pour la prévention et le traitement des maladies atopiques comme la rhinite allergique, la dermatite, la conjonctivite et, en particulier, l'asthme bronchique.
EP97925399A 1996-04-26 1997-04-24 Recepteur d'eotaxine eosinophile Withdrawn EP1012190A4 (fr)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US1711396P 1996-04-26 1996-04-26
US1615896P 1996-04-26 1996-04-26
US16158P 1996-04-26
US17113P 1996-04-26
GB9700894 1997-01-17
GBGB9700894.0A GB9700894D0 (en) 1997-01-17 1997-01-17 Eosinophil eotain receptor
PCT/US1997/006568 WO1997041154A1 (fr) 1996-04-26 1997-04-24 Recepteur d'eotaxine eosinophile

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EP1012190A1 true EP1012190A1 (fr) 2000-06-28
EP1012190A4 EP1012190A4 (fr) 2004-04-28

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US6537764B1 (en) 1995-01-19 2003-03-25 Children's Medical Center Corporation Method of identifying inhibitors of C—C chemokine receptor 3
WO1998050434A1 (fr) * 1997-05-07 1998-11-12 Schering Corporation Methodes relatives a un appauvrissement des eosinophiles et compositions afferentes
US6207155B1 (en) 1997-05-07 2001-03-27 Schering Corporation Method of eosinophil depletion with antibody to CCR 3 receptor
CA2235420A1 (fr) * 1998-06-17 1999-12-17 Paolo Renzi Oligonucleoside antisens pour traiter ou prevenir les maladies atopiques et la proliferation de cellules neoplastiques
US6358697B2 (en) 1999-04-21 2002-03-19 Children's Hospital Medical Center Intracellular pharmaceutical targeting
WO2003014153A2 (fr) * 2001-08-10 2003-02-20 Topigen Pharmaceutique Inc. Recepteurs viraux cellulaires et procedes d'utilisation correspondants
WO2006045202A1 (fr) 2004-10-29 2006-05-04 Topigen Pharmaceuticals Inc. Oligonucleotides antisens pour le traitement d'allergies et de proliferation de cellules neoplastiques
EP2285961A4 (fr) 2008-05-15 2012-10-17 Topigen Pharmaceuticals Inc Oligonucléotides pour le traitement de l inflammation et de la prolifération des cellules néoplasiques

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US5401629A (en) * 1990-08-07 1995-03-28 The Salk Institute Biotechnology/Industrial Associates, Inc. Assay methods and compositions useful for measuring the transduction of an intracellular signal
US6806061B1 (en) * 1995-01-19 2004-10-19 Children's Medical Center Corporation G protein-coupled receptor gene and methods of use therefor
US7265201B1 (en) * 1995-06-23 2007-09-04 Millennium Pharmaceuticals, Inc. Human chemotactic cytokine
US6265184B1 (en) * 1995-12-20 2001-07-24 Icos Corporation Polynucleotides encoding chemokine receptor 88C

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JP2002503950A (ja) 2002-02-05

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