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WO1993008469A1 - Polypeptides recombines de canaux calciques - Google Patents

Polypeptides recombines de canaux calciques Download PDF

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Publication number
WO1993008469A1
WO1993008469A1 PCT/US1992/009109 US9209109W WO9308469A1 WO 1993008469 A1 WO1993008469 A1 WO 1993008469A1 US 9209109 W US9209109 W US 9209109W WO 9308469 A1 WO9308469 A1 WO 9308469A1
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WIPO (PCT)
Prior art keywords
polypeptide
seq
immunoabsorbent
human
amino acid
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PCT/US1992/009109
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English (en)
Inventor
Stanley C. Froehner
Elizabeth L. R. Barry
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Trustees Of Dartmouth College
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Publication of WO1993008469A1 publication Critical patent/WO1993008469A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57423—Specifically defined cancers of lung
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • SCCL Small cell (or "oat cell”) carcinoma of the lung (SCCL) is a highly malignant tumor of epithelial cell origin, the development of which is often linked to cigarette smoking. These carcinoma are the most aggressive of lung tumors: they metastasize widely, and are essentially incurable by surgical means (see Robbins e_t al. - ⁇ Pathologic Basis of Disease (3rd Ed.) 1984 by the W.B. Saunders Co., Philadelphia, PA, p. 753).
  • This marker is a membrane-spanning, multisubunit protein that allows for the controlled entry of calcium ions into cells from the extracellular environment.
  • the opening of the channel to allow influx of calcium ions requires a depolarization to a certain level of the potential difference between the inside of the cell and the exteral medium, the rate of influx depending on this voltage difference.
  • the presence of SCCL may be detected in tissue specimens with antibodies specific for this protein (U.S. Patent No. 4,954,436). Patients with SCCL have a high incidence of
  • LES Lambert-Eaton Syndrome
  • LES a neuromuscular disease characterized by deficient neurotransmitter release from the neuromuscular presynaptic terminal (Lambert et al. (1982) Muscle Nerve 5:529-535). This condition appears to be the result of the action of anti-calcium channel autoantibodies developed in response to the presence of calcium channel protein on SCCL cells. In some cases, the appearance of LES precedes the diagnosis of the accompanying SCCL, suggesting that these calcium channel antibodies may be present before the tumor can be detected.
  • This invention pertains to an essentially pure recombinant alpha-1 polypeptide subunit, or portion thereof, of the human voltage-dependent calcium channel on small cell lung carcinoma.
  • An "essentially pure polypeptide” refers to a polypeptide that is substantially free of other peptide components such that it is considered homogeneous by SDS-PAGE, and/or can be unambiguously sequenced.
  • this recombinant polypeptide, or portion thereof is immunoreactive, and may have, as at least a portion of its amino acid sequence, the sequences set forth in the Sequence Listing as SEQ ID NO:l, 2, or 3.
  • the recombinant polypeptide of the invention may also be in the form of a fusion protein.
  • This invention also provides an isolated nucleic acid, such as DNA or expression vector, having a nucleotide sequence encoding the SCCL alpha-1 calcium channel polypeptide, or portion thereof.
  • the nucleic acid is a DNA having the sequences listed in the Sequence Listing as SEQ ID NOS:4, 5, and 6.
  • the recombinant polypeptides of the invention can be used as a part of an immunochemical assay for detecting autoantibodies to the human voltage- dependent calcium channel, and for diagnosing SCCL, neuroblastoma, and other tumors which express a surface protein recognized by antibodies specific for the voltage-sensitive calcium channel.
  • a preferred assay is a solid phase immunometric assay.
  • an immunoabsorbent is provided which includes a solid phase to which is attached the polypeptide of the invention.
  • the immunoabsorbent is incubated with a sample of a biological fluid (e.g., blood, serum, ascites, or plasma) taken from the subject, under conditions conducive for the binding of an anti-human calcium channel antibody to the immunoabsorbent-bound polypeptide.
  • the binding of the antibody to the immunoabsorbent is determined after separation of the immunoabsorbent from the sample.
  • the presence of the bound antibody is indicative of the presence of autoantibodies against the voltage- dependent calcium channel, and hence of SCCL in the subject.
  • the polypeptides of the invention can also be a part of a kit useful for detecting SCCL or Lambert-Eaton Syndrome.
  • This kit includes the solid-phase-bound polypeptide with which a sample from a potentially afflicted subject is contacted, and a labelled anti-human antibody, or a human antibody- binding portion thereof, used to detect the presence of human anti-calcium channel antibodies.
  • the recombinant polypeptides of the invention can also be used as a part of a method for detecting, diagnosing, and treating Lambert-Eaton Syndrome.
  • an immunoreactive, recombinant polypeptide of the invention, or immunoreactive portion thereof is provided and administered to a subject in an amount sufficient to block the binding of anti-calcium channel protein antibody to calcium channel protein on neurotransmitter-producing cells in the subject.
  • FIG. 1 is a schematic representation of the primers used for PCR, indicating the positions of the residues corresponding to the numbering in the skeletal muscle calcium channel and their locations in the putative domain structure of the channel;
  • 2 is a schematic representation of the overlapping products of PCR using (a) primers 1 (SEQ ID NO:7) and 3 (SEQ ID NO:9), (b) primers 1 (SEQ ID NO:7) and 4 (SEQ ID NO:10), (c) primers 2 (SEQ ID NO:8) and 3 (SEQ ID NO:9), and (d) primers 2 (SEQ ID NO:8) and 4 (SEQ ID NO:10).
  • the human voltage-dependent calcium channel protein consist of two large subunits, alpha-1 and alpha-2, having molecular weights of about 170 - 210 kilodaltons (kD) , and 150 - 170 kD, respectively, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions.
  • the invention is a DNA which codes for recombinant forms of the al ⁇ ha-1 subunit found on human SCCL.
  • Partial nucleic acid sequences and the corresponding amino acid sequences of the alpha-1 subunit of the voltage-dependent calcium channel expressed on human SCCL cells are shown in the Sequence Listing as SEQ ID NOS:l-3 and NOS:4-6, respectively. These partial sequences can be used as probes to isolate the entire gene sequence for the SCCL alpha-1 subunit. In addition, the partial and complete sequences may also be used to produce recombinant forms of the alpha-1 subunit but standard methodologies, including ligating the sequence into an expression vector, transforming an appropriate prokaryotic or eucaryotic host cell, culturing the cell to produce the polypeptide, possibly as a fusion protein, and then purifying the subunit from the cells.
  • the DNA encoding the recombinant SCCL alpha-1 polypeptides can be prepared as follows. Total cellular RNA is isolated from SCCL cells according to standard methods such as those described in Sambrook et al. (Molecular Cloning, A laboratory Manual,- 2nd Ed., Cold Spring Harbor Laboratory Press (1989) pp. 7.6-7.35). Poly A+ RNA is isolated and reversed transcribed and amplified by the polymerase chain reaction. Using the sequences available for other alpha-1 calcium channel polypeptides, such as the rabbit skeletal and cardiac muscle calcium channels, primers have been assigned that correspond to highly conserved regions of the alpha-1 subunit that are only poorly conserved with sodium channel sequences. These are depicted in FIG.
  • PCR polymerase chain reaction
  • the cDNA reversed transcribed from the SCCL RNA can be cloned directly into and amplified in a prokaryotic or eucaryotic host for amplification.
  • Expression systems such as expression vectors and host cells transformed with this alpha-1 calcium channel subunit-encoding DNA are also provided by the invention.
  • this invention is an essentially pure, recombinant form of the SCCL alpha-1 subunit.
  • An "essentially pure polypeptide" refers to a polypeptide that is substantially free of other peptide components such that it is considered homogeneous by SDS-PAGE, and/or can be unambiguosly sequenced. This polypeptide may be apart of a fusion protein containing, for example, a signal sequence or other peptide sequences.
  • the recombinant alpha-1 subunit of the voltage-dependent calcium channel can be used in immunochemical methods of detecting and diagnosising SCCL in a human subject.
  • immunochemical assays employing the polypeptides can take a variety of forms.
  • the preferred type is a solid phase immunometric assay.
  • the purified, recombinant SCCL alpha-1 polypeptide is immobilized on a solid phase to form an immunoabsorbent.
  • the immunoabsorbent is incubated with the biological sample to be tested, such as blood, serum, plasma, or any body fluid likely to have antibodies in it.
  • the incubation is performed under consitions suitable for the formation of an antigen-antibody complex between an anti-human calcium channel antibody in the fluid and an immunoabsorbent- bound polypeptide.
  • the immunoabsorbent is then separated from the sample, and a labeled anti-(human IgG) antibody is used to detect human anti-calcium channel antibody bound to the immunoabsorbent.
  • the amount of label associated with the immunoabsorbent is compared to positive and negative controls to assess the presence or absence of anti-calcium channel antibody, the presence of the bound antibody being indicative of the presence of SCCL in the subject.
  • the immunoabsorbent can be prepared by adsorbing or coupling purified polypeptide to a solid phase.
  • Various solid phases can be used, such as beads formed of glass, polystyrene, polypropylene dextran, or other material.
  • Other suitable solid phases include tubes or immunotitre plates formed from or coated with these materials.
  • the recombinant polypeptides can be either covalently or non-covalently bound to the solid phase.
  • the polypeptides can be covalently bonded to the solid phase using techniques involving amide or ester linkage or adsorption.
  • the solid phase can be post-coated with an animal protein, e.g., 3% fish gelatin or bovine serum albumin. This provides a blocking protein which reduces nonspecific adsorption of protein to the immunoabsorbent surface.
  • the immunoabsorbent functions to insolubilize anti-calcium channel antibody in the liquid sample tested.
  • the immunoabsorbent is incubated with blood plasma or serum. Before incubation, plasma or serum is diluted with normal animal plasma or serum.
  • the diluent plasma or serum is derived from the same animal species that is the source of the anti-(human IgG) antibody.
  • the preferred anti-(human IgG) antibody is goat anti-(human IgG) antibody.
  • the diluent would be goat serum or plasma.
  • the optical dilution factor for human plasma and serum is about 10-11 fold.
  • incubation e.g. pH and temperature
  • duration of incubation can be optimized by routine experimentation. Generally, the incubation will be run for 1-2 hours at about 45°C, in a buffer having a pH of about 7 - 8.
  • the immunoabsorbent and the sample are separated. Separation can be accomplished by any conventional technique such as sedimentation or centrifugation. The immunoabsorbent then is washed free of sample to eliminate any interfering substances.
  • the immunoabsorbent is incubated with the labelled anti-(human IgG) antibody (tracer). Generally, the immunoabsorbent is incubated with a solution of the labeled anti-(human IgG) antibody which contains a small amount (about 1%) of the serum or plasma of the animal species which serves as the source of the anti-(human IgG) antibody.
  • Anti-(human IgG) antibody can be obtained from any animal source. However, goat or rabbit anti-(human IgG) antibody is preferred.
  • the anti-(human IgG) antibody can be an antibody against the Fc fragment of human IgG, for example, goat anti-(human IgG) Fc antibody.
  • the anti-(human IgG) antibody or anti-(human IgG)Fc can be labelled with a radioactive material such as 125 Iodine; labeled with an optical label, such as a fluorescent material; or labeled with an enzyme such as horseradish peroxidase.
  • a radioactive material such as 125 Iodine
  • an optical label such as a fluorescent material
  • an enzyme such as horseradish peroxidase.
  • the anti-human antibody can also be biotinylated and labeled avidin used to detect its binding to the immunoabsorbent.
  • the immunoabsorbent After incubation with the labeled antibody, the immunoabsorbent is separated from the solution and the label associated with the immunoabsorbent is evaluated. Depending upon the choice of label, the evaluation can be done in a variety of ways.
  • the label may be detected by a gamma counter if the label is a radioactive gamma emitter. Alternatively, the label may be detected by a fluorimeter, if the label is a fluorescent material. In .the case of an enzyme label detection may be done colorimetrically employing a substrate for the enzyme.
  • the amount of label associated with the immunoabsorbent is compared with positive and negative controls in order to determine the presence of anti-calcium channel antibody.
  • the controls are generally run concomitantly with the sample to be tested.
  • a positive control is a serum containing antibody against calcium channel;
  • a negative control is a serum from healthy individuals which do not contain antibody against calcium channel protein.
  • reagents for the performance of the immunometric assay can be assembled in assay kits.
  • a kit for screening blood can include: (a) an immunoabsorbent such as, e.g., a polystyrene bead coated with a recombinant alpha-1 subunit of the calcium channel protein;
  • a diluent for the serum or plasma sample such as, e.g., normal goat serum or plasma
  • an anti-(human IgG) antibody such as, e.g., goat anti-(human IgG) antibody in buffered, aqueous solution containing about 1% goat serum or plasma
  • a positive control i.e., serum containing antibody against the calcium channel protein
  • a negative control such as, e.g., serum from healthy individuals which does not contain antibody against the calcium channel protein.
  • an additional element of the kit can be the substrate for the enzyme.
  • Another type of assay for anti-calcium channel antibody is an antigen sandwich assay. In this' assay, a labelled recombinant calcium channel polypeptide of this invention is used in place of anti-(human IgG) antibody to detect anti-calcium channel antibody bound to the immunoabsorbent.
  • the assay is based in principle on the bivalency of antibody molecules. One binding site of the antibody binds the antigen affixed to the solid phase; the second is available for binding the labeled antigen.
  • the assay procedure is essentially the same as described for the immunometric assay except that after incubation with the sample, the immunoabsorbent is incubated with a solution of labeled core polypeptide.
  • the calcium channel polypeptide can be labeled with radioisotope, an enzyme, etc. for this type of assay.
  • Protein A which binds the Fc segment of an IgG molecule without interfering with the antigen antibody interaction can be used as the labeled tracer to detect anti-calcium channel antibody adsorbed to the immunoabsorbent. Protein A can be readily labeled with a radioisotope, enzyme, or other detectable species.
  • an additional use of the recombinant alpha-1 polypeptide is in the treatment of Lambert-Eaton syndrome.
  • the polypeptide is administered to an afflicted subject, with a pharmaceutically aceptible carrier, in an amount sufficient to block the binding of anti-calcium channel protein antibody to calcium channel protein on neurotransmitter producing cells in the subject.
  • Effective dosages of the recombinant. polypeptide and modes of its administration in the treatment of LES can be determined by routine experimentation.
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile.
  • the carrier can be a solvent or dispersion medium.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and " antifungal agents.
  • Prolonged absorption of the injectable therapeutic agents can be brought about by the use in the compositions of agents delaying absorption.
  • Sterile injectable solutions are prepared by incorporating the recombinant polypeptide in the required amount in the appropriate solvent, followed by filtered sterilization.
  • the recombinant polypeptide may be administered parenterally or intraperitoneally.
  • Solutions of the therapeutic agent as pharmacologically acceptable salts can be prepared in water or some other physiologically acceptable solvent.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • Supplementary active ingredients can also be incorporated into the compositions.
  • RNA analyzed on a formaldehyde/ agarose gel migrated as a broad smear from much greater than 9.5 kilobases (kb) to about 0.25 kb.
  • kb kilobases
  • several discrete bands of RNA were seen within the smear.
  • the RNA was analyzed by Northern blotting (see, e.g., Goldberg (1980) Proc. Natl. Acad. Sci. (U.S.A.) 5794) and hybridized to a radioactive probe for actin. A single sharp band was obtained, indicating that the RNA was undegraded.
  • This RNA was used for the determination of SCCL calcium channel sequences using the Polymerase Chain Reaction (PCR) as described below.
  • RNA from a mouse muscle cell line (C2) which were induced to form myotubes in culture (Silberstein (1981) Nature 295:143-145). This RNA was used as a control in the PCR reaction since it is expected to express a muscle calcium channel closely related to the rabbit muscle channel.
  • Each PCR reaction requires a pair of primers which flank the region of DNA to be amplified and hybridize to opposite strands.
  • primers were designed, which could be used in four combinations for PCR reactions. Their sequences were based on regions which are perfectly or almost perfectly conserved between the rabbit skeletal muscle and cardiac calcium channels (94 to 100% idential with any differences near the 5' end of the primer). The sequence homology between calcium and sodium channels in these regions ranges from approximately 50% to 60% (Tanabe (1987) Nature 228:313-318 and Trimmer (1989) Neuron 3.:33-49). Thus, these primers are specific for calcium channels relative to other types of cation channels. The position of the residues indicated in FIG. 1 below correspond to the numbering in the skeletal muscle calcium channel (Tanabe (1987) Nature 328:313-318) . Their location of these primers in the putative domain structure of the channel is noted in parenthesese.
  • restriction sites One of two types of restriction sites (underscored) were included near the 5* ends of the primers to facilitate directional subclong: ECP Rl sites for the two primers hybridizing to the sense strand; and BAM HI sites for the two primers hybridizing to the antisense strand.
  • ECP Rl sites for the two primers hybridizing to the sense strand
  • BAM HI sites for the two primers hybridizing to the antisense strand.
  • four nucleotides were included 5' to the restriction sites to insure that cutting could occur at these sites.
  • Other features which were taken into consideration in the design of these primers were the G + C composition (close to 50% to maximize specificity) , and complementarity of primer pairs which was minimized to avoid formation of primer dimers. These primers were synthesized locally, and purified on Nensorb prep columns (Johnson (1990) Biotechniques 8:424-429).
  • RNA purified from cultured cells as described above, was reverse transcribed in order to make cDNA templates for PCR Current Protocols in Molecular Biolo ⁇ v, (1989) Ausubel et al. (eds.)), John Wiley and Sons, New York, pp. 5.5.2-5.5.4). Random hexamers were used to prime the reverese transcriptase reaction. The cDNAs were purified by phenol extraction and aliquots were used for PCR reactions. PCR was performed on an automated instrument
  • Positive and negative controls were included in each PCR run.
  • a clone (1 ng) containing the entire rabbit skeletal muscle calcium channel cDNA (Ellis (1988) Science 141:1661-1664) was used as template for a positive control.
  • cDNA synthesized from poly A+RNA isolated from C2 cells was used as template since this muscle cell line is expected to express a calcium channel with high homology to the rabbit skeletal muscle channel.
  • Two negative controls were also used: in one no template DNA was included in the reaction mixture, and in the other the template was the products isolated from a mock reverse transcriptase reaction in which only the enzyme was left out. The products of the PCR reactions were analyzed by electrophoresis on 3% Nuseive, 1% Seakem agarose gels in order to visualize small DNA products.
  • primers 1 SEQ ID NO:l
  • 3 SEQ ID NO:3
  • primers 1 SEQ ID NO:l
  • 4 SEQ ID NO:4
  • primers 2 SEQ ID NO: . and 3 (SEQ ID NO:3):
  • H146- a doublet at around 390 bp and another band at approximately 250 bp
  • the PCR products from two of the sets of primer pairs (1 (SEQ ID NO:l) and 3 (SEQ ID NO:3) and 2 (SEQ ID NO:2) and 3 (SEQ ID NO:3)) were subcloned and sequenced. This region corresponds to the entire fourth domain of the calcium channel.
  • the PCR products were purified by phenol extraction and ethanol precipitation and digested with ECO Rl and Bam HI. The products of the restriction digest were then isolated as a band from a low melt
  • the Bluescript vector (Stratagene) was also digested with ECO Rl and BAM HI and isolated on a low melt agarose gel. The vector and PCR product were ligated and used to transform XL-1 Blue bacteria. The sequences of the recombinant clones were determined by the dideoxynucleotide chain termination procedure (Sanger e al. (1971) Proc. Natl. Acad. Sci. (USA) 74.:5463-5467) . Initially, the SK and KS Bluescript primers were used to sequence in from the ends of the inserts.
  • the SCCL-B (SEQ ID NO:6) and SCCL-C (SEQ ID NO:7) clones are.highly homologous to each other and to the skeletal muscle calcium channel.
  • the homology between the two SCCL clones is 90% (nucleotide) and 95% (amino acid) .
  • Both of these clones are approximately 90% (nucleotide) and 95% (amino acid) homologous to the skeletal muscle calcium channel in the region betweenprimers 1 and 3.
  • the degree of homology to the cardiac channel is considerably less: 79% (nucleotide) and 75% (amino acid) .
  • the gap between domains IVS3 and IVS4 is larger: a stretch of 19 amino acids is missing from the SCCL clones but present in the skeletal channel. Compared to the cardiac sequence, the SCCL clones are missing only 11 amino acids in this region since the cardiac is already 8 short of the skeletal channel. The 2 amino acids between IVS5 and IVS6 unique to the cardiac channel are also not found in the SCCL clones. Finally, SCCL-B (SEQ ID NO:9) is missing a single isolated amino acid which is found in all the other types of calcium channels. Thus, while the SCCL clones are highly homologous to the skeletal channel, they appear to have features unique to SCCL. Furthermore, it is expected that the region sequenced here will be one of the most conserved in the channels. The amino and carboxy- terminal portions of the channels may be much less strongly conserved, as is seen in a comparison of the cardiac and skeletal muscle channels.
  • SCCL-A The third type of SCCL clone (SCCL-A) was significantly less homologous to cardiac and skeletal muscle calcium channels than were SCCL-B and SCCL-C. It was found as a product of only one primer pair (2 (SEQ ID NO:2) and 3 (SEQ ID NO:3)) but not the other (1 (SEQ ID NO:l and 3 (SEQ ID NO:3)), and therefore only the sequence in this smaller region could be compared. In addition, primer 2 (SEQ ID NO:2) was able to prime at a second site giving rise to both the 250 bp product and the larger product of the doublet around 390 basepairs seen when PCR was performed on SCCL cDNA.
  • the degreee of homology to the skeletal channel was 70% (nucleotide) and 60% (amino acid). However, there are two gaps in the sequence comparison in this region: (1) 8 amino acids which are found only in the SCCL-A clone; and (2) 3 amino acids which are absent in the SCCL-A clone.
  • the SCCL clone (SCCL-A) has sequence homology with a more recently characterized rabbit brain calcium channel (Mori et al. (1991) Nature 3 ⁇ :398-402). The nucleic acid sequence identity is 89.7%, and the amino acid sequence identity is 98.2%. Therefore, this clone is thought to represent a human neuronal calcium channel.
  • ORGANISM Oryctolagus cuniculus
  • F TISSUE TYPE: Skeletal muscle
  • ORGANISM Oryctolagus cuniculus
  • ORGANISM Oryctolagus cuniculus
  • TISSUE TYPE skeletal muscle
  • FEATURE FEATURE:
  • ORGANISM Oryctolagus cuniculus
  • TISSUE TYPE skeletal muscle
  • FEATURE FEATURE:
  • ORGANISM Homo sapiens
  • TISSUE TYPE small cell lung carcinoma
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:6:
  • MOLECULE TYPE peptide
  • HYPOTHETICAL YES
  • FRAGMENT TYPE internal
  • ORIGINAL SOURCE
  • ORGANISM Homo sapiens
  • TISSUE TYPE Small Cell Lung Carcinoma •
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:9:

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Abstract

L'invention concerne une sous-unité polypeptidique alpha-1 immunoréactive, recombinée ou une partie de celle-ci, de la protéine de canaux calciques sensible à la tension, exprimée sur l'épithélioma à petites cellules des bronches de l'homme. L'invention concerne également un acide nucléique isolé codant cette polypeptide et un vecteur d'expression contenant un tel acide nucléique. En outre, des procédés sont décrits, selon lesquels on utilise cette polypeptide pour la détection d'anticorps dirigés contre la protéine de canaux calciques, pour le diagnostic de l'épithélioma à petites cellules des bronches chez l'homme, et également pour la détection et le traitement du syndrome de Lambert-Eaton.
PCT/US1992/009109 1991-10-23 1992-10-21 Polypeptides recombines de canaux calciques WO1993008469A1 (fr)

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US5618720A (en) * 1988-04-04 1997-04-08 Sibia Neurosciences, Inc. Cells expressing calcium channel α2 subunit-encoding DNA, optionally with a reporter gene for screening assays
US5686241A (en) * 1988-04-04 1997-11-11 Sibia Neurosciences, Inc. Probes and assays for calcium channel α2 subunit-encoding nucleic acids
US5726035A (en) * 1990-02-20 1998-03-10 Sibia Neurosciences, Inc. Recombinant production of mammalian calcium channel gamma subunits
US5792846A (en) * 1988-04-04 1998-08-11 Sibia Neurosciences, Inc. Human calcium channel compositions and methods
US5846757A (en) * 1988-04-04 1998-12-08 Sibia Neurosciences, Inc. Human calcium channel α1, α2, and β subunits and assays using them
US5851824A (en) * 1988-04-04 1998-12-22 Sibia Neurosciences, Inc. Human calcium channel α-1C/α-1D, α-2, β-1, and γsubunits and cells expressing the DNA
US5874236A (en) * 1988-04-04 1999-02-23 Sibia Neurosciences. Inc. DNA encoding human calcium channel α-1A, β1, β-2, and β-4 subunits, and assays using cells that express the subunits
US6090623A (en) * 1993-08-11 2000-07-18 Merck & Co., Inc. Recombinant human calcium channel β4 subunits
US6096514A (en) * 1988-04-04 2000-08-01 Sibia Neurosciences, Inc. Human calcium channel compositions and methods
US6387696B1 (en) 1988-04-04 2002-05-14 Merck & Co., Inc. Human calcium channel compositions and methods
US6528630B1 (en) 1997-12-03 2003-03-04 Merck & Co., Inc. Calcium channel compositions and methods
US6653097B1 (en) 1991-08-15 2003-11-25 Merck & Co., Inc. Human calcium channel compositions and methods
US7414110B2 (en) 1988-04-04 2008-08-19 Merck & Co., Inc. Human calcium channel compositions and methods

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US6013474A (en) * 1988-04-04 2000-01-11 Sibia Neurosciences, Inc. Calcium channel compositions and methods
US6096514A (en) * 1988-04-04 2000-08-01 Sibia Neurosciences, Inc. Human calcium channel compositions and methods
US7414110B2 (en) 1988-04-04 2008-08-19 Merck & Co., Inc. Human calcium channel compositions and methods
US5792846A (en) * 1988-04-04 1998-08-11 Sibia Neurosciences, Inc. Human calcium channel compositions and methods
US5846757A (en) * 1988-04-04 1998-12-08 Sibia Neurosciences, Inc. Human calcium channel α1, α2, and β subunits and assays using them
US5851824A (en) * 1988-04-04 1998-12-22 Sibia Neurosciences, Inc. Human calcium channel α-1C/α-1D, α-2, β-1, and γsubunits and cells expressing the DNA
US5686241A (en) * 1988-04-04 1997-11-11 Sibia Neurosciences, Inc. Probes and assays for calcium channel α2 subunit-encoding nucleic acids
US7063950B1 (en) 1988-04-04 2006-06-20 Harpold Michael M Nucleic acids encoding human calcium channel and methods of use thereof
US5618720A (en) * 1988-04-04 1997-04-08 Sibia Neurosciences, Inc. Cells expressing calcium channel α2 subunit-encoding DNA, optionally with a reporter gene for screening assays
US5876958A (en) * 1988-04-04 1999-03-02 Sibia Neurosciences, Inc. Assays of cells expressing human calcium channels containing α1 β subunits
US5874236A (en) * 1988-04-04 1999-02-23 Sibia Neurosciences. Inc. DNA encoding human calcium channel α-1A, β1, β-2, and β-4 subunits, and assays using cells that express the subunits
US6387696B1 (en) 1988-04-04 2002-05-14 Merck & Co., Inc. Human calcium channel compositions and methods
US5726035A (en) * 1990-02-20 1998-03-10 Sibia Neurosciences, Inc. Recombinant production of mammalian calcium channel gamma subunits
US6653097B1 (en) 1991-08-15 2003-11-25 Merck & Co., Inc. Human calcium channel compositions and methods
US6090623A (en) * 1993-08-11 2000-07-18 Merck & Co., Inc. Recombinant human calcium channel β4 subunits
US6528630B1 (en) 1997-12-03 2003-03-04 Merck & Co., Inc. Calcium channel compositions and methods

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