[go: up one dir, main page]

WO2001083551A1 - Reactif destine a la detection de biomolecules et son utilisation - Google Patents

Reactif destine a la detection de biomolecules et son utilisation Download PDF

Info

Publication number
WO2001083551A1
WO2001083551A1 PCT/SE2001/000941 SE0100941W WO0183551A1 WO 2001083551 A1 WO2001083551 A1 WO 2001083551A1 SE 0100941 W SE0100941 W SE 0100941W WO 0183551 A1 WO0183551 A1 WO 0183551A1
Authority
WO
WIPO (PCT)
Prior art keywords
domain
reagent according
receptor
reagent
binding domain
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.)
Ceased
Application number
PCT/SE2001/000941
Other languages
English (en)
Inventor
Lena Carlsson
Björn CARLSSON
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sahltech i Goteborg AB
Original Assignee
Sahltech i Goteborg AB
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Sahltech i Goteborg AB filed Critical Sahltech i Goteborg AB
Priority to AU2001256892A priority Critical patent/AU2001256892A1/en
Publication of WO2001083551A1 publication Critical patent/WO2001083551A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • 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
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • 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
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • 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
    • G01N33/531—Production of immunochemical test materials
    • 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
    • G01N33/566—Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/485—Epidermal growth factor [EGF] (urogastrone)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/475—Assays involving growth factors
    • G01N2333/505—Erythropoietin [EPO]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/61—Growth hormones [GH] (Somatotropin)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/71—Assays involving receptors, cell surface antigens or cell surface determinants for growth factors; for growth regulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72—Assays involving receptors, cell surface antigens or cell surface determinants for hormones

Definitions

  • the present invention relates to a new cell-free reagent for detection of biomolecules, and in particular of receptor ligands.
  • the invention also relates to use of said reagent .
  • re- ceptors where activation is dependent receptor dimerization include receptors of the cytokine receptor superfa- mily and the tyrosine kinase superfamily.
  • Receptors of the receptor tyrosine kinase family are single-pass transmembrane proteins with intrinsic enzymatic activity.
  • the receptors consist of an extracellular domain which binds ligands, a transmembrane domain and a cytoplasmatic or intracellular domain which has enzymatic activity.
  • the cytoplasmatic domain of the tyrosine kinase receptors can catalyze the transfer of phosphate from ATP to tyrosine residues in protein substrates. Activation of the receptors is initiated by ligand binding, which facilitates receptor dimerization and results in phosphorylation of tyrosine residues in the intracellular domain of the receptor protein.
  • the phosphorylated tyrosine residues en- hance the catalytic activity of the receptor or serve as docking sites for downstream signaling molecules.
  • the tyrosine kinase receptor superfamily includes several growth factor receptors, e.g. receptors for epidermal growth factor (EFG) , fibroblast growth factor, platelet derived growth factor, and vascular endothelial growth factor.
  • Receptors of the cytokine superfamily are single chain transmembrane receptors consisting of an extracellular ligand-binding domain, a transmembrane domain and an intracellular cytoplasmatic domain.
  • the GH receptor (GH-R) is a member of the cytokine receptor super-family, which also includes, amongst others, the receptors for prolactin, leptin, erythropoietin, macrophage colony- stimulating factor, interleukins and leukemia inhibitor factor.
  • the cytoplasmatic domain of the cytokine receptors lacks intrinsic enzymatic activity.
  • WO 94/29458 describes hybrid receptor molecules wherein one domain of the receptor is derived from the cytokine superfamily of receptors and another domain is derived from an unrelated family of receptors. One of the domains is a receptor extracellular domain and the other is a receptor intracellular domain. These hybrid receptor molecules also comprises an transmembrane domain derived from the same receptor as either the intracellular or extracellular domain, or it may be derived from a third receptor source.
  • hybrid receptor molecules according to WO 94/29458 are then transfected into host cell lines before they can be used for, for example, screening for receptor ligands.
  • a major disadvantage with these hybrid receptor molecules is that they are transfected into cells and must be used as a cell culture, and thus cannot be used in solution.
  • the object of the present invention is to provide a reagent based on type of hybrid receptor that can be used in solution.
  • the method according to the present invention is based on the use of a dimerizable, or optionally oli- gomerizable, molecule, preferably a biomolecule, comprising or consisting of a soluble chimeric receptor or part of such a receptor linked to a molecule which has an activity or other feature that is changed after dimeriza- tion of two such biomolecules .
  • the reagent according to the present invention has the advantage, compared to hitherto utilized reagents such as antibodies, that it recognizes molecules that activate or inhibit receptors.
  • Another advantage of the invention is that it is possible to use the chimeric receptor in solution instead of on cells or transfected into cells to obtain a signal caused by a ligand for the receptor.
  • the inventors have also for the first time shown that is that it is not necessary to include the transmem- brane domain of a membrane-bound receptor in a reagent.
  • the reagent according to the invention can be used to measure the concentration of biochemically active ligand.
  • the reagent according to the present invention may replace the cell based high throughput screening systems commonly used in pharmaceutical research to identify candidates for drug development.
  • the present invention relates to a cell-free reagent for the detection of target molecules, said reagent comprising a binding domain linked to a reporter domain, wherein said binding domain has affinity to said target molecule and will bind to said target molecule when brought in contact therewith, and wherein the binding of said binding domain to said target molecule will induce dimerization or oligomerization of the reagent molecule with another reagent molecule with a similar binding domain, and wherein said reporter domain will induce a measurable signal upon said dimerization or oli- gomerization
  • the invention also relates to a method for determination of the presence and/or the concentration of a specific target molecule in a sample, such as a body fluid, or more precisely to the use of the above mentioned rea- gent for the determination of the presence and/or the concentration of a specific target molecule in a sample.
  • the invention also relates to a method for screening of potentially pharmaceutical active biomolecules, or more precisely to the use of the above mentioned reagent for the screening of potentially pharmaceutical active biomolecules .
  • the invention relates to reagent for detection of a target molecule, said reagent comprising a binding domain linked to a reporter domain, wherein said binding domain has affinity to said target molecule and will bind to said target molecule when brought in contact therewith, and wherein the binding of said binding domain to said target molecule will induce dimerization or oligomerization of the reagent molecule with another reagent molecule with binding domain, and wherein said reporter domain will induce a measurable signal upon said dimerization or oligomerization.
  • the binding domain of the reagent according to the invention may be any kind of molecule or structure that has affinity for the target molecule to be detected. It may, for example, be any kind of receptor or part of receptor. When the binding domain is constituted of a part of a receptor, this part should comprise the specific binding site of the receptor in question.
  • a receptor suitable for forming the binding domain of the reagent according to the invention are growth hormone receptors (GH-R) , erythropoietin receptors and EGF receptors (EGF-R) .
  • the reporter domain of the reagent according to the invention may be any kind of molecule or structure that in some way will lead to a measurable signal upon dimeri- zation, or oligomerization, of the reagent.
  • the reporter domain should thus have an intrinsic activity or other feature which is possible to detect after dimerization. It may also have a measurable activity prior to dimerization, and in this case the activity will be altered by the dimerization.
  • the target molecule is a biomolecule, which must be capable of dimerizing or oli- gomerizing the reagent.
  • Non-limiting examples of such ligands are hormones, receptor agonists, receptor antagonists, antigens, antibodies, and non-peptide molecules.
  • binding domain and the reporter domain of the reagent according to the invention may be linked directly to each other. They may also be linked to each other via any kind of spacer, provided that this spacer does not have any negative influence on the affinity of the bind- ing domain to the target molecule or on the dimerization or oligomerization of the reporter domain or on the inducement of the measurable signal .
  • the measurable signal induces upon dimerization or oligomerization may e.g. be the activation of an enzyme, which modifies a substrate.
  • the modified subtrate may be detected by several methods known to man skilled in the art, e.g. spectrophotometrically or as incorporated radioactivity.
  • the sample analyzed with the reagent according to the invention may be any fluid sample, such as a body fluid or a solution of different biomolecules.
  • the reagent consists of a fusion protein formed between the extracellular domain of the human growth hormone receptor linked to the intracellular domain of the human epidermal growth factor receptor, for which the intrinsic enzymatic activity is activated when two such molecules are dimerized by a ligand.
  • Another example of specific reagent according to the invention is a fusion protein between the extracellular domain of a cytokine receptor linked to the intracellular domain of a tyrosine kinase receptor, for which the intrinsic enzymatic activity is activated when two such molecules are dimerized by a ligand.
  • the reagent may be used in the form of a solution. However, in some cases it may be advantageous to attach the reactant to a solid carrier, such as a plate or test tube .
  • the reagent according to the invention is suitable for detection and concentration determination of a spe- cific target molecule in a sample.
  • the reagent according to the invention may also be used in assays for measuring the concentration of the target molecule, wherein the reagent replaces the traditionally used antibodies.
  • the reagent is exposed to unknown amounts of the target molecule in a sample and the signal produced is compared to the signal in a standard curve constructed by activation of the fusion protein by known amounts of a target molecule.
  • Fig. 1 illustrates Xba-Kpn I and Hind III-Kpn I digestion of pAL-1-48. Clone number identifies the specific colony from which the plasmid was purified and "uncut" refers to undigested plasmid;
  • Fig. 2 illustrates Xba I -Hind III digestion of pAL-1-48;
  • Fig. 3 illustrates detection of GH using cell lysates containing a recombinant reagent consisting of a
  • GH-R extracellular domain/EGF-R intracellular domain fusion protein GH-R extracellular domain/EGF-R intracellular domain fusion protein
  • Fig. 4 illustrates detection of GH using purified recombinant reagent consisting of a GH-R extracellular domain/EGF-R intracellular domain fusion protein.
  • GH-R growth hormone receptor
  • hGH-R human growth hormone receptor
  • EGF-R epidermal growth factor receptor
  • Example I a reagent consisting of a
  • GH-R/EGF-R fusion protein was produced.
  • a DNA construct was made with a cDNA sequence corresponding to the extracellular domain of the human GH-R (hGH-R) ligated to a cDNA se- quence corresponding to the intracellular domain of the human epidermal growth factor receptor (hEGF-R) .
  • the recombinant GH-R/EGF-R chimeric receptor was expressed in a baculovirus system using methods known to man skilled in the art .
  • Taq extender buffer (10 mM KCl, 20 mM Tris-HCl (pH 8,8), 10 mM NH 4 S0 4/ 2 mM MgS0 4 , 0,1% triton X-100, 0.1 mg/ml BSA) , a mixture of Taq polymerase (2.5 U) (Boehringer Mannheim, Mannheim, Germany) and Pfu (1.25 U) (Stratagene, La Jolla, CA, U.S.A.), primers (50 ⁇ M each), human liver cDNA, dNTP (0.2 mM each), using a PCR GeneAmp System 9600 (Perkin Elmer, Foster City, CA, U.S.A.), programmed for 20 cycles with a denaturation step at 94°C for 20 s, annealing at 55°C for 20 s and an extension step at 72 °C for 1 min and 30 s.
  • PCR GeneAmp System 9600 Perkin Elmer, Foster City, CA, U.
  • the hEGF-R was cloned by PCR using primers 5 ' - AGGTACCCGAAGGCGCCACATC GTT-3 ' and 5'-
  • TCTCAAGCTTTCAGTGATGGTGATGGTGATGTGCTCCAATAA ATTCACT-3 ' (the underlined sequences contain restriction enzyme sites for Kpn I and Hind III, respectively and the bold sequence encodes a repeat of six histidine residues) , based on the nucleotide sequence of the hEGF-R, using cDNA as template. Amplification was done as described above. The PCR GeneAmp System was this time programmed for 20 cycles with a denaturation step at 94°C for 20 s, annealing at 55°C for 20 s and an extension step at 72 °C for 3 min. The primers used were purchased from Scandinavian Gene Synthesis AB, K ⁇ ping, Sweden.
  • the hGH-R and EGF-R DNA fragments were cloned into pCRII (Invitrogen, San Diego, CA, U.S.A.), yielding pAL ⁇ 1-26 and pAL-1-32, respectively.
  • the E. coli strain DH5- alpha was used for amplification of plasmid DNA, using the expression of plasmidic lacZ indicator gene for selection of colonies containing the insert. Transformation and plasmid purification (QIAprep Spin Miniprep Kit,
  • the plasmid pAL-1-26 was digested with Kpn I and Xba I and the released fragment was gel-purified and sub- cloned into a Kpn I- Xba I digested pFAST (Gibco, Pais- ley, UK.), yielding the plasmid pAL-1-38.
  • E. coli strain DH5-alpha was used for amplification of plasmid DNA.
  • restriction enzyme digestion was performed. Hind III-Bam HI digestion revealed an insert of expected size and Hind III-Eco RV digestion verified correct orientation of the fragment .
  • the pAL-1-32 plasmid was digested with Kpn I- Hind III, the released fragment was gel purified and subcloned into a Kpn I- Hind III digested pAL-1-38 yielding the plasmid pAL-1-48.
  • digestion was done with Xba I -Kpn I (releasing the GH-R fragment) , Kpn I -Hind III (releasing the EGF-R fragment) (Fig. 1) and Xba I-Hind III that released the fusion protein DNA construct (Fig. 2) . Clone 9 was selected for further work.
  • Recombinant protein was expressed using baculovirus, Bac-to-Bac expression system (GIBCO, Paisley, U.K.) using methods known to man skilled in the art . All reagents for cell culture and transfection were from GIBCO.
  • a cell free bioassay for human GH was developed using a reagent according to the invention.
  • a reagent according to the invention In contrast to the cytokine receptors (e.g. GH-R), receptors of the tyrosine kinase family (e.g. EGF-R) possess intrinsic tyrosine kinase activity and are therefore not dependent on other proteins for enzyme activity. Therefore, a GH-R extracellular domain/EGF-R intracellu- lar domain fusion protein was used as the reagent to detect GH in a sample.
  • Lysates from cells expressing the recombinant GH- R/EGF-R fusion protein was added to samples containing recombinant human GH and to control samples containing no GH.
  • a solution containing a biotinylated peptide substrate and radiolabeled ATP was added to the samples. After incubation at 30°C for 15 min, the reaction was stopped by adding termination buffer. The samples were pipetted on to streptavidin coated membranes. The mem- branes were washed and the radioactivity was counted. As shown in Fig. 3, the presence of GH in the samples resulted in increased phosphorylation of the substrate.
  • the recombinant GH-R extracellular domain/EGF-R intracellular domain fusion protein was purified from lysates from cells expressing the fusion protein using Ni-NTA columns (Qiagen, Germany) and the repeats of histidine residues (see Example I) as affinity tags.
  • Purified recombinant GH-R/EGF-R fusion protein was added to samples containing recombinant human GH and to control samples containing no GH.
  • a solution containing a biotinylated peptide substrate and radiolabeled ATP was added to the samples. After incubation at 30°C for 15 min, the reaction was stopped by adding termination buffer. The samples were pipetted on to streptavidin coated membranes. The membranes were washed and the ra- dioactivity was counted. As shown in Fig. 4, the presence of GH in the samples resulted in increased phosphoryla- tion of the substrate.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • Organic Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • Endocrinology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un réactif acellulaire destiné à la détection d'une molécule cible. Ce réactif comprend un site de liaison lié à un site rapporteur. Le site de liaison a une affinité à la molécule cible et s'y lie lorsqu'il est à son contact. La liaison du site de liaison à la molécule cible va induire la dimérisation d'une molécule réactif avec une autre molécule réactif avec un site de liaison similaire, et comme résultat le site rapporteur va induire un signal mesurable. Le site de liaison peut notamment être le site extracellulaire d'un récepteur, et la molécule rapporteur peut être le site intracellulaire d'un autre récepteur. L'invention concerne enfin l'utilisation de ce récepteur pour la détermination de la présence et/ou du niveau de la molécule cible présent(e) dans un échantillon fluide et pour le criblage de biomolécules éventuellement pharmaceutiquement actives.
PCT/SE2001/000941 2000-05-04 2001-05-03 Reactif destine a la detection de biomolecules et son utilisation Ceased WO2001083551A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001256892A AU2001256892A1 (en) 2000-05-04 2001-05-03 Reagent for detection of biomolecules, and use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
SE0001642-8 2000-05-04
SE0001642A SE0001642D0 (sv) 2000-05-04 2000-05-04 Reagent for detection of biomolecules, and use thereof

Publications (1)

Publication Number Publication Date
WO2001083551A1 true WO2001083551A1 (fr) 2001-11-08

Family

ID=20279543

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/SE2001/000941 Ceased WO2001083551A1 (fr) 2000-05-04 2001-05-03 Reactif destine a la detection de biomolecules et son utilisation

Country Status (3)

Country Link
AU (1) AU2001256892A1 (fr)
SE (1) SE0001642D0 (fr)
WO (1) WO2001083551A1 (fr)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7404950B2 (en) 2003-02-18 2008-07-29 Baylor College Of Medicine Induced activation in dendritic cell
US8691210B2 (en) 2006-10-19 2014-04-08 David M Spencer Methods and compositions for generating an immune response by inducing CD40 and pattern recognition receptors and adaptors thereof
US9089520B2 (en) 2010-05-21 2015-07-28 Baylor College Of Medicine Methods for inducing selective apoptosis
US9315559B2 (en) 2008-09-22 2016-04-19 Baylor College Of Medicine Methods and compositions for generating an immune response by inducing CD40 and pattern recognition receptor adapters
US9434935B2 (en) 2013-03-10 2016-09-06 Bellicum Pharmaceuticals, Inc. Modified caspase polypeptides and uses thereof
GB2549351A (en) * 2015-11-13 2017-10-18 Asterion Ltd Fusion polypeptide
US9944690B2 (en) 2013-03-14 2018-04-17 Bellicum Pharmaceuticals, Inc. Methods for controlling T cell proliferation
US10189880B2 (en) 2014-11-03 2019-01-29 Leiden University Medical Center T cell receptors directed against Bob1 and uses thereof
US10525110B2 (en) 2013-06-05 2020-01-07 Bellicum Pharmaceuticals, Inc. Methods for inducing partial apoptosis using caspase polypeptides
US10888608B2 (en) 2014-09-02 2021-01-12 Bellicum Pharmaceuticals, Inc. Costimulation of chimeric antigen receptors by MyD88 and CD40 polypeptides
US10934346B2 (en) 2014-02-14 2021-03-02 Bellicum Pharmaceuticals, Inc. Modified T cell comprising a polynucleotide encoding an inducible stimulating molecule comprising MyD88, CD40 and FKBP12

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994029458A1 (fr) * 1993-06-07 1994-12-22 Amgen Inc. Molecules receptrices hybrides
WO1997040381A1 (fr) * 1996-04-19 1997-10-30 Xenogen Biodetecteurs ciblant des ligands specifiques
EP0950710A2 (fr) * 1998-04-03 1999-10-20 Jcr Pharmaceuticals Co., Ltd. Cellules exprimant un récepteur chimérique et leurs utilisations

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994029458A1 (fr) * 1993-06-07 1994-12-22 Amgen Inc. Molecules receptrices hybrides
WO1997040381A1 (fr) * 1996-04-19 1997-10-30 Xenogen Biodetecteurs ciblant des ligands specifiques
EP0950710A2 (fr) * 1998-04-03 1999-10-20 Jcr Pharmaceuticals Co., Ltd. Cellules exprimant un récepteur chimérique et leurs utilisations

Cited By (22)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9572835B2 (en) 2003-02-18 2017-02-21 Baylor College Of Medicine Induced activation in dendritic cells
US8771671B2 (en) 2003-02-18 2014-07-08 Baylor College Of Medicine Induced activation in dendritic cells
US8999949B2 (en) 2003-02-18 2015-04-07 Baylor College Of Medicine Induced activation in dendritic cells
US10420824B2 (en) 2003-02-18 2019-09-24 Baylor College Of Medicine Induced activation in dendritic cells
US7404950B2 (en) 2003-02-18 2008-07-29 Baylor College Of Medicine Induced activation in dendritic cell
US8691210B2 (en) 2006-10-19 2014-04-08 David M Spencer Methods and compositions for generating an immune response by inducing CD40 and pattern recognition receptors and adaptors thereof
US9315559B2 (en) 2008-09-22 2016-04-19 Baylor College Of Medicine Methods and compositions for generating an immune response by inducing CD40 and pattern recognition receptor adapters
US9976122B2 (en) 2008-09-22 2018-05-22 Baylor College Of Medicine Methods and compositions for generating an immune response by inducing CD40 and pattern recognition receptor adapters
US9428569B2 (en) 2008-09-22 2016-08-30 Baylor College Of Medicine Methods and compositions for generating an immune response by inducing CD40 and pattern recognition receptor adapters
US9089520B2 (en) 2010-05-21 2015-07-28 Baylor College Of Medicine Methods for inducing selective apoptosis
US9393292B2 (en) 2010-05-21 2016-07-19 Baylor College Of Medicine Methods for inducing selective apoptosis
US11077176B2 (en) 2010-05-21 2021-08-03 Baylor College Of Medicine Methods for inducing selective apoptosis
US9434935B2 (en) 2013-03-10 2016-09-06 Bellicum Pharmaceuticals, Inc. Modified caspase polypeptides and uses thereof
US9932572B2 (en) 2013-03-10 2018-04-03 Bellicum Pharmaceuticals, Inc. Modified Caspase polypeptides and uses thereof
US9944690B2 (en) 2013-03-14 2018-04-17 Bellicum Pharmaceuticals, Inc. Methods for controlling T cell proliferation
US10525110B2 (en) 2013-06-05 2020-01-07 Bellicum Pharmaceuticals, Inc. Methods for inducing partial apoptosis using caspase polypeptides
US11839647B2 (en) 2013-06-05 2023-12-12 Bellicum Pharmaceuticals, Inc. Methods for inducing partial apoptosis using caspase polypeptides
US10934346B2 (en) 2014-02-14 2021-03-02 Bellicum Pharmaceuticals, Inc. Modified T cell comprising a polynucleotide encoding an inducible stimulating molecule comprising MyD88, CD40 and FKBP12
US10888608B2 (en) 2014-09-02 2021-01-12 Bellicum Pharmaceuticals, Inc. Costimulation of chimeric antigen receptors by MyD88 and CD40 polypeptides
US10918705B2 (en) 2014-09-02 2021-02-16 Bellicum Pharmaceutics, Inc. Costimulation of chimeric antigen receptors by MYD88 and CD40 polypeptides
US10189880B2 (en) 2014-11-03 2019-01-29 Leiden University Medical Center T cell receptors directed against Bob1 and uses thereof
GB2549351A (en) * 2015-11-13 2017-10-18 Asterion Ltd Fusion polypeptide

Also Published As

Publication number Publication date
AU2001256892A1 (en) 2001-11-12
SE0001642D0 (sv) 2000-05-04

Similar Documents

Publication Publication Date Title
Sleeman et al. Identification of a new fibroblast growth factor receptor, FGFR5
Ebner et al. Determination of type I receptor specificity by the type II receptors for TGF-β or activin
Heim et al. Contribution of STAT SH2 groups to specific interferon signaling by the Jak-STAT pathway
Lin et al. The carboxyl terminus of B class ephrins constitutes a PDZ domain binding motif
KR100210183B1 (ko) 세포-유리된 수용체 결합 검정물, 이의 제조방법 및 용도
Flanagan et al. Alkaline phosphatase fusion proteins for molecular characterization and cloning of receptors and their ligands
Zhu et al. Receptor chimeras indicate that the met tyrosine kinase mediates the motility and morphogenic responses of hepatocyte growth/scatter factor
US20060228346A1 (en) Constitutively active phosphatidylinositol 3-kinase and uses thereof
EP1003863A1 (fr) Recepteur 11 de cytokines de mammiferes
WO2001083551A1 (fr) Reactif destine a la detection de biomolecules et son utilisation
AU673141B2 (en) Purification and cloning of P62
EP0832230B1 (fr) Compositions destinees au recepteur neuronal humain de l'acetylcholine nicotinique et leurs procedes d'utilisation
Sayeski et al. The Angiotensin II-dependent nuclear translocation of STAT1 Is mediated by the JAK2 protein motif231YRFRR
CA2165953C (fr) Recepteur ep3 de la prostaglandine et adn codant ce recepteur
AU2013229528B2 (en) Membrane span-kinase fusion protein and the uses thereof
US20080268460A1 (en) Assays to Identify Irreversibly Binding Inhibitors of Receptor Tyrosine Kinases
US5874230A (en) Assays using TRAF2-associated protein kinase polypeptides
US5756700A (en) Nucleic acid encoding human signal transducer and activator of transcription 4
Clark et al. Loss of three major auto phosphorylation sites in the EGF receptor does not block the mitogenic action of EGF
US6045797A (en) Treatment or diagnosis of diseases or conditions associated with a BLM domain
EP0528878A1 (fr) Recepteur cellulaire hybride
JP2001078781A (ja) 多発性骨髄腫の細胞増殖の阻害手段
Muntasir et al. Identification of amino acid residues important for sarpogrelate binding to the human 5-hydroxytryptamine2A serotonin receptor
EP1294929B1 (fr) Methode permettant de detecter des modulateurs du domaine kinase du recepteur du vegf
Seibert et al. Activation of the rod G‐protein Gt by the thrombin receptor (PAR1) expressed in Sf9 cells

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AE AG AL AM AT AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ CZ DE DE DK DK DM DZ EE EE ES FI FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ PL PT RO RU SD SE SG SI SK SK SL TJ TM TR TT TZ UA UG US UZ VN YU ZA ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: JP