US20020045186A1 - Inhibition of protein binding to mast cells - Google Patents

Inhibition of protein binding to mast cells Download PDF

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Publication number
US20020045186A1
US20020045186A1 US09/756,899 US75689901A US2002045186A1 US 20020045186 A1 US20020045186 A1 US 20020045186A1 US 75689901 A US75689901 A US 75689901A US 2002045186 A1 US2002045186 A1 US 2002045186A1
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Prior art keywords
compound
immunoglobulin
light chain
free light
binding
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Abandoned
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US09/756,899
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English (en)
Inventor
Franciscus Redegeld
Aletta Kraneveld
Franciscus Nijkamp
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Fornix Biosciences NV
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Fornix Biosciences NV
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Assigned to FORNIX BIOSCIENCES N.V. reassignment FORNIX BIOSCIENCES N.V. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: KRANEVELD, ALETTA D., NIJKAMP, FRANCISCUS P., REDEGELD, FRANCISCUS A.M.
Publication of US20020045186A1 publication Critical patent/US20020045186A1/en
Priority to US10/888,552 priority Critical patent/US7056511B2/en
Abandoned 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the current invention relates to the field of immunology, and more specifically, to components which sensitize mast cells.
  • Ig LC immunoglobulin
  • the present invention relates to a compound which inhibits the binding of the free light chain of immunoglobulin to mast cells, wherein the compound, in the presence of an equimolar quantity of free light chain (LC) of immunoglobulin reduces its binding by at least 5%, said compound not being Tamm-Horsefall glycoprotein (THP), or LC-binding peptide fragments thereof.
  • LC free light chain
  • Such a compound is of major importance for use as a means for suppressing the unpleasant effects resulting from sensitization experienced by a patient.
  • the compounds can be detected in a simple manner, for example, by incubating a compound to be tested, together with fluorescent labeled Ig LC and mast cells. With the aid of a fluorescence microscope or, for quantitative measurement, a Fluorescence Activated Cell Sorter (FACA), inhibition may be assessed. This inhibition may occur due to competition between the compound and Ig LC for binding to the mast cell.
  • FACA Fluorescence Activated Cell Sorter
  • the compound can bind to the free light chain of immunoglobulin, while the compound is capable of competing with a peptide binding to the free light chain having the amino acid sequence (AHWSGHCCL) SEQ ID NO: 1 and that in the presence of an equimolar quantity of the peptide, the compound reduces its binding by at least 5%.
  • AHWSGHCCL amino acid sequence
  • Huang Z. -Q. et al. (ref. 2) describe a unique urinary protein, Tamm-Horsefall glycoprotein (THP, also known as uromoduline) causing aggregation of immunoglobulin light chains and THP. This aggregate causes renal failure due to clogging of the distal nephron of the kidney.
  • THP Tamm-Horsefall glycoprotein
  • the publication discloses tryptic peptides of THP also causing aggregation.
  • the use thereof or THP as a drug is not disclosed, nor is the binding thereof to mast cells disclosed.
  • the compound reduces the binding of the peptide by at least 10%, preferably by at least 25%, more preferably by at least 50%, even more preferably by at least 75% and most preferably by at least 90%.
  • such compounds are very useful as an active component for a pharmaceutical composition, particularly if binding is reduced by more than 50%.
  • the peptide may also be used as the active component. It is also possible to use peptides with unusual and/or modified amino acids. According to a preferred embodiment, the compound is a peptidomimeticum.
  • a suitable peptidomimeticum is, for example, a peptoid such as a peptoid corresponding with the peptide, but in which the side chains are located on the nitrogen atoms of the peptide backbone. In comparison with the original peptide, such a peptoid has a longer half-life in the blood.
  • the synthesis of peptoids is well-documented in the art. The most important difference with the synthesis of peptides is the different starting materials corresponding to the amino acids.
  • the present invention also relates to a method of screening a series of compounds for their capability to bind the free light chain of immunoglobulin using a labeled compound capable of binding the free light chain of immunoglobulin, and capable of competing with the peptide with the amino acid sequence (AHWSGHCCL) of the formula sheet, wherein the screening is performed using a test comprising a competition reaction between the compound to be tested and the labelled compound.
  • the test is suitably a homogenous test, making it possible to quickly screen compounds and to select active compounds.
  • a homogenous test is understood to be a test wherein for detection it is not necessary to separate a non-complexed labelled peptide (or peptidomimeticum) from a complexed-labelled peptide.
  • a labelled peptide or peptidomimeticum
  • AHWSGHCCL amino acid sequence
  • the present invention also relates to a method of screening a series of compounds for their capability of reducing the sensitization of mast cells, wherein the screening is performed by incubating a compound to be tested and a labelled free light chain of immunoglobulin with a mast cell, and detecting reduced binding of the labelled free light chain of immunoglobulin.
  • the screening occurs under physiological conditions, as the compound will have to be active when used as a drug under those conditions.
  • the compound may be used for pharmaceutical purposes, especially if the compound is pharmaceutically acceptable.
  • the present invention also relates to an application of a compound (obtained) according to the present invention or Tamm-Horsfall glycoprotein (THP) or LC-binding peptide fragments thereof for the preparation of a drug for a disease having as a symptom i) a concentration of the free light chain of immunoglobulin in serum of at least 8 mg/l, in particular of at least 15 mg/l and more in particular 20 mg/l; and/or ii) a concentration of the free light kappa-chain of immunoglobulin in spinal fluid of at least 70 ⁇ g/l, in particular at least 100 ⁇ g/l, and more in particular 150 ⁇ g/l; and/or iii) a concentration of the free lambda-chain of immunoglobulin in spinal fluid of at least 300 ⁇ g/l, in particular at least 400 ⁇ g/l, and more in particular 500 ⁇ g/l.
  • THP Tamm-Horsfall glycoprotein
  • a compound which is a peptide or peptidomimeticum with a mass of less than 10 kDal, preferably less than 2 kDal.
  • the mass is of less importance, although the peptide is then preferably non-immunogenic.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound according to the invention or Tamm-Horsfall glycoprotein (THP) or LC-binding peptides thereof together with a pharmaceutically acceptable carrier or excipient.
  • THP Tamm-Horsfall glycoprotein
  • the invention relates to a method of diagnosing a disease in a patient having an elevated level of the free light chain of immunoglobulin in a bodily fluid, wherein a foreign antigen specific for the disease is contacted with the bodily fluid from the patient, and subsequently the presence is determined of a complex of the foreign antigen and the free light chain of immunoglobulin.
  • the bodily fluid is suitably urine, serum or plasma. Spinal fluid, lung washing and sputum are considered as well.
  • a bodily fluid also comprises liquids prepared 0006on the basis of the bodily material.
  • the presence of a complex can be detected by using one of many methods known in the state of the art such as, for example, a sandwich ELISA wherein the complex is detected using a labeled antibody directed against the free light chain.
  • Such a labeled antibody is suitably directed against a conserved part of the free light chain.
  • FIG. 1 represents a graph of the relative fluorescence as a measure for the amount of bound Ig LC against the number of mast cells;
  • FIG. 2 portions A and B, show Western blots after SDS-PAGEelectrophores is.
  • FIG. 3 shows a graph for an ELISA-based binding assay.
  • the peptide Ac-AHWSGHCCL-NH 2 (SEQ ID NO: 1) was prepared using a 430A Applied Biosystems Instruments (Foster City, Calif., U.S.A.) using solid-phase FastMoc chemistry.
  • a Tentagel-S-RAM resin was used as carrier material. Sensitive side chains were protected using His(Trt), Cys(TrT), Trp(boc), Ser(tBu).
  • the peptide was released from the resin and the protective groups were removed using a mixture of trifluoroacetic acid, ethane dithiol and water (95:2.5:2.5 v/v).
  • the raw peptide was precipitated using ether and purified by means of preparative HPILC. The purity of LCBP was verified using analytical HPLC and mass spectrometry.
  • BALB/c mice (RIVM, Bilthoven, the Netherlands) were skin-sensitized using picrylchloride (PLC), dinitrofluorobenzene or oxazolone as described earlier (ref. 1).
  • PLC picrylchloride
  • dinitrofluorobenzene or oxazolone as described earlier (ref. 1).
  • spleen cells (10 ⁇ 10 6 cells/ml) were cultured for 24-48 hours in RPMI medium supplemented with penicillin, streptomycin and gentamycin.
  • the supernatant was harvested and antigen-binding proteins were isolated using hapten-affinity chromatography (bovine gamma globulin or BSA provided with hapten immobilized to Affigel-10 (Bio Rad I,abs., Veenendaaj., the Netherlands)) as described by Ferguson. T. A. et al. (ref. 3)
  • BSA bovine gamma globulin
  • the proteins were eluted with 5 ml 5 M guanidine solution. Subsequently, extensive dialysis against PBS took place. Proteins of biologically active samples, such as determined with an ear swelling test (see hereinafter), were fractionated using 15% Tricine SDS-PAGE, blotted onto PVDF and subsequently subjected to an Edman degradation for amino acid sequence analysis.
  • hapten-binding proteins were fractionated using 12.5% SDS-PAGE, blotted onto PVDF and tested with horseradish peroxidase-labeled anti-Ig kappa LC (The Binding Site, Birmingham, U.K.) in a dilution of 1:2000. Immunoreactive proteins were visualized using ECL (Amersham Pharmacia Biotech Benelux, Roosendaal, the Netherlands) according to the manufacturer's recommendations (FIG. 2, portion A). This showed that in lymphocyte factors specific for picric acid, dinitrofluorobenzene and exazolon respectively, the presence of Ic LC could be demonstrated using an anti-kappa Ig LC-specific antibody.
  • FIG. 2 portion B shows that lymphocyte factor comprises a large variety of antigen-binding proteins.
  • the lanes labeled A eluted with 0.2 MN Na2CO 3
  • B void volume of column
  • fraction A exhibited the biological activity demonstrated in Example 3.
  • DIQMTQSPPSLSAXLG the amino acid sequence was determined, which corresponded to the sequence of Ig LC (DIQMTQSPSSLSASLG)(SEQ ID NO: 3) known from the literature.
  • Basophilic leukemia cells RBL-2H3 (a gift of C. Fewtrell. Ithaca, N.Y., U.S.A.) of the rat, an established model for mast cells, were incubated with 200 ng/10 5 cells Ig LC labeled with fluorescein isothiocyanate. They were incubated for 30 minutes at 4° C. in the presence or absence of 250 ⁇ g/ml of the peptide LCBP prepared in preparation 1 (peptide binding to the light chain). Subsequently they were washed using a phosphate-buffered saline supplemented with 1% v/v foetal calf serum and 0.01% w/v sodium azide. Binding of FITC-labelled Ig LC to RBL-2H3 cells was analyzed using a FACScan flow cytometer.
  • curve 1 of FIG. 1 shows that the free light chain of Ig binds to mast cells.
  • curves 2 and 3 of FIG. 1 show that this binding can be inhibited using LCBP (0.25 mg/ml and 0.50 mg/ml respectively).
  • Curve 4 represents the autofluorescence of unlabelled RBL-2H3 cells.
  • mice Lightly anaesthetized with halothane, mice were passively sensitized by injection with trinitrophenyl (TNP)-specific Ig LC (2 ⁇ g in 50 ⁇ l of sterile saline) in the retroorbital plexus. Control mice received only 50 ⁇ l of sterile saline. Thirty min. after injection, while being lightly anaesthetized with halothane, all mice received intranasally 50 ⁇ l PSA-solution (picrylsulphonic acid dissolved in phosphate-buffered saline).
  • TNP trinitrophenyl
  • Bronchoconstriction was measured as described by Kraneveld A. D. et al. (ref. 3) and Zuany-Amorim et al. (ref. 6).
  • mice were placed in a plethysmographic chamber (Buxco Electronics Inc., Shanon, Conn.) in order to analyze respiration and to obtain basal line readings. After the intranasal administration the animals were directly returned to the chamber.
  • the respiratory resistance was measured for a period of 45 minutes.
  • the respiratory resistance is expressed as a dimensionless value calculated by using the formula for the Penh (ref. 4). For each mouse the maximum Penh values were measured during an interval of 1 minute at the moments shown in Table 1.
  • mice were, as described by Example 2.1, passively sensitized by injection with a lymphocyte factor PLC-F obtained from a mouse sensitized with picrylchloride or Ig LC specific for trinitrophenyl. Control mice received either only PBS or TNP-specific Ig HC (heavy chain of immunoglobulin). Thirty minutes after injection picrylchloride (50 ⁇ l 0.8% picrylchloride (PCL) dissolved in olive oil) was applied to the ear. After 2 hours, the thickness of the ear was measured (Table 2).
  • PLC-F lymphocyte factor obtained from a mouse sensitized with picrylchloride or Ig LC specific for trinitrophenyl. Control mice received either only PBS or TNP-specific Ig HC (heavy chain of immunoglobulin).
  • picrylchloride 50 ⁇ l 0.8% picrylchloride (PCL) dissolved in olive oil
  • TNP-specific Ig LC has the same effect as lymphocyte factor.
  • the heavy chain does not show this effect.
  • BSA Bovine Serum Albumin
  • uromodulin concentration within, for example, 4-40 ⁇ g/ml, is an excellent concentration for repeating the above ELISA to detect novel compounds according to the present invention.
  • uromodulin and the compound (preferably at several concentrations) to be investigated are incubated simultaneously in order to compete with each other.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
US09/756,899 1998-07-09 2001-01-09 Inhibition of protein binding to mast cells Abandoned US20020045186A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US10/888,552 US7056511B2 (en) 1998-07-09 2004-07-09 Inhibition of protein binding to mast cells

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
NL1009601A NL1009601C2 (nl) 1998-07-09 1998-07-09 Verbinding welke de binding van een eiwit aan mestcellen kan remmen, toepassing van de verbinding voor de bereiding van een geneesmiddel, een farmaceutisch preparaat, een werkwijze voor het diagnostiseren van een ziekte en een selectiewerkwijze.
NL1009601 1998-07-09
PCT/NL1999/000430 WO2000002915A1 (en) 1998-07-09 1999-07-07 Compound capable of inhibiting the binding of a protein to mast cells, use of the compound for the preparation of a drug, a pharmaceutical composition, a method of diagnosing a disease, and a method of selection

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PCT/NL1999/000430 Continuation WO2000002915A1 (en) 1998-07-09 1999-07-07 Compound capable of inhibiting the binding of a protein to mast cells, use of the compound for the preparation of a drug, a pharmaceutical composition, a method of diagnosing a disease, and a method of selection

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US10/888,552 Continuation US7056511B2 (en) 1998-07-09 2004-07-09 Inhibition of protein binding to mast cells

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US (2) US20020045186A1 (de)
EP (1) EP1093462B1 (de)
AT (1) ATE443080T1 (de)
AU (1) AU4804799A (de)
CA (1) CA2332561C (de)
DE (1) DE69941431D1 (de)
ES (1) ES2333493T3 (de)
NL (1) NL1009601C2 (de)
WO (1) WO2000002915A1 (de)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050191290A1 (en) * 2002-03-06 2005-09-01 Nijkamp Franciscus P. Means and methods for manipulating hypersensitivity-like responses

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1342779A1 (de) * 2002-03-06 2003-09-10 Fornix Biosciences N.V. Mittel und Verfahren zur Manipulation hypersensitivitätähnlicher Antworten
WO2005103717A1 (en) * 2004-04-21 2005-11-03 Fornix Biosciences N.V. Measuring free light chains of immunoglobulin
CN111249441A (zh) * 2020-04-10 2020-06-09 北京大学 一种小分子寡肽在制备治疗银屑病药物中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3907502A (en) * 1973-12-11 1975-09-23 Miless L Brink Method for identifying Bence Jones proteins
US4654325A (en) * 1984-05-24 1987-03-31 Selenke William M Medicament for reducing nephrotoxicity caused by positively charged agents such as aminoglycosides and methods of use thereof
US4977244A (en) * 1985-06-27 1990-12-11 The United States Of America As Represented By The Department Of Health And Human Services Uromodulin and a process of purifying it
US5824784A (en) * 1994-10-12 1998-10-20 Amgen Inc. N-terminally chemically modified protein compositions and methods

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3907502A (en) * 1973-12-11 1975-09-23 Miless L Brink Method for identifying Bence Jones proteins
US4654325A (en) * 1984-05-24 1987-03-31 Selenke William M Medicament for reducing nephrotoxicity caused by positively charged agents such as aminoglycosides and methods of use thereof
US4977244A (en) * 1985-06-27 1990-12-11 The United States Of America As Represented By The Department Of Health And Human Services Uromodulin and a process of purifying it
US5824784A (en) * 1994-10-12 1998-10-20 Amgen Inc. N-terminally chemically modified protein compositions and methods

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20050191290A1 (en) * 2002-03-06 2005-09-01 Nijkamp Franciscus P. Means and methods for manipulating hypersensitivity-like responses

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AU4804799A (en) 2000-02-01
EP1093462A1 (de) 2001-04-25
EP1093462B1 (de) 2009-09-16
CA2332561A1 (en) 2000-01-20
US7056511B2 (en) 2006-06-06
NL1009601C2 (nl) 2000-01-11
DE69941431D1 (de) 2009-10-29
US20050049183A1 (en) 2005-03-03
WO2000002915A1 (en) 2000-01-20
ES2333493T3 (es) 2010-02-22
ATE443080T1 (de) 2009-10-15
CA2332561C (en) 2010-02-02

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