Anti-adrenomedullin (ADM) antibody or Anti-adm antibody fragment or Anti-adm non-ig scaffold for use in intervention and therapy of congestion in a patient in need thereof

An adrenomedullin, antibody fragment technology, applied in the direction of antibodies, antibody medical components, blood diseases, etc., can solve harmful problems

Pending Publication Date: 2019-08-23
ADRENOMED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, ADM can also be harmful if it is completely neutralized, as certain amounts of ADM may be required for several biological functions

Method used

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  • Anti-adrenomedullin (ADM) antibody or Anti-adm antibody fragment or Anti-adm non-ig scaffold for use in intervention and therapy of congestion in a patient in need thereof
  • Anti-adrenomedullin (ADM) antibody or Anti-adm antibody fragment or Anti-adm non-ig scaffold for use in intervention and therapy of congestion in a patient in need thereof
  • Anti-adrenomedullin (ADM) antibody or Anti-adm antibody fragment or Anti-adm non-ig scaffold for use in intervention and therapy of congestion in a patient in need thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0347] Production of antibodies and determination of their affinity constants

[0348] Several human and murine antibodies were generated and their affinity constants determined (see Tables 1 and 2).

[0349] Peptides / Conjugates for Immunization:

[0350] Peptides for immunization were synthesized, see Table 1 (JPT Technologies, Berlin, Germany) with an additional N-terminal cysteine ​​(if no cysteine ​​is present within the chosen ADM sequence) residue Used to couple the peptide to bovine serum albumin (BSA). The peptides were covalently linked to BSA using Sulfolink coupling gels (Perbio-science, Bonn, Germany). The coupling procedure was performed according to the manual of Perbio.

[0351]Mouse antibodies were generated as follows:

[0352] Balb / c mice were treated with 100 μg of peptide-BSA conjugate (emulsified in 100 μl Freund’s complete adjuvant) on days 0 and 14 and 50 μg (in 100 μl Freund’s incomplete adjuvant) on days 21 and 28 Immunization. Three days prior t...

Embodiment 2

[0403] Effect of selected anti-ADM antibodies on anti-ADM biological activity

[0404] The effect of selected ADM antibodies on ADM biological activity was tested in a human recombinant adrenomedullin receptor cAMP functional assay (adrenomedullin bioassay).

[0405] Testing of Antibodies Targeting Human or Mouse Adrenomedullin in Human Recombinant Adrenomedullin Receptor cAMP Functional Assay (Adrenomedullin Bioassay)

[0406] Material:

[0407] Cell line: CHO-K1

[0408] Receptor: Adrenomedullin (CRLR+RAMP3)

[0409] Recipient cell line accession numbers: CRLR: U17473; RAMP3: AJ001016

[0410] CHO-K1 cells expressing human recombinant adrenomedullin receptor (FAST-027C) grown in antibiotic-free medium prior to the assay were detached by gentle washing with PBS-EDTA (5 mM EDTA) and recovered by centrifugation , and resuspended in assay buffer (KRH: 5mM KCl, 1.25mM MgSO 4 , 124mM NaCl, 25mM HEPES, 13.3mM Glucose, 1.25mM KH 2 PO 4 , 1.45mM CaCl 2 , 0.5g / l BSA).

[04...

Embodiment 3

[0420] Stabilization of hADM data by anti-ADM antibody

[0421] The stabilizing effect of human ADM antibodies on human ADM was tested using the hADM immunoassay.

[0422] Immunoassay for the quantification of human adrenomedullin

[0423] The technique used is based on acridinium ester labeled sandwich-coated tube luminescence immunoassay.

[0424] Labeled compounds (tracers):

[0425] 100 μg (100 μl) of CT-H (1 mg / ml in PBS, pH 7.4, AdrenoMed AG Germany) was mixed with 10 μl of acridine NHS ester (1 mg / ml in acetonitrile, InVent GmbH, Germany) (EP 0353971), And incubate at room temperature for 20min. Marked CT-H in Purification was performed by gel filtration HPLC on SEC 400-5 (Bio-Rad Laboratories, Inc., USA). The purified CT-H was diluted in (300mmol / L potassium phosphate, 100mmol / L NaCl, 10mmol / L Na-EDTA, 5g / L bovine serum albumin, pH 7.0). The final concentration of labeled compound was about 800.000 relative light units (RLU) per 200 μL (about 20 ng of labeled an...

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Abstract

Subject matter of the present invention is an anti-Adrenomedullin (ADM) antibody or an anti-Adrenomedullin antibody fragment or an anti-ADM non-lg scaffold for use in intervention and therapy of congestion in a patient in need thereof.

Description

technical field [0001] The subject matter of the present invention is an anti-adrenomedullin (ADM) antibody or anti-adrenomedullin antibody fragment or anti-ADM non-Ig scaffold for the intervention and treatment of hyperemia in patients in need thereof. Background technique [0002] The peptide adrenomedullin (ADM) was first described in 1993 ( Kitamura et al., 1993. Biochem Biophys Res Comm192(2):553-560 ) is a novel hypotensive peptide comprising 52 amino acids, which has been isolated from a human pheochromocytoma cell line (SEQ ID No.: 20). In the same year, a cDNA encoding a precursor peptide comprising 185 amino acids and the complete amino acid sequence of this precursor peptide were also described. The precursor peptide, in particular comprising a signal sequence of 21 amino acids at the N-terminus, is called "pro-adrenomedullin pre-peptide" (pre-proADM). In this specification, all amino acid positions specified generally relate to said pre-proADM comprising 185 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/22G01N33/50A61P7/10
CPCC07K16/22A61K2039/505C07K2317/21C07K2317/24C07K2317/34C07K2317/54C07K2317/55C07K2317/76C07K2317/92A61P1/16A61P13/12A61P43/00A61P7/10A61P9/00A61P9/04A61P9/12A61P9/14A61K39/3955C07K2317/94
Inventor 阿德里安·沃斯
Owner ADRENOMED
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