Agonistic antibody directed against human thrombopoietin receptor

A thrombopoietin and agonist technology, applied in the direction of antibodies, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, anti-animal/human immunoglobulin, etc. Antibodies, low antigenicity, long-lasting half-life, etc.

Inactive Publication Date: 2009-06-03
KIRIN PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] Thus, intact antibodies possess properties useful as pharmaceuticals, such as low antigenicity or prolonged half-life in blood; however, as described above, it is difficult to produce agonist human antibodies with sufficient activity in intact antibody form

Method used

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  • Agonistic antibody directed against human thrombopoietin receptor
  • Agonistic antibody directed against human thrombopoietin receptor
  • Agonistic antibody directed against human thrombopoietin receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0250] Antigen preparation

[0251] 1-1: Preparation of human c-Mpl-expressing cells

[0252] In general, when an antigenic protein expressing cell line is used for immunization, it is more advantageous to use a cell line showing a higher expression level for antibody production. Various types of human megakaryocyte cell lines or forced expression cell lines are known to be human c-Mpl-expressing cell lines; but the expression level of c-Mpl in such cell lines is only several thousand molecules per cell, so such Cell lines are not suitable for use as antigens. When immunized with the expressing cell line FDCP-hMpl (see FEBS Lett. Oct 21, 1996; 395(2-3): 228-234) comprising human c-Mpl introduced into FDCP2 (ie, a murine hematopoietic cell line), Human antibody mouse (such as KM mouse TM ), the increase in antibody titer was actually not sufficient, and the hMpl-specific human antibody could not be obtained. Antibodies reactive with other membrane molecules were also induce...

Embodiment 2

[0282] Preparation of monoclonal antibodies

[0283] Mice (KM mice) that produce human antibodies were immunized with the antigen TM ) to prepare monoclonal antibodies, thereby obtaining the antibodies of the present invention, and the above-mentioned mice can produce human antibodies through genetic modification. KM mice lack endogenous mouse immunoglobulin (Ig) heavy chains and mouse kappa light chains, while carrying a chromosome 14 fragment with a human Ig heavy chain gene (SC20) and a human Ig kappa chain transgene (KCo5). In particular, KM mice are capable of producing human antibodies and lack murine Ig heavy and kappa chains. The mice were prepared by crossing lineage A mice with the human Ig heavy chain locus and lineage B mice with the human Ig kappa chain transgene. Lineage A mice are homozygous for deficient endogenous Ig heavy chain and kappa light chain genes and carry a chromosome 14 segment (SC20) repertoire that is transmitted to offspring (see Tomizuka. et ...

Embodiment 3

[0297] Preparation of purified antibodies from hybridoma culture supernatants

[0298] Anti-human c-Mpl monoclonal antibody was purified from hybridoma culture supernatant in the following manner. Using rmp protein A (Amersham Pharmacia Biotech), 0.8 × 40cm column (Bio-Rad), PBS as adsorption buffer and 0.02M glycine buffer (pH 3) as elution buffer, the culture supernatant containing antibody Perform affinity purification. The pH of the eluted fractions was adjusted to around 7.2 by adding 1M Tris (pH 9.0). The prepared antibody solution was replaced with PBS using a dialysis membrane (10,000 cutoff, Spectrum Laboratories), and passed through a membrane filter MILLEX-GV (Millipore), pore size 0.22 μm, to obtain purified anti-human c-Mpl monoclonal antibody . The concentration of the purified antibody was determined by measuring the absorbance at 280 nm and calculating by specifying 1 mg / ml to be equal to 1.4 OD.

[0299] Culture supernatants containing anti-human c-Mpl mon...

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Abstract

The present invention discloses an agonistic antibody directed against human thrombopoietin receptor (also referred to as 'human c-Mpl). Specifically, the antibody has a constant region having a set of amino acid sequences selected from the following items (1) to (3): (1) amino acid sequences for a heavy-chain constant region and a light-chain constant region of a human antibody; (2) an amino acid sequence for a human antibody heavy-chain constant region in which the domain is replaced by one of other human antibody subclass and an amino acid sequence for a human antibody light-chain constant region; and (3) a set of amino acid sequences having the deletion, substitution, addition or insertion of one or several amino acid residues in each of the amino acid sequences shown in (1) and (2), and the antibody has a variable region capable of binding to a human thrombopoietin receptor to activate the receptor. The antibody also has the following properties: (a) the antibody can induce the formation of a colony at a concentration of 10,000 ng/mL or less in the CFU-MK colony formation assay using a human umbilical cord blood CD34+ cell; and (b) the antibody has the maximum activity higher than that of PEG-rHuMGDF by 50% or more and a 50% effective concentration (EC50) of 100 nM or less in the cell growth assay using an UT7/TPO cell. Also disclosed is a pharmaceutical composition for the treatment of thrombocytopenia, which comprises the antibody.

Description

technical field [0001] The present invention relates to an agonist antibody against human thrombopoietin receptor (alias: human c-Mpl). [0002] The present invention further relates to a therapeutic agent for patients / diseases, especially for thrombocytopenia, in the case of a clinical need to increase platelets, comprising as an active ingredient, said anti-human c-Mpl agonist Antibodies. Background of the invention [0003] <TPO and TPO receptors> [0004] Thrombopoietin (TPO) is a hematopoietic factor that promotes the proliferation of megakaryocytes and platelets in vivo. Human TPO is a glycoprotein with a full length of 332 amino acid residues, and its N-terminal sequence is known to play an important role in human TPO activity. Human TPO exhibits its function of binding to TPO receptors on cell membranes. [0005] c-Mpl is the only known TPO receptor. Human c-Mpl is a glycoprotein with one transmembrane domain, comprising 635 amino acids if the glycoprotein...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09A61K39/395A61P7/00A61P7/04A61P43/00C07K16/28C07K16/46C12N15/02C12P21/08
Inventor 甲斐正之元木一宏片冈之郎吉田英明萩原哲也
Owner KIRIN PHARMA
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