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Humanized antibody specific for human 4-1BB and pharmaceutical compsn comprising same

A technology of humanized antibodies and compositions, which is applied in the direction of drug combinations, antibody mimics/stents, and medical preparations containing active ingredients, etc., which can solve the problem of blocking antibody functions, reducing antibody half-life, and mouse antibody half-life. and other issues to achieve a high affinity effect

Inactive Publication Date: 2002-07-03
LG CHEM LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The HAMA reaction not only blocks the function of the antibody as a new drug, but also forms an immune complex with the murine antibody, which causes some side effects and reduces the half-life of the antibody
Second, in vivo, murine antibodies have a shorter half-life than human antibodies, even without forming immune complexes
All of the above factors can reduce the efficacy of murine mAbs, and the common problems in human immunotherapy are also caused by heterologous mAbs

Method used

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  • Humanized antibody specific for human 4-1BB and pharmaceutical compsn comprising same
  • Humanized antibody specific for human 4-1BB and pharmaceutical compsn comprising same
  • Humanized antibody specific for human 4-1BB and pharmaceutical compsn comprising same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1: Design of humanized antibody Hz4B4-1

[0038] To construct a humanized antibody, the amino acid sequences of the light and heavy chain variable regions of the mouse monoclonal antibody 4B4-1-1 (Korean Laid-Open Application No. 96-37064) were compared with the human sequences in the gene database Compare. The human heavy chain variable region sequence M17750 (Dersimonian, H. et al., J. Immunol., 139, 2496 (1987)) which is very similar to the heavy chain sequence of the mouse 4B4-1-1 antibody was selected, and the sequence of the mouse 4B4-1-1 1-1 The antibody light chain is very similar to the human light chain variable region sequence X82934 (Esposito, G. et al., Arch. Vitrol., 142, 601 (1997)). To humanize the mouse monoclonal antibody 4B4-1-1, the CDR regions of the mouse antibody were transferred to a human antibody. At the same time, 10 key amino acid residues in the FR region of the humanized light chain and 11 key amino acid residues in the FR region ...

Embodiment 2

[0040] Example 2: Construction of gene encoding humanized antibody Hz4B4-1 and expression plasmid

[0041] Primers are prepared including the base sequence in the region where substitution is desired. These primers are: KXA (SEQ ID NO: 5), KXB (SEQ ID NO: 6), KXC (SEQ ID NO: 7), KXD (SEQ ID NO: 8), KXE (SEQ ID NO: 9), KXF ( SEQ ID NO: 10), KXG (SEQ ID NO: 11), KXH (SEQ ID NO: 12), AMH (SEQ ID NO: 13), BMH (SEQ ID NO: 14), CMH (SEQ ID NO: 15 ), DMH (SEQ ID NO: 16), EMH (SEQ ID NO: 17), FMH (SEQ ID NO: 18), GMH (SEQ ID NO 19), HMH (SEQ ID NO: 20).

[0042] Among the above primers, primers KXA (SEQ ID NO: 5) to KXH (SEQ ID NO: 12) were used to construct a gene encoding the humanized κ-light chain variable region. Primers AMH (SCQ ID NO: 13) to HMH (SEQ ID NO: 20) were used to construct the gene encoding the variable region of the humanized heavy chain. Figure 2 shows the humanized region in the humanized antibody Hz4B4-1 by the location of the primers, including genes encoding...

Embodiment 3

[0048] Example 3: Expression of humanized antibody Hz4B4-1 and screening of cells

[0049] 5% CO in GDMEM medium containing 10% permeable bovine serum (FBS) 2, CHO-K1 cells (ATCC CCL61) cultured at 37°C were inoculated into a petri dish with a diameter of 6 cm to obtain 5×10 5 cell. GDMEM medium containing DMEM (Gibco), 4.5g / L glucose, 15mg / L phenol red, 1mM sodium pyruvate, 1.75g / L sodium bicarbonate, 500μm asparagine, 30μm adenosine, 30μm guanosine, 30μm cytidine , 30 μM uridine, 10 μM thymidine and non-essential amino acids (GIBCO). 2.5 μg of the pRc-Hz4B4-k-gs plasmid prepared from Example 2 and 2.5 μg of the pCI-Hz4B4-H plasmid were combined, and 0.3ml OPTI-MEM I TM (GIBCO) diluted with 0.3ml of OPTI-MEM I TM Dilute 15 μl of Lipofectamine TM (GIBCO), then mixed and left for 15 minutes.

[0050] Pre-cultured cells with OPTI-MEM I TM Wash 3 times. Then the plasmid-Lipofectamine prepared in the previous step TM The mixture was added evenly to the cells. Cells in 5%...

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Abstract

The present invention is directed to humanized antibodies that specifically bind the protein 4-1BB. The antibodies can be made by grafting of the complementarity determining regions (CDR's) of mouse monoclonal antibody to human 4-1BB to the remaining portions of a human antibody and by making further amino acid replacements. In addition, a pharmaceutical composition that includes the humanized antibody can be made and can be used to treat autoimmune diseases to suppress an immune response. The humanized antibody of the invention has high affinity for human 4-1BB, and exhibits sequence similarity to human antibody. As a result, the pharmaceutical composition of the present invention can be used to treat autoimmune disease and act as an immunosuppressant in humans without much side-effect.

Description

field of invention [0001] The present invention relates to humanized antibodies that specifically bind to 4-1BB receptor protein, preferably human 4-1BB receptor protein. Such humanized antibodies can be used as diagnostic reagents or in the formulation of pharmaceutical compositions for administration to patients. background of the invention [0002] The immune system varies in diversity, and since B cells and T cells randomly and specifically express various components, that component specifically binds to many components, including itself. Therefore, self-immune tolerance mechanisms must be established in vivo to discriminate between self and non-self determinants to avoid self-reactions. But all mechanisms are at risk of being disrupted. The self-recognition mechanism is no exception, and it has been proved that many diseases are caused by the large amount of autoantibodies and autoreactive T cells produced by autoimmunity. [0003] There are at least 30 kinds of dise...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/50A61K38/00A61K39/395A61P19/02A61P29/00A61P37/06C07K16/18C07K16/28C07K16/46C12N1/15C12N1/19C12N1/21C12N5/10C12N15/09C12N15/12G01N33/15
CPCC07K2319/00A61K2039/505C07K16/2878C07K2317/24A61P19/02A61P29/00A61P37/06C07K16/18
Inventor 洪孝贞朴圣燮姜荣峻姜昌律尹圣宽
Owner LG CHEM LTD
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