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Antibody of anti human CD20 from human resources functionally, and application

A technology of humanization and antibody, applied in application, antibody, anti-tumor drug, etc., can solve the problem of minimizing immunogenicity and achieve the effect of eliminating immunogenicity

Active Publication Date: 2009-01-28
SINOMAB BIOSCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So this can only be regarded as a humanized antibody on the surface, but it fails to minimize immunogenicity functionally

Method used

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  • Antibody of anti human CD20 from human resources functionally, and application
  • Antibody of anti human CD20 from human resources functionally, and application
  • Antibody of anti human CD20 from human resources functionally, and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] Example 1, Designing the Amino Acid Sequence and DNA Sequence of the Light Chain Variable Region and the Heavy Chain Variable Region of the Functional Humanized Antibody SMO9

[0084] 1. Clone the DNA sequences of the light chain variable region and the heavy chain variable region of the murine maternal antibody 1F5 of the humanized antibody SMO9

[0085] Antibody 1F5 is a murine antibody against CD20 (Nadler LM, Ritz J, Hardy R, et al. 1981. Aunique cell surface antigen identifying lymphoid malignancies of B cell origin. J. Clin. Invest. 67: 134; Clark EA, Shu G, Ledbetter JA: Role of the Bp35 cell surface polypeptide in human B-cell activation. PNAS USA 82: 1766, 1985; Press OW, Howell-Clark J, Anderson S, Bernstein I: Retention of B-cell-specific monoclonal antibodies by human lymphoma cells. Blood 83:1390, 1994).

[0086] Hybridomas secreting the murine 1F5 antibody are commercially available from the American Type Culture Collection (ATCC). Using the Track mRNA I...

Embodiment 2

[0122] Example 2, Expression of Functional Humanized Antibody SMO9

[0123] 1. Construction of VK and VH stage vectors (Staging Vector)

[0124] To express the functional humanized antibody SMO9, the VK and VH segment DNA encoded by SMO9 must first be inserted into an expression vector implanted with DNA encoding the human light chain and heavy chain regions. To do this, first insert the VK or VH segment DNA into the appropriate stage vector. In the synthesized stage vector, an IgG expression promoter (Promoter) and a signal peptide (Signal Peptide) sequence are connected upstream of the VK or VH segment DNA.

[0125] The VK stage vector was modified from M13VKPCR1 (Orlandi et al. 1989. PNAS 86:3833-3837). The BamHI / HindIII segment of M13VKPCR1 carrying the IgG expression promoter (Promoter) sequence and a signal peptide (Signal Peptide) sequence was cloned between the BamHI and HindIII sites of the pBR322 (ΔPvuII) vector. pBR322(ΔPvuII) was originally a pBR322 vector (Stra...

Embodiment 3

[0146] Embodiment 3, analyze SMO9 with flow cytometry (flow cytometry)

[0147] In order to test whether SMO9 still maintains its specificity (specificity) and affinity (affinity), the purified SMO9 and mouse-derived maternal antibody 1F5 were analyzed by flow cytometry to test its specific binding ability to Raji human lymphoma cells. The specific method is as follows: human Hodgkin's lymphoma Raji cells (5 × 10 5 1) mixed with 0.1ug, 0.01ug or 0.001ug of SMO9 or 1F5 in 100ul PBS (PBS-FA) solution containing 1% FCS and 0.01% sodium azide (sodium azide), and placed in 4°C for 30 minute. The treated Raji cells were first washed three times with PBS to remove excess antibodies not attached to the Raji cells. Antibodies attached to Raji cells were measured with 20-fold diluted goat anti-human IgG1 Fc fragment (measurement of SMO9) or goat anti-mouse IgG1 Fc fragment (measurement of 1F5) antibody containing FITC-conjugate, and the results were as follows Figure 6 As shown, in ...

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Abstract

This invention discloses functional humanized antibody against CD20 molecules and its application. The functional humanized antibody specifies human CD20 molecules, and is composed of a light chain and a heavy chain, each of which comprises a constant region and a variable region. The constant region of the heavy chain is isotypic human IgG1 constant region, and that of the light chain is isotypic human kappa constant region. The variable region of the heavy chain is shown in SEQ ID No.2, and that of the light chain is shown in SEQ ID No.1. The functional humanized antibody has low immunogenicity. Experiments show that the functional humanized antibody has the same specificity and affinity as the original murine antibody. Drugs containing the functional humanized antibody and its derivatives can be used for treating cancers or immune system diseases with the high-expression of CD20, such as rheumatic arthritis and lupus erythematosus.

Description

technical field [0001] The invention relates to a functional humanized anti-human CD20 antibody and its application. Background technique [0002] CD20 antigen is a human B-lymphocyte-specific restricted antigen (Bcell lineage restricted antigen), CD20 molecular weight 33kD, is a non-glycosylated protein composed of 4 transmembrane regions and N- / C-terminal in the cytoplasm, CD20 The cell density is about 50,000-200,000. The function of CD20 is not very clear, it may be through Ca 2+ Transmembrane conduction, tyrosine kinase signal transduction, regulation of transcription factors helix-loop-helix unit (helix-loop-helix) and leucine zipper, promote DNA synthesis, regulate the proliferation and differentiation of B cells. CD20 is widely expressed in pre-B cells, mature B cells, and activated B cells, but not in hematopoietic stem cells (stem cells), T cells, dendritic cells (dendritic cells), normal plasma cells, and other normal tissues. Malignant B cells often overexpres...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/18C07K16/24C12N15/13C12N15/63C12N5/10A61K39/395A61K51/10A61P37/02A61P35/00A61K101/02A61K103/10A61K103/20A61K103/32
Inventor 杨蕾李正东黄佩芬
Owner SINOMAB BIOSCI
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