Human-derived anti-human CD146 monoclonal antibody with efficient neutralizing activity

A monoclonal antibody and humanized technology, applied in the field of biomedicine, can solve the problems of rejection, weak ADCC effect, short half-life, etc., and achieve the effect of high-efficiency neutralization activity

Inactive Publication Date: 2015-05-27
RENOPHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that these antibodies recognize different epitopes. P1H12 recognizes a linear epitope located in the N-terminal domain, while some anti-CD146 antibodies recognize epitopes based on disulfide bond-based spatial conformations, thus resulting in different endothelial tissue-binding activities. difference
In addition, the above two antibodies are mouse-derived monoclonal antibodies, which have low binding ability to human complement components and weak

Method used

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  • Human-derived anti-human CD146 monoclonal antibody with efficient neutralizing activity

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Humanization strategy of anti-human CD146 human-mouse chimeric antibody

[0041] The best-fit CDR construction technology was used to humanize the anti-human CD146 human-mouse chimeric antibody. The murine CDRs are grafted into a human-acceptable framework with high sequence homology to the framework region of the chimeric antibody. The best matching FR frameworks of graftable CDRs were searched in the human germline sequence database. Antibody primary structural sequences and 3D structural data were used to identify key framework residues required to maintain the correct conformation of the murine CDRs to preserve binding affinity and specificity. In order to maintain the affinity of the antibody, back mutations are carried out in different humanized molecular frameworks, and finally a highly humanized monoclonal antibody is obtained.

[0042] By BLAST analysis of the human V region gene database, human IGHV3-33 and anti-human CD146 human-mouse chimeric antibody heav...

Embodiment 2

[0045] Figure 1 shows the ELISA results of the binding of anti-CD146 murine antibody to CD146. Determination of specificity of humanized designed antibody binding to human CD146 receptor by ELISA

[0046] Synthesize the VH domain and VL domain according to the results of computer simulation humanization design, and clone the synthetic VH domain and VL domain into a mammalian expression vector, which enables the corresponding domains to be placed on the complete human IgG backbone in the expression. Humanized antibodies are produced in small batches by co-transfecting the heavy chain construct with the light chain construct into mammalian cells. Transient transfection supernatants were subjected to ELISA detection. IgG is purified to homogeneity by protein A or G resin for detection in cell-based assays.

[0047] To determine the specificity of humanized antibodies binding to CD146, ELISA was performed against CD146 and control protein. The above-mentioned transient transfe...

Embodiment 3

[0071] BIACORE analysis of binding of humanized antibody series to CD146

[0072] The purpose of this example is to use BIACORE to analyze the binding affinity of the humanized engineered antibody to human CD146. The humanized engineered antibody is an H5L0 antibody, and the H5L0 antibody comprises the amino acid sequence of SEQ ID NO: 14 and SEQ ID NO: 24); the H10L2 antibody comprises the amino acid sequence of SEQ ID NO: 19 and SEQ ID NO: 26; H14L0 The antibody comprises the amino acid sequences of SEQ ID NO: 23 and SEQ ID NO: 24; the H11L3 antibody comprises the amino acid sequences of SEQ ID NO: 20 and SEQ ID NO: 27; the H11L4 antibody comprises the amino acid sequences of SEQ ID NO: 20 and SEQ ID NO: 28; The H13L0 antibody comprises the amino acid sequences of SEQ ID NO: 22 and SEQ ID NO: 24; the H0L2 antibody comprises the amino acid sequences of SEQ ID NO: 9 and SEQ ID NO: 26; the H11L2 antibody comprises the amino acid sequences of SEQ ID NO: 20 and SEQ ID NO: 26 The...

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Abstract

The invention discloses a human-derived anti-human CD146 monoclonal antibody with efficient neutralizing activity, which comprises a light chain variable region, wherein the amino acid sequence of the light chain variable region is any one of the amino acid sequences disclosed as SEQ ID NO:24 to SEQ ID NO:29. The human-derived anti-human CD146 monoclonal antibody with efficient neutralizing activity also comprises a heavy chain variable region, wherein the amino acid sequence of the heavy chain variable region is any one of the amino acid sequences disclosed as SEQ ID NO:9 to SEQ ID NO:23. On the premise of keeping or enhancing the combination activity of the antibody molecule with the human CD146 receptor molecule, the monoclonal antibody with maximum human derivation degree is prepared, thereby preventing the antibody from causing human anti-mouse immunoreaction when entering the human body as a therapeutic antibody.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a humanized anti-human CD146 monoclonal antibody with high-efficiency neutralizing activity. Background technique [0002] CD146 is an adhesion molecule with a transmembrane structure, a type I membrane protein composed of 646 amino acid residues, and a member of the Ig superfamily of immunoglobulins. Studies have shown that CD146 is a co-receptor of the vascular endothelial cell growth factor receptor VEGFR-2, which can regulate the activation of VEGF-induced VEGFR-2 and the transmission of downstream signals, thereby promoting tumor angiogenesis MicroRNA329Suppresses Angiogenesis by Targeting CD146.Molecular and Cellular Biology , 2013; 33(18):3689-99. In addition, the CD146 molecule is an important factor that promotes the transformation of inflammatory bowel disease into colorectal tumors. The pro-inflammatory cytokines TNFα and IL1β can activate the transcription of CD146 and mak...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P35/00A61P37/02A61P9/10A61P27/06A61P27/02G01N33/577G01N33/574
Inventor 梅岩李涛
Owner RENOPHARMA INC
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