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High-affinity anti-EGFR (epidermal growth factor receptor) monoclonal antibody

A monoclonal antibody and affinity technology, applied in the field of genetic engineering products, can solve the problem of high tumor lethality and achieve obvious anti-tumor effects

Active Publication Date: 2011-10-19
SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The treatment of stage I and stage II patients is based on surgery, and most patients are suitable for radical resection, but 10% to 15% of patients are accompanied by metastatic tumors, and the postoperative recurrence or tumor-related mortality is very high

Method used

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  • High-affinity anti-EGFR (epidermal growth factor receptor) monoclonal antibody
  • High-affinity anti-EGFR (epidermal growth factor receptor) monoclonal antibody
  • High-affinity anti-EGFR (epidermal growth factor receptor) monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1. Cloning of human antibody light and heavy chain constant region genes

[0043] Lymphocyte separation solution was used to separate healthy human lymphocytes, and Trizol reagent was used to extract total RNA. According to the literature (Clonedhuman and mouse kappa immunoglobulin constant and J region genes conserve homology infunctional segments.Hieter PA, Max EE, Seidman JG, Maizel JV Jr, Leder P. Cell. 1980 Nov; 22(1Pt 1): 197-207.) and the literature (The nucleotide sequence of a human immunoglobulin C gammal gene. Ellison JW, Berson BJ, Hood LE. Nucleic Acids Res. 1982 Jul 10; 10 (13): 4071-9.) According to the reported sequence, primers were designed to amplify the antibody heavy chain constant region and light chain constant region genes by RT-PCR reaction. The PCR product was purified and recovered by agarose gel electrophoresis and cloned into the pGEM-T vector. After sequencing verification, it was confirmed that the correct clone was obtained. Among th...

Embodiment 2

[0044] Example 2. Construction of expression vector for anti-EGFR chimeric antibody C225

[0045] Refer to the anti-EGFR monoclonal antibody data and sequence published in the US patent US7060808, and entrust Shanghai Shenggong Bioengineering Co., Ltd. to fully synthesize the anti-EGFR monoclonal antibody C225 heavy chain variable region gene (C225VH) and light chain variable region gene (C225VL) . figure 1 The nucleotide sequence and amino acid sequence of the C225 heavy chain and light chain variable regions are shown.

[0046] Using C225VH gene and pGEM-T / CH vector as templates, the chimeric antibody heavy chain gene was synthesized by overlapping PCR. The reaction conditions were: 95°C for 15 minutes; 94°C for 50 seconds, 58°C for 50 seconds, 72°C for 50 seconds, and 30 cycles ; 72°C for 10 minutes. The PCR product C225VHCH is obtained, the 5'end contains the restriction enzyme site HindIII and the signal peptide gene sequence, and the 3'end contains the translation terminatio...

Embodiment 3

[0048] Example 3. Stable expression and purification of chimeric antibodies

[0049] Inoculate 3×10 in a 3.5cm tissue culture dish with serum-containing medium 5 CHO-K1 cells are transfected when the cells are cultured to 90%-95% confluence. The specific transfection process is as follows:

[0050] Take 10μg of plasmid (plasmid pcDNA3.1(+)(C225VHCH) 4μg and plasmid pcDNA3.1(C225VLCL) 6μg) and 20μl Lipofectamine2000 Reagent respectively and dissolve them in 500μl serum-free DMEM medium. Let stand at room temperature for 5 minutes, and mix the above two liquids. Mix and incubate at room temperature for 20 minutes to form DNA-liposome complexes. During this time, replace the serum-containing medium in the petri dish with 3ml of serum-free DMEM medium, and then add the formed DNA-liposome complexes to the culture In a dish, place CO 2 After 4 hours of incubation, add 2ml of DMEM complete medium containing 10% serum and place in CO 2 Continue cultivation in the incubator.

[0051] After ...

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Abstract

The invention relates to the field of genetic engineering products, more particularly, discloses a high-affinity anti-EGFR (epidermal growth factor receptor) monoclonal antibody and an application in preparing drugs for treating tumors, wherein the amino acid sequence of a heavy chain variable region (VH) of the antibody is SEQ ID NO:14, and the amino acid sequence of a light chain variable region (VL) of the antibody is SEQ ID NO:6 or SEQ ID NO:12. The affinity of the antibody to the EGFR is improved by at least ten times more than the affinity of Cetuximab to EGFR.

Description

Technical field [0001] The present invention relates to the field of genetic engineering products. More specifically, the present invention discloses an anti-EGFR monoclonal antibody with high affinity and its use in preparing anti-tumor drugs. Background technique [0002] Malignant tumor is a disease that seriously endangers human health in the world today. It ranks second in deaths caused by various diseases. Among them, metastatic colorectal cancer (CRC) is the third most common tumor type worldwide, with new diagnoses every year Estimated to reach 950,000, it is also the second leading cause of cancer deaths in Europe and North America. An investigation showed that in 2005, 280,000 new cases and 150,000 deaths were expected in the European Community. The treatment of stage I and stage II patients is based on surgery. Most patients are suitable for radical resection, but 10% to 15% of patients are accompanied by metastatic tumors, and the postoperative recurrence or tumor-re...

Claims

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Application Information

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IPC IPC(8): C07K16/28A61K39/395A61P35/00A61P1/00
Inventor 郭亚军戴建新侯盛寇庚赵健李博华王皓
Owner SHANGHAI NAT ENG RES CENT OF ANTIBODY MEDICINE
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