Human single chain antibody and application of antitumor vascular endothelium marker molecule 8-cell outer region

A tumor blood vessel, single-chain antibody technology, used in anti-tumor drugs, anti-animal/human immunoglobulins, antibodies, etc.

Inactive Publication Date: 2009-12-02
HANGZHOU IMMUSOURCE BIOTECHN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report of TEM8-specific human-derived antibody. Therefore, the first direct screening of high-affinity TEM8 single-chain antibody from the human-derived single-chain antibody library constructed by tumor patients will undoubtedly provide a new way for tumor-specific imaging diagnosis and targeting. Significant contribution to sex therapy drug research

Method used

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  • Human single chain antibody and application of antitumor vascular endothelium marker molecule 8-cell outer region
  • Human single chain antibody and application of antitumor vascular endothelium marker molecule 8-cell outer region

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] (1) Isolation of lymphocytes

[0023] A total of 1000ml of peripheral blood was collected from about 200 patients with a variety of tumors, added an equal volume of lymphocyte separation medium, and centrifuged to separate layers, 2500g, 10min. Carefully absorb the middle lymphocytes and centrifuge at 2500g for 10min. . Discard the supernatant, and use 1ml Trizol / 5ml peripheral blood to lyse the lymphocytes until the solution is completely transparent to obtain a lymphocyte lysate, which is stored at -80°C.

[0024] (2) Extraction of RNA

[0025] Thaw the lymphocyte lysate stored at -80°C to room temperature, add 0.2ml chloroform / ml Trizol, shake with Votex for 15s, let stand at room temperature for 3min, 12000g, 5min. Take the upper clear water phase. Add 0.5ml isopropanol / ml Trizol, invert and mix well, precipitate at -20°C for 30-60min, and centrifuge at 12000g for 10min. Keep the precipitate, wash it once with 70% ethanol, air dry, and dissolve the precipitated ...

Embodiment 2

[0080] The single-chain antibody of the present invention is identified according to the above-mentioned method, and the results are as follows: figure 1 . according to figure 1 , we draw the following conclusion: the affinity between the single chain antibody of the present invention and recombinant human tumor vascular endothelial marker molecule 8 (TEM8) extracellular region protein reaches Kd=6nM.

[0081] Therefore, the humanized single-chain antibody of the present invention has the function of high affinity and specific binding to the extracellular region of human tumor vascular endothelial marker molecule 8 (TEM8) and the potential value of tumor diagnosis and targeted therapy.

Embodiment 3

[0082] Example 3. Identification of binding activity of soluble antibody to natural TEM8 expressed on cell surface

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Abstract

The invention discloses a human single chain antibody and application of an antitumor vascular endothelium marker molecule 8-cell outer region. The amino acid sequence of the single chain antibody is SEQ ID NO:1. The amino acid sequence of a heavy chain variable region of the antibody is SEQ ID NO:2, and the amino acid sequence of a light chain variable region of the antibody is SEQ ID NO:3. The human single chain antibody of the antitumor vascular endothelium marker molecule 8-cell outer region can be applied to early diagnosis and target treatment of tumor.

Description

technical field [0001] The invention belongs to genetic engineering antibodies, and relates to a humanized single-chain antibody against the extracellular region of human tumor vascular endothelial marker molecule 8 (Tumor Endothelial Marker 8, TEM8) and its application in tumor imaging diagnosis and therapeutic drug development . Background technique [0002] The early specific diagnosis and targeted therapy of tumors have been the research goals of researchers for a long time. Studies have shown that the neovascular endothelium of tumors is different from that of normal tissues, and expresses some specific tumor-specific marker molecules. TEM8 is one of the more specific tumor antigens, which is highly expressed in the vascular endothelium of tumors, but not or lowly expressed in normal tissues. With the continuous improvement of phage surface display antibody library technology, it provides an efficient platform for the preparation of tumor-specific antibodies for early...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/30A61K39/395A61P35/00G01N33/577G01N33/574
Inventor 韩森韩月恒刘向昕
Owner HANGZHOU IMMUSOURCE BIOTECHN
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