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A humanized CD70 antibody LD70 and a preparing method and application thereof

A humanized and antibody technology, applied in the field of bioengineering, can solve the problem of CD27 lack of cytoplasmic death domain

Active Publication Date: 2018-12-18
ZHEJIANG BLUE SHIELD PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, unlike the Fas receptor, CD27 lacks a cytoplasmic death domain

Method used

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  • A humanized CD70 antibody LD70 and a preparing method and application thereof
  • A humanized CD70 antibody LD70 and a preparing method and application thereof
  • A humanized CD70 antibody LD70 and a preparing method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Comparing the expression levels of CD70 in tumor cells and normal cells, CD 70 is highly expressed in breast cancer. Such as figure 1 , Western Blot found that CD70 was highly expressed in breast cancer cells and low in normal cells. This phenomenon shows that CD70 may be used as a marker of tumor cells.

Embodiment 2

[0032] Example 2: Preparation of Antibodies

[0033] (1) Antigen preparation

[0034] Extraction of membrane proteins

[0035]1 Construct the plasmid for full-length expression of CD70-Fc;

[0036] 2. Transfect CHO cells to construct a CHO cell line overexpressing CD70;

[0037] 3. Cultivate CHO-CD70 overexpressed cells, homogenize, separate and extract membrane protein. Methods as below:

[0038] 1) Sow the cells in T75 or T175 culture flasks and culture them for several days. After the cells cover the bottom of the flasks, absorb the culture medium. Add PBS / EDTA solution (NaCl: 0.1M, NaH2PO4: 0.01M, EDTA: 0.04%) to cover the cells, put it in an incubator or digest it in an ultra-clean bench for 3-5 minutes, if it still does not fall off the bottom of the bottle , blow with a pipette to completely detach the cells.

[0039] 2) Collect the cell suspension in a 10 ml or 50 ml centrifuge tube, centrifuge at 1000 rpm for 10 minutes, and remove the supernatant.

[0040] 3) ...

Embodiment 3

[0057] Example 3, CD 70 Affinity Determination

[0058] The LD70 sequence was cloned into the pET28a vector, transformed into Escherichia coli for expression, and the LD70-Scfv protein was obtained. Such as Figure 4 . The binding force of LD70-ScFv to CD70 protein is higher than that of the existing CD70 antibody SNG70 ( Figure 5 ).

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PUM

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Abstract

The invention belongs to the field of bioengineering and relates to a humanized CD70 antibody LD70 and a preparing method and application thereof. The heavy chain variable region (VH) sequence of thehumanized CD70 antibody LD70 is shown as SEQ ID No.1 and the light chain variable region (VL) sequence of the humanized CD70 antibody LD70 is shown as SEQ ID No.2. CD70 protein is expressed in eukaryocytes and then the humanized CD70 antibody LD70 is obtained through screening. Verification proves that the antibody LD70 has CD70 affinity better than that of a reported CD70 antibody (SNG70). The disclosed antibody is used for treatment of animal models having breast cancer for the first time, and has obvious antitumor effects superior to antitumor effects of the reported antibody (SNG70).

Description

technical field [0001] The technology belongs to the field of bioengineering and relates to a CD70 antibody preparation method and a CD70 antibody sequence. The antibody can be used alone to inhibit tumor growth. Background technique [0002] CD70 belongs to the tumor necrosis factor (tumor necrosis factor, TNF) family member, is a type II transmembrane protein, also known as tumor necrosis factor receptor superfamily (tumor necrosis factor receptor superfamily, TNFSF) factor. Mainly expressed in activated T cells, B cells and mature dendritic cells (DCs), has the ability to regulate the activation, proliferation and differentiation of T cells and B cells, and plays an important role in maintaining the body's immune response role. [0003] Early studies have found that CD70 is mainly expressed in B cells in the germinal center and T cells in local lymphoid tissue, and CD70 can also be expressed at a low level in epithelial cells of the thymus medulla, interleukin-1α (interl...

Claims

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Application Information

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IPC IPC(8): C07K16/28A61K39/395A61P35/00
CPCA61K2039/505A61P35/00C07K16/2875C07K2317/24C07K2317/56
Inventor 郭志刚沈炳辉吴劳生
Owner ZHEJIANG BLUE SHIELD PHARM CO LTD