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Development and application of tumor treatment agent containing antibody

An antibody, bispecific antibody technology, applied in anti-tumor drugs, applications, antibodies, etc., can solve problems such as deletion

Active Publication Date: 2020-07-28
CHENGDU CONMED BIOSCI CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although B7-H3 mRNA transcripts can be detected in many normal tissues (Collins M, 2005), evidence shows that the expression of B7-H3 protein level is strictly regulated, and it is absent or extremely low on the cell surface of normal tissues (Yi KH, 2009)

Method used

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  • Development and application of tumor treatment agent containing antibody
  • Development and application of tumor treatment agent containing antibody
  • Development and application of tumor treatment agent containing antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0154] Embodiment 1, the preparation of B7-H3 recombinant protein

[0155] Using the plasmid pMD18-B7-H3 (Sino Biological Inc.) containing the cDNA sequence of the human B7-H3 gene (SEQ ID NO: 1) as a template, using the forward primer 5'-CTGAGAGGTGCCAGATGTATGCTGCGTCGGCGGGGC-3', the reverse primer 5'-TCCGCCTCCGCCGCTAGCTGTCATAGGCTGCCCTGTG-3 ', the human B7-H3 extracellular region fragment (Met1-Thr461) was amplified by PCR, and the amplified fragment was digested with BspQI and then cloned into the self-constructed eukaryotic expression plasmid system (pSect). HEK293E cells were transfected with this plasmid, and after 6 days, the culture supernatant was collected and purified by nickel column affinity chromatography to obtain the recombinant protein of human B7-H3 extracellular region (SEQ ID NO: 2, the results are as follows: figure 1 shown).

Embodiment 2

[0156] Example 2, cell lines expressing the full length of human B7-H3 or the IgC domain

[0157] B7-H3 is widely and highly expressed in tumor cell lines, and the common cell line HEK293 cells also highly express B7-H3 (ShiJ, 2016). Therefore, in the preliminary screening, B7-H3 recombinant protein and HEK293 cells can be used alternately. In order to further confirm the binding specificity of the candidate antibody, the full-length B7-H3 (B7-H3-4Ig) or IgC (B7-H3-IgC) was constructed as a eukaryotic cell expression plasmid and transiently transfected into CHO cells.

[0158] Spread CHO-k1 cells into a 10cm cell culture dish 16-24 hours in advance, make the confluence reach about 90% during transfection, collect 5×10 6 cells, washed 3 times with PBS and resuspended in Opti-MEM medium, the voltage of the electroporation instrument was set to 300V, and the capacitance was 950μF, and the constructed human B7-H3 full-length sequence (SEQ ID NO: 1), B7-H3 IgC sequence The eukaryo...

Embodiment 3

[0159] Embodiment 3, preparation of anti-human B7-H3 monoclonal antibody

[0160] 1) Immune mice

[0161] Mix 400 μL human B7-H3 recombinant protein with a concentration of 1 mg / mL as an antigen with an equal volume of immune adjuvant (Titermax adjuvant, Sigma-Aldrich), and take six 6-week-old female Balb / c mice (Beijing Weitongli Hua) for subcutaneous immunization. After the primary immunization, a booster immunization of the same dose was given every two weeks.

[0162] 2) Cell fusion and hybridoma screening

[0163] Three days before fusion, 10 μg / 100 μL / human B7-H3 recombinant protein was used for tail vein pulse immunization. Three days later, the spleen and lymph nodes of the mice were taken and ground in DMEM to obtain a B cell suspension. After mixing an appropriate amount of the B cell suspension with SP2 / 0, the cells were fused using an electrofusion apparatus. The fused cells were placed in DMEM complete medium containing HAT in 5% CO 2 , cultured at 37°C.

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Abstract

The invention relates to a antibody for identifying a B7-H3 protein, a double-specific antibody for identifying the B7-H3 protein and CD3, and a manufacturing method and application thereof.

Description

technical field [0001] The present disclosure relates to an antibody recognizing B7-H3 protein, its production method and application. Background technique [0002] B7-H3, also known as CD276, is a type I transmembrane protein that belongs to the B7-CD28 superfamily. The extracellular region of B7-H3 contains two tandem IgV-IgC domains (4Ig-B7-H3, namely IgV-IgC-IgV-IgC) (Collins M, 2005), which is similar to the murine B7-H3 (2Ig-B7- H3) was shown to have a similar function (Hofmeyer K, 2008). It was initially thought that B7-H3, as a co-stimulatory molecule, co-stimulates the proliferation of CD4 and CD8 T cells, selectively enhancing interferon production in the presence of TCR signaling (Chapoval, Al. 2001; Zhang, GB.2004). However, most research results in recent years have shown that B7-H3 is an inhibitory molecule, such as B7-H3 can inhibit T cell proliferation mediated by antibodies against TCR or alloantigen-presenting cells (Sub WK, 2003; Veenstra RG, 2015), th...

Claims

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Application Information

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IPC IPC(8): C07K16/28C07K16/46C12N15/13C12N15/85C12N5/10A61K39/395A61P35/00
CPCC07K16/2827C07K16/2809C12N15/85A61P35/00C07K2317/565C07K2317/31A61K2039/505
Inventor 王景坤
Owner CHENGDU CONMED BIOSCI CO LTD
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