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Anti-CD123 (anti-cluster of differentiation 123) single-chain antibody, chimeric antigen receptor combined by same and application thereof

A technology of chimeric antigen receptors and single-chain antibodies, applied in the field of genetic engineering, can solve the impact of no reports showing stable CD123 myeloid tumor curative effect, no good solution for the stability of antigen chimeric receptors, and efficacy Limited safety and other issues, to achieve the effect of strengthening the ability to proliferate and kill tumors, no toxic and side effects, and a wide range of choices

Active Publication Date: 2018-08-17
CHONGQING PRECISION BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows an artificially created type called chimeras to combine two different parts - one part contains specific proteins like monocarboxylating enzyme or gamma ray sensor polypeptide fragments linked together while another part consists mainly of other small peptides involved in metabolism such as histidase or trypsin. These tiny components help regulate gene expression within cancer tissue without causing harmful changes during treatment procedures. By combining these specialized portions from both parts at once, this system makes possible precise control over how certain genes get turned off when they've been exposed to their targets inside healthy ones.

Problems solved by technology

This patented technical problem addressed in this patents relates to improving the response rates and selectivity of cytotoxins towards different types of targets while maintaining their effectiveness against solid tissue tumours like sarcoma and breast carcinomas. Current treatments involve modifying existing ones either alone or combined together multiple agents, making these techniques challenges because they may lead to side effects caused by non-target responses.

Method used

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  • Anti-CD123 (anti-cluster of differentiation 123) single-chain antibody, chimeric antigen receptor combined by same and application thereof
  • Anti-CD123 (anti-cluster of differentiation 123) single-chain antibody, chimeric antigen receptor combined by same and application thereof
  • Anti-CD123 (anti-cluster of differentiation 123) single-chain antibody, chimeric antigen receptor combined by same and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0058] Example 1 Construction of Chimeric Antigen Receptor Virus

[0059] In order to screen better CAR combinations, the following 6 chimeric antigen receptors need to be constructed:

[0060] CD123-CAR-1, CD123-CAR-2, CD123-CAR-3, CD123-CAR-4, CD123-CAR-5, CD123-CAR-6.

[0061] 1. Synthesize the gene sequence of chimeric antigen receptors targeting CD123 containing different scFvs and synthesize different single-chain antibody ScFvs containing leader peptide (also known as signal peptide) (abbreviated as LP) and anti-human CD123 antigen in turn, IgG4 hinge region (abbreviated as IgG4), CD28 transmembrane region or CD8 transmembrane region (abbreviated as TM), its structure is as follows figure 1 shown.

[0062] 2. Construction of lentiviral vectors expressing chimeric antigen receptors

[0063] 1) Design the following primers and synthesize them by Nanjing GenScript Biotechnology Co., Ltd. The specific primers are as follows:

[0064] Primer 1: 5'-ag gctagca tgggatggagct...

Embodiment 2

[0080] Example 2 Detection of Chimeric Antigen Receptor Transfection Efficiency in T Cells

[0081] 1) Isolation of human peripheral blood mononuclear cells

[0082] Collect about 60ml of peripheral blood with anticoagulant-added blood collection tubes, divide into 30ml of 50ml centrifuge tubes, add 7.5ml of hydroxyethyl starch to dilute; naturally settle at room temperature (18-25°C) for about 30min, collect the upper layer of plasma, and The collected upper plasma was centrifuged at 1400rpm / min for 15min; then the pellet was resuspended with normal saline, added to the lymphocyte separation medium at a volume ratio of 1:1, gradient centrifuged at 400g / min, and the speed of the centrifuge was set to 1 , centrifuged for 20min; after centrifugation, the centrifuge tube was divided from top to bottom: the first layer: plasma layer; the second layer: annular milky white lymphocyte layer; the third layer: transparent separation liquid layer; the fourth layer: red blood cell layer;...

Embodiment 3

[0085] Example 3 Detection of the positive rate of CAR molecules and the ratio of CD4 and CD8

[0086] CD123-CAR-T cells cultured for 11 days were centrifuged at 300g / min for 5min, discarded the supernatant and resuspended with 1ml PBS, repeated this step twice, then added labeled CD3, CD4, CD8 (BD company) and The flow detection antibody of Protein-L (Beijing Yiqiao Shenzhou Co., Ltd.) was incubated at 4 degrees for 30 minutes, washed 3 times with PBS, and then AF647-labeled mouse anti-Protein-L antibody (Nanjing GenScript Company) was added, and incubated at 4 degrees for 30 minutes. Wash twice with PBS for 1 minute, discard the supernatant, add 200ulPBS to resuspend the marked cells, and perform flow cytometry results as shown in Table 1 and image 3 shown.

[0087] Table 1 CD4 / CD8 flow cytometry results and statistical results of different CD123-CAR-T cells

[0088] CAR name

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Abstract

The invention belongs to the field of genetic engineering and particularly relates to an anti-CD123 single-chain antibody, a chimeric antigen receptor combined by the same, and application thereof. The anti-CD123 single-chain antibody is a CD123 antigen-recognizing peptide with an amino acid sequence shown as SEQ ID NO: 1. The anti-CD123 antigen chimeric antigen receptor is formed by sequentiallyconnecting the CD123 antigen-recognizing peptide (ScFv), a hinge region, a transmembrane region and an intracellular signal region. When being expressed in immune cells, the anti-CD123 antigen chimeric antigen receptor can not only effectively eliminate target tumor cells, which can express CD123 antigens, without toxicity to tumor cell with negative antigens/incapable of expressing CD123, but also maintain the positive rate of a CD123-targeted antigen chimeric antigen receptor (CAR) during patient cell culturing, which can propagate for a long time after being stimulated by target antigens and accordingly achieves targeted treatment on tumors.

Description

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Claims

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Application Information

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Owner CHONGQING PRECISION BIOTECH CO LTD
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