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Fusion protein of anti-cluster of differetiation (CD3) antibody Fv segment and interleukin 3, method for preparing fusion protein and application of fusion protein

A fusion protein and application technology, applied in the fields of oncology and biopharmaceuticals, can solve the problems of inability to kill tumor stem cells, unsatisfactory tumor treatment effect, poor selectivity, etc., and achieve strong tumor cell specificity and good application prospects

Inactive Publication Date: 2012-11-28
中国医学科学院血液病医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The traditional treatment of leukemia is usually combined chemotherapy with cytotoxic drugs and hematopoietic stem cell transplantation. However, this method has poor selectivity. While killing tumor cells, it also damages certain types of normal cells in the body, and can only kill most of the differentiated cells. Tumor cells can not kill tumor stem cells, so the effect of tumor treatment is very unsatisfactory

Method used

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  • Fusion protein of anti-cluster of differetiation (CD3) antibody Fv segment and interleukin 3, method for preparing fusion protein and application of fusion protein
  • Fusion protein of anti-cluster of differetiation (CD3) antibody Fv segment and interleukin 3, method for preparing fusion protein and application of fusion protein
  • Fusion protein of anti-cluster of differetiation (CD3) antibody Fv segment and interleukin 3, method for preparing fusion protein and application of fusion protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1 Recombinant expression plasmid pAYZ anti-CD3 * - IL3, construction of pAYZ anti-CD3-IL3.

[0080] PCR amplified CD3 * V L , CD3V L Fragment:

[0081] Anti-CD3 due to recombinant plasmid pB11d1 * V L / anti-CD19V H , pB11d1 anti-CD3V L / anti-CD19V H CD3 containing anti-CD3 mAbs, respectively * V L and CD3V L gene, and the recombinant plasmid pB11d1 is anti-CD3 * V L / anti-CD19V H , pB11d1 anti-CD3V L / anti-CD19V H Only the upstream of the VL region contains a mluI restriction site, so the present invention uses the recombinant plasmid pB11d1 containing the anti-CD3 monoclonal antibody VL gene to resist CD3 * V L / anti-CD19V H , pB11d1 anti-CD3V L / anti-CD19V H (constructed by the applicant's laboratory) as a template to obtain CD3 * V L , CD3V L . PCR primers were synthesized by Shanghai Yingjun Company.

[0082] Specifically, the recombinant plasmid pB11d1 anti-CD3 * V L / anti-CD19V H , pB11d1 anti-CD3V L / anti-CD19V H As a templat...

Embodiment 2

[0111] Example 2 Recombinant expression plasmid pAYZ anti-CD3 * - ΔIL3, construction of pAYZ anti-CD3-ΔIL3.

[0112] PCR amplification of CD3V L * -ΔIL3, CD3V L - ΔIL3 gene fragment:

[0113] CD3V L * -IL3, CD3V L -IL3 intermediate vector pB11d1 as template, with primer P 1 is the 5' end primer, P 9 Used as a 3' primer to amplify CD3V with the codons encoding the last eight amino acids of IL3 deleted L * -ΔIL3, CD3V L - ΔIL3 gene fragment. Reaction conditions: pre-denaturation at 94°C for 10 minutes, denaturation at 94°C for 1 minute, annealing at 62°C for 1 minute, extension at 72°C for 1 min 30s, a total of 30 cycles, and extension at 72°C for 10 minutes. get CD3V L * -ΔIL3(K), CD3V L - ΔIL3(L) gene fragment. The K and L fragments were subjected to 1% agarose gel electrophoresis, and were recovered and purified with a type A gel recovery kit from Treasure Bioengineering (Dalian) Co., Ltd.

[0114] CD3V L * -ΔIL3, CD3V L - ΔIL3 gene fragment downstream pri...

Embodiment 3

[0126] Example 3 pAYZ anti-CD3 * -IL3, pAYZ anti-CD3-IL3 and pAYZ anti-CD3 * Expression and validation of -ΔIL3, pAYZ anti-CD3-ΔIL3.

[0127] 3.1 The plasmid pAYZ anti-CD3-IL3, pAYZ anti-CD3-IL3 and pAYZ anti-CD3 obtained in Examples 1 and 2 *-ΔIL3, pAYZ A single colony of Escherichia coli 16C9 resistant to CD3-ΔIL3 was inoculated in 5 ml of 2×YT medium containing 100 μg / ml of ampicillin (Amp), and cultured with shaking overnight at 37° C. and 200 rpm in a constant temperature shaker flask; Transfer to 500ml of 2×YT medium containing Amp 100 μg / ml (1L of 2×YT medium contains 1.6% tryptone, 1.0% yeast extract, 0.5% sodium chloride, pH7.4), at 37°C, After shaking at 200rpm for 8 hours, centrifuge at 6000rpm and 4°C for 10 minutes on a low-temperature high-speed vacuum centrifuge to collect the bacteria, resuspend the bacteria in 1000ml of AP5 medium containing 100ug / ml ampicillin, and shake at 30°C for 24h at 200rpm . Centrifuge at 8000rpm×10min in a low-temperature high-spe...

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Abstract

The invention discloses a fusion protein of an anti-cluster of differetiation (CD3) antibody Fv segment and an interleukin 3, a method for preparing the fusion protein and an application of the fusion protein, relates to the fusion proteins of anti-CD3 star-delta IL3 and anti-CD3-delta IL3 and encoded genes thereof, and further relates to a genetic engineering construction method and an application of the strengthened fusion protein.

Description

technical field [0001] The present invention relates to the fields of oncology and biopharmaceuticals. Specifically, the present invention provides a fusion protein capable of targeting and killing leukemia stem cells, a preparation method thereof, and an application thereof in drugs for targeting and treating tumor stem cells. Background technique [0002] The fundamental reason for the occurrence of leukemia, drug resistance in treatment and even leukemia relapse is that there is a group of leukemia stem cells (LSC) in the patient's body, which has the same self-renewal and differentiation potential as normal stem cells, and it only accounts for 0.25-1.0% of the whole blood cells . [0003] The traditional treatment of leukemia is usually combined chemotherapy with cytotoxic drugs and hematopoietic stem cell transplantation. However, this method has poor selectivity. While killing tumor cells, it also damages certain types of normal cells in the body, and can only kill mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/63C12N1/15C12N1/19C12N1/21A61K38/20A61K47/48A61P35/00A61P35/02
Inventor 杨纯正熊冬生张秀丽张砚君李真真胡蕴慧丁亚辉
Owner 中国医学科学院血液病医院
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