Anti-human EGFR (epidermal growth factor receptor) nano antibody and application thereof

A nanobody and antibody technology, applied in the field of anti-human EGFR nanobody, can solve the problems of ineffective removal of small lesions, poor penetration ability, large molecular weight, etc., and achieve the effects of easy transformation, good stability, and application promotion.

Active Publication Date: 2019-02-15
深圳新锐基因科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, antibodies targeting EGFR have achieved good results in the clinical treatment of non-small cell lung cancer, but because they are chimeric antibodies, they have certain immunogenicity, and due to their large molecular weight and poor penetrability, they cannot effectively clear micro lesions

Method used

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  • Anti-human EGFR (epidermal growth factor receptor) nano antibody and application thereof
  • Anti-human EGFR (epidermal growth factor receptor) nano antibody and application thereof
  • Anti-human EGFR (epidermal growth factor receptor) nano antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 preparation of helper phage

[0048] (1) Take out the glycerol-preserved TG1 Escherichia coli clone strain from the -80°C refrigerator, spread it on a TYE non-resistant plate using the four-section method, and incubate it upside down at 37°C for 13 hours.

[0049] (2) Pick a TG1 monoclonal strain from the plate and inoculate it into 5 mL of 2×TY non-resistance liquid medium, and culture it at 37° C. and 230 rpm for 13 hours.

[0050] (3) Transfer the TG1 bacterial solution to 5mL 2×TY non-resistant liquid medium at a volume ratio of 1:100, and culture it at 37°C and 230rpm for 2h (bacterial solution OD 600 = around 0.5).

[0051] (4) Take 200 μL TG1 bacterial solution (OD 600 =0.5 or so) into a 1.5mL centrifuge tube, add 10μL of KM13 helper phage (1×10 13 pfu / mL), placed in preheated 37°C water bath for 30min.

[0052] (5) Prepare soft agar, let it cool to about 40°C, pour the TG1 bacterial solution treated in step (4), mix, and then pour the pre-prepared ...

Embodiment 2

[0057] Example 2 Amplification of Phage Nanobody Library

[0058] (1) Thaw the TG1 bacteria containing the antibody plasmid (i.e., the human nanobody phage library, SourceBioscience, London, UK) on ice, and transfer it to 500mL 2×TY liquid medium (containing 0.1% ampicillin by mass volume ratio) and mass volume ratio of 1% glucose), 37 ° C, 230rpm shaker culture 2.5h (OD 600 =0.5), and then add 2×10 12 The helper phage, 37 ℃ water bath for 30min.

[0059] (2) Divide the bacterial solution into 250 mL / bottle, centrifuge at 3200 g for 10 min, remove the supernatant, and use 500 mL of 2×TY medium (containing 0.1% ampicillin by mass volume ratio and 0.05% kanamycin by mass volume ratio) The pellet was resuspended, and cultured with shaking at 25°C and 220rpm for 20h.

[0060] (3) The bacterial liquid was centrifuged at 3200 g for 20 min, and the supernatant was collected, and the phage was purified by the same method as steps (8)-(9) in Example 1, and the prepared library phage...

Embodiment 3

[0062] Example 3 Screening Nanobodies Against EGFR Fragments from a Phage Nanobody Library

[0063] (1) Screening of anti-EGFR nanobody phage

[0064] 1) Coat the EGFR polypeptide (purchased from Shanghai Botai Biological Co., Ltd., with the amino acid sequence shown in SEQ ID NO.15) in the immunotube (nunc) (the first round: 0.1 mg / mL; the second and third rounds : 0.05mg / mL; the fourth and fifth rounds: 0.025mg / mL;), set the PBS (control) tube. overnight at 4°C.

[0065] 2) Pour out the coating solution, wash each tube with 4.5mL PBS three times (after adding the liquid each time, discard without stopping), add 4.5mL of BSA blocking solution with a concentration of 2% by mass volume ratio to each tube, and place at room temperature 2h.

[0066] 3) Pour off the BSA blocking solution, and wash each tube with 4.5mL PBS three times (after adding the liquid each time, discard it directly without stopping).

[0067] 4) Add 4mL to each tube containing 5×10 12 2% BSA solution o...

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Abstract

The invention discloses an anti-human EGFR (epidermal growth factor receptor) nano antibody and an application of the nano antibody. The nano antibody is one or a combination of at least two of an aEG1B4 nano antibody with an amino acid sequence shown as SEQ ID NO: (sequence identifier number) 1, an aEG2C7 nano antibody with an amino acid sequence shown as SEQ ID NO: 2, an aEG2E12 nano antibody with an amino acid sequence shown as SEQ ID NO: 3, an aEG4D9 nano antibody with an amino acid sequence shown as SEQ ID NO: 4 and an aEG6B2 nano antibody with an amino acid sequence shown as SEQ ID NO: 5. The nano antibody has high affinity, specificity and low immunogenicity and the advantages of better stability, simple structure and easy engineering transformation, and can be better applied to development of antibody medicines.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an anti-human EGFR nanobody and its application. Background technique [0002] The term cancer was proposed in more than 400 BC, and it has not been conquered after more than 2,000 years. Today, the threat to human health is becoming more and more obvious. In recent years, studies have found that epidermal growth factor (EGFR) is overexpressed in a variety of solid tumor cells, and plays an important role in the occurrence and development of cancer, and is positively correlated with poor prognosis and drug resistance. The overexpression of EGFR in cancer cells can lead to the continuous activation of EGFR / EGF signaling pathway, promote the proliferation, migration and invasion of cancer cells, and can also promote the secretion of VEGF factors, leading to the formation of blood vessels in the cancer tissue microenvironment. At present, antibodies targeting EGFR have achieved good resu...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13A61K39/395A61P37/02A61P35/00
CPCA61P35/00A61P37/02C07K16/2863A61K2039/505C07K2317/569C07K2317/92C07K2317/76
Inventor 魏星刘雪陈涛
Owner 深圳新锐基因科技有限公司
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