Anti-CLD18A2 nanobody and application thereof

A nanobody, FR1 technology, applied in the direction of antibody medical components, recombinant DNA technology, antibody mimics/scaffolds, etc.

Active Publication Date: 2020-07-21
ZHEJIANG DOER BIOLOGICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, there is currently no specific nanobody targeting the CLD18A2 epitope. Therefo

Method used

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  • Anti-CLD18A2 nanobody and application thereof
  • Anti-CLD18A2 nanobody and application thereof
  • Anti-CLD18A2 nanobody and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0143] Example 1: Construction and detection of cell lines expressing CLD18A2

[0144] The pCDNA3.1 vectors (Life Technologies) containing the full-length genes of CLD18A1 (amino acid sequence, SEQ ID NO.96) and CLD18A2 (amino acid sequence, SEQ ID NO.97), respectively, were extracted with a plasmid extraction kit (Biomiga), The expression plasmid was sterile-filtered and electrotransfected into CHO-S cells, and G418 (sigma) was added for 96-well plating to construct CHO-S-CLD18A1 and CHO-S-CLD18A2 stably transfected cell lines, respectively. Pick the stable cell lines on the 96-well plate and gradually amplify them under the culture condition of adding G418, and use the Anti-Claudin18 antibody [34H14L15] (abcam) to detect the positive expression cell line CLD18A1 by dot blotting. and CLD18A2. The same method was used to construct NUGC-4-CLD18A1 and NUGC-4-CLD18A2. Gastric adenocarcinoma NUGC-4 was purchased from Wuhan Jinkairui Co., Ltd. The gastric adenocarcinoma NUGC-4 did...

Embodiment 2

[0145] Example 2: Anti-CLD18A2 nanobody library construction

[0146] The CHO-S-CLD18A2 in Example 1 was stably transfected with 1.0×10 7 A healthy alpaca (Vicugna pacos) was immunized with cells per ml, and immunized with 1ml of Freund's complete adjuvant (sigma), and then immunized again after 21 days. A total of 3 times of immunization stimulated B cells to express antigen-specific nano Antibody. One week after the third immunization, 30ml of alpaca blood was collected with a vacuum blood collection tube, lymphocytes were separated by lymphocyte separation medium (Tianjin Haoyang Huake Biotechnology Co., Ltd.), and total RNA was extracted by Trizol method. 3 μg of total RNA was reverse-transcribed into cDNA using a reverse transcription kit (Invitrogen) according to the instructions, and VHH was amplified using nested PCR and the following primers: the upstream primer of the first round of PCR 5'-CTTGGTGGTCCTGGCTGC-3' (SEQ ID NO.110) and downstream primer 5'-GGTACGTGCTGTT...

Embodiment 3

[0147] Example 3: Screening and Identification of Anti-CLD18A2 Nanobodies

[0148] 3.1 Screening of Anti-CLD18A2 Nanobodies:

[0149] The constructed Anti-CLD18A2 nanobody library was packaged with the helper phage M13KO7 (NEB), and the titer of the recombinant phage displayed in the display library was 5.7×10 13 PFU / ml. Take 18ml, 7×10 5 pcs / ml of CHO-S-CLD18A2 and 15ml, 3×10 6 CHO-S cells / ml, after centrifugation at 300g for 5 minutes at 4°C, remove the medium supernatant, resuspend the cells in PBS, centrifuge again and block with 2% skimmed milk powder (diluted in PBS) for 1 hour at room temperature. The recombinant phage library was approximately 5.7×10 11 Add PFU to the closed CHO-S cells (about 4.5×10 7 ), and incubated at room temperature for 30 minutes to perform two whole-cell subtractive screening methods. Add the supernatant to about 1.5 x 10 7 A well-blocked CHO-S-CLD18A2 stably transfected cells were incubated at room temperature for 1 hour for binding, an...

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Abstract

The invention relates to the field of biology, in particular to an anti-CLD18A2 nanobody. According to the anti-CLD18A2 nanobody provided by the invention, a complementary determining region (CDR) ofthe anti-CLD18A2 nanobody includes CDR1-CDR3 of which the amino acid sequences are shown as follows: CDR1 of which the amino acid sequence is shown by one of SEQ ID NO.4-11, CDR2 of which the amino acid sequence is shown by one of SEQ ID NO.19-26 and CDR3 of which the amino acid sequence is shown by one of SEQ ID NO.33-39. The anti-CLD18A2 nanobody provided by the invention can be specifically bound to an epitope present on CLD18A2, and fusion protein corresponding to the nanobody has good specificity and affinity on CLD18A2, and has an obvious tumor suppression effect.

Description

technical field [0001] The invention relates to the field of biology, in particular to an anti-CLD18A2 nanobody and an application thereof. Background technique [0002] Claudin 18 (Claudin 18, CLD18) is a transmembrane protein with a molecular weight of about 28kD, located in the tight junction of epithelium and endothelium, and is tightly connected between adjacent cells. In normal epithelial tissue, claudin on the cell surface is difficult to access due to the tight intercellular space, while the intercellular space in tumor cells is relatively loose. Therefore, claudin on tumor cells has become a potential target for extracellular antibodies and immunotherapy. CLD18 has four hydrophobic regions, which form two extracellular domains as transmembrane regions, in which hydrophobic region 1 and hydrophobic region 2 surround to form extracellular domain 1, and hydrophobic region 3 and hydrophobic region 4 surround to form extracellular domain 2. Due to the different splicing...

Claims

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Application Information

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IPC IPC(8): C07K16/30C07K16/28C07K19/00C07K16/46C12N15/13C12N15/62C12N5/10A61K39/395A61K47/64A61K35/17A61P35/00
CPCC07K16/3046C07K16/28C07K16/2809C07K14/7051C12N5/0636A61P35/00C07K2317/569C07K2317/565C07K2317/567C07K2317/24C07K2317/31C07K2317/52C07K2317/92C07K2317/732C07K2317/734C07K2317/72C07K2319/31C07K2319/21C07K2319/00C07K2319/33C12N2510/00A61K2039/505A61K47/643A61K39/001102C07K2319/03A61K2039/5156C07K2319/74A61K35/17C12N15/62A61K9/2004A61K9/0019A61K9/19A61K39/0011C07K14/705A61K47/68Y02A50/30
Inventor 姚高锋陆亚丽周振兴方艺琳朱丽娜黎常魁章宏塔温晓芳董佳里黄岩山
Owner ZHEJIANG DOER BIOLOGICS CO LTD
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