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Nanometer antibody targeting calreticulin and application thereof

A technology of calreticulin and nano-antibody, which is applied in the field of immunology or molecular biology, can solve the problems of insensitivity and off-target detection, and achieve the effects of low preparation cost, high affinity and good tissue penetration

Active Publication Date: 2022-04-26
SHENZHEN PEOPLES HOSPITAL
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  • Description
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Problems solved by technology

However, the affinity of the 7-peptide selected in this study to the CRT protein is only 1.868 μM, and there are risks of insensitivity and off-target detection

Method used

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  • Nanometer antibody targeting calreticulin and application thereof
  • Nanometer antibody targeting calreticulin and application thereof
  • Nanometer antibody targeting calreticulin and application thereof

Examples

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Embodiment Construction

[0039] The preferred embodiments of the present invention will be further described in detail below.

[0040] 1. Verify the translocation of CRT protein after ICD induction in vivo and in vitro

[0041] (1) In vitro verification (immunofluorescence)

[0042] HCT116 and A549 cells were resuscitated, and after three passages, the cells were seeded in 24-well plates, and ICD was induced with 300 μM OXP and 1 μM MTX, and a MOCK control group without treatment was set. After 4 hours of induction, immunofluorescence detection was carried out, and the specific steps were as follows:

[0043] a), the cells were placed on ice, washed 3 times with PBS, and fixed with 0.25% paraformaldehyde for 5 minutes;

[0044] b), washed 3 times with PBS, blocked with 2% donkey serum for 1 hour;

[0045] c) Dilute CRT antibody 1:200 with blocking solution and incubate with cells for 1 hour;

[0046] d), wash with pre-cooled PBS for 3 times, dilute the Alexa 594-coupled secondary antibody with blo...

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Abstract

The invention provides a nano antibody for targeting calreticulin and application of the nano antibody. Complementary determining regions of amino acid sequences of the nano antibody for targeting calreticulin are CDR1 as shown in SEQ ID NO: 2, CDR2 as shown in SEQ ID NO: 4 and CDR3 as shown in SEQ ID NO: 6; the CDR1 is as shown in SEQ ID NO: 9, the CDR2 is as shown in SEQ ID NO: 11, and the CDR3 is as shown in SEQ ID NO: 13. According to the present invention, the 204 and 215 nanometer antibodies are verified through the CRT binding condition, such that the 204 and 215 nanometer antibodies have strong binding force with the CRT protein, the affinity with the CRT protein is in the nanomole level, and the affinity is high; and the nano antibody has better tissue penetrability and can be expressed and purified by using an escherichia coli system, so that the preparation cost is greatly reduced.

Description

technical field [0001] The invention belongs to the technical field of immunology or molecular biology, and in particular relates to a nanobody targeting calreticulin and its application. Background technique [0002] A number of monoclonal antibodies targeting solid tumor associated antigens (tumor associated antigens, TAAs) have been widely used in the targeted therapy of tumors. Classic monoclonal antibodies of this type, such as EGFR antibody, Her2 antibody and VEGF antibody, are expressed in the corresponding molecules Positive colorectal cancer, lung cancer, head and neck cancer, gastric cancer, glioma, ovarian cancer, cervical cancer, kidney cancer and breast cancer have been proven to have good curative effect. At the same time, derivative drugs based on the above-mentioned monoclonal antibodies, such as bispecific antibodies (bispecfic antibodies), antibody drug conjugates (antibody drug conjugates, ADC) and chimeric receptor T cells (chimeric antigen receptor T cel...

Claims

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Application Information

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IPC IPC(8): C07K16/18C07K19/00C12N15/13C12N5/10A61K49/00A61K39/00A61P35/00G01N33/68G01N33/574
CPCC07K16/18A61K49/0058A61K39/0011A61P35/00G01N33/6893G01N33/57484C07K2317/565C07K2317/567C07K2317/569C07K2317/92C07K2319/33G01N2333/47
Inventor 李志杰王继刚郑六海杨传彬袁吉民陈昆
Owner SHENZHEN PEOPLES HOSPITAL
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