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EpCAM-CD16-NKG2D trispecific antibody and application thereof

An epcam-cd16-nkg2d, specific technology, applied in application, antibody, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, etc., can solve the problem of weak lethality of immune cells and achieve large killing force effect

Active Publication Date: 2021-10-29
THE THIRD AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, current bispecific antibody-mediated immune cell killing is weak, especially for metastatic tumors

Method used

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  • EpCAM-CD16-NKG2D trispecific antibody and application thereof
  • EpCAM-CD16-NKG2D trispecific antibody and application thereof
  • EpCAM-CD16-NKG2D trispecific antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 EpCAM+HMA+CD16+hinge+NKG2D recombinant protein expression

[0045] 1. Expression vector construction

[0046] 1.1 Sequence information

[0047] The amino acid sequence is as follows:

[0048]

[0049] Note: The sequence in bold is mutated human IgG1hinge, the sequence in underline is EpCAM+HMA+CD16, and the sequence in italics is NKG2D. It can be understood that the role of signal peptide and histidine tag is only to facilitate expression and purification, but not necessary for its specific binding ability. The optimized DNA sequence is as follows:

[0050]

[0051]

[0052] 1.2 Recombinant expression amino acid sequence and vector map

[0053] Such as figure 1 As shown, it is the vector map of pET28a-EPCAM+HMA+CD16+hinge+NKG2D.

[0054] 1.3 Experimental process

[0055] 1.3.1 Cloning of fragments

[0056] The fragmented PCR product was amplified by PCR, and recombined into the target vector pET-28a(+) (NcoI-BamHI restriction vector) by multi-...

Embodiment 2

[0147] Example 2 Detection of Antibody Activity

[0148] The purpose of the experiment: biotin-labeled antibody, coated with the corresponding antigen protein, the combination of antibody and antigen was detected by Elisa method, and the bispecific antibody (comparative example and EpCAM+HAM+CD16+hinge+Claudin18.2) and trispecific antibody ( Example 1) Whether the corresponding antigen can be recognized.

[0149] experiment process:

[0150] 1. Indirect ELISA

[0151] (1) Coating: Dilute EpCAM, CD16, and ULBP1 proteins with coating solution (2 μg / mL, 100 μL per well), add 100 μL to the microplate, seal the reaction well with plastic wrap, overnight at 4 °C, wash Wash the plate 3 times.

[0152] (2) Blocking: Add 300 μL of blocking solution to the washed reaction wells, seal the plastic wrap, place at room temperature for 1 hour, and wash the plate 4 times with the washing solution.

[0153] (3) Adding samples: Dilute the biotin-labeled bispecific and trispecific antibodies...

Embodiment 3

[0164] Example 3 Cell Killing Experiment

[0165] 1. Experimental materials

[0166] 1.1 Cell lines

[0167] English name Chinese name source HT-29 human colorectal adenocarcinoma cells China Center for Type Culture Collection SK-OV-3 human ovarian adenocarcinoma cells China Center for Type Culture Collection NK92 human natural killer cells ATCC

[0168] 1.2 Reagents

[0169] name factory McCoy's 5A Medium Gibco MEM Alpha Medium Gibco trypsin Sigma fetal bovine serum ExCell Bio PBS self-provided CCK8 Solarbio Na 2 CrO 4` 4H 2 o

aladdin Cr in water sample 6+ Concentration Detection Kit

Solarbio

[0170] 1.3 Instruments

[0171] name factory biological safety cabinet Haier Carbon dioxide incubator Shanghai Yiheng Scientific Instruments Medical centrifuge Beijing Aries Medical Devices UV spectrophotometer Shang...

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Abstract

The invention relates to an EpCAM-CD16-NKG2D trispecific antibody and an application thereof. The EpCAM-CD16-NKG2D trispecific antibody comprises an EpCAM binding domain, a CD16 binding domain and an NKG2D ligand binding domain. According to the EpCAM-CD16-NKG2D trispecific antibody constructed by the invention, an NKG2D receptor is added on the basis of an EpCAM * CD16 bispecific antibody, and the EpCAM-CD16-NKG2D trispecific antibody can be used for immune cells not only to target tumor cells expressing EpCAM in an epithelial state, but also to target tumor cells expressing NKG2D ligands in a mesenchymal state, or target tumor cells expressing both the EpCAM and the NKG2D ligands in a mixed state, so that stronger ability of immune cells to resist migration tumors can be mediated.

Description

technical field [0001] The invention relates to the technical field of biochemical molecules, in particular to an EpCAM-CD16-NKG2D trispecific antibody and its application. Background technique [0002] The adoptive immunotherapy of tumors is to inject autologous or allogeneic immunocompetent cells into the patient's body after in vitro expansion, directly kill tumor cells, regulate and enhance the immune function of the body, mainly including LAK cells, TIL cells, activated T lymphocytes and Immunotherapy of CIK cells. However, immunotherapy can only remove a small amount of scattered tumor cells, and has limited curative effect on advanced solid tumors. Therefore, it is often used as an adjuvant therapy in combination with conventional methods such as surgery, chemotherapy, and radiotherapy. After removing a large number of tumor cells by conventional methods, immunotherapy is used to remove the remaining tumor cells to improve the effect of comprehensive tumor treatment....

Claims

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Application Information

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IPC IPC(8): C07K16/46C12N15/13C12N15/70C12N1/21A61K39/395A61P35/00
CPCC07K16/30C07K16/283C07K16/2851C12N15/70A61P35/00C07K2317/31C07K2317/14C07K2317/73
Inventor 骆玉梅朱德途
Owner THE THIRD AFFILIATED HOSPITAL OF GUANGZHOU MEDICAL UNIVERSITY
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