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Anti-ccr4 antibody and its application in the treatment of cancer

An antibody and cancer technology, applied in the field of biomedicine, can solve problems such as the great demand for anti-CCR4 antibodies, and achieve the effect of promoting ADCC

Active Publication Date: 2021-03-30
TIANJIN KINGMED CENT FOR CLINICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There are some disclosed CCR4 antibodies in the prior art, such as CN107250160A, CN104436191A, CN103328513B, US20170290911, etc., but there is still a great demand for new anti-CCR4 antibodies

Method used

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  • Anti-ccr4 antibody and its application in the treatment of cancer
  • Anti-ccr4 antibody and its application in the treatment of cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1. Preparation and purification of anti-CCR4 antibody

[0028] C57BL / 6 mice were immunized with CHO cells stably expressing CCR4 at high levels (the cells express CCR4 on their cell membranes). will be 10 7 CCR4 + CHO cells were injected into the peritoneal cavity of female C57BL / 6 mice. Immunization once every two weeks, a total of 5-8 immunizations; the last immunization CCR4 + After 4 days of CHO cells, the mice were sacrificed, and the spleens of the mice were taken for cell fusion. The cells were mixed according to the ratio of splenocytes: SP2 / 0 cells = 1:1, and the supernatant was discarded after centrifugation at 1500 rpm for 7 minutes. The cells were resuspended with 20mL electroporation buffer and centrifuged at 1500rpm for 7min. Discard the supernatant and repeat once. Resuspend the cells with an appropriate amount of electroporation buffer to ensure a cell concentration of 2×10 7cells / mL. Add the cell suspension to a 9mL electroporation fusio...

Embodiment 2

[0029] Example 2. Antibody humanization

[0030] The subcloned positive hybridoma cells were expanded and cultured, and an appropriate amount of cells was taken to extract total RNA according to the instructions of the RNeasy PlusMini Kit (Qiagen, 74134) kit, and TransScript One-Step gDNARemoval and cDNA Synthesis SuperMix (TRANS, AT311-02) The reverse transcription kit synthesizes the first strand of cDNA. Design specific primers according to the variable region of the mouse antibody subtype (the 5' end contains the homology arm sequence for homologous recombination with the eukaryotic expression vector), and use cDNA as a template to perform PCR amplification of the antibody variable region gene , so as to obtain the gene fragments of the mouse antibody light chain and heavy chain variable region respectively. For the humanization of antibodies, first use the currently effective public databases (ie, NCBI's Blast for IgG and MRC's V-base), and compare the amino acid sequenc...

Embodiment 3

[0031] Example 3. Hu-11F6-IgG1 specifically binds to CCR4 on the surface of tumor cells

[0032] The CCR4-positive cell line human breast cancer cell line MCF-7 was selected, and the binding activity of the anti-CCR4 antibody to the CCR4 protein expressed on the surface of tumor cells was detected, and a cell-based ELISA method was adopted. MCF-7 cells were seeded in 96-well cell culture plates, 1 × 10 per well 5 cells were cultured overnight at 37°C. The CCR4 antibody Hu-11F6-IgG1 to be tested and the control antibody 503 (the sequence of the control antibody was obtained from line 23 on page 3 of the patent specification of CN103328513B, and the control antibody 503 was obtained according to the method in Example 2) were mixed with 10% FBS The DMEM medium was serially diluted 3 times, with a concentration ranging from 1.83ng / mL to 4μg / mL, and then added to the corresponding wells at a volume of 50μL per well, and the cell culture plate was incubated at 4°C for 1h. Remove t...

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Abstract

The invention relates to an antibody specifically binding to CCR4 and an application thereof in treatment of cancers. The antibody can be combined with CCR4 positive cells with relatively high affinity, block CCL17 and CCL22 from being combined with CCR4 on the surfaces of tumor cells, promote the ADCC effect of effector cells on the tumor cells and provide new possibilities for treatment and / or prevention of cancers.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to an anti-CCR4 antibody and its application in treating various hematological malignancies and solid tumors. Background technique [0002] Chemokine receptors are seven transmembrane receptors expressed on the surface of some specific cells, and their encoded proteins belong to the G protein-coupled receptor family, which can be divided into four types according to their ligands: CXC type, XC type , CC type, CX 3 Type C. CC-type chemokine receptor 4 (CCR4), also known as CD194, is mainly expressed on T cells (especially Th2 cells and Treg cells), and on dendritic cells, macrophages, NK cells, platelets, mast cells and Expression was also found on some tumor cells. CCR4 has three natural specific ligands: thymus activation-regulated chemokine (TRAC / CCL17), macrophage-derived chemokine (MDC / CCL22), and chemokine-like factor 1 (CKLF1). [0003] With the discovery of CCR4 and its three l...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28A61K39/395A61P35/00
CPCA61K2039/505A61P35/00C07K16/2866C07K2317/56C07K2317/565
Inventor 彭菲顾超
Owner TIANJIN KINGMED CENT FOR CLINICAL CO LTD
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