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Cluster of differentiation 19 (CD19) antibody and application thereof

An antibody and antigen technology, applied in the field of biomedicine, can solve the problems of limited tumor inhibition ability and low specificity of CD19 antibody, and achieve the effect of good target killing effect, high purity and high stability

Pending Publication Date: 2020-06-09
SUNSHINE LAKE PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] However, the currently developed CD19 antibodies have low specificity and limited ability to inhibit tumors, so it is urgent to develop new anti-CD19 drugs for new drug development

Method used

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  • Cluster of differentiation 19 (CD19) antibody and application thereof
  • Cluster of differentiation 19 (CD19) antibody and application thereof
  • Cluster of differentiation 19 (CD19) antibody and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1CD19

[0125] The preparation of embodiment 1CD19 antibody

[0126] 1.1 Immunization of experimental animals

[0127] To generate CD19 antibodies, purified recombinant CD19 protein (ACRO, CD9-H52H2) and CD19 +Ramos cells (ATCC) were used to immunize six-week-old Babl / c female mice (Hunan Slack Biotechnology Co., Ltd.), and the mice used were SPF grade (specific pathogen free). Mice can be immunized according to subcutaneous immunization of recombinant protein and intraperitoneal injection of Ramos cells (purchased from ATCC), and the mice are six weeks old after the first infusion of antigen. For the first immunization, the recombinant CD19 protein and complete Freund's adjuvant (purchased from Sigma) were used for immunization at the ratio of v:v=1:1, and then subcutaneous immunization was carried out on the limbs, 50 μg per mouse. A week later, the second immunization was carried out, and the recombinant CD19 protein and Freund's incomplete adjuvant (purchased from Sigma) were ...

Embodiment 2CD19

[0140] Example 2 CD19 Antibody Activity Detection (ELISA)

[0141] The CD19 antibody prepared in Example 1 was detected by ELISA method. Take 100 μg of CD19 protein (purchased from acro), and add 300 μL of ultrapure water (purchased from PALL for ultrapure water instrument) to fully dissolve to obtain a CD19 protein dilution with a concentration of 0.33 μg / μL. The 96-well assay plate was coated with 100 ng per well.

[0142] Dilute CD19 protein to 1μg / μL with PBS buffer solution, take 40mL; and take 120μL of CD19 protein dissolved in ultrapure water above into 40mL PBS buffer solution, mix the two thoroughly and use 100μL per well Add to 96-well assay plate and incubate at 37°C for 2 hours. After incubation, the remaining protein solution in the 96-well plate was removed, and 280 μL (1% BSA+PBS) blocking solution was added to each well, incubated at 37°C for 2 hours, and then stored at 4°C for use.

[0143] The antibodies 16F10, 25C5-2, and 10H10-1 prepared in Example 1 wer...

Embodiment 3CD19

[0146] Example 3 Detection of CD19 Antibody Activity

[0147] 3.1 Cell Treatment

[0148] Collect NALM-6 cells into a 50mL centrifuge tube, centrifuge at 400g for 5 minutes to collect the cells, take 20mL of PBS pre-cooled to 4°C to resuspend the cells, and collect the cells by centrifugation, wash the cells three times with PBS according to this method, and then wash the cells with 13mL Count the cells after resuspending them in PBS, and the cell concentration is 3.92×10 6 cells / mL, the cell survival rate was 98%. The cells were dispensed into flow detection tubes according to 600 μL per tube, and there were 25 tubes in total.

[0149] 3.2 CD19 Antibody Dilution

[0150] The CD19 antibody was diluted with PBS according to the following gradient. The specific operation is as follows: Take the antibodies 16F10, 10H10-1, and 25C5-2 prepared in Example 1 and dilute them to 50 μg / mL, 10 μg / mL, 5 μg / mL, 2.5 μg / mL, and 1.25 μg / mL, respectively. Take 200 μL and add it to the c...

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Abstract

The application relates to an antibody which can be specifically bound to a cluster of differentiation 19 (CD19) or an antigen-binding fragment of the antibody, and the anti body is bound to CD19 protein with 3.5 [mu]g / mL or a smaller half maximal inhibitory concentration (IC50) value. The application further relates to a use of the antibody or the antigen-binding fragment thereof in preparation of drugs.

Description

technical field [0001] The present application relates to the field of biomedicine, in particular to an antibody or an antigen-binding fragment thereof that specifically binds to the CD19 protein. Background technique [0002] B lymphocyte antigen CD19, also known as CD19 molecule (cluster of differentiation 19). In normal lymphoid tissue, CD19 is expressed in B cells in the germinal center and dendritic cells in the follicular dendritic cells, mantle cells, and dendritic cells in the interfollicular T cell zone. CD19 plays two main roles in human B cells, acting as an adapter protein to recruit cytoplasmic signaling proteins to the membrane, and acting within the CD19 / CD21 complex to lower the threshold of the B cell receptor signaling pathway . CD19 protein is a target protein that exists stably on the surface of B lymphocytes. It exists in all stages of B cell maturation. Most hematological tumor-related tumors (B-ALL / CLL / B-NHL, etc.) highly express CD19. It is not exp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K19/00C12N15/13C12N15/62A61K39/00A61P35/00A61P35/02A61P37/02
CPCC07K16/2803C07K14/7051C07K14/70578A61K39/0008A61K39/001112A61P35/00A61P35/02A61P37/02C07K2317/92C07K2317/565C07K2317/56C07K2317/622C07K2317/76C07K2319/03C07K2319/33C07K2319/74A61K39/4631A61K39/00A61K39/464412A61K39/4611C07K16/28C07K19/00C12N15/62C07K17/00A61K38/17C12N5/10
Inventor 董军纪陈超于婷婷徐乐危喜玲李志广李想曾怡李少燕陈小锋李文佳
Owner SUNSHINE LAKE PHARM CO LTD
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