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Monoclonal antibody against CD19 as well as preparation method and application thereof

A monoclonal antibody and sequence technology, applied in the fields of botany equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of human anti-mouse and other problems, and achieve low immunogenicity, good stability and safety , good targeting and lethal effect

Active Publication Date: 2018-11-20
CELLYAN THERAPEUTICS WUHAN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A disadvantage of using murine antibodies to treat human subjects is the development of human anti-mouse (HAMA) responses after administration to patients

Method used

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  • Monoclonal antibody against CD19 as well as preparation method and application thereof
  • Monoclonal antibody against CD19 as well as preparation method and application thereof
  • Monoclonal antibody against CD19 as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Preparation of specific single-chain antibody (scFv) binding to human CD19

[0043] 1. Screening of anti-human CD19 specific antibody based on phage display

[0044] 1. In order to achieve this purpose, inoculate glycerol bacteria (self-built library) with phage displaying the natural library of fully human single-chain antibody in 400ml 2×YT / ampicillin medium, so that the cell density reaches OD600=0.1, at 37°C and Shake culture at 200 rpm until the cell density reaches OD600=0.5. use 10 12 Pfu's M13KO7 helper phage (purchased from ThermoFisher) was infected, and incubated at 30° C. and 50 rpm for 30 minutes. After adding 50mg / L kanamycin, shake and culture at 37°C and 200rpm for 30 minutes, separate the precipitate by centrifugation (15 minutes, 1600×g, 4°C), and resuspend in 400ml 2×YT / ampicillin / cardiac Namycin culture medium was shaken at 37° C. and 200 rpm for 16 hours. Finally, the precipitate was separated by centrifugation (20 minutes, 5000 × g, 4...

Embodiment 2

[0053] Example 2 Expression and purification of single-chain antibody against CD19

[0054] 1. Expression of human CD19 single chain antibody

[0055] The selected single-chain antibody was constructed by recombinant eukaryotic expression vector, transfected with HEK293F to induce recombinant expression and purified. Use primer pair 3B5-F (SEQ ID NO:10) and 3B5-R (SEQ ID NO:11) to amplify the ScFv-3B5 fragment from clone pCantab-3B5 obtained in embodiment 1; Through NheI / BamHI (purchased from Takara Company) double enzyme digestion, and T4 DNA ligase in the same double digestion vector plasmid pCMV-V5-Fc with Nhe I / BamH I (this vector is fused to express the Fc fragment of human IgG1 antibody downstream of the multiple cloning site, hereinafter referred to as V5 -Fc) was ligated and transformed into the host strain TOP10, and the clones were picked to identify positive clones by PCR and confirmed by sequencing to obtain the scFv-3B5-Fc eukaryotic expression plasmid.

[0056]...

Embodiment 3

[0058] Example 3. Antigen-binding activity analysis of single-chain antibody against human CD19

[0059] 1. ELISA analysis of the binding activity of recombinant antibody to human CD19 antigen

[0060] The binding activity of the screened antibodies to the antigen human CD19 was determined by concentration gradient ELISA experiment. For this purpose, the antigen human Fc-CD19 was diluted with 0.1M NaHCO3 (pH 9.6) coating solution, coated with 100ng per well, 50μl / well, overnight at 4°C, and coated with 2% BSA and 0.01% (v / v ) Tween-20 in PBST blocking solution for 2 hours at room temperature. Plates were then rinsed three times with PBST and removed. Subsequently, 100 μl of PBST solution containing a series of concentrations (initial concentration 10 nM, 3-fold serial dilution, until diluted to 1:729) of each antibody protein was added to each well plate, and the determination of each sample was performed using parallel triplicate analysis. . After incubation at 37°C for 2...

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Abstract

The invention provides a monoclonal antibody against CD19. The monoclonal antibody is characterized in that a complementary determining region (CDR) of a VH chain has amino acid sequences selected from the following groups: a heavy chain CDR1 shown as SEQ ID NO: 3, a heavy chain CDR2 shown as SEQ ID NO: 4 and a heavy chain CDR3 shown as SEQ ID NO: 5; a complementary determining region (CDR) of a VL chain has amino acid sequences selected from the following groups: a light chain CDR1 shown as SEQ ID NO: 6, a light chain CDR2 shown as SEQ ID NO: 7 and a light chain CDR3 shown as SEQ ID NO: 8. The invention further provides a preparation method and application of the monoclonal antibody. The monoclonal antibody can specifically recognize human CD19 proteins on the cell surface, is combined with the human CD19 by using 1*10<-7> M or lower KD and has the advantages that the immunogenicity is low, the HAMA reaction is not caused, and the like.

Description

technical field [0001] The invention relates to the technical field of biological immunity, in particular to an anti-CD19 monoclonal antibody and its preparation method and application. Background technique [0002] CD19 is a transmembrane protein on the surface of B cells. It is closely related to B cell activation, signal transduction and growth regulation. It is a functional receptor molecule on the surface of B lymphocytes. When B cell antigen receptor (BCR) recognizes antigens, it constitutes a B cell double antigen binding model and participates in Inner Ca 2+ It regulates the activation and proliferation of B cells. As an important marker, CD19 is widely used in the diagnosis and prognosis of leukemia, lymphoma and immune system diseases. At the same time, CD19 is used as an important target of immunotherapy, and CD19 is an important target of B cell malignancies. CD19 is expressed at almost all stages of B-cell development, as well as in various B-cell tumors, but...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13C12N5/10A61K35/17A61P35/00
CPCA61P35/00A61K35/17C12N5/0636C07K14/7051C07K16/2803C07K2319/02C12N2510/00A61K39/4611A61K39/464412A61K39/4631C07K2319/03
Inventor 华权高沈鹤霄金丹罗绍祥李璐李国龙唐文
Owner CELLYAN THERAPEUTICS WUHAN CO LTD
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