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Anti-CD19 antibody and preparation method and use thereof

An antibody and monoclonal antibody technology, applied in the direction of antibody medical components, antibody mimics/stents, anti-tumor drugs, etc., can solve the problems of difficult to obtain curative effects and prolong patient survival.

Active Publication Date: 2018-03-13
HUADAO SHANGHAI BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, the treatment of B cell tumors mainly includes small molecule targeted drugs such as imatinib (BCR-Abl kinase inhibitor) and ibrutinib (brutonkinase inhibitor), antibody drugs such as rituximab (CD20antibody) and bone marrow Bonemarrow transplantation: In clinical application, small molecule drugs and antibody drugs can significantly prolong the survival period of patients and buy time for bone marrow transplantation, but some patients will relapse with drug resistance. It is difficult for relapsed patients to obtain curative effect

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  • Anti-CD19 antibody and preparation method and use thereof
  • Anti-CD19 antibody and preparation method and use thereof
  • Anti-CD19 antibody and preparation method and use thereof

Examples

Experimental program
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Embodiment 1

[0066] Construction of the CDR1, CDR2, and CDR3 mutation libraries of the heavy chain (H) and light chain (L) of FMC63scFv:

[0067] The selection of FMC63 heavy chain and light chain CDR regions is based on the amino acid counting method of the variable region (the template is monoclonal antibody FMC63, VH: Y14283.1, VL: Y14284.1), Kabat counting (Kabat number scheme.Bioinf.org. uk) list as follows:

[0068] Table 1

[0069]

[0070]

[0071]Using the pCAN-FMC63scFv plasmid (constructed by inserting the template sequence into the multiple cloning site of the pCANTAB 5E plasmid (purchased from GE), the template sequence is: VH: Y14283.1, VL: Y14284.1) as a template, the mutation was introduced by PCR with random primers , the primers are shown in Table 2. The obtained PCR products of the CDR1, CDR2, and CDR3 mutant libraries of the heavy chain (H) and light chain (L) of FMC63 scFv were named H1, H2, H3, L1, L2, and L3, respectively. After the PCR product and pCANTAB 5...

Embodiment 2

[0075] Panning of phage antibody library:

[0076] Add 20nM CD19-his-biotin antigen and incubate with the phage antibody library at room temperature for 2h, then transfer the mixture to streptavidin magnetic beads and incubate for 15min at room temperature. Unbound phages were washed away with PBST-PBS, and trypsin was added for 30 min at 37°C to elute bound phages. Infect 4ml of TG1 cells in the logarithmic phase with the phages digested and eluted by trypsin, let stand at 37°C for 30 minutes, take part of the bacterial solution to serially dilute for plate counting, and spread the rest of the bacterial solution on 2xYT(GA) plates for next A round of packaging. The packaged phage can be used for the next round of panning. A total of 4 rounds of panning and enrichment were carried out. Each round of panning was 10 times diluted and the antigen concentration was gradually reduced, and the number of PBST-PBS washings was increased round by round (the CD19 -his-biotin antigen c...

Embodiment 3

[0078] Screening and identification of high-affinity scFv:

[0079] After four rounds of panning, single clones were randomly selected, and after IPTG induction, the supernatant was taken for ELISA. After preliminary screening by ELISA, clones with positive signals at least 2 times greater than negative signals were selected and sent for sequencing. clones corresponding to more CDR regions. The concrete steps of described ELISA preliminary screening are as follows:

[0080] Pick colony clones in 4ml 2xYT medium, culture overnight at 37°C 200rpm, transfer to 500ml 2xYT medium at a ratio of 1:100, and culture at 37°C 200rpm until logarithmic growth phase; add M13K07 helper phage (MOI=20), 37 Incubate at ℃ for 30 minutes, then incubate at 37℃ and 200rpm for 30 minutes; remove the supernatant by centrifugation, and resuspend the bacteria in 500ml 2xYT / Kan / Amp resistance medium for overnight culture; use 1 / 5 volume of the overnight culture supernatant The PEG / NaCl solution was pr...

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Abstract

The invention relates to the technical field of biotechnology and particularly relates to an anti-CD19 antibody and a preparation method and use thereof. CDR in a heavy chain variable region of the anti-CD19 antibody comprises CDR-H1 with an amino acid sequence shown in the formula of SEQ ID No. 1, CDR-H2 with an amino acid sequence shown in the formula of SEQ ID No. 2 and CDR-H3 with an amino acid sequence shown in the formula of SEQ ID No. 3. The CDR of a light chain variable region of the anti-CD19 antibody comprises CDR-L1 with an amino acid sequence shown in the formula of SEQ ID No. 4, CDR-L2 with an amino acid sequence shown in the formula of SEQ ID No. 5 and CDR-L3 with an amino acid sequence shown in the formula of SEQ ID No. 6. The FMC63 scFv is screened in affinity maturation through a phage display technology so that a high affinity single chain antibody against CD19 is obtained.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an anti-CD19 antibody and its preparation method and application. Background technique [0002] CD19 antigen (B lymphocyte antigen CD19, CD19) is a protein specifically expressed on the surface of human B cells. CD19 is widely expressed on the surface of B cells at various developmental stages and plays an important role: CD19 acts as a B cell receptor (B cell receptor, BCR) co-receptor (co-receptor), which can reduce antigen-mediated B cell receptor Signaling threshold required for antigen receptor-dependent stimulation; B-cell receptor activation is dependent on phosphorylation of the intracellular domain of CD19 followed by recruitment of Src kinase and PI3K kinase to fully activate B cells. Fully mature B cells are called plasma cells, and plasma cells will lose the expression of CD19 antigen after fully mature. [0003] Hematologic tumors associated with malignant proliferatio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K19/00C12N15/13C12N5/20C12N15/63A61K39/395A61P35/00A61P35/02A61P37/00G01N33/68G01N33/577
CPCC07K16/2803A61K2039/505C07K2317/565A61P35/02A61K2039/5158A61K2039/5156C07K14/7051C07K2319/03A61K39/0011A61K35/17
Inventor 牟男张云马泽龙袁纪军曹跃琼
Owner HUADAO SHANGHAI BIOPHARMA CO LTD
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