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Anti-CD19 antibody and preparation method and use thereof

A CDR-H1, CDR-L1 technology, applied in the field of anti-CD19 antibody and its preparation, can solve the problems of poor persistence of CART cells, decreased affinity, and inability of CART cells to clear plasma cells

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

AI Technical Summary

Problems solved by technology

2. CD19 is still expressed on the surface of tumor cells. The persistence of CART cells in the body is poor, and they cannot continue to expand in patients. Cell activity is blocked or cleared; before the patient achieves complete remission, the murine framework induces a normal B cell immune response in the body and develops into plasma cells (Plasma Cells), which secrete a large amount of HAMA antibodies; since plasma cells do not express CD19 antigen, CART cells Unable to clear plasma cells, resulting in antibody-mediated CART cell activity blocking (Blocking) and elimination (Elimination)
In the patent No. US20140271635, FMC63 has been humanized (CDR transplantation under the framework of human Germline), its heavy chain variable region is VH4 4-59 (Vbase2), and its light chain variable region is VK3_L25 (Vbase2). Through the in vitro binding test, a total of 12 clones (C2136-C2147) were obtained to maintain binding activity to CD19, and the C2146 clone was the preferred clone, and a single amino acid mutation (YNS A L→YNS S L); the scFv cell binding experiment showed that the affinity of C2146 clone was 2-3 times lower than that of FMC63 clone

Method used

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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
Comparison scheme
Effect test

Embodiment 1

[0094] Construction of CDR1, CDR2, and CDR3 mutation libraries of heavy chain (H) and light chain (L) of FMC63 scFv and C2146 scFv:

[0095] The selection of FMC63 and C2146 heavy chain and light chain CDR regions is based on the amino acid counting method of the variable region (the template is the monoclonal antibody FMC63, VH: Y14283.1, VL: Y14284.1; the C2146 single-chain antibody sequence is selected from US20140271635), Kabat counts (Kabat number scheme.Bioinf.org.uk) are listed below:

[0096] Table 1

[0097]

[0098]

[0099] Constructed with pCAN-FMC63 scFv plasmid and pCAN-C2146 scFv (the template sequence is inserted into the multiple cloning site of pCANTAB 5E plasmid (purchased from GE), the FMC63 template sequence is: VH: Y14283.1, VL: Y14284.1, C2146 template See US20140271635 for the sequence) as a template, and introduce mutations by PCR with random primers. The primers for the FMC63 mutation library and the C2146 mutation library primers are shown in ...

Embodiment 2

[0107] Panning of phage antibody library:

[0108] Add 20nM CD19-his-biotin antigen and FMC63 scFv or C2146 scFv phage antibody library and incubate at room temperature for 2h, then transfer the mixture to streptavidin magnetic beads (Dynabeads M-280Streptavidin) 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 eluted by trypsin digestion, let stand at 37°C for 30min, and take part of the bacterial liquid to serially dilute (1 / 10, 1 / 100, 1 / 1000) for plate counting, and the remaining bacteria The solution was all coated on 2xYT(GA) plates for the next 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 PB...

Embodiment 3

[0110] Screening and identification of high-affinity scFv:

[0111]After four rounds of panning, single clones were randomly selected, and the supernatant was taken for ELISA screening after IPTG induction: 1ug / ml Avidin was coated overnight on a 96-well plate, washed twice with PBST, and 100ul 1ug / ml CD19-biotin was added Avidin-coated 96-well plates were incubated for 1 hour, washed twice with PBST, incubated with supernatant after IPTG induction for 1 hour, washed 4 times with PBST, added 100ul diluted anti-Myc antibody at 1:5000, incubated for 30 minutes, washed with PBST After 4 times, add TMB substrate to react for 10 minutes, add 1M sulfuric acid solution to terminate the reaction, and read OD450nm light absorption. Pick the clones whose positive signal (OD450nm light absorption) is at least 2 times greater than the negative signal and send them for sequencing, analyze the sequencing results, and extract the clones corresponding to the more enriched CDR regions. The re...

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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. The anti-CD19 antibody comprises a heavy chainvariable region and a light chain variable region. The complementarity determining region of the heavy chain variable region 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 or 3 and CDR-H3 with an amino acid sequence shown in the formula of SEQ ID No. 4. The complementarity determining region of the light chain variable region comprises CDR-L1 with an amino acid sequence shown in the formula of SEQ ID No. 5, CDR-L2 with an amino acid sequence shown in the formula of SEQ ID No. 6 and CDR-L3 with an amino acid sequence shown in the formula of SEQ ID No. 7. The FMC63 scFv is screened in affinity maturation through a phage display technology so that a high affinity single chain antibodyagainst 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] Hematological tumors associated with malignant proliferat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/28C07K19/00C12N15/13C12N5/10A61K35/17A61P35/02G01N33/68
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 SHANGHAI GENBASE BIOTECH CO LTD
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