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Specific antibody taking CD19 as target point, CAR-NK cell as well as preparation and application thereof

An antibody and cell technology, applied in the field of biomedicine, can solve the problems of non-allogeneic reinfusion, large immune rejection risk, etc., and achieve the effect of stable traits

Active Publication Date: 2018-05-18
ASCLEPIUS SUZHOU TECH CO GRP CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example: first, the T cells used to prepare CAR-T can only come from the patient itself, and cannot be reinfused; immune rejection risk

Method used

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  • Specific antibody taking CD19 as target point, CAR-NK cell as well as preparation and application thereof
  • Specific antibody taking CD19 as target point, CAR-NK cell as well as preparation and application thereof
  • Specific antibody taking CD19 as target point, CAR-NK cell as well as preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Screening of CD19 Human Antibody

[0065] 2×YT liquid medium: 16g peptone, 10g yeast extract, 5g sodium chloride, 800mL water, adjust pH to 7.0 with sodium hydroxide, add water to 1000mL, sterilize at 121°C for 20min.

[0066] 2×YT-G: Add 2% glucose to 2×YT medium.

[0067] 2×YT-AK: Add 100 g / mL ampicillin and 50 g / mL kanamycin to 2×YT medium.

[0068] 1. Preparation of M13KO7 helper phage

[0069] 1. Under the condition of 37° C., the TG1 bacterial cells in logarithmic growth phase were infected with different dilution concentrations of helper phage M13K07 for 30 minutes, and then spread on the agar plate.

[0070] 2. Pick the TG1 plaque clone into 3mL liquid 2YT medium for culture. Incubate at 37°C for 2 hours.

[0071] 3. Transfer the culture in step 2 to 1L 2YT medium, add kanamycin to 50 μg / m, and culture at 37°C for 16 hours.

[0072] 4. Centrifuge (10min at 5000g) to remove bacterial cells, add phage precipitation agent to the supernatant to collec...

Embodiment 2

[0088] Example 2: Expression and Identification of CD19 Antibody

[0089] The single-stranded phage DNA that embodiment 1 obtains is cloned on the pCDNA3.4 carrier, constructs such as figure 1 For the pCDNA3.4-ScFV (anti-CD19) expression vector shown, the vector was transiently transferred to CHO cells for expression, and the single-chain antibody sequence was purified by nickel strain for subsequent analysis.

[0090] The affinity of the screened single-chain antibody to CD19 protein was identified by SPR method, and the single-chain antibody with the highest affinity to CD19 protein was selected and named as single-chain antibody 2-27. The molecular affinity between single chain antibody 2-27 and CD19 is shown in Table 1.

[0091] Table 1: Molecular affinity of scFv 2-27 to CD19

[0092] sequence

[0093] Wherein, the amino acid sequence of the VH chain of the single-chain antibody 2-27 is shown in SEQ ID NO.7, and its nucleotide sequence is shown in SEQ ID NO.10...

Embodiment 3

[0094] Example 3 Preparation of lentiviral expression vector

[0095] Gene synthesis SCFV(2-27)-CD8-4-1BB-CD3ζ fusion gene sequence (the amino acid sequence is shown in SEQ ID NO: 13, and the gene sequence is shown in SEQ ID NO: 14). Through enzyme digestion, it was transformed and connected to the PRRSL IN vector, and the upstream of the gene was the EP-1α promoter. Transform the Stbl3 E. coli strain with the vector, screen with ampicillin, obtain positive clones, extract the plasmids, identify the clones by enzyme digestion, and obtain the PRRLSIN-SCFV(2-27)-CD8-4-1BB-CD3ζ lentiviral transfection vector (such as figure 2 shown).

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Abstract

The invention provides an anti-CD19 antibody taking CD19 as a target point or an antigen binding fragment, wherein the antibody contains a heavy chain variable area (VH) and / or a light chain variablearea (VL); the heavy chain variable area contains heavy chains VHCDR1, VHCDR2 and VHCDR3; and the light chain variable area contains light chains VLCDR1, VLCDR2 and VLCDR3. The anti-CD19 antibody taking CD19 as the target point or the antigen binding fragment, which is provided by the invention, an anti-CD19 CAR-NK cell structured thereby is obtained through monoclonal cell cultivation and amplification, has stable characteristic and can be applied to large-scale production and preparation. The anti-CD19 CAR-NK cell takes a CD19 molecule as a target antigen, can specifically kill or damage lymphoma cells, can serve as a medicine for treating lymphoma diseases and is applied to treatment on tumor highly expressed by the CD19 molecules.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a specific antibody targeting CD19, a CAR-NK cell targeting CD19, and its preparation and application. Background technique [0002] Chimeric antigen receptor (chimeric antigen receptor, CAR) modified immune cells use genetic engineering to modify immune cells to express exogenous anti-tumor genes. The CAR gene mainly includes an extracellular recognition domain and an intracellular signal transduction domain: the former is used to recognize specific molecules on the tumor surface and the latter is used to initiate immune cell responses after recognizing tumor surface molecules and exert cytotoxicity. T-cells are the vectors. [0003] CAR-T, the full name is Chimeric AntigenReceptor T-Cell Immunotherapy, chimeric antigen receptor T cell immunotherapy. Chimeric antigen receptor T cells (CAR-T cells) combine the antigen-binding part of an antibody that can recognize a certain tumor ant...

Claims

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Application Information

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IPC IPC(8): C07K16/28C12N15/13C12N5/10C12N15/62C12P21/02A61K35/17A61P35/00
CPCC07K14/7051C07K16/2803C07K2317/92C07K2317/56C07K2317/622C07K2319/33A61K39/464412A61K39/4613A61K39/4631
Inventor 李华顺
Owner ASCLEPIUS SUZHOU TECH CO GRP CO LTD
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