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Specific antibody targeting CD19, car-nk cell and its preparation and application

An antibody and cell technology, applied in the field of biomedicine, can solve the problems of inability to reinfuse the allogeneic body, the risk of large immune rejection, etc., and achieve the effect of stable traits

Active Publication Date: 2021-08-24
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 targeting CD19, car-nk cell and its preparation and application
  • Specific antibody targeting CD19, car-nk cell and its preparation and application
  • Specific antibody targeting CD19, car-nk cell and its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] Example 1 Screening of CD19 Human Source Antibodies

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

[0066] 2 × YT-G: 2 × YT medium was added 2% glucose.

[0067] The 2 × YT-AK: 2 x YT medium was added 100 g / ml of ampicillin and 50 g / ml kanamycin.

[0068] First, prepare M13KO7 auxiliary phage

[0069] 1. Under 37 ° C, the long-term TG1 bacterial cells were infected with different dilution concentrations for 30 minutes, then coated it on the agar plate.

[0070] 2. TG1 bacteria collealed to 3 ml of liquid 2YT medium for culture. It was cultured for 2 hours at 37 ° C.

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

[0072] 4. Centrifugation (10min AT 5000g) Remove bacterial cells, adding phage precipitates to the supernatant to collect phage.

[0073] 5. Ca...

Embodiment 2

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

[0089] The single-stranded phage DNA obtained in Example 1 was cloned to the PCDNA3.4 vector, and constructed Figure 1 The PCDNA3.4-SCFV (Anti-CD19) expression vector was transiently expressed to CHO cells, purified by nickel strains for subsequent analysis.

[0090] The fractional single-chain antibody to the CD19 protein was identified by the SPR method, and the highest affinity of the CD19 protein was selected from which the affinity of the CD19 protein was selected, and it was named a single chain antibody 2-27. Single-chain antibody 2-27 and CD19 molecular affinity are shown in Table 1.

[0091] Table 1: Single-chain antibody 2-27 and CD19 molecular affinity

[0092] Sequence KA (1 / ms) KD (1 / s) KD (M) 2-27 4.984E+4 0.004223 8.473E-8

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

Embodiment 3

[0094] Example 3 Preparation of a slow viral expression vector

[0095] Gene Synthesis SCFV (2-27) -CD8-4-1BB-CD3ζ Fusion Gene sequence (which is shown in SEQ ID NO: 13, shown in SEQ ID NO: 14). Through the digestion, it is converted to the PRRSL IN vector, and the gene is upstream of the EP-1α promoter. The vector transformed STBL3 Escherichia coli strain, ampicillin screening, gaining positive clones, extracting plasmid, and cloning, obtained Prrlsin-SCFV (2-27) -CD8-4-1BB-CD3ζ slow virus transfection carrier (eg figure 2 Indicated).

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Abstract

The present invention provides an anti-CD19 antibody or an antigen-binding fragment thereof targeting CD19, wherein the antibody comprises a heavy chain variable region (VH) and / or a light chain variable region (VL), wherein the heavy chain The variable region comprises heavy chain VHCDR1, VHCDR2, VHCDR3; the light chain variable region comprises light chain VLCDR1, VLCDR2, VLCDR3. The present invention provides an anti-CD19 antibody or an antigen-binding fragment thereof targeting CD19, and the Anti CD19 CAR-NK cells constructed therefrom are cultured and expanded monoclonal cell lines, which have stable properties and can be used for large-scale production preparation. Anti CD19 CAR‑NK cells use CD19 molecule as the target antigen, can specifically kill or kill lymphoma cells, and can be used as a therapeutic drug for lymphoma diseases, and can be used for the treatment of tumors with high expression of CD19 molecules.

Description

Technical field [0001] The present invention relates to the field of biopharmaceutical, and more particularly to CD19 as a target of CAR-NK cells and its preparation and application thereof in CD19. Background technique [0002] Chimeric antigen receptor, abbreviated Car) modified immune cells use genetic engineering means to modify immunocytes to express exogenous anti-tumor genes. The Car gene mainly includes an extracellular identification domain and intracellular signal transduction domain: the former is used to identify tumor surface-specific molecules and latter for initiating immune cell responses after identifying tumor surface molecules, and plays cytotoxic effects. T-cells are vector. [0003] CAR-T, full name is Chimeric Antigenreceptor T-Cell Immunterapy, chimeric antigen receptor T cell immunotherapy. The chimeric antigen receptor T cell (CAR-T cell) is an intracellular element such as an antigen binding portion that recognizes a tumor antibody and a CD3-ζ chain or 4...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13C12N5/10C12N15/62C12P21/02A61K35/17A61P35/00
CPCA61K35/17C07K14/7051C07K16/2803C07K2317/56C07K2317/622C07K2317/92C07K2319/33
Inventor 李华顺
Owner ASCLEPIUS SUZHOU TECH CO GRP CO LTD
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