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CAR-NK cell as well as preparation method and application thereof

A technology of NK cells and CD5, applied in biochemical equipment and methods, DNA/RNA fragments, genetically modified cells, etc., to achieve the effect of eliminating tumor immune escape, promoting expression, and improving killing effect

Pending Publication Date: 2022-06-10
JIANGSU MOBILI BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, chimeric antigen receptors are widely used in T cells, but there are only a few attempts in NK cells, how to choose a chimeric antigen receptor structure suitable for NK cells, and how to cooperate with immune checkpoint anti-tumor mechanisms Still to be confirmed and excavated

Method used

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  • CAR-NK cell as well as preparation method and application thereof
  • CAR-NK cell as well as preparation method and application thereof
  • CAR-NK cell as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Chimeric Antigen Receptor Design

[0033] In this example, a bispecific antibody including an anti-BCMA target was constructed, and the sequence diagram is as follows figure 1 As shown, its structure is S-BCMA scFv-CDx scFv-H-TM-C-CD3ζ, wherein BCMA scFv is the antigen-binding domain targeting BCMA; CDxscFv is the antigen-binding domain targeting CDx, specifically including CD3 scFv, CD5 scFv, CD7 scFv, CD19 scFv, CD20scFv; "-" means connecting peptide or peptide bond; S means signal peptide; H means hinge region; TM means transmembrane domain; C means co-stimulatory signal molecule; CD3ζ means intracellular signal transduction sequence.

[0034] The antigen-binding domain sequences of each target were obtained by the inventors in the previous research, taking the bispecific chimeric antigen receptor targeting BCMA and CD5 as an example, wherein the BCMA scFv includes the heavy sequence shown in SEQ ID NO: 1-3 Chain CDR region and light chain CDR region sho...

Embodiment 2

[0035] Embodiment 2: NK cell preparation

[0036] In this embodiment, the peripheral blood mononuclear cells (PBMC) of patients were obtained by density gradient centrifugation. Specific steps include:

[0037] Draw 20mL of human peripheral blood into a centrifuge tube with anticoagulant added, centrifuge at 2000rpm for 10min; collect the upper plasma, add an equal volume of pre-warmed normal saline to the remaining blood cell pellet, and mix thoroughly; Gently add the mixed blood cell pellet to the surface of the lymphocyte separation medium according to the volume of 1:1, centrifuge at 18000rpm for 25min; carefully absorb the white lymphocyte layer; transfer the buffy coat layer to a new centrifuge tube, and use Make up to 45 mL with PBS, centrifuge at 1500 rpm for 5 min, wash twice; add an appropriate amount of RPMI1640 (containing 10% FBS) complete medium to resuspend the cells and count them.

[0038] NK cells were sorted out using NK cell sorting kit (purchased from bi...

Embodiment 3

[0039] Example 3: NK cell immune checkpoint gene knockout

[0040] According to the sequence structure of human PD-1, CTLA4, LAG-3 and Tim-3 genes, and referring to the existing research content of related CRISPR gene editing tools, design their respective sgRNA sequences, wherein the sgRNA of PD-1 gene is as shown in SEQ The nucleotide sequence shown in ID NO.19, the sgRNA of CTLA4 gene is the nucleotide sequence shown in SEQ ID NO.20, the sgRNA of LAG-3 gene is the nucleotide sequence shown in SEQ ID NO.21 Sequence, the sgRNA of Tim-3 gene is the nucleotide sequence shown in SEQ ID NO.22. Entrust Sangon Bioengineering (Shanghai) Co., Ltd. to synthesize the above sgRNA nucleic acid sequence and insert it into a standard vector and connect it to the CRISPR / CAS9 expression vector pX330A to obtain pX330A-TIGIT vector and pX330A-PD-1, pX330A-CTLA4, pX330A-LAG-3 , pX330A-Tim-3 vector.

[0041] Electrotransfect NK cells with the above vectors, the specific steps include: take 1×1...

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Abstract

The invention relates to a CAR-NK cell as well as a preparation method and application thereof. The invention firstly provides a bispecific chimeric antigen receptor targeting BCMA and CD5, the bispecific chimeric antigen receptor has the following structure: S-BCMA scFv-CD5scFv-H-TM-CF-CD3zeta, the BCMA scFv is a BCMA antigen binding domain, the BCMA scFv-CD5scFv-H-TM-CF-CD3zeta is a BCMA antigen binding domain, and the BCMA The CD5 scFv is a CD5 antigen binding domain; s is a signal peptide; h is a hinge area; tM is a transmembrane area; cF is a costimulatory factor; cD3zeta is a CD3zeta intracellular signal domain, the chimeric antigen receptor can be combined with two anti-tumor targets, namely BCMA and CD5 at the same time, and the targeting property and effectiveness can be remarkably improved; by knockout of immune checkpoint inhibitor genes in NK cells, it is possible to prevent tumor cells from triggering immune escape through specific binding with related immune checkpoint inhibitor sites, and experimental data proves that CTLA4 is selected as a knockout immune checkpoint and has more advantages compared with other immune checkpoint genes such as PD-1, Tim-3 and LAG-3. The chimeric antigen receptor is introduced into an NK cell to prepare a CAR-NK cell, and the CAR-NK cell can inhibit leukemia cell proliferation, regulate expression levels of cell factors such as IL-6, IL-12 and IFN-gamma, inhibit growth of tumors in an animal model and play a synergistic anti-tumor role.

Description

Technical field: [0001] The invention belongs to the field of tumor immunotherapy, and specifically provides a CAR-NK cell and its preparation method and application. Background technique: [0002] Malignant tumors are the number one "killer" that threatens human health. The diagnosis and treatment of malignant tumors has always been a hot and difficult issue to be explored at home and abroad. It is difficult for tumors to obtain a long-term prognosis, which makes the research on new anti-tumor methods more urgent and meaningful. Among the known malignant tumors, multiple myeloma (multiple myeloma, MM) is a bone marrow plasma cell clonal proliferative disease. Two common hematological malignancies. Monoclonal immunoglobulin or its fragments appear in the blood and urine of patients with myeloma, causing functional damage to related target organs such as bone marrow, kidney, and bone. The main clinical manifestations are anemia, bone pain, osteolytic destruction, renal insu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/10A61K35/17A61P35/00
CPCC07K14/7051C07K16/2878C07K16/2896A61K35/17A61P35/00C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N2510/00
Inventor 沈健葛永刘正明杨淑青
Owner JIANGSU MOBILI BIOTECH CO LTD
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