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Natural killer cell expressing anti-cotinine chimeric antigen receptor

A technology of natural killer cells and chimeric antigen receptors, applied in the direction of receptors/cell surface antigens/cell surface determinants, antibodies, animal cells, etc., to achieve high-efficiency anti-cancer effects

Active Publication Date: 2019-11-19
KOREA RES INST OF BIOSCI & BIOTECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The present inventors thus determined that natural killer cells may solve the problems of existing CAR-T therapeutic agents while facilitating the development of general therapeutic agents, thus completing the present invention

Method used

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  • Natural killer cell expressing anti-cotinine chimeric antigen receptor
  • Natural killer cell expressing anti-cotinine chimeric antigen receptor
  • Natural killer cell expressing anti-cotinine chimeric antigen receptor

Examples

Experimental program
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Effect test

Embodiment 1

[0094] Example 1. Carrier Skeleton

[0095] As the vector used in the present invention, a lentiviral vector (Clontech, 632155) was used. Specifically, using figure 1 pLVX-AcGFP-C1 as indicated. The Kozak sequence (CTCGAG; nucleotides 2801-2806) and AcGFP1 (Aequorea coerulescens green fluorescent protein; nucleotides 2807-3604) were deleted for use in the experiment, then XhoI was used as a restriction enzyme. The specific related sequences are shown in Figure 4 .

Embodiment 2

[0096] Example 2. Preparation of Target Antigen and Cotinine Conjugate

[0097] Cotinine (trans-4-cotinine carboxylic acid) is a small molecular substance used as a target antigen, and its chemical structure is shown in Formula 1 below. Cotinine from Sigma-Aldrich was used.

[0098] [Formula 1]

[0099]

[0100] In addition, a conjugate in which cotinine is fused to a binding substance is prepared by the following method.

[0101] Specifically, for the HER2-cotinine conjugate, a conjugate of cotinine and anti-HER2 antibody was prepared using Trastuzumab (Genentech, USA) as an anti-HER2 antibody. Here, the conjugate was coupled by the 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) coupling method. First, an anti-HER2 antibody was prepared by dissolving in PBS at a concentration of 25 μM. On the other hand, prepare trans-4-cotinine carboxylic acid by dissolving in 1 ml of MES buffer [0.1M 2-[morpholino]ethanesulfonic acid (MES) and 0.5M NaCl, pH 6.0] (Sigma-Aldrich ...

Embodiment 3

[0104] Example 3. Anti-cotinine chimeric antigen receptor

[0105] A plasmid containing a nucleic acid encoding each domain of the chimeric antigen receptor of the present invention that specifically binds cotinine was prepared by the following method.

[0106] (1) Signal peptide

[0107] Based on the human T cell surface glycoprotein CD8α chain (GenBank: AK300089.1), two types of primers (forward primer: SEQ ID NO: 18, reverse primer: SEQ ID NO: 19) Perform polymerase chain reaction followed by cloning.

[0108] (2) Target-specific recognition domain-scFv

[0109]As an antigen-binding domain capable of specifically binding cotinine, it is desired to obtain an anti-cotinine chimeric antibody or an antibody fragment thereof. For the sequence of ScFv, refer to information related to ScFv in Korean Patent No. 10-1648960. Specifically, the antigen-binding domain includes the nucleotide sequence shown in SEQ ID NO: 17, and is constructed as VH-linker-VL.

[0110] (3) Junction ...

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Abstract

The present invention relates to a natural killer cell expressing an anti-cotinine chimeric antigen receptor (CAR) specifically binding to cotinine, and a cell therapeutic agent comprising same. The natural killer cell expressing a chimeric antigen receptor specifically binding to cotinine, according to the present invention, can effectively move to tumor tissue, regardless of the kind of cancer,depending on the binding substance bound to cotinine. Therefore, the natural killer cell according to the present invention can be usefully employed as a gene therapy exhibiting a highly efficient anticancer effect.

Description

technical field [0001] The present invention relates to natural killer cells expressing an anti-cotinine chimeric antigen receptor (CAR) that specifically binds cotinine, and cell therapeutics comprising the same. Background technique [0002] Over the decades, approaches to treating cancer have changed and evolved steadily. From the 19th century to the 20th century, methods such as surgery, chemotherapy, and radiotherapy were mainly performed, and the limitations of these methods began to appear. Most typically, existing treatments are effective only in the early stages of cancer that has not metastasized; in the case of metastases, there is a high probability of recurrence even after surgery. In addition, it has been reported that chemotherapy has low curative effect in solid tumors and causes side effects in that the growth of normal cells other than cancer cells is also inhibited. Recently, in order to overcome these problems, immunotherapies against cancer are being a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783C12N15/115C07K16/44C07K14/705C07K14/725A61K35/17G01N33/569G01N33/574
CPCC07K16/44G01N33/574G01N33/5047G01N33/56972C12N2740/16043A61P35/00C07K14/7051C07K2319/03C07K2317/24C07K2317/622C07K16/32C07K2319/33A61K39/464404A61K39/464406A61K2239/55A61K39/4613A61K39/4631A61K2239/38A61K35/17A61K38/00A61K2039/505C07K14/70503C07K14/70517C07K14/70521C07K16/16C07K16/2863C07K2317/30C07K2317/732C07K2317/734C07K2317/76C07K2319/02C07K2319/30
Inventor 崔仁杓金泰暾李洙义李秀然郑埈昊金起铉
Owner KOREA RES INST OF BIOSCI & BIOTECH
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