CAR-T cells with inhibited sterol o-acyltransferase 1 (SOAT1) as well as preparation method and application of CAR-T (Chimeric Antigen Receptor-T-cell Immunotherapy) cells

A technology of SOAT1 and cholesterol, which is applied in the field of medical biology, can solve problems such as nutritional deficiency and poor therapeutic effect, and achieve the effects of increasing content, enhancing therapeutic activity, and great application value

Active Publication Date: 2017-08-18
SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] CAR-T is currently effective in the treatment of several types of hematological tumors such as B-ALL, but in myeloma, lymphoma and even solid tumors, the current treatment effect is not very good. The main reasons may be as follows: 1. Tumor microenvironment characterized by oxidative stress, nutrient deficiency, acidic pH and hypoxia; 2. Existence of immunosuppressive factors secreted by tumor cells; 3. Inhibition of suppressive immune cells, such as regulatory T cells (Treg), myelotrophic precursor suppressor cells (MDSC), tumor-associated macrophages (TAM), immature dendritic cells (iDC), etc.; 4. The inherent immune checkpoint regulation mechanism of T cells

Method used

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  • CAR-T cells with inhibited sterol o-acyltransferase 1 (SOAT1) as well as preparation method and application of CAR-T (Chimeric Antigen Receptor-T-cell Immunotherapy) cells
  • CAR-T cells with inhibited sterol o-acyltransferase 1 (SOAT1) as well as preparation method and application of CAR-T (Chimeric Antigen Receptor-T-cell Immunotherapy) cells
  • CAR-T cells with inhibited sterol o-acyltransferase 1 (SOAT1) as well as preparation method and application of CAR-T (Chimeric Antigen Receptor-T-cell Immunotherapy) cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0154] Embodiment 1 hCAR19-KO SOAT1 - T cell construction.

[0155] see Figure 4 , the hCAR19-KO of the present invention SOAT1 -The construction method of T cells is as follows:

[0156]1. Construction, purification and detection methods of SOAT1 knockout recombinant lentiviral vectors lvCas9-SOAT1-1-lvCas9-SOAT1-7.

[0157] 1. Ligate the synthesized SOAT1-target1~SOAT1-target7 fragments into pLenti-Cas9-monoKO plasmids respectively to obtain SOAT1 knockout recombinant lentiviral plasmids pCas9-SOAT1-1~pCas9-SOAT1-7.

[0158] (1) The recombinant lentiviral plasmid pLenti-Cas9-monoKO was digested with BsmB I restriction endonuclease, the product was subjected to 1.5% agarose gel electrophoresis, and the fragment V1 of 11127bp was confirmed, and recovered by tapping the gel and placed in an Eppendorf tube, Reclaim the corresponding fragment (see Table 1) with the agarose gel recovery kit of MN company, and measure the purity and concentration of product;

[0159]

[01...

Embodiment 2

[0257] Example 2 hCAR19-shRNA SOAT1 - T cell construction.

[0258] see Figure 5 , the hCAR19-shRNA of the present invention SOAT1 -The construction method of T cells is as follows:

[0259] 1. Recombinant knockdown lentiviral vector lv-hCAR19-shRNA1 SOAT1 ~lv-hCAR19-shRNA13 SOAT1 Construction, purification and detection methods.

[0260] 1. The synthesized shRNA1 SOAT1 ~shRNA13 SOAT1 The fragments were co-ligated with the hU6 fragment into the p-hCAR19 recombinant lentiviral vector plasmid to obtain the SOAT1 recombinant knockdown lentiviral plasmid p-hCAR19-shRNA1 SOAT1 ~p-hCAR19-shRNA13 SOAT1 .

[0261] (1) The p-hCAR19 recombinant lentiviral vector plasmid was single-digested with Sal I restriction endonuclease, and the product was subjected to 1.5% agarose gel electrophoresis to confirm the 8519bp fragment V2, which was recovered by tapping the gel and placed in an Eppendorf tube. Recover the corresponding fragments (see Table 1 in Example 1) with the agarose g...

Embodiment 3

[0304] Example 3 hCAR19-T and hCAR19-Inhibitor SOAT1 - T cell construction.

[0305] see Figure 6 , the hCAR19-Inhibitor of the present invention SOAT1 -The construction method of T cells is as follows:

[0306] 1. Isolation of PBMCs.

[0307] (1) Take 50ml of fresh peripheral blood from a healthy donor;

[0308] (2) Spray the blood collection bag with alcohol twice and dry it.

[0309] (3) Use a 50ml syringe to suck out the blood cells in the bag and transfer them to a new 50ml tube.

[0310] (4) Centrifuge at 400g for 10 minutes at 20°C.

[0311] (5) Transfer the upper layer of plasma to a new 50ml centrifuge tube, inactivate the plasma at 56°C for 30 minutes, return to room temperature, centrifuge at 2000g for 30 minutes, and take the supernatant into a 50ml centrifuge tube for later use.

[0312] (6) Make up to 50ml with D-PBS(-), tighten the cap, and mix evenly by inversion.

[0313] (7) Take two new 50ml centrifuge tubes and add 15ml Ficoll lymphocyte separation...

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Abstract

The invention discloses CAR-T cells with inhibited sterol o-acyltransferase 1 (SOAT1). The CAR-T cells with inhibited SOAT1 comprise the following cells: a T cell subjected to SOAT1 gene knockout at DNA level and for expression of a hCAR19 receptor, a T cell subjected to SOAT1 gene knock-down at mRNA level and for expression of a hCAR19 receptor, and a T cell subjected to SOAT1 gene inhibiting effect by an inhibitor at protein level and for expression of a hCAR19 receptor. The invention also discloses a preparation method of the CAR-T cells with inhibited SOAT1 and application of the CAR-T cells in preparation of medicine for treating cells of tumors. A series of pre-clinical experiments prove that the CAR-T cells with inhibited SOAT1 have killing ability superior to that of CAR-T cells and have an extremely high application value in treatment of the cells of the tumors.

Description

technical field [0001] The invention belongs to the field of medical biology, and in particular relates to a CAR-T cell for tumor immunotherapy, in particular to a CAR-T cell in which cholesterol transferase SOAT1 (sterol O-acyltransferase 1, gene ID: 6646) is inhibited. In addition, the present invention also relates to the preparation method and application of the cell. Background technique [0002] The theoretical basis of tumor immunotherapy is that the immune system has the ability to recognize tumor-associated antigens and regulate the body's ability to attack tumor cells (highly specific cytolysis). In the 1950s, Burnet and Thomas put forward the theory of "immune surveillance", thinking that the mutated tumor cells that often appear in the body can be recognized and eliminated by the immune system, which laid the theoretical foundation for tumor immunotherapy [Burnet FM. Immunological aspects of malignant disease. Lancet, 1967; 1:1171-4]. Subsequently, various tumo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C12N15/867A61K35/17A61P35/00
CPCA61K35/17C07K14/7051C07K16/2803C07K2317/622C07K2319/33C12N9/1029C12N15/86C12N2510/00C12N2740/15043C12Y203/01026
Inventor 祁伟余宙康立清俞磊
Owner SHANGHAI UNICAR THERAPY BIOPHARM TECH CO LTD
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