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Modified T cells and preparation method and application thereof

A technology of cells and cell receptors, applied in the fields of gene editing and tumor immunotherapy, can solve problems such as limited clinical response

Pending Publication Date: 2019-05-24
北京东方略细胞技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite the success of CAR-T cell therapy in early clinical studies for the treatment of CD19-positive hematologic malignancies (Daviala et al, 2014; Lee et al, 2015; Maude et al, 2014), targeting solid tumors with CAR-T cells Very limited clinical response to antigen

Method used

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  • Modified T cells and preparation method and application thereof
  • Modified T cells and preparation method and application thereof
  • Modified T cells and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0213] Example 1. Knockout of the LAG-3 gene in CAR-T cells

[0214] 1. Screen the most effective sgRNA targeting LAG-3 on T cells

[0215] To abolish LAG-3 expression in T cells, five sgRNAs were designed targeting the first exon of LAG-3. figure 1 A takes sgRNA5 as an example, illustrating the position of the sgRNA in the LAG-3 locus.

[0216] The targeting sequences of the involved sgRNAs are shown in Table 1.

[0217] Table 1. sgRNA targeting LAG-3

[0218] sgRNA

target sequence

SEQ ID NO

sgRNA1

ATGTGGGAGGCTCAGTTCCT

1

sgRNA2

GCTGCAGAAACAGCAAGCCC

2

sgRNA3

TGCTGTTTCTGCAGCCGCTT

3

sgRNA4

GCTGTTTCTGCAGCCGCTTT

4

sgRNA5

GTTTCTGCAGCCGCTTTGGG

5

[0219] Cas9 protein (3 μg) was complexed with in vitro transcribed sgRNA (3 μg), and then electroporated into primary CD3 + in T cells. The gene editing efficiency of each sgRNA was quantified by TIDE analysis, and the most effective sgRNA was ...

Embodiment 2

[0236] Example 2. Knocking out the CTLA-4 gene in CAR-T cells

[0237] 1. Screening sgRNA targeting CTLA-4

[0238] Design five kinds of sgRNA targeting exon 1 coding region of CTLA-4 locus, target sequence such as SEQ ID NO:6-10 (Table 2), such as Figure 6 In A, the targeting sequence of sgRNA1 is in green, and the PAM sequence is in blue.

[0239] Table 2. sgRNA targeting CTLA-4

[0240]

[0241]

[0242] Cas9 protein (3 μg) and in vitro transcribed sgRNA (3 μg) were complexed to form Cas9-sgRNA ribonucleoprotein (RNP), and then electroporated into primary 1×10 6 CD3+ T cells (3 days after activation).

[0243] Knockdown efficiency using each sgRNA was quantified by TIDE analysis, and indel frequency in CTLA-4 was analyzed by sequencing. The result is as Figure 6 As shown in B, sgRNA1 has the highest knockout efficiency, and gene editing with sgRNA1 in another donor (donor 2) also obtained a significant knockout effect.

[0244] PCR products from each sample we...

Embodiment 3

[0262] Example 3, knockout of the Foxp3 gene in CAR-T cells

[0263] 1. Screening of sgRNA targeting Foxp3

[0264] Design six kinds of sgRNA targeting Foxp3 gene locus exon 2 coding region, the sequence is as SEQ ID NO:11-15 (Table 3), such as Figure 11 In A, the targeting sequence of sgRNA3 is in green, and the PAM sequence is in blue.

[0265] Table 3. sgRNA targeting Foxp3

[0266] sgRNA

Foxp3 target sequence

SEQ ID NO

sgRNA1

GGGCCGAGATCTTCGAGGCG

11

sgRNA2

TCGAAGATCTCGGCCCTGGA

12

sgRNA3

GCAGCTGCGATGGTGGCATG

13

sgRNA4

AGGGCCGAGATCTTCGAGGC

14

sgRNA5

GGCCCTGGAAGGTTCCCCCT

15

sgRNA6

TTTGGGTGCAGCCTCCAGC

16

[0267] Cas9 protein (3 μg) and in vitro transcribed sgRNA (3 μg) were complexed to form Cas9-sgRNA ribonucleoprotein (RNP), and then electroporated into primary 1×106 In CD3+ T cells (3 days after activation).

[0268] The knockout efficiency of each sgRNA was quanti...

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Abstract

The invention relates to the field of genetic editing and tumor immunotherapy, in particular to a method of preparing modified T cells, such as CAR-T cells, through genetic editing, modified T cells prepared via the preparation method, and application of the modified T cells.

Description

[0001] This application is a divisional application of the Chinese invention patent application with the application number 201880002752.8, the application date is May 8, 2018, and the invention title is "Modified T cells, their preparation method and use". technical field [0002] The invention relates to the fields of gene editing and tumor immunotherapy. In particular, the present invention relates to a method for preparing modified T cells such as CAR-T cells by gene editing, as well as the modified T cells prepared by the method and uses thereof. Background technique [0003] T cells play an important role in anti-tumor immunity. However, in tumor patients, the local specific cytotoxic T lymphocytes (CTL) content is very small, it is difficult to obtain and expand in vitro, and the low affinity of CTL limits its application in the clinical treatment of tumors. [0004] Adoptive transfer of T cells is a specific, low-toxicity anti-tumor method that has received high att...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N9/22C12N5/10A61K35/17A61P35/00A61P35/02
CPCA61P35/00A61K39/464468A61K2239/38A61K39/4611A61K2239/55A61K2239/31A61K2239/48A61K39/4631
Inventor 王皓毅张永平刘晓娟程晨张兴颖李娜
Owner 北京东方略细胞技术有限公司
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