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DC cells and CTL cells loaded with tax antigen and their preparation method and application

An antigen and cell technology, applied in the field of CTL cells and their preparation, DC cells loaded with Tax antigens, can solve the problems of non-regeneration, exhaustion and insufficient accuracy of cells, and achieve long-term survival, improve growth state, and improve growth. effect of speed

Active Publication Date: 2021-10-22
CELARTICS BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of this method are: (1) the prediction accuracy is not enough; (2) different HLAs (such as HLA-A11, A24, B**) need to be predicted by similar methods, the workload is heavy and the efficiency is not good. High; (3) The predicted polypeptide is presented by DC cells, and DC cells are only suitable for HLA-A2 positive patients, and the utility is affected by prediction accuracy, DC cell number, activity and peptide loading efficiency; (4) Cells are non-renewable and are a depleted product
In addition, the loading efficiency of DC cells and the activation of T cells are affected by the activity of DC cells, but the current DC cells generally have problems such as poor activity and difficulty in expansion.

Method used

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  • DC cells and CTL cells loaded with tax antigen and their preparation method and application
  • DC cells and CTL cells loaded with tax antigen and their preparation method and application
  • DC cells and CTL cells loaded with tax antigen and their preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Construction of lentiviral vectors for pX and CD3-EGFR

[0039] (1) Cloning the pX synthetic gene and CD3-EGFR fusion gene into the lentiviral backbone vector pCDH to obtain a recombinant plasmid; the nucleic acid sequence and amino acid sequence of the pX gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The fusion gene and amino acid sequence of CD3-EGFR are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Among them, the CD3-EGFR gene is obtained by cloning the CD3ζ fragment (1-70 amino acids) and the intracellular fragment of EGFR respectively, and connecting them with blunt ends. CD3 primers are 5'-gaggaattcgccaccatgaagtggaaggcgcttttcacc-3' (SEQ ID NO:5) and 5'-ctggttctggccctgctggtacgc-3 (SEQ ID NO:6)'; EGFR primers are 5'-aggcgccacatcgttcggaagcgc-3' (SEQ ID NO: 7) and 5'-gagtctagatcatgctccaataaattcactgctttg-3' (SEQ ID NO: 8).

[0040] pX nucleotide sequence (SEQ ID NO: 1)

[0041] atggcccatttcccagggtttggacagagtcttcttttcggatacccagtcta...

Embodiment 2

[0050] Example 2 pX+CD3-EGFR activates and expands DC cells

[0051] (1) Collect 10 mL of venous blood from healthy donors into heparin anticoagulant tubes.

[0052] (2) Transfer 10 mL of blood in the anticoagulant tube to a 15 mL centrifuge tube, 600 g, 10 min.

[0053] (3) Take the upper layer of plasma and transfer it to a new 15mL centrifuge tube, and freeze it; the lower layer of blood cells is diluted with an equal volume of normal saline and mixed.

[0054] (4) Add 15 mL of Ficoll-Paque PLUS (GE Healthcare) solution into a 50 mL centrifuge tube.

[0055] (5) Gently add the diluted blood to the surface of the Ficoll along the wall of the centrifuge tube; centrifuge at 800 g for 20 min at room temperature.

[0056] (6) Discard as much of the upper layer of plasma as possible with a pipette, and the cell layer of peripheral blood mononuclear cells (PBMC) is located at the diluted plasma / Ficoll interface.

[0057] (7) Carefully collect the buffy coat, add an appropriate ...

Embodiment 3

[0073] Example 3 DCX1-1 induced expansion of antigen-specific CTL cells and detection of their activity

[0074] (1) Take DCX1-1 cells and ligand PBMC cells (HLA-A2.1) and count them.

[0075] (2) Use RPMI1640 medium containing 5% human serum, mix DC cells and PBMC cells according to the amount of DC cells: PBMC = 1:100, and culture overnight.

[0076] (3) The next day, add 200 unit / mL IL2 to the culture system, mix and culture.

[0077] (4) According to the growth status of the cells, add appropriate amount of medium and IL2.

[0078] (5) On day 12-14, some cells were collected for flow cytometric analysis.

[0079] (6) Take 1-5×10 5 cells, centrifuged at 2500rpm for 5min.

[0080] (7) Wash twice with PBS, centrifuge at 2500rpm for 5min.

[0081] (8) Add 100 μL BSA-containing blocking solution to the cells, and block on ice for 10-15 minutes.

[0082] (9) Divide the cells into two parts, add CD3+CD56 antibody to one part, and add the isotype control antibody of the two ...

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Abstract

The invention provides a DC cell loaded with Tax antigen, a CTL cell, a preparation method and application thereof. The present invention firstly provides a DC cell loaded with Tax antigen, which is a DC cell presenting Tax antigen obtained by cloning CD3‑EGFR fusion gene and pX gene encoding Tax antigen into a lentiviral vector and transfecting DC cells. In the present invention, DC cells loaded with Tax antigen are co-cultured with autologous PBMC cells to obtain a large number of highly active CTL cells. The killing experiment proved that this group of CTL cells can efficiently kill the target cells carrying the Tax antigen.

Description

technical field [0001] The invention belongs to the technical field of cellular immunotherapy, and in particular relates to a DC cell loaded with Tax antigen, a CTL cell and a preparation method and application thereof. Background technique [0002] Adult T-cell leukemia / lymphoma (ATL) is a recalcitrant mature T-cell malignancy with diverse clinical features that is etiologically related to the retrovirus known as human T-cell leukemia virus type I (HTLV-1) relevant. HTLV-1 virus infection is more common in Southwest Japan, Central and South America, Central Africa, Middle East, Far East, Central Australia and Romania. China belongs to the low prevalence area of ​​HTLV, but local concentration of HTLV-1 infection has been found in coastal areas such as Fujian and Guangdong in China. [0003] HTLV-1 is an oncogenic retrovirus that infects 10 to 20 million people worldwide. Of these infected populations, 1-6% will develop T-cell leukemia / lymphoma (ATL / ATLL), and another 2-3...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/62C12N15/867C12N15/48C12N5/0783C07K14/15C07K19/00A61K39/21A61P35/00A61P35/02A61P25/00
CPCA61K39/12A61P25/00A61P35/00A61P35/02C07K14/005C07K14/7051C07K14/71C07K2319/00C12N5/0638C12N5/0639C12N15/86C12N2501/2302C12N2502/11C12N2510/00C12N2740/10022C12N2740/15043
Inventor 张欢于洋程铧
Owner CELARTICS BIOPHARMA CO LTD
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