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Replication-defective recombinant lentivirus CAR-T transgenic vector targeting CD152 and construction method of replication-defective recombinant lentivirus CAR-T transgenic vector

A technology of recombinant lentivirus and transgenic vector, which is applied in the field of medical biology, can solve problems such as slow sales growth, no response, and large side effects, and achieve the effects of reducing endotoxin content, avoiding tediousness and mistakes, and improving safety

Active Publication Date: 2019-06-21
EAST CHINA NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But it should be soberly aware that at present, only about 5% to 10% of patients can actually benefit from this expensive treatment, and most patients either do not respond or relapse after treatment. Sales growth is slow and there is no more development

Method used

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  • Replication-defective recombinant lentivirus CAR-T transgenic vector targeting CD152 and construction method of replication-defective recombinant lentivirus CAR-T transgenic vector
  • Replication-defective recombinant lentivirus CAR-T transgenic vector targeting CD152 and construction method of replication-defective recombinant lentivirus CAR-T transgenic vector
  • Replication-defective recombinant lentivirus CAR-T transgenic vector targeting CD152 and construction method of replication-defective recombinant lentivirus CAR-T transgenic vector

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Experimental program
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Effect test

Embodiment 1

[0110] The construction methods of recombinant lentiviral plasmids pCAR152-0, pCAR152-1, pCAR152-2 and pvCAR152-3 are as follows:

[0111] The promoters and sequences of CAR152-0, CAR152-1, CAR152-2, CAR152-3 (see Table 1) and human EF1α were all synthesized by Gene Synthesis Company. Then, as shown in Table 2, the synthetic human EF1α promoter and CAR structure (CAR152-0, CAR152-1, CAR152-2, CAR152-3) were cloned into the recombinant lentiviral backbone plasmid pLenti-3G ​​basic2 (see attached figure 1 In A), the recombinant lentiviral plasmids pCAR152-0~pCAR152-3 were respectively obtained (pCAR152-0 is a negative control, so the structural diagram is not shown; the structural diagram of pCAR152-1~pCAR152-3 is shown in figure 1 B. figure 1 C and figure 1 D).

[0112] Table 1 Chimeric Antigen Receptor Structure

[0113]

[0114] Table 2 Composition of recombinant lentiviral plasmids

[0115] recombinant lentiviral plasmid Recombinant Lentiviral Backbone Pl...

Embodiment 2

[0132] Obtaining supernatant of lvCAR152-0~lvCAR152-3 recombinant lentiviral vector

[0133]The recombinant lentiviral plasmids pCAR152-0~pCAR152-3 obtained in Example 1 (see the structure diagram of pCAR152-1 figure 1 B. Schematic diagram of pCAR152-2 structure, see figure 1 C. The structure diagram of pCAR152-3 is shown in figure 1 C; because pCAR152-0 is a negative control, so it is not shown in the figure) and the packaging plasmid (see the schematic diagram of the structure) respectively figure 1 E. figure 1 F. figure 1 G) co-transfect into 293T cells, and collect the cell supernatant containing the recombinant lentiviral vectors lvCAR152-0-lvCAR152-3 after 72 hours of transfection.

[0134] 2.1. Complete medium: Take out the preheated fresh medium, add 10% FBS + 5ml Pen-Srep, and mix upside down;

[0135] 2.2. 1XPBS solution: Weigh NaCl 8g, KCl 0.2, NaCl 2 HPO 4 .12H 2 O 3.58g, KH 2 PO4 0.24g

[0136] 2.3. Put it in a 1000ml beaker, add 900ml Milli-Q grade ultr...

Embodiment 3

[0157] Purification of recombinant lentiviral vector lvCAR152-0~lvCAR152-3

[0158] Purification flow chart such as figure 2 As shown, firstly, the cell supernatant collected in 2.21 is filtered by a vacuum pump, and then the impurities and endotoxins are removed through the ion exchange column and the endotoxin removal column, and then injected into the ion exchange column at a certain flow rate by the peristaltic pump. , and eluted to obtain a harvested solution of replication-deficient lentiviral vectors. The lvCAR152 eluted by this method is more pure and easier to operate.

[0159] The specific operation steps are as follows:

[0160] 3.1. Use a Thermo vacuum pump to filter the collected supernatant through a 0.22μm-0.8μm PES filter to remove impurities;

[0161] 3.2. Add 1.5M NaCl 250mM Tris-HCl (pH 6-8) to the supernatant in an appropriate proportion;

[0162] 3.3. Place two ion-exchange columns and endotoxin removal columns in series, and pass through the columns ...

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Abstract

The invention discloses a replication-defective recombinant lentivirus CAR-T transgenic vector targeting CD152 and a construction method of the replication-defective recombinant lentivirus CAR-T transgenic vector. The transgenic vector comprises a promoter for controlling a replication initiation site, a prokaryotic replicon for plasmid replication, a viral replicon for enhancing replication in eukaryotic cells, a resistance gene for detection, a regulatory element after viral transcription, a packaging cis element cis for lentiviral packaging and a chimeric antigen receptor CAR for recognition, transmission and initiation; the construction of the replication-defective recombinant lentiviral CAR-T transgenic vector comprises construction of a recombinant lentiviral plasmid, and packaging,concentration and purification of the recombinant lentiviral vector to obtain the replication-defective recombinant lentiviral CAR-T transgenic vector targeting CD152 with high purity. The recombinantlentivirus CAR-T transgenic vector provided by the invention can remarkably improve secretion of cytokines and specifically kill target cells CTLA-4, and provides a new treatment direction for inhibition of tumor microenvironment and attack of solid tumors.

Description

technical field [0001] The invention belongs to the field of medical biology, and in particular relates to a vector, which is a CD152-targeted replication-defective recombinant lentiviral CAR-T transgene vector. In addition, the present invention also relates to the construction method of the vector. Background technique [0002] In October 2018, the Nobel Prize in Physiology or Medicine was awarded to American scientist James P. Allison and Japanese scientist Tasuku Honjo for their discovery of "Suppressive Immunomodulatory Therapy for Cancer" contribution made. The contributions of them and countless researchers in immunology have completely changed the pattern of human anti-cancer. Before tumor immunotherapy, the most common treatment methods for cancer were surgery, chemotherapy, radiotherapy and targeted therapy. The former removed tumor cells surgically, but usually the removal was not complete; while chemotherapy and radiotherapy can cure many tumors, they are harmf...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/62C12N15/66
Inventor 王依婷许姣姣康丽清张金芳徐南李明昊刘秧刘丽谭靖雯沈鸿伟骆声根闫志强王镜朱建中俞磊
Owner EAST CHINA NORMAL UNIVERSITY
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