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Targeting-CD33 chimeric antigen receptor (CAR) and application thereof

A technology of single-chain antibody and fusion protein, applied in the field of cell therapy

Pending Publication Date: 2019-12-10
HRAIN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The General Hospital of the Chinese People's Liberation Army applied for the first clinical trial of CD33 CAR-T cells in the treatment of multiple myeloma (NCT01864902) in 2013, and reported the treatment of one of the patients in 2015, proving that the anti-CD33 CAR-T The clinical effectiveness of the cells, but its long-term effectiveness and related toxicity still need to accumulate more clinical treatment data for further analysis

Method used

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  • Targeting-CD33 chimeric antigen receptor (CAR) and application thereof
  • Targeting-CD33 chimeric antigen receptor (CAR) and application thereof
  • Targeting-CD33 chimeric antigen receptor (CAR) and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1: Determination of CD33-scFv-CD8α-41BB-CD3ζ gene sequence

[0081] The gene sequence information of human CD8α hinge region, human CD8α transmembrane region, 41BB intracellular region and human CD3ζ intracellular region was searched from the NCBI website database. The anti-CD33 single-chain antibody clone number is HIM3-4. These sequences are available on the website http: / Codon optimization is performed on / sg.idtdna.com / site to ensure that it is more suitable for expression in human cells without changing the encoded amino acid sequence.

[0082] Using overlapping PCR, the above sequences were sequentially connected according to anti-CD33 scFv, human CD8α hinge region gene, human CD8α transmembrane region gene, 41BB intracellular region gene, and human CD3ζ intracellular region gene sequence, and different enzyme cutting sites were introduced at the junction of each sequence Click to form the complete CD33-CAR gene sequence information.

[0083] The nucleot...

Embodiment 2

[0089] Example 2: Retroviral packaging

[0090] 1. Day 1: 293T cells should be less than 20 passages, not overgrown. Take 0.6×10 6 For cell / ml plating, add 10ml of DMEM medium to a 10cm dish, mix the cells well, and culture overnight at 37°C;

[0091] 2. Day 2: 293T cell confluency reaches about 90% for transfection (usually about 14-18 hours after plating); prepare plasmid complexes, the amount of various plasmids is RV backbone 12.5ug, Gag-pol 10ug, VSVg 6.25 Ug, CaCl 2 250ul,H 2 O1ml, the total volume is 1.25ml; add HBS equal to the volume of the plasmid complex in another tube, and vortex for 20 seconds while adding the plasmid complex. Gently add the mixture to the 293T dish along the side, incubate at 37°C for 4 hours, remove the medium, wash with PBS, and add pre-warmed fresh medium again.

[0092] 3. Day 4: 48 hours after transfection, collect the supernatant and filter it with a 0.45um filter, store in -80°C, and continue to add preheated fresh DMEM medium.

Embodiment 3

[0093] Example 3: Retrovirus infection of human T cells

[0094] 1. Use Ficcol separation medium (Tianjin Haoyang) to separate and obtain relatively pure CD3+ T cells, and use X-VIVO (LONZA) medium containing 5% AB serum to adjust the cell density to 1×10 6 / mL. Inoculate the cells with 1ml / well into the anti-human 50ng / ml CD3 antibody (Beijing Tongli Haiyuan) and 50ng / ml CD28 antibody (Beijing Tongli Haiyuan), and then add 100IU / ml interleukin 2 (Beijing Tongli Haiyuan) Heron), the virus infection that prepares with embodiment 3 after stimulating culture for 48 hours;

[0095] 2. The next day after T cell activation culture, PBS was diluted to a final concentration of 15 μg / ml, and Retronectin (Takara) was used to coat a non-tissue-treated culture plate, 250 μl per well of a 24-well plate. Protected from light, overnight at 4°C for later use.

[0096] 3. After T cell activation and culture for two days, two coated 24-well plates were taken out, the coating solution was dis...

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Abstract

The invention relates to a targeting-CD33 chimeric antigen receptor (CAR) and application thereof. Specifically, the invention provides a polynucleotide sequence which is selected from a polynucleotide sequence (1) containing a coding sequence of anti-CD33 single-chain antibodies connected in sequence, a coding sequence of a human CD8 alpha hinge region, a coding sequence of a human CD8 transmembrane region (TM), a coding sequence of a human 41BB intracellular region and a coding sequence of a human CD3 zeta intracellular region, and a complementary sequence (2) of the polynucleotide sequence(1). The invention further provides a relevant fusion protein, a carrier containing the coding sequences, and application of the fusion protein, the coding sequences and the carrier.

Description

technical field [0001] The invention belongs to the field of cell therapy, and in particular relates to a chimeric antigen receptor targeting CD33 and its application. Background technique [0002] Acute myeloid leukemia is a kind of hematological malignancy that seriously threatens human health. At present, the curative effect of AML other than acute promyelocytic leukemia is not optimistic, and the prognosis is mostly poor. Traditional chemotherapy drugs cannot fundamentally solve the huge problem of recurrence and drug resistance, and hematopoietic stem cell transplantation is expensive, risky, and there is a possibility of recurrence after transplantation, so it is urgent to deeply study the occurrence, development, recurrence and drug resistance of AML. Mechanisms, development of new therapeutic drugs to improve efficacy. [0003] CD33 is a type I transmembrane receptor protein with a molecular weight of 67KD. It consists of 364 amino acids and is specifically express...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C07K19/00C12N15/867C12N5/10A61K35/17A61P35/02
CPCC07K16/2803C07K14/70517C07K14/70578C07K14/7051C12N15/86C12N5/0636A61K35/17A61P35/02C07K2317/622C07K2317/56C07K2319/00C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N2740/10043C12N2510/00
Inventor 王海鹰金涛
Owner HRAIN BIOTECHNOLOGY CO LTD
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