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Engineered CD20 targeting NKT cell and its preparation method and application

An NKT cell engineering technology, applied in biochemical equipment and methods, chemical equipment and methods, botany equipment and methods, etc., can solve the problems of drug resistance in patients, achieve good industrial application prospects, and prolong survival time , the effect of enhanced ability

Active Publication Date: 2014-05-28
SHANGHAI CELLULAR BIOPHARMACEUTICAL GROUP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows scientists to create specific types of T lymphocytes called natural killer (NK) cell lines with high efficiency against cancerous tissue through genetic engineering techniques or chemical modification methods. These Nk lineages could then use them effectively at treatments such as chemotherapy drugs or radiation therapies aimed towards developing new ways to kill these diseased areas without causing severe side effectes like other forms of cancer.

Problems solved by technology

This patents describes methods used by immunotherapists to treat cancer with high success rates without causing side effects such as recurrence or reduced efficacy over time. These techniques involve administering modified autologous T cells called CRACs, where they act like natural killer cells, allowing them to attack abnormal ones from healthy individuals while sparing others who may be harmed during chemo/immunotherapeusis procedures. Additionally, there exist various ways how these modified T cells work: 1) directly engage with certain types of targets found inside tumour cells;2) use this ability alone when combined with another type of agent against those targets, resulting in effective destruction of tumours and reducing offending agents.

Method used

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  • Engineered CD20 targeting NKT cell and its preparation method and application
  • Engineered CD20 targeting NKT cell and its preparation method and application
  • Engineered CD20 targeting NKT cell and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1 Preparation of NKT cells

[0053] (1) Take human venous blood in a vacuum tube containing heparin. Mononuclear cells (PBMCs) were obtained by using lymphocyte separation medium and density gradient centrifugation.

[0054] (2) After PBMCs were washed three times, the final concentration of cells was adjusted to 2×10 with NKT cell culture medium GT-T551 containing 0.6% conventional serum. 6 cell / mL; cells were inoculated in 75 cm cell culture flasks pre-coated with a final concentration of 5 μg / mL LCD3 monoclonal antibody and a final concentration of 10 μg / mL of retronectin (purchased from TAKORA). Add recombinant human protein interferon-γ with a final concentration of 1000U / mL and recombinant human interleukin-2 at a final concentration of 1000U / mL to the culture medium, at 37°C, with a saturated humidity of 5% CO 2 Incubator cultivation.

[0055] (3) On the fourth day, add 100 mL of NKT cell culture medium containing 0.6% conventional serum to the bottle,...

Embodiment 2

[0056] Example 2: Construction of chimeric antigen receptor (pWPT-CD20ScFv-CD8-CD137-CD3ζ) lentiviral expression vector

[0057] (1) Preparation of NKT cell cDNA

[0058] The NKT cells cultured in Example 1 were centrifuged to precipitate, and the total RNA of the cells was extracted with RNAiso Reagent, a total RNA extraction kit, and stored at -80°C for future use. Extracted total RNA with RevertAid Reverse Transcription Kit TM NKT cell cDNA was reverse transcribed with First Strand cDNA Synthesis Kit and stored at -20°C for future use.

[0059] (2) Preparation of lentiviral plasmid pWPT-CD8-CD137-CD3ζ

[0060] The primers used were synthesized by Beijing Tianyi Huiyuan Biotechnology Co., Ltd., and the primer sequences are as follows (wherein, the underline marks are protected bases, and the boxes are restriction sites):

[0061]

[0062] Using the NKT cell cDNA in step (1) as a template, PCR amplification was performed with primers P1 and P2 to obtain the hinge region...

Embodiment 3

[0075] Example 3 Preparation of NKT cells modified by chimeric antigen receptor pWPT-CD20ScFv-CD8-CD137-CD3ζ

[0076] (1) Packaging and concentration of lentivirus

[0077] The lentiviral expression plasmid pWPT-CD20ScFv-CD8-CD137-CD3ζ and the helper plasmids psPAX2 and pMD2.G were respectively extracted with the plasmid mini-extraction kit. The concentration of plasmid was measured by spectrophotometer, and the mass ratio of three plasmids was 4:2:1 with Lipofectamine TM 2000Transfection Reagent transfection reagent co-transfected 293T packaging cells. Collect virus supernatants in EP tubes at 48h and 72h of transfection, centrifuge at 2000g for 10min at 4°C, transfer the supernatants to new EP tubes, filter the virus supernatants with a 4.5μm filter; filter the virus supernatants with 5× Mix PEG6000-NaCl according to the volume ratio of 4:1, let stand at 4°C for 2h, then centrifuge at 10,000g for 20min at 4°C, discard the supernatant, and dissolve the precipitate with 4°C ...

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Abstract

The invention relates to an engineered CD20 targeting NKT cell and its preparation method and application. The cell is an NKT cell modified by a chimeric antigen receptor CD20ScFv-CD8-CD137-CD3zeta. An amino acid sequence of the chimeric antigen receptor is as shown in SEQID NO.1 in a sequence table. The preparation method comprises the following steps: firstly constructing a chimeric antigen receptor pWPT-CD20ScFv-CD8-CD137-CD3zeta, carrying out infection of the NKT cell, inducing in vitro and carrying out amplification culture so as to obtain the engineered CD20 targeting NKT cell. During advanced CD20 positive malignant tumors, the engineered CD20 targeting NKT cell can be used to obviously prolong survival time of immune cells in a patient's body, enhance the immune cells' targeting recognition capability of tumor antigen and reinforce the killing activity of tumor cells.

Description

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Claims

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Application Information

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Owner SHANGHAI CELLULAR BIOPHARMACEUTICAL GROUP LTD
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