Engineering CD20 targeting NKT cell and preparation method and application thereof

Abstract

The invention relates to an engineering CD20 targeting NKT cell and a preparation method and application thereof. The cell is an NKT cell modified by chimeric antigen receptor CD20ScFv-CD8-CD137-CD3 sigma, an amino acid sequence of the chimeric antigen receptor is as shown by SEQID NO. 1 in a sequence list. The preparation method comprises the following steps: firstly establishing a chimeric antigen receptor pWPT-CD20ScFv-CD8-CD137-CD3 sigma, infecting the NKT cell, performing in-vitro induction and multiplication culture, and obtaining the engineering CD20 targeting NKT cell. When the engineering CD20 targeting NKT cell is used for treating a CD20 positive malignant tumor at a progressive stage, the survival time of the immune cell in the body of a patient can be obviously prolonged, the capacity of the immune cell for target identifying a tumor antigen is improved, and the killing activity for the tumor cell is improved.

Description

[0001] This application is a divisional application of an invention application with a filing date of February 24, 2014, an application number of 201410062069.7, and an invention title of "Engineered CD20-targeted NKT cells and its preparation method and application". technical field

[0002] The invention belongs to the field of tumor biological products, and relates to an engineered CD20-targeted NKT cell (CAR20-NKT cell) in adoptive immunotherapy, its preparation and application. Background technique

[0003] TCR - , mIg - , CD56 + , CD16 + , CD3 + Lymphoid cells were identified as NKT cells. NKT cells express both T cell surface markers and NK cell surface markers. NKT cells can recognize and kill mutated tumor cells and virus-infected cells, but have no cytotoxic effect on normal self-tissue cells. NKT cells bind to the Fc region of specific antibodies through CD16 on their surface, and play the role of ADCC (antibody-dependent cell-mediated cytotoxicity). However...

Examples

Embodiment 1

[0053] The preparation of embodiment 1 NKT cell

[0054] (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.

[0055] (2) After PBMCs were washed three times, the final concentration of cells was adjusted to 2×10 by using NKT cell culture medium GT-T551 containing 0.6% conventional serum. 6 cell / mL; the cells were inoculated in a 75 cm cell culture flask 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 1000U / mL recombinant human protein interferon-γ and 1000U / mL recombinant human interleukin-2 to the culture medium, at 37°C, with a saturated humidity of 5% CO 2 Incubator cultivation.

[0056] (3) On the fourth day, add 100 mL of NKT cell medium containing 0.6% conventional serum to the bottle, and add recombinant human interleukin-2 with a final c...

Embodiment 2

[0057] Embodiment 2: the construction of chimeric antigen receptor (pWPT-CD20ScFv-CD8-CD137-CD3ζ) lentiviral expression vector

[0058] (1) Preparation of NKT cell cDNA

[0059] 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 obtained by reverse transcription with First Strand cDNASynthesis Kit, and stored at -20°C for future use.

[0060] (2) Preparation of lentiviral plasmid pWPT-CD8-CD137-CD3ζ

[0061] The primers used were synthesized by Beijing Tianyi Huiyuan Biotechnology Co., Ltd., and the primer sequences are as follows (wherein, the underlined marks are protected bases, and the boxes are enzyme cleavage sites):

[0062]

[0063]Using NKT cell cDNA in step (1) as a template, PCR amplification is carried out with primers P1 and P2 to ...

Embodiment 3

[0076] Example 3 Preparation of NKT cells modified by chimeric antigen receptor pWPT-CD20ScFv-CD8-CD137-CD3ζ

[0077] (1) Packaging and concentration of lentivirus

[0078] 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...

[0001] This application is a divisional application of an invention application with a filing date of February 24, 2014, an application number of 201410062069.7, and an invention title of "Engineered CD20-targeted NKT cells and its preparation method and application". technical field

[0002] The invention belongs to the field of tumor biological products, and relates to an engineered CD20-targeted NKT cell (CAR20-NKT cell) in adoptive immunotherapy, its preparation and application. Background technique

[0003] TCR - , mIg - , CD56 + , CD16 + , CD3 + Lymphoid cells were identified as NKT cells. NKT cells express both T cell surface markers and NK cell surface markers. NKT cells can recognize and kill mutated tumor cells and virus-infected cells, but have no cytotoxic effect on normal self-tissue cells. NKT cells bind to the Fc region of specific antibodies through CD16 on their surface, and play the role of ADCC (antibody-dependent cell-mediated cytotoxicity). However...

Examples

Embodiment 1

[0053] The preparation of embodiment 1 NKT cell

[0054] (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.

[0055] (2) After PBMCs were washed three times, the final concentration of cells was adjusted to 2×10 by using NKT cell culture medium GT-T551 containing 0.6% conventional serum. 6 cell / mL; the cells were inoculated in a 75 cm cell culture flask 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 1000U / mL recombinant human protein interferon-γ and 1000U / mL recombinant human interleukin-2 to the culture medium, at 37°C, with a saturated humidity of 5% CO 2 Incubator cultivation.

[0056] (3) On the fourth day, add 100 mL of NKT cell medium containing 0.6% conventional serum to the bottle, and add recombinant human interleukin-2 with a final c...

Embodiment 2

[0057] Embodiment 2: the construction of chimeric antigen receptor (pWPT-CD20ScFv-CD8-CD137-CD3ζ) lentiviral expression vector

[0058] (1) Preparation of NKT cell cDNA

[0059] 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 obtained by reverse transcription with First Strand cDNASynthesis Kit, and stored at -20°C for future use.

[0060] (2) Preparation of lentiviral plasmid pWPT-CD8-CD137-CD3ζ

[0061] The primers used were synthesized by Beijing Tianyi Huiyuan Biotechnology Co., Ltd., and the primer sequences are as follows (wherein, the underlined marks are protected bases, and the boxes are enzyme cleavage sites):

[0062]

[0063]Using NKT cell cDNA in step (1) as a template, PCR amplification is carried out with primers P1 and P2 to ...

Embodiment 3

[0076] Example 3 Preparation of NKT cells modified by chimeric antigen receptor pWPT-CD20ScFv-CD8-CD137-CD3ζ

[0077] (1) Packaging and concentration of lentivirus

[0078] 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...

Images