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Therapeutic composition for treating B cell leukemia and B cell lymphoma

A lymphocyte and cell surface technology, applied in the field of biomedicine, can solve problems that need to be further developed

Active Publication Date: 2017-08-11
BEIJING MARINO BIOTECH PTY LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, adoptive T cell therapy remains to be further developed

Method used

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  • Therapeutic composition for treating B cell leukemia and B cell lymphoma
  • Therapeutic composition for treating B cell leukemia and B cell lymphoma
  • Therapeutic composition for treating B cell leukemia and B cell lymphoma

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0160] Cell lines and basic experimental techniques used in the embodiments of the present invention are as follows:

[0161] cell line

[0162] Use following cell line in the embodiment: OCI-LY3 cell (CD19 + Diffuse large B-cell lymphoma cell line (DLBCL), Jurkat cells (CD19 - T lymphoma cell line), and K562 (target cell of natural killer cells). The above cells are all from ATCC (American cell bank) and DSMZ (German cell bank), and cultured in RPMI-1640 medium (purchased from Gibco-BRL, San Francisco, CA, USA).

[0163] Generation of lentivirus and transduction of human T lymphocytes

[0164] Replication-defective lentiviral vectors were generated and collected by centrifugation for transduction of human T lymphocytes. The following is a brief introduction to the production and collection of lentiviral vectors: 293T cells were placed in a cell culture dish with a bottom area of ​​150-cm2, and Express-In (purchased from Open Biosystems / Thermo Scientific, Waltham , MA) f...

Embodiment 2

[0173] Example 2 Construction of a vector co-expressing PD1-shRNA and anti-CD19 chimeric antigen receptor

[0174] In this example, the inventor cloned the sequence encoding the single-chain antibody against human CD19, the ζ-chain sequence of the combination of the intracellular segment of CD28 and the T cell receptor into the LV lentiviral vector. During the cloning process, the selection restriction The specific restriction enzyme digestion is XbaI and NotI double enzyme digestion, and NotI and XhoI double enzyme digestion. Through enzyme digestion, ligation, screening and amplification of the target plasmid, a slow DNA sequence linked to the nucleotide sequence encoding the anti-CD19 chimeric antigen receptor is generated. Viral plasmid (LV-CD19 CAR); the sequence of U6 promoter and PD1-shRNA (shPD1) was cloned into the lentiviral plasmid of the above LV-CD19 CAR to generate the nuclear sequence linked with shPD1 sequence and encoding anti-CD19 chimeric antigen receptor Le...

Embodiment 3

[0175] Example 3 Enhanced targeted killing ability of T cells co-expressing PD1-shRNA and anti-CD19 chimeric antigen receptor

[0176] In this example, peripheral blood lymphocytes were obtained from anonymous blood donors. Peripheral blood lymphocytes were separated by gradient centrifugation using Ficoll-Hypaque. T lymphocytes and T cell activator magnetic beads CD3 / CD28 (purchased from Invitrogen, Carlsbad, CA) in 5% CO 2 , Incubated at 37 degrees Celsius for 72 hours, the medium was added with 2mmol / L glutamine, 10% high temperature inactivated fetal calf serum (FCS) (purchased from Sigma-Aldrich Co.) and 100U / ml of penicillin / chain RPMI medium 1640 (purchased from Invitrogen Gibco Cat. no. 12633-012) with antimycin double antibody. After activating and culturing for 72 hours, the cells were rinsed with washing solution to wash away the magnetic beads. The T cells were planted on cell culture dishes covered with recombinant fibronectin fragments (FN ch-296; Retronectin)...

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Abstract

The invention provides a transgenic lymphocyte, a construct and a therapeutic composition for treating cancers. A cell surface immune checkpoint of the transgenic lymphocyte provided by the invention is silenced and is applied to expression of a chimeric antigen receptor. The transgenic lymphocyte provided by the invention is high in cytokine production and strong in targeted killing effect on CD19+ tumor cells; and especially, a directional killing effect on the CD19+ tumor cells of B cell leukemia and B cell lymphoma is obviously improved.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular, the present invention relates to a T lymphocyte, a lentivirus, a transgenic lymphocyte, a construct, a therapeutic composition for treating cancer and a lymphatic method of cell activity. Background technique [0002] Tumors can avoid immune surveillance by inducing the expression of immunosuppressive receptors, which are turned off by tumor cells from the immune response. In terms of immunoregulatory mechanisms, activated cytotoxic T lymphocytes (CTLs) express negatively regulated regulatory mechanisms, that is, express immune checkpoint molecules on the cell surface, and one of the main immune checkpoint molecules, programmed cell death, is regulated by PD1 is expressed on activated CTLs, which interacts with programmed death ligand 1 (PD-L1B7-H1) expressed on tumor cells, and can inhibit anti-tumor T cell responses. Many tumors, including lymphoma, lung cancer, ovarian cance...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12N15/86C12N15/12C12N15/113C12N7/01
CPCC07K14/70596C12N7/00C12N15/113C12N2740/15041A61K39/4631A61K39/464412A61K39/4611A61K31/7088A61K35/76C12N5/10C12N15/86C12N15/867
Inventor 严勇朝朱益林陈思毅
Owner BEIJING MARINO BIOTECH PTY LTD
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