Improved anti-CD19 CAR-T cells

A technology of lymphocytes and extracellular regions, applied in the field of biomedicine, can solve problems such as cytokine release syndrome and toxic reactions, and achieve the effect of safe anti-tumor activity and mild proliferation

Active Publication Date: 2019-12-03
BEIJING MARINO BIOTECH PTY LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the abstract of the American Society of Hematology (ASH) annual meeting (December 2017), Novartis announced the data of the global pivotal phase 2 clinical trial (Juliet trial) of CTL019 in the treatment of diffuse large B-cell lymphoma (DLBCL). Efficacy in refractory DLBCL patients, 99 patients who received a single dose of CTL019 infusion, the best overall response rate (ORR) was 53.1%, complete response rate (CR) was 39.5%, however, 86% of patients Gra

Method used

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Examples

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Embodiment 1

[0112] Cell lines and basic experimental techniques used in the embodiments of the present invention are as follows:

[0113] Generation of lentivirus and transduction of human T lymphocytes

[0114] 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 on a cell culture dish with a bottom area of ​​150-cm2, and according to the instructions, Express-In (purchased from Open Biosystems / ThermoScientific, Waltham, MA) Viral transduction of 293T cells. Add 15 μg of lentiviral transgenic plasmid, 5 μg of pVSV-G (VSV glycoprotein expression plasmid), 10 μg of pCMVR8.74 plasmid (Gag / Pol / Tat / Rev expression plasmid) and 174 μl of Express- In (concentration is 1 μg / μl). The supernatants were collected at 24 hours and 48 hours, and centrifuged for 2 hours using an ultracentrifuge at 28,000 rpm (B...

Embodiment 2

[0121] Example 2 Construction of vectors co-expressing non-functional EGFR and anti-CD19 chimeric antigen receptor

[0122] In this example, the inventors combined the XbaI and BstBI cloning site sequences at both ends and the sequence encoding the single-chain antibody against human CD19, human CD8α sequence, 4-1BB intracellular segment and T cell receptor combination ζ -Strand sequence was artificially synthesized, cloned into lentiviral vector shuttle plasmid containing EF-1 promoter (PCDH-EF1-MCS-IRES-GFP (System Biosciences, Palo Alto, CA). The inventor then artificially synthesized two ends connected with BspEI and SalI cloning site sequences and sequences encoding and expressing non-functional tEGFR, through double enzyme digestion, connection, screening and amplification of the target plasmid , to generate a lentiviral vector shuttle plasmid (named LV-CD19-BBz(86)) co-expressing anti-CD19 chimeric antigen receptor and non-functional tEGFR sequences.

[0123] figure 1...

Embodiment 3

[0124] Example 3 The ability of improved LV-CD19-BBz(86) CAR-T lymphocytes to produce and secrete cytokines is significantly weakened

[0125]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; Retro...

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Abstract

The invention provides a chimeric antigen receptor. The chimeric antigen receptor comprises an extracellular region, a transmembrane region and an intracellular region, wherein the extracellular region comprises a single-chain antibody and a hinge region, the single-chain antibody comprises a heavy chain variable region of the single-chain antibody and a light chain variable region of the single-chain antibody, the single-chain antibody specially recognizes antigen human CD19, the hinge region comprises a human CD8 alpha extracellular segment containing 55 amino acid residues and 3 additionalamino acid residues AAA, and the AAA is located at the N terminal of the human CD8 alpha extracellular segment; the transmembrane region comprises a human CD8 alpha transmembrane segment, the human CD8 alpha transmembrane segment is connected with the hinge region of the extracellular region, and is embedded in the cell membrane of T lymphocyte; and the intracellular region comprises a human CD8 alpha intracellular segment containing 7 amino acid residues, a 4-1BB intracellular region and a CD3 zeta chain intracellular segment, and the intracellular region is connected with the human CD8 alphamolecule transmembrane segment.

Description

technical field [0001] The present invention relates to the field of biomedicine, specifically, the present invention relates to anti-CD19 chimeric antigen receptor, T lymphocytes, lentivirus, transgenic lymphocytes, constructs, therapeutic compositions for treating cancer and improving the safety of lymphocyte therapy Methods. Background technique [0002] Chimeric antigen receptor (CAR)-T cell therapy is a class of immunotherapy products that can effectively treat malignant tumors, especially hematological tumors. Chimeric antigen receptors (CARs) are fusion proteins that include a single-chain antibody and an intracellular T-cell signaling domain connected to the hinge and transmembrane regions. The hinge region and transmembrane region of CAR may be critical for the function of T cells to express CAR. Recent clinical trials have shown that anti-CD19 CAR T cells have been used in the treatment of advanced B-cell malignancies, resulting in complete remission in many pati...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N5/10C12N15/867A61K35/17A61P35/00
CPCC07K16/2803C07K14/7051C07K14/70517C12N5/0636C12N15/86A61K35/17A61P35/00C07K2317/622C07K2319/03C12N2740/15043C12N2510/00C07K2319/33C07K2317/73C07K16/2863A61K39/001112A61K2039/5156C12N5/0638C12N2740/16043C07K14/70578C07K14/70503A61K2039/505C07K14/71
Inventor 陈思毅严勇朝朱益林
Owner BEIJING MARINO BIOTECH PTY LTD
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