Polypeptide, nucleic acid for coding polypeptide, polypeptide-modified T lymphocyte and application of T lymphocyte

Abstract

The invention belongs to the technical field of biology, and discloses a polypeptide containing two chimeric antigen receptor structures, nucleic acid for coding the polypeptide, a modified T lymphocyte, and a preparation method and application of the T lymphocyte. The polypeptide provided by the invention contains the two chimeric antigen receptor (CAR) structures, wherein one chimeric antigen receptor structure specifically targets a tumor cell, and the other one specifically targets a normal B lymphocyte, so that a CAR-T cell is effectively amplified through recognizing the B lymphocyte ofa blood circulation system, and more CAR-T cells arrive at a solid tumor part so as to specifically kill the tumor cell for improving a treatment effect on the solid tumor. The polypeptide further comprises an ScFv structure of an anti-PD-L1 monoclonal antibody, and the CAR-T cells can simultaneously secrete a PD-L1 monoclonal antibody which interdicts an immunosuppressive action medicated by a PD1 / PDL1 signal pathway and T cell failure, so that the treatment effect on the solid tumor is improved, the functional activity of immune cells in vivo is prolonged at the same time, and finally the tumor recurrence is retarded and even avoided.

Description

[0001] This application requires a Chinese patent application submitted to the China Patent Office on October 30, 2017, with the application number 201711034769.5, and the title of the invention is "a polypeptide, a nucleic acid encoding it, T lymphocytes modified by it, and its preparation method and application" priority, the entire contents of which are incorporated in this application by reference. technical field

[0002] The invention belongs to the field of biotechnology, and relates to a polypeptide, a nucleic acid encoding it, a modified T lymphocyte and its preparation method and application, in particular to a polypeptide comprising two chimeric antigen receptor structures, a nucleic acid encoding it, and its Modified T lymphocyte and its preparation method and application. Background technique

[0003] After entering the 21st century, with breakthroughs in the two major technical fields of adoptive cell therapy represented by CAR-T and immune checkpoint inhibitor...

Examples

Embodiment 1

[0068] Example 1, construction of CAR(CD30)-CAR(CD22)-aPDL1 lentiviral vector

[0069] The inventors designed three target gene structures according to genetic engineering technology, and their sequences were sequentially spliced ​​from the amino terminal to the carboxyl terminal as follows:

[0070] 1. CAR(CD30):ScFv(CD30)-Hinge(CD8)-TM(CD8)-CD137-CD3ζ

[0071] 2. CAR(CD22):ScFv(CD22)-Hinge(IgG4-short)-TM(CD28)-CD137-CD3ζ

[0072] 3. CAR(CD30)-CAR(CD22)-aPDL1:

[0073] ScFv(CD30)-Hinge(CD8)-TM(CD8)-CD137-CD3ζ-T2A-ScFv(CD22)-Hinge(IgG4-short)-TM(CD28)-CD137-CD3ζ-P2A-ScFv(PD-L1) .

[0074] Among them, ScFv(CD30)-Hinge(CD8)-TM(CD8)-CD137-CD3ζ expresses a chimeric antigen receptor targeting CD30, and its gene sequence is as follows: CD30 antibody single-chain variable region, CD8a hinge Region and transmembrane region, CD137 signal domain, CD3ζ chain intracellular region. The above sequence targets the antigen chimeric receptor sequence of CD22 in series through the self-cle...

Embodiment 2

[0081] Embodiment 2, lentiviral packaging

[0082] The operation steps of packaging are as follows:

[0083] The 293FT cell culture flask (T175) that had grown to 80%-90% was cooled from 37°C in 5% CO 2 Take it out from the cell culture incubator, add 2 mL of EDTA-free-0.25% Trypsin to digest, collect and wash the cells, add 4.5×10 per 10 cm cell culture dish 6 For each cell, add 9mL DMEM medium, shake gently, put in 37℃5%CO 2 cultured in an incubator.

[0084] On day 2, 500 μL per plate Buffer, 6 μg target gene, 3 μg psPAX2, 1.5 μg pMD2.G, mix the above solutions evenly. Add to the mixture 25μL / 10cm plate, mix well again, and let stand at room temperature for 10min. Warm the 293FT cells used for packaging virus from 37°C 5% CO 2 Take it out of the cell culture incubator, add the above mixture evenly to each plate, shake gently, and put it in 37°C 5% CO 2 in the incubator. After 4 hours, discard the old medium, add 10mL of preheated PBS to wash the cells, then add 9...

Embodiment 3

[0086] Example 3, preparation of CAR-T cells

[0087] 1. Cell expansion (Day0)

[0088] Aseptically collect 50-100ml of venous blood from the patient, and perform density gradient centrifugation on the blood sample to obtain peripheral blood mononuclear cells (PBMC). CD3+ cells were sorted with CD3 sorting magnetic beads (Miltenyi, Germany). The sorted positive cells were first resuspended with 2ml of medium, and T cell activation magnetic beads (Gibco, USA) were added according to the ratio of positive cells to magnetic beads at 1:1. Then spread a 24-well plate according to 3×10^5 cells / 500 μl medium / well, and move to 37°C, 5% CO 2 The cells were cultured in an incubator for 2 days.

[0089] 2. Cell transfection (Day2)

[0090] The 24-well plate was taken out of the incubator in advance and placed in a biological safety cabinet, and allowed to cool to room temperature. Then add Protamine Sulfate according to 1‰ (V / V). After the virus was melted, it was added to the 24-w...

[0001] This application requires a Chinese patent application submitted to the China Patent Office on October 30, 2017, with the application number 201711034769.5, and the title of the invention is "a polypeptide, a nucleic acid encoding it, T lymphocytes modified by it, and its preparation method and application" priority, the entire contents of which are incorporated in this application by reference. technical field

[0002] The invention belongs to the field of biotechnology, and relates to a polypeptide, a nucleic acid encoding it, a modified T lymphocyte and its preparation method and application, in particular to a polypeptide comprising two chimeric antigen receptor structures, a nucleic acid encoding it, and its Modified T lymphocyte and its preparation method and application. Background technique

[0003] After entering the 21st century, with breakthroughs in the two major technical fields of adoptive cell therapy represented by CAR-T and immune checkpoint inhibitor...

Examples

Embodiment 1

[0068] Example 1, construction of CAR(CD30)-CAR(CD22)-aPDL1 lentiviral vector

[0069] The inventors designed three target gene structures according to genetic engineering technology, and their sequences were sequentially spliced ​​from the amino terminal to the carboxyl terminal as follows:

[0070] 1. CAR(CD30):ScFv(CD30)-Hinge(CD8)-TM(CD8)-CD137-CD3ζ

[0071] 2. CAR(CD22):ScFv(CD22)-Hinge(IgG4-short)-TM(CD28)-CD137-CD3ζ

[0072] 3. CAR(CD30)-CAR(CD22)-aPDL1:

[0073] ScFv(CD30)-Hinge(CD8)-TM(CD8)-CD137-CD3ζ-T2A-ScFv(CD22)-Hinge(IgG4-short)-TM(CD28)-CD137-CD3ζ-P2A-ScFv(PD-L1) .

[0074] Among them, ScFv(CD30)-Hinge(CD8)-TM(CD8)-CD137-CD3ζ expresses a chimeric antigen receptor targeting CD30, and its gene sequence is as follows: CD30 antibody single-chain variable region, CD8a hinge Region and transmembrane region, CD137 signal domain, CD3ζ chain intracellular region. The above sequence targets the antigen chimeric receptor sequence of CD22 in series through the self-cle...

Embodiment 2

[0081] Embodiment 2, lentiviral packaging

[0082] The operation steps of packaging are as follows:

[0083] The 293FT cell culture flask (T175) that had grown to 80%-90% was cooled from 37°C in 5% CO 2 Take it out from the cell culture incubator, add 2 mL of EDTA-free-0.25% Trypsin to digest, collect and wash the cells, add 4.5×10 per 10 cm cell culture dish 6 For each cell, add 9mL DMEM medium, shake gently, put in 37℃5%CO 2 cultured in an incubator.

[0084] On day 2, 500 μL per plate Buffer, 6 μg target gene, 3 μg psPAX2, 1.5 μg pMD2.G, mix the above solutions evenly. Add to the mixture 25μL / 10cm plate, mix well again, and let stand at room temperature for 10min. Warm the 293FT cells used for packaging virus from 37°C 5% CO 2 Take it out of the cell culture incubator, add the above mixture evenly to each plate, shake gently, and put it in 37°C 5% CO 2 in the incubator. After 4 hours, discard the old medium, add 10mL of preheated PBS to wash the cells, then add 9...

Embodiment 3

[0086] Example 3, preparation of CAR-T cells

[0087] 1. Cell expansion (Day0)

[0088] Aseptically collect 50-100ml of venous blood from the patient, and perform density gradient centrifugation on the blood sample to obtain peripheral blood mononuclear cells (PBMC). CD3+ cells were sorted with CD3 sorting magnetic beads (Miltenyi, Germany). The sorted positive cells were first resuspended with 2ml of medium, and T cell activation magnetic beads (Gibco, USA) were added according to the ratio of positive cells to magnetic beads at 1:1. Then spread a 24-well plate according to 3×10^5 cells / 500 μl medium / well, and move to 37°C, 5% CO 2 The cells were cultured in an incubator for 2 days.

[0089] 2. Cell transfection (Day2)

[0090] The 24-well plate was taken out of the incubator in advance and placed in a biological safety cabinet, and allowed to cool to room temperature. Then add Protamine Sulfate according to 1‰ (V / V). After the virus was melted, it was added to the 24-w...

Images