CN110964121A

CN110964121A - CD19-CAR-T cell secreting IL-7 and IL-15 factors and application thereof

Info

Publication number: CN110964121A
Application number: CN201911324848.9A
Authority: CN
Inventors: 谭毅; 陈倩; 张慧慧; 韩镇
Original assignee: Qilu Cell Therapy Technology Co ltd
Current assignee: Qilu Cell Therapy Technology Co ltd
Priority date: 2019-12-20
Filing date: 2019-12-20
Publication date: 2020-04-07
Anticipated expiration: 2039-12-20
Also published as: CN110964121B

Abstract

The invention provides a CD19-CAR-T cell secreting IL-7 and IL-15 factors, wherein the chimeric antigen receptor comprises an antigen binding domain that specifically binds CD19, a transmembrane domain and an intracellular domain, and further comprises an IL-7 element and an IL-15 element that are linked to and co-expressed with the intracellular domain. The CD19-CAR-T cells secreting IL-7 and IL-15 factors improve the characteristics of cell killing, promotion of secretion of IL-2 and INF-gamma cytokines and the like, can improve the number of CD8+ T cells, prolong the survival time of the cells after reinfusion and the formation of memory T cells, and increase the effectiveness of the cells; can be used for treating acute lymphocytic leukemia and chronic lymphocytic leukemia.

Description

CD19-CAR-T cell secreting IL-7 and IL-15 factors and application thereof

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a CD19-CAR-T cell for secreting IL-7 and IL-15 factors.

Background

Chimeric antigen receptor-modified T cells directed against CD19 (anti-CD19 polymeric antigen receptors cells, CD19CAR-T cells for short) have had great success in the treatment of refractory B-cell malignancies; and the CAR-T technology targeting other tumor targets also shows good application prospect in the treatment of other solid tumors. Affecting the effectiveness of CD19CAR-T therapy is the survival time of the cells after their reinfusion and the number of memory T cell formations.

For the time that the reinfused CAR-T cells survive in the patient, current studies indicate that the use of CD28 or 4-1BB in the CAR-T construction may affect the life of the cells in vivo. It has also been suggested that the cells that are returned are mainly terminally differentiated effector T cells, while the memory T cells that can survive for a long period are less abundant. There is a great deal of literature that exogenous addition of specific cytokines has important roles in promoting proliferation, activating and regulating the homeostasis of immune cells.

At present, IL-7 and IL-15 are expensive, which can significantly increase the cost of cell culture and increase the economic burden of patients. Therefore, we would like to be able to let CAR-T cells secrete this antibody by themselves.

Therefore, it is a problem to be solved to modify T cells to autocrine cells and increase the effectiveness of CAR-T cells and the proportion of memory T cells, so that the long-term survival and effectiveness of the reinfused CAR-T cells in patients is achieved.

The invention aims at the problems of low memory T cell ratio and poor CAR-T cell effectiveness, and provides a CD19 phenotype-targeted CAR-T cell capable of self-expressing IL-7 and IL-15, which can effectively improve the ratio of memory T cells and the effectiveness of the CAR-T cells after reinfusion.

In order to achieve the purpose, the invention adopts the following technical scheme.

A chimeric antigen receptor comprising an antigen binding domain that specifically binds CD19, a transmembrane domain, and an intracellular domain; the chimeric antigen receptor also includes an IL-7 element and an IL-15 element linked to and coexpressed with the intracellular domain.

The nucleic acid sequence structure of the chimeric antigen receptor is shown as the following formula:

Z1-T-H-TM-C-Z2-Z3-P1-Z4-P2；

in the formula (I), the compound is shown in the specification,

"-" is a connecting bond;

z1 is null or a signal peptide;

t is a single chain antibody targeting CD 19;

h is a null or hinge region;

TM is a transmembrane domain;

c is a co-stimulation signal;

z2 is the cytoplasmic signal of CD3 ζ;

z3 is a 2A peptide;

p1 is IL-7;

z4 is a ribosome entry site;

p2 is IL-15.

Z1 is a signal peptide of a protein selected from the group consisting of: CD8, CD28, GM-CSF, CD4 or CD 137.

The H is a hinge region of the following proteins: CD8, CD28, CD137, or a combination thereof.

The TM is selected from the transmembrane regions of the following proteins: CD3epsilon, CD4, CD8, CD9, CD16, CD22, CD33, CD137, CTLA-4, PD-1 or LAG-3.

C is a costimulatory signal molecule selected from the following proteins: OX40, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, or a combination thereof.

The 2A is selected from T2A, P2A, E2A and F2A.

Preferably, the structure of the chimeric antigen receptor is as follows:

CD8 signal peptide-single chain antibody targeting CD 19-CD 8 transmembrane region-4-1 BB signal-cytoplasmic signal of CD3 ζ -P2A peptide-IL-7-IRES-IL-15.

The chimeric antigen receptor comprises a nucleotide sequence shown as SEQ ID NO. 1.

A gene expression cassette comprising a structure comprising a promoter and the chimeric antigen receptor. Preferably, the promoter is SFFV.

A vector comprising the gene expression cassette. The vector is selected from plasmids, phages, viruses or transposons. Preferably, the vector is a virus; more preferably a lentivirus.

A T lymphocyte comprising the vector.

The chimeric antigen receptor, the gene expression cassette, the vector or the T lymphocyte can be used for preparing medicaments for preventing and/or treating and/or adjunctively treating malignant tumors. The malignant tumor is selected from acute lymphocytic leukemia and chronic lymphocytic leukemia.

The invention has the following advantages:

the CD19-CAR-T cell provided by the invention can autocrine IL-7 and IL15, improves the characteristics of cell killing, promotion of secretion of IL-2 and INF-gamma cytokines and the like, can improve the number of CD8+ T cells, prolongs the survival time of the cell after reinfusion and the formation of memory T cells, and increases the effectiveness of the cell.

Some common terms involved in the present invention are described below:

"chimeric antigen receptors" are artificially engineered receptors that can anchor specific molecules (e.g., antibodies) that recognize tumor cell surface antigens to immune cells (e.g., T cells) such that the immune cells recognize the tumor antigens or viral antigens and kill the tumor cells or virally infected cells;

"Single-chain antibody" (scFv) refers to an antibody fragment which is formed by connecting an amino acid sequence of a light chain variable region (VL region) and an amino acid sequence of a heavy chain variable region (VH region) through a hinge and has the ability of binding antigen;

a "hinge" is a polypeptide segment that links different proteins or polypeptides, with the purpose of maintaining the linked proteins or polypeptides in their respective spatial conformations, to maintain the function or activity of the protein or polypeptide;

"Gene expression cassette" refers to the smallest unit capable of expressing a gene of interest, including promoter-gene of interest-terminator;

"CD 19" refers to human leukocyte differentiation antigen 19 with ID number 930 in NCBI GenBank, 2 isoforms (cDNA sequence/protein sequence), NM-001178098.1/NP-001171569.1, NM-001770.5/NP-001761.3, respectively. When referring to the amino acid sequence of CD19, it includes the full length of the CD19 protein or a fragment of CD19 with CD19 function; fusion proteins of the full-length or fragment are also included. Also, it is understood by those skilled in the art that mutations or variations (including but not limited to substitutions, deletions and/or additions) may be naturally occurring or artificially introduced into the amino acid sequence of CD19 without affecting its biological function. And, when describing a protein sequence fragment of CD19, also includes the corresponding sequence fragment in its natural or artificial variants;

"IL-7 factor" refers to an IL-7 protein containing 177 amino acids and is a glycoprotein with a relative molecular weight of 25 kDa. Plays an important role in the growth, survival and differentiation of T cells;

"IL-15 factor" refers to an IL-15 protein of 162 amino acids, a glycoprotein with a relative molecular weight of 18kDa, which has ID number 3600 in NCBI GenBank. Plays an important role in the growth of T cells and the differentiation of memory T cells;

the 2A peptide is self-splitting polypeptide, is a section of polypeptide with the length between 18 and 22 amino acids, is firstly found in small RNA virus, and then is found in insect virus, rotavirus and the like, can be applied to the construction of a polycistronic vector, has the advantages of short structure, good upstream and downstream gene expression balance, capability of co-expressing a plurality of proteins and the like, and is an effective tool for constructing the polycistronic vector;

"IRES" refers to an Internal ribosome entry site (Internal ribosome entry site), a secondary structure-rich RNA sequence that folds into a structure similar to that of an initiator tRNA, and mediates ribosome binding to RNA, thereby initiating protein translation. After translation of a protein preceding an IRES, the ribosome does not detach from the mRNA and can bind to the IRES, allowing translation to proceed, so that one mRNA can translate both proteins.

Drawings

FIG. 1: the structure of the expression vector pHR-anti CD19CAR-4-1BB-CD3 ζ -P2 a-IL-7-IRES-IL-15;

FIG. 2: titer flow assay of lentiviral CD19CAR-IL-7-IL-15 infected with K562;

FIG. 3: flow assay graph for positive rate of CD19CAR-IL-7-IL-15-T cells;

FIG. 4: flow-through assay of effector memory T cells after 8 days of CD19CAR-IL-7-IL-15-T cell culture;

FIG. 5: CD 8T cell flow assay after 8 days of CD19CAR-IL-7-IL-15-T cell culture;

FIG. 6: CD19CAR-IL-7-IL-15-T cell proliferation fold map;

FIG. 7: flow assay of CD19CAR-IL-7-IL-15-T cell killing function (24h cell killing rate for each group);

FIG. 8: ELISA detects the IL-2 secretion pattern of CD19CAR-IL-7-IL-15-T cells;

FIG. 9: ELISA detects CD19CAR-IL-7-IL-15-T cell IFN-gamma secretion pattern;

FIG. 10: ELISA detects the IL-7 secretion pattern of CD19CAR-IL-7-IL-15-T cells;

FIG. 11: ELISA detects the IL-15 secretion pattern of CD19CAR-IL-7-IL-15-T cells;

FIG. 12: survival profiles of tumor-bearing mice after transplantation of CD19CAR-IL-7-IL-15-T cells into B-cell lymphoma transplanted tumor mouse models.

Detailed Description

The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.

The instruments, reagents, materials and the like used in the examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal way unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art. The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruker et al, Huang Petang et al) or according to the product instructions.

Example 1 construction of IL-7 Gene-containing CD19CAR Lentiviral expression vector

1. Construction of CD19CAR plasmid containing IL-7 and IL-15 genes

Genes are synthesized according to the sequence of a CD8 transmembrane signal peptide, anti-CD19 scFv, a CD8 transmembrane region, a 4-1BB costimulatory signal region, a CD3 zeta TCR activation region, P2a and IL-7, an MluI enzyme cutting site (ACGCGT) and a NotI enzyme cutting site (CGCCGGCG) are respectively added at the 5 'end and the 3' end, and then the genes are linked into the lentiviral backbone plasmid pHR-Mherry which is also cut in two times after MluI and NotI double enzyme cutting. The detection positive plasmid is named as pHR-anti CD19CAR-4-1BB-CD3 zeta-P2 a-IL-7 for standby, IRES and IL-15 are sequentially synthesized into genes, NotI enzyme cutting sites (CGCCGGCG) are respectively added at the 5 'and 3' ends, the genes are connected to pHR-anti CD19CAR-4-1BB-CD3 zeta-P2 a-IL-7 which is also subjected to NotI enzyme cutting after the NotI enzyme cutting, and the detection positive plasmid is named as pHR-anti CD19CAR-4-1BB-CD3 zeta-P2 a-IL-7-IRES-IL-15, wherein the structure diagram of the detection positive plasmid is shown in figure 1.

2. Lentivirus packaging and concentration

And (2) mixing the CD19CAR plasmid containing IL-7 and IL-15 genes, the pCMV vector and the pMD.2G vector obtained in the step (1), transfecting into 293FT cells, replacing with a complete culture medium for culture after 6h after transfection, collecting a culture solution after 48h, centrifuging, retaining a supernatant, filtering the supernatant by using a 0.45 mu m filter, and retaining a filtrate, wherein the filtrate is a solution of the recombinant lentivirus. Lenti-XTMConcentor (Takara, Cat.631231) instructions for lentivirus concentration.

3. Lentiviral titer detection

1640 medium to resuspend cells to 1X 10⁶/mL。37℃、5％CO₂Culturing in an incubator for 6-8h, and centrifuging to change the culture solution into fresh K562 cell proliferation culture solution. Adding fresh K562 cell proliferation culture solution every 2-3 days to maintain cell density at 0.5 × 10⁶about/mL. Each hole of the 24-hole plate is paved with 1 multiplied by 10⁵Taking virus stock solution from K562 cells, respectivelyAfter 48H of viral infection with 1. mu.L, 3. mu.L, 10. mu.L of virus was added, the proportion of cells positive for CAR was determined by flow cytometry, FITC-laboratory HumanCD19(20-291) Protein (brand: Acro, Cat.: CD9-HF2H 210. mu.L) was added to each sample, incubated for 1H at 4 ℃ in the absence of light, washed 3 times, centrifuged, and the pellet was resuspended in 200. mu.L of PBS and tested on the machine (Millipore guava easy Cyte HT), as shown in FIG. 3, the proportion of positive cells increased gradually with increasing amount of virus added. When the positive rate is less than or equal to 10%, the number of virus particles is considered to be equal to the number of cells, so that the virus titer is 7.47 multiplied by 10⁶pfu/mL。

Example 2 preparation of CD19CAR-IL-7-IL-15-T cells

CTS for freshly isolated PBMC^TMAIM V^TMSFM Medium (GIBCO, cat # A3021002). The medium was supplemented with 5% ICS (GIBCO, A2596101). Simultaneously adding CD3 monoclonal antibody (ebioscience, cat # 160037-85) and CD28 monoclonal antibody (ebioscience, cat # 160288-85) to activate T lymphocyte with final concentration of 50ng/mL, and activating T lymphocyte with 5% CO at 37 ℃₂The culture was carried out for 48 hours. Take 2X 10⁶The lentivirus concentrated in step 2 was added to the cells according to MOI 2.5, and IL-2 (spring harbor) and polybrene (Sigma) were added to final concentrations of 250U/mL, mixed well, and 5% CO was added at 37 deg.C₂Culturing for 6-8 hours, centrifuging for 5min at 300g, and changing the liquid into fresh CTS^TMAIM V^TMSFM medium (containing 250U/mL IL-2). Adding fresh T cell proliferation culture solution every 2-3 days to maintain cell density at 1 × 10⁶about/mL, 10-12 days of amplification.

48h after viral infection, the proportion of cells positive for CAR was determined by flow cytometry (FITC-LabeledHumanCD19(20-291) Protein). As shown in FIG. 4, the proportion of CD19CAR-T cells containing IL-7 and IL-15 genes was about 63.3%. Indicating that the CD19CAR-T cells containing IL-7 and IL-15 genes have been successfully obtained. Designated as CD19 CAR-IL-7-IL-15-T.

Example 3 CD19CAR-IL-7-IL-15-T cell function assay

1. Promotion of memory T cell formation

CD19CAR-IL-7-T cells were obtained in the same manner as in example 1 and example 2, and then were compared with CD19CAR-IL-7-IL-15-T cells obtained in example 2The cells were cultured until day 8, and about 1X 10 cells were collected⁶And (3) detecting the proportion of CD8 cells and memory T cells in the T cells by using a flow cytometer: CD8-PE (brand: Biolegend, Cat.: 344706) and CCR7-FITC (brand: Biolegend, Cat.: 353216), CD45RA-APC (brand: Biolegend, Cat.: 304112) were added at 1. mu.L each, incubated 15min at room temperature in the absence of light, washed 1 time, centrifuged, and the pellet was resuspended in 200. mu.L PBS and tested on the machine (Millipore guava easy Cyte HT), as shown in FIG. 5. The results show that: the proportion of memory T cells in CD19CAR-T cells at day 8 was about 38.9%, the proportion of memory T cells in CD19CAR-IL-7-T cells was about 59.9%, and the proportion of memory T cells in CD19CAR-IL-7-IL-15-T cells was 70.1%, i.e., CAR-T had the effect of promoting memory T cell formation in vitro culture after expression of IL-7 and IL-15. As shown in fig. 6. The results show that: the proportion of CD 8T cells in CD19CAR-T cells was about 58.8%, the proportion of CD 8T cells in CD19CAR-IL-7-T cells was about 78.6%, and the proportion of CD 8T cells in CD19CAR-IL-7-IL-15-T cells was 91.8% at day 8, i.e. after expression of IL-7 and IL-15, CAR-T had the effect of promoting CD 8T cell formation in vitro culture.

The CD19CAR-IL-7-IL-15-T cell group and the CD19CAR-IL-7-T cell group were cultured at 0d, 7d, 9d, 11d, 14d during cell culture to count the number of T cells, respectively, and a T cell proliferation curve was plotted. As shown in fig. 7, the CD19CAR-IL-7-IL-15-T cell group, CD19CAR-IL-7-T cell group, and CD19CAR-T cell group cells proliferated 778-fold, 566-fold, and 122-fold, respectively, with significant differences between the three groups. Namely after IL-7 and IL-15 genes are expressed, CAR-T has the obvious function of promoting the proliferation of memory T cells in vitro culture.

Detection of the killing function of CD19CAR-IL-7-IL-15-T

2.1 preparation of K562 cells expressing CD19

(1) Synthesizing DNA sequences (SEQ ID NO:2) of an extracellular region of CD19 and a transmembrane region of CD8, adding MluI and NotI enzyme cutting sites at two ends, inserting a pHR-Mchery vector subjected to the same enzyme cutting after enzyme cutting, and constructing the obtained vector and naming the pHR-CD 19;

(2) mixing a pHR-CD19 vector, a pCMV vector and a pMD.2G vector, transfecting into 293FT cells, replacing with a complete culture medium for culture 6-8h after transfection, collecting a culture solution 48h after transfection, centrifuging, retaining a supernatant, filtering the supernatant by using a 0.45 mu m filter, and retaining a filtrate, wherein the filtrate is a solution of the recombinant lentivirus. Lenti-XTM Concentrator (Takara, Cat.631231) instructions for lentivirus concentration;

(3)1640(Gibco) medium resuspended K562 cells to 1X 10⁶PermL, lentivirus (MOI 5), 5% CO at 37 ℃₂Culturing for 6h in an incubator, and centrifuging to change the culture solution into a fresh K562 cell proliferation culture solution. Adding fresh K562 cell proliferation culture solution every 2-3 days to maintain cell density at 0.5 × 10⁶About one/mL. After 5 generations, monoclonal screening was performed using limiting dilution. After the monoclonal cells grow to a certain amount, screening by a flow cytometer (anti-CD19PE, biolegend, Cat: 302254), and obtaining cell clones with high expression level and high purity, namely K562 cells stably expressing CD19, which are named as K562-CD19 and serve as target cells.

2.2 killing function assay of CD19CAR-IL-7-T

K562 cells and K562-CD19 cells were seeded at 1:1 in 96-well plates, and CD19CAR-IL-7-IL-15-T, CD19CAR-IL-7-T cells, CD19CAR-T cells, and T cells, which were cultured for 9 days, were seeded at E: T ═ 1:1, and 3:1, respectively. 5% CO at 37 ℃₂Culturing for 24 hr, collecting supernatant of each well, and freezing to-80 deg.C in refrigerator for use. mu.L each of PE anti-humanCD19(Biolegend Cat.: 302254) and FITC anti-humanCD 3(Biolegend Cat.: 300306) was added to each of the remaining cells in each well, incubated for 15min at room temperature in the absence of light, centrifuged, and the pellet was resuspended in 200. mu.L PBS and then tested on the machine (Millipore guava easy CyteHT). The T cell group was used as a negative control group, and the CD19CAR-IL-7-T cell group and the CD19CAR-T cell group were used as positive control groups.

As shown in fig. 7, the group of T cells without specific killing was used as a negative control group, and the specific killing of the group was theoretically 0. When T is 3:1, the killing rates of the CD19CAR-IL-7-T cell group and the CD19CAR-IL-7-IL-15-T cell group are both 100%, and the killing rates of the CD19CAR-T cell group are both 95%. When T is 1:1, the killing rate of the CD19CAR-IL-7-T cell group is 90.7%, the killing rate of the CD19CAR-IL-7-IL-15-T cell group is 92%, no obvious difference between groups exists, the killing rate of the CD19CAR-T cell group is 41.2%, and obvious difference between groups exists. The statistical results show that the killing rates of the CD19CAR-IL-7-IL-15-T cell group and the CD19CAR-T cell group are different significantly, and the killing rates are not different from those of the CD19CAR-IL-7-T cell group. Thus, CD19CAR-IL-7-IL-15-T cells have a distinct killing advantage over CD19CAR-T cells, i.e., CD19CAR-T cells expressing IL-7, IL-15, promote the killing function of CAR-T cells compared to the CD19CAR-T cell group, and CD19CAR-T cells expressing IL-7, IL-15, do not affect the killing function of CAR-T cells compared to the CD19CAR-IL-7-T cell group.

CD19CAR-IL-7-IL-15-T cell cytokine secretion

The killer supernatant from step 2.2 was used for ELISA to detect cytokine secretion (human IL-2ELISA-Kit, Biolegend Cat.: 431804; human IFN-. gamma.ELISA-Kit, Biolegend Cat.: 430104; human IL-7ELISA-Kit, Union, Cat.: EK 1072; human IL-15ELISA-Kit, brand R & D Cat.: DY 247-05). The T cell group and the K562+ K562-CD19 cell group were used as negative controls, and the CD19CAR-IL-7-T cell group and the CD19CAR-T cell group were used as positive controls. As shown in fig. 8, the release amounts of IL-2 for the CD19CAR-IL-7-IL-15-T cell group, CD19CAR-IL-7-T cell group, and CD19CAR-T cell group were 4022pg/mL, 3627pg/mL, and 1535pg/mL, respectively. The CD19CAR-IL-7-IL-15-T cell group was not significantly different from the CD19CAR-IL-7-T cell group, and was significantly different from the CD19CAR-T cell group. That is, compared to the CD19CAR-IL-7-T cell group, CD19CAR-T expressing IL-15 did not affect release of IL-2; CD19CAR-T cells expressing IL-7 and IL-15 promoted the release of IL-2 compared to the CD19CAR-T cell group. As shown in fig. 9, the release of INF- γ for the CD19CAR-IL-7-IL-15-T cell group, CD19CAR-IL-7-T cell group, and CD19CAR-T cell group was 45170pg/mL, 31902pg/mL, and 12510pg/mL, respectively. That is, IL-7 and IL-15 expressing CD19CAR-T cells increased IFN- γ release compared to the CD19CAR-IL-7-T cell group, but the effect was not significant; CD19CAR-T cells expressing IL-7 and IL-15 significantly increased IFN- γ release compared to the CD19CAR-T cell group. As shown in figure 10, the release of IL-7 was 1382.7pg/mL and 1362.5pg/mL for the CD19CAR-IL-7-IL-15-T cell group, CD19CAR-IL-7-IL-15-T cell group, respectively, with no significant difference, i.e., the expression of IL-15 by the CD19CAR-IL-7-IL-15-T cell group did not affect the expression of IL-7 compared to the CD19CAR-IL-7-T cell group; and the CD19CAR-T cell group had no IL-7 release. As shown in FIG. 11, the release amounts of IL-15 in the CD19CAR-T cell group, the CD19CAR-IL-7-T cell group and the CD19CAR-IL-7-IL-15-T cell group were 2.3pg/mL, 2.37pg/mL and 1139.3pg/mL, respectively, which were significantly different. That is, the release amount of IL-15 in the CD19CAR-IL-7-IL-15-T cell group was significantly increased compared to the CD19CAR-T cell group and the CD19CAR-IL-7-T cell group.

CD19CAR-IL-7-IL-15-T cell animal assay

Mixing CD19CAR-IL-7-IL-15-T cells, CD19CAR-IL-7-T cells, CD19CAR-T cells, GFP-T cells at 37 deg.C with 5% CO₂The incubator is used for 144h for standby. Of 40 male SPF-grade NCG mice (breed strain: genetically engineered mice), 32 mice were inoculated with 0.3 mL/model of human Raji cells (day 0) by tail vein injection, and the 32 mice surviving on day 4 after model creation were grouped into 4 groups by body weight, i.e., Group1(CD19CAR-T cell Group), Group2(CD19CAR-IL-7-T cell Group), Group3(CD19CAR-IL-7-IL-15-T cell Group), and Group4(Mock-GFP CAR-T cell Group); another 8 mice were not inoculated with human Raji cells and were designated Group5 (blank control). On the day of dosing, mice in the negative control Group (Group1) were injected with 0.9% sodium chloride injection via tail vein. Animals were observed daily for general signs and recorded on days 0-60. Moribund or mid-dead mice and cervical dislocation of mice observed to day 60 were sacrificed. The change of the physical signs of each group of animals during the experiment is observed, and a survival curve is drawn. Statistical analysis and curve plotting were performed using GraphPadPrism 5.0 software, as shown in figure 11.

Sign observations during the trial showed that Group4 animals all died within 25 days after molding. Under the test conditions, compared with the negative control group diseased mouse model, the CD19CAR-T cell group, CD19-CAR-IL-7-T cells and CD19-CAR-IL-7-IL-15-T cells were administered with 0.5 mL/mouse by single tail vein injection (CAR positive T cell count 3X 10)⁷cells/mL) can obviously improve the sign performance of a disease model of a mouse inoculated with Raji cells NCG, and can prolong the survival time of the mouse of the disease model, and the survival rate of the mouse of a CD19-CAR-IL-7-IL-15-T cell group is superior to that of the mouse of a CD19CAR-IL-7-T cell groupSurvival rate of mice, no significant difference; the survival rate of mice superior to the CD19CAR-T cell group was significantly different.

Sequence listing

<110> Qilu cell therapy engineering Co., Ltd, Shandong province

<120> CD19-CAR-T cell secreting IL-7 and IL-15 factors and application thereof

<130>20191211

<160>2

<170>PatentIn version 3.5

<210>1

<211>3208

<212>DNA

<213>Artificial Sequence

<220>

<223>IL 7-IL 15-antiCD19 CAR

<400>1

atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60

ccggagcaga agctgatcag cgaggaggac ctggacatcc agatgacaca gactacatcc 120

tccctgtctg cctctctggg agacagagtc accatcagtt gcagggcaag tcaggacatt 180

agtaaatatt taaattggta tcagcagaaa ccagatggaa ctgttaaact cctgatctac 240

catacatcaa gattacactc aggagtccca tcaaggttca gtggcagtgg gtctggaaca 300

gattattctc tcaccattag caacctggag caagaagata ttgccactta cttttgccaa 360

cagggtaata cgcttccgta cacgttcgga ggggggacca agctggagat cacaggtggc 420

ggtggctcgg gcggtggtgg gtcgggtggc ggcggatctg aggtgaaact gcaggagtca 480

ggacctggcc tggtggcgcc ctcacagagc ctgtccgtca catgcactgt ctcaggggtc 540

tcattacccg actatggtgt aagctggatt cgccagcctc cacgaaaggg tctggagtgg 600

ctgggagtaa tatggggtag tgaaaccaca tactataatt cagctctcaa atccagactg 660

accatcatca aggacaactc caagagccaa gttttcttaa aaatgaacag tctgcaaact 720

gatgacacag ccatttacta ctgtgccaaa cattattact acggtggtag ctatgctatg 780

gactactggg gccaaggaac ctcagtcacc gtctcctcaa ccacgacgcc agcgccgcga 840

ccaccaacac cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc 900

cggccagcgg cggggggcgc agtgcacacg agggggctgg acttcgcctg tgatatctac 960

atctgggcgc ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt 1020

tactgctccc taaaacgggg cagaaagaaa ctcctgtata tattcaaaca accatttatg 1080

agaccagtac aaactactca agaggaagat ggctgtagct gccgatttcc agaagaagaa 1140

gaaggaggat gtgaactgag agtgaagttc agcaggagcg cagacgcccc cgcgtacaag 1200

cagggccaga accagctcta taacgagctc aatctaggac gaagagagga gtacgatgtt 1260

ttggacaaga gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct 1320

caggaaggcc tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt 1380

gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt 1440

acagccacca aggacaccta cgacgccctt cacatgcagg ccctgccccc tcgcggcgaa 1500

ttcggaagcg gagctactaa cttcagcctg ctgaagcagg ctggagacgt ggaggagaac 1560

cctggaccta tgttccatgt ttcttttagg tatatctttg gacttcctcc cctgatcctt 1620

gttctgttgc cagtagcatc atctgattgt gatattgaag gtaaagatgg caaacaatat 1680

gagagtgttc taatggtcag catcgatcaa ttattggaca gcatgaaaga aattggtagc 1740

aattgcctga ataatgaatt taactttttt aaaagacata tctgtgatgc taataaggaa 1800

ggtatgtttt tattccgtgc tgctcgcaag ttgaggcaat ttcttaaaat gaatagcact 1860

ggtgattttg atctccactt attaaaagtt tcagaaggca caacaatact gttgaactgc 1920

actggccagg ttaaaggaag aaaaccagct gccctgggtg aagcccaacc aacaaagagt 1980

ttggaagaaa ataaatcttt aaaggaacag aaaaaactga atgacttgtg tttcctaaag 2040

agactattac aagagataaa aacttgttgg aataaaattt tgatgggcac taaagaacac 2100

taagcggccg ccgcccctct ccctcccccc cccctaacgt tactggccga agccgcttgg 2160

aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg tcttttggca 2220

atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg ggtctttccc 2280

ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt cctctggaag 2340

cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac cccccacctg 2400

gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca aaggcggcac 2460

aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg ctctcctcaa 2520

gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg ggatctgatc 2580

tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa cgtctaggcc 2640

ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataagct tgccacaacc 2700

cacaaggaga cgaccttcca tgagaatttc gaaaccacat ttgagaagta tttccatcca 2760

gtgctacttg tgtttacttc taaacagtca ttttctaact gaagctggca ttcatgtctt 2820

cattttgggc tgtttcagtg cagggcttcc taaaacagaa gccaactggg tgaatgtaat 2880

aagtgatttg aaaaaaattg aagatcttat tcaatctatg catattgatg ctactttata 2940

tacggaaagt gatgttcacc ccagttgcaa agtaacagca atgaagtgct ttctcttgga 3000

gttacaagtt atttcacttg agtccggaga tgcaagtatt catgatacag tagaaaatct 3060

gatcatccta gcaaacaaca gtttgtcttc taatgggaat gtaacagaat ctggatgcaa 3120

agaatgtgag gaactggagg aaaaaaatat taaagaattt ttgcagagtt ttgtacatat 3180

tgtccaaatg ttcatcaaca cttcttga 3208

<210>2

<211>960

<212>DNA

<213>Artificial Sequence

<220>

<223>CD19-CD8

<400>2

atgccacctc ctcgcctcct cttcttcctc ctcttcctca cccccatgga agtcaggccc 60

gaggaacctc tagtggtgaa ggtggaagag ggagataacg ctgtgctgca gtgcctcaag 120

gggacctcag atggccccac tcagcagctg acctggtctc gggagtcccc gcttaaaccc 180

ttcttaaaac tcagcctggg gctgccaggc ctgggaatcc acatgaggcc cctggccatc 240

tggcttttca tcttcaacgt ctctcaacag atggggggct tctacctgtg ccagccgggg 300

cccccctctg agaaggcctg gcagcctggc tggacagtca atgtggaggg cagcggggag 360

ctgttccggt ggaatgtttc ggacctaggt ggcctgggct gtggcctgaa gaacaggtcc 420

tcagagggcc ccagctcccc ttccgggaag ctcatgagcc ccaagctgta tgtgtgggcc 480

aaagaccgcc ctgagatctg ggagggagag cctccgtgtc tcccaccgag ggacagcctg 540

aaccagagcc tcagccagga cctcaccatg gcccctggct ccacactctg gctgtcctgt 600

ggggtacccc ctgactctgt gtccaggggc cccctctcct ggacccatgt gcaccccaag 660

gggcctaagt cattgctgag cctagagctg aaggacgatc gcccggccag agatatgtgg 720

gtaatggaga cgggtctgtt gttgccccgg gccacagctc aagacgctgg aaagtattat 780

tgtcaccgtg gcaacctgac catgtcattc cacctggaga tcactgctcg gccagtacta 840

tggcactggc tgctgaggac tggtggctgg aaggtctcag ctgtgacttt ggcttatctg 900

atcttctgcc tgtgttccct tgtgggcatt cttcatcttc aaagagccct ggtcctgagg 960

Claims

1. A chimeric antigen receptor comprising an antigen binding domain, a transmembrane domain and an intracellular domain, wherein the antigen binding domain specifically binds CD 19; the chimeric antigen receptor also includes an IL-7 element and an IL-15 element linked to and coexpressed with the intracellular domain.

2. The chimeric antigen receptor according to claim 1, wherein the nucleic acid sequence structure comprises a sequence of the formula:

Z1-T-H-TM-C-Z2-Z3-P1-Z4-P2；

in the formula (I), the compound is shown in the specification,

"-" is a connecting bond;

z1 is null or a signal peptide;

t is a single chain antibody targeting CD 19;

h is a null or hinge region;

TM is a transmembrane domain;

c is a co-stimulation signal;

z2 is the cytoplasmic signal of CD3 ζ;

z3 is a 2A peptide;

p1 is IL-7;

z4 is a ribosome entry site;

p2 is IL-15.

3. The chimeric antigen receptor according to claim 2, wherein Z1 is a signal peptide of a protein selected from the group consisting of: CD8, CD28, GM-CSF, CD4, or CD 137;

the H is a hinge region of the following proteins: CD8, CD28, CD137, or a combination thereof;

the TM is selected from the transmembrane regions of the following proteins: CD3 ζ, CD4, CD8, CD9, CD16, CD22, CD33, CD137, CTLA-4, PD-1 or LAG-3;

c is a costimulatory signal molecule selected from the following proteins: OX40, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, or a combination thereof;

the 2A is selected from T2A, P2A, E2A and F2A.

4. The chimeric antigen receptor according to claim 2, wherein the structure of the chimeric antigen receptor is as follows: CD8 signal peptide-single chain antibody targeting CD 19-CD 8 transmembrane region-4-1 BB signal-cytoplasmic signal of CD3 ζ -P2A peptide-IL-7-IRES-IL-15.

5. The chimeric antigen receptor according to claim 2, wherein the chimeric antigen receptor comprises the nucleotide sequence shown as SEQ ID NO. 1.

6. A gene expression cassette comprising the chimeric antigen receptor of any one of claims 1-5.

7. A vector comprising the gene expression cassette of claim 6.

8. The vector of claim 7, wherein the vector is selected from the group consisting of a plasmid, a phage, a virus, and a transposon; preferably, the vector is a virus; more preferably a lentivirus.

9. A T lymphocyte comprising the vector of claims 7-8.

10. Use of the chimeric antigen receptor of any one of claims 1 to 5, the gene expression cassette of claim 6, the vector of claims 7 to 8 and the T lymphocyte of claim 9 for the preparation of a medicament for the prophylaxis and/or treatment and/or adjuvant treatment of malignancies; preferably, the malignant tumor is selected from acute lymphocytic leukemia and chronic lymphocytic leukemia.