WO2020000526A1

WO2020000526A1 - Shp-1-knockout t-cell and construction method therefor

Info

Publication number: WO2020000526A1
Application number: PCT/CN2018/095635
Authority: WO
Inventors: 胡边; 张琳琳
Original assignee: 奥妙生物技术（广州）有限公司
Priority date: 2018-06-26
Filing date: 2018-07-13
Publication date: 2020-01-02
Also published as: CN108866004A

Abstract

Provided are an SHP-1-knockout T-cell and a construction method therefor. The killing effect of the T-cell on tumors can be effectively improved by the endogenous blockade of SHP-1 in the T-cell, thereby providing a new target for tumor immunotherapy.

Description

SHP-1 knockout T cells and construction method thereof

Technical field

The invention relates to the technical fields of tumor immunology, molecular biology, cell biology and gene editing, and in particular relates to a method for preparing SHP-1 knockout T cells by using a CRISPR / Cas9 system.

Background technique

Immune checkpoint blocking therapy has shown amazing results in the treatment of tumors. Clinical trials have found that immune checkpoint blocking therapy can reactivate the effector functions of depleted T cells and increase the anti-tumor activity of T cells. Immune checkpoint blocking therapy mainly uses antibodies to block the interaction between immune checkpoint molecules and their corresponding antibodies to improve the anti-tumor ability of T cells. The most common immune checkpoint molecules are PD-1 and CTLA-4. Immune checkpoint blocking therapy has achieved significant results in the treatment of a variety of tumors, including melanoma, non-small cell lung cancer, Hodgkin's lymphoma, head and neck cancer, ovarian cancer, kidney cancer, bladder cancer, and mismatch repair Defective tumors [1]. CTLA-4-targeting antibody ipilimumab has been approved for use in the treatment of patients with advanced melanoma, 20% of whom have received antibody therapy and have a survival period of more than three years. Recently, PD-1 antibodies nivolumab and pembrolizumab and PD-L1 antibody atezolizumab have also been approved by the FDA for the treatment of advanced melanoma, non-small cell lung cancer and kidney cancer. Some patients show partial or complete remission after receiving immune checkpoint blockade. However, not all patients respond to the treatment of CTLA-4 and PD-1 inhibitors.

The presence of phosphatase in T cells has important regulatory effects on the function of T cells. After T cells recognize the antigen through the TCR-MHC complex, a large number of intracellular signaling molecules will be phosphorylated and activated by kinases such as Lck, ZAP70, and participate in signal transmission. However, in order to maintain homeostasis and prevent excessive activation of T cells, a large number of inhibitory phosphatases in T cells will also be activated, causing dephosphorylation of signal molecules and inactivation, affecting the transmission of T cell activation signals, and ultimately weakening T cells. Killing function of cells [2]. There are more than a hundred types of PTP phosphatases in cells, even human T cells contain at least 45 species, and there is increasing experimental evidence that these phosphatases often have independent and irreplaceable effects. Therefore, although theoretically inhibiting phosphatase activity in T cells may increase the ability of T cells to clear tumor cells, the same, improper inhibition of phosphatase activity in T cells, especially key phosphatase activity, may Leading to unpredictable adverse consequences, the impact on the immune system is unknown, and even trigger the occurrence of autoimmune diseases. Choosing which one to use as our target is also a common problem [2]. In addition, because PTP phosphatase is an intracellular protein, its function cannot be blocked by antibody blocking agents such as PD-1 and CTLA-4; small molecule inhibitors of phosphatase often lack the ability to inhibit enzymes or cells. Specificity, leading to great clinical side effects. These reasons also increase the difficulty for people to make choices and operations.

T cells are terminally differentiated primary cells, and it is difficult to perform genetic manipulations such as transfection or infection, coupled with their limited proliferation capacity, which limits the genetic manipulation of T cells in vitro, especially gene knockout. In recent years, the revolutionary development of gene editing technology has provided an opportunity for T cell immunotherapy. Gene editing with CRISPR / Cas9 technology is simple, efficient, and specific, which makes gene editing of T cells in vitro gradually possible from possible.

references:

1.Topalian SL, Drake CG, Pardoll DM. (2015) Immune checkpoint blockade: a common denominator approach therapy. Cancer Cell. 27 (4): 450-461

2. Mustelin T, Vang T, Bottini N. (2005) Protein tyrosine phosphatises and the immune response. Nat Rev Immunol. 5 (1): 43-57.

Summary of the invention

The purpose of the present invention is to overcome the shortcomings of the prior art and provide an SHP-1 knockout T cell and a method for constructing the same.

Another object of the present invention is to provide a SHP-1 knockout CAR-T cell and a method for constructing the same.

The technical solutions adopted by the present invention are:

The T cells of tumor cells are efficiently killed, and the SHP-1 gene of T cells is endogenously silenced.

As a further improvement of the above T cells, the SHP-1 gene of T cells or its regulatory gene is at least partially knocked out.

As a further improvement of the aforementioned T cells, the third exon of the SHP-1 gene of the T cells was knocked out.

As a further improvement of the above T cells, the T cells are CAR-T cells.

As a further improvement of the above T cells, CAR-T cells include CD133 CART, CD19 CART, CD20 CART, BMSA CART, MSLN CART, EGFRVIII CART, Her2 CART, GD2 CART, CEA CART.

The method for constructing the T cell includes introducing into the T cell a plasmid DNA expressing Cas9 and sgRNA, a Cas9 protein or a sgRNA-Cas9 protein complex, and knocking out at least a part of the sequence of SHP-1 or at least a part of its expression control sequence to make SHP-1 Gene endogenous silencing.

As a further improvement of the above T-cell construction method, the sgRNA sequence targets the third exon of the SHP-1 gene.

As a further improvement of the above T cell construction method, CAR-T cells include CD19 CART, CD20 CART, BMSACART, MSLN CART, EGFRVIIICART, Her2CART, GD2CART, CEACART.

A method for treating a tumor includes the following steps:

1) Isolate T cells from tumor patients;

2) CAR modification of T cells and endogenous silencing of SHP-1 gene in T cells;

3) Transfuse the transformed T cells into the patient.

As a further improvement of the above method, the SHP-1 gene of T cells or its regulatory gene is at least partially knocked out.

As a further improvement of the above method, the third exon of the SHP-1 gene of T cells was knocked out.

As a further improvement of the above method, the CAR modification is selected from the group consisting of CD133, CD19, CD20, BMSA, MSLN, EGFRVIII, Her2, GD2, and CEA modifications.

The beneficial effects of the present invention are:

The invention breaks through the limitations of the prior art, creatively blocks SHP-1 in T cells endogenously, and effectively improves the killing effect of T cells on tumors. In particular, CAR-T cells, such as CD133 CAR-T, CD19 CAR T, CD20 CAR T, BMSA CAR T, MSLN CAR T, EGFRVIII CAR T, Her2 CAR T, GD2 CAR T, CEA SHP in CAR T cells -1 knockout can effectively improve the ability of CAR-T cells to kill tumor cells and provide a new target for tumor immunotherapy.

By electroporation of plasmid DNA, Cas9 and sgRNA-expressing plasmids were transferred to T cells to achieve efficient knockout of the SHP-1 gene. Compared with the previously reported use of Cas9 protein or Cas9 mRNA and electrotransduction of sgRNA transcribed in vitro , Saving time and cost of preparation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a structure of a third-generation CAR used in the present invention, including a CSF2RA chimeric receptor signal peptide, an extracellular antigen-binding region (scFv), a c-Myc tag peptide, a CD8 hinge region, and an intracellular signaling region;

FIG. 2 shows the knockout of the SHP-1 gene in T cells after T cells were transfected by the plasmid expressing Cas9 and sgRNA in Example 1 of the present invention; FIG.

3 is the result of overexpression of CD133 CAR and knockout of the SHP-1 gene in T cells by plasmid electrotransformation in the present invention, and the expression of the CAR gene and the knockout of the SHP-1 gene in the T cells were detected, respectively;

Figure 4 is a comparison of the efficiency of different sgRNAs;

FIG. 5 is a detection of the ability to kill tumor cells and detection of cytokine secretion by SHP-1 knockout CD133 CAR-T cells prepared in Example 3 of the present invention; FIG.

Figure 6 shows the results of the safety experiment of SHP-1 gene knockout in vitro mediated by CRISPR / Cas9.

detailed description

Gene silencing refers to a decrease in the expression level of a gene or a lack of expression. Endogenous silencing means that the expression of the gene itself is reduced or is not expressed.

As a further improvement of the above T cells, the SHP-1 gene of T cells or its regulatory gene is at least partially knocked out. So that SHP-1 cannot be expressed normally, or active SHP-1 cannot be obtained after expression.

As a further improvement of the aforementioned T cells, the third exon of the SHP-1 gene of the T cells was knocked out. Experimental data show that knockout of this exon has higher efficiency, better targeting, and correspondingly better safety.

CAR-T cells have specific lethality to specific cells. As a further improvement of the above T cells, the T cells are CAR-T cells.

As a further improvement of the above T cells, CAR-T cells include but are not limited to CD133 CAR T, CD19 CAR T, CD20 CAR T, BMSA CAR T, MSLN CAR T, EGFRVIII CAR T, Her2 CAR T, GD2 CAR T, CEA CAR T etc.

The technical solutions of the present invention will be further exemplified below in combination with examples and experiments. Knockout of other CAR T cells can be performed with reference to the method described below.

Example 1

Knockout of SHP-1 gene in T cells by Cas9 protein and sgRNA electroporation

1) Experimental PBMCs need to be activated by human anti-CD3 / CD28 and Dynabeads, and cultured in AIM-V medium containing 10% FBS and 300U / ml IL-2;

2) After two days, remove Dynabeads and perform electric transfer:

3) Take 4μg of sgRNA transcribed in vitro and mix with 16μg Cas9 protein, and incubate at room temperature for 20 minutes;

4) Add 82 μl electroporation buffer (Lonza, V4XP-3024), 18 μl supplementation 1 to the above system, and mix well;

5) Take 1 million PBMC cells, wash them twice with PBS, resuspend them in the above system, transfer them to an electric rotor, and use the Lonza-4D electrorotator EO-115 program to electrotransform;

6) Immediately after the electroporation, transfer the cells to a pre-warmed medium for cultivation.

7) Count 10 days after electroporation, and perform T7EN1 digestion identification and Sanger sequencing to determine the knockout efficiency.

The results are shown in Figure 1. Figure 1a shows the position of the sgRNA-targeted sequence on the SHP1 gene. T7EN1 digestion showed (Figure 1b) that cleavage of DNA occurred at the sgRNA targeting site. Sanger sequencing results showed (Figure 1c) that the mutation rate of SHP-1 gene was 87.5% (sevenths of eight).

Example 2:

Overexpression of CD133CAR and knockout of SHP-1 gene in T cells were achieved by plasmid electroporation.

1) The CAR-targeted antigen used in this example is CD133, and the structure of the CAR is shown in Figure 2. In this order, it includes the CSF2RA chimeric receptor signal peptide (SEQ ID NO: 1), the extracellular antigen-binding region (scFv, SEQ ID:

2), c-Myc tag peptide (SEQ ID NO: 3), CD8 hinge region, intracellular signaling region (SEQ ID NO:

4) Puromycin, a resistance gene linked to a T2A short peptide. The entire CAR gene is loaded into the PiggyBac plasmid and constitutes PiggyBac-CD133 CAR. The CAR gene initiates gene expression driven by the EF1α promoter. The integration of the CAR gene into the genome is dependent on the transposition of the transposon;

2) 5 μg of PiggyBac-CD133 CAR plasmid, transposase plasmid, Cas9 plasmid (Addgene plasmid # 44758) and SHP-1 gene knockout (sgRNA sequence shown in SEQ ID NO: 5) and Amaxa electroporation kit The electrotransformation reagents in the solution were mixed, and 2 × 10 ⁷ PBMC cells were resuspended in the mixed solution and transferred to an electrorotation cup, and electrotransformed using the Lonza-2B electrorotator U-014 program;

3) Immediately transfer the cells to the pre-warmed medium for cultivation immediately;

4) One day later, PBMCs were activated with human anti-CD3 / CD28 and Dynabeads, and cultured in AIM-V medium containing 10% FBS and 300U / ml IL-2. After 5 days of cell activation, Dynabeads were removed and cultured, and 0.5 μg / ml puromycin drug sieve to enrich CAR positive T cells;

5) On the fourteenth day, knockout efficiency was determined by T7EN1 digestion identification and Sanger sequencing; CAR expression was detected by flow cytometry.

The results are shown in Figure 3. The sgRNA sequence used for SHP-1 knockout is consistent with Figure 1a. T7EN1 digestion showed (Figure 3a) that DNA cleavage occurred at the sgRNA targeting site. Sanger sequencing results showed (Figure 3b) that the mutation rate of SHP-1 gene was 87.5% (sevenths of eight). CAR expression was above 95% in both the knockout group and the non-knockout group (Figure 3c).

In contrast to the sgRNA used above, the inventors also selected sgRNAs targeted at other sites for comparison (Figure 4a and Figure 4b). When using T7EN1 to detect and compare the cleavage effect of different sgRNAs, it can be found that the sg1 used above has the best effect, while the other sgRNA (sg2- 4), the editing efficiency is lower than sg1 (editing efficiency is proportional to the brightness of the smaller bands generated, Figure 4b). The experimental data show that the sequence selected and optimized in the present invention has better targeting and cutting efficiency, and has unexpected effects.

Example 3:

In vitro experiments demonstrated the killing effect of CD133 CAR-T cells knocked out by SHP-1 on tumors.

In vitro killing experiments and cytokine secretion experiments demonstrated the effect of SHP-1 knockout on the antitumor ability of CD133 CAR-T cells.

The method used for cell killing detection is the Luciferase-Luciferin chemiluminescence detection method. Luciferase is overexpressed in the target cells for killing. After T cells are killed, the number of target cells is reduced and the chemiluminescence detection value is reduced. By comparing the chemiluminescence detection values of target cells before and after killing, the killing ability of T cells is estimated. The target cells used in the present invention are U251 cells (U251-CD133-luc) that overexpress CD133 antigen and Luciferase, and the non-target cells are U251 cells (U251-CD133) that overexpress luciferase. The specific operations are as follows:

1) Take 2 × 10 ⁵ target cells or non-target cells and spread them in a 48-well plate;

2) According to the effect target ratio of 1: 1, 4: 1, 16: 1, add un-transduced T cells (T cells), CAR T cells (CD133 CAR), and gene-edited CAR T cells ( SHP-1 KO CD133 CAR), in which the control group was cultured without T cells, with a total volume of 300 μl / well, three replicates were set for each effect target ratio, and the cells were cultured in a 5% CO ₂ cell incubator at 37 ° C for 16- 18 hours; Photomicrograph to detect T cell killing ability;

3) The cultured cells were collected, washed once with PBS, and resuspended in 100 μl of PBS. The cells were transferred to an opaque 96-well plate, 100 μl of D-luciferin at a concentration of 150 μg / ml was added, and the luminal at 560 nm was detected by a microplate reader;

4) Calculate the killing capacity of T cells: Take the well containing only PBS as a blank control, the reading is N0, the reading in the well containing only tumor cells is set to N, and the reading in the experimental group is set to N1, then the cell killing efficiency is 1- ( N1-N0) / (N-N0) x 100%.

Cytokine release experiments were tested using AlphaLISA series kits:

1) Spread 2 × 10 ⁵ target cells U251-CD133-luc in a 48-well plate, and add a certain amount of CAR-T cells or SHP-1 to each well according to a 16: 1 target ratio. In addition to CAR-T cells, each T cell was divided into three duplicate wells, and a total of 300 μl of AIM-V medium was added and cultured in a 37 ° C incubator;

2) After 16 hours, pipette 100 μl of the supernatant, centrifuge at 500 g for 5 minutes, and collect the supernatant for subsequent detection. PerkinElmer's AlphaLISA series kits were used to detect the release of IFN-γ and TNF-α cytokines.

The results are shown in Figure 5. SHP-1 knockout significantly enhanced the ability of CART cells to kill tumor cells (Figures 5a, 5b), and enhanced the ability of CAR T cells to secrete cytokines TNF-α and IFN-γ (Figure 5c).

Example 4:

In vitro cell experiments demonstrated the safety of CRISPR / Cas9-mediated SHP-1 gene knockout.

T cell electroporation uses plasmid electrotransformation, which may cause random insertion of the genome. Simultaneous insertion of Cas9 and sgRNA will cause multiple cuts of the genome and improve off-target effects. Because the Cas9 protein is fused with a GFP fluorescent protein, the integration of Cas9 can be roughly judged by detecting whether there is a GFP signal. The insertion of Cas9 was detected by flow cytometry one day and 24 days after electric rotation (Figure 6a). The results showed that no Cas9 gene was integrated into the genome.

Another safety feature of the CRISPR / Cas9 system for gene editing is the mop effect. Find the highest scoring off-target site based on the sgRNA sequence targeting SHP-1 on www.crispr.mit.edu. Finally, the top ten sites with the highest scores were selected as potential off-target sites (Figure 6b). The first four potential off-target sites were further detected by PCR and T7EN1. T7EN1 digestion analysis (Figure 6c) showed no off-target effect at the corresponding site. Experimental data show that the system of the present invention has good security.

Other CAR T cells can be constructed in a similar way without creative effort.

Claims

T cells that efficiently kill tumor cells are characterized in that the SHP-1 gene of the T cells is endogenously silenced.
The T cell according to claim 1, wherein the SHP-1 gene of the T cell or a regulatory gene thereof is at least partially knocked out.
The T cell according to claim 1, wherein the third exon of the SHP-1 gene of the T cell is knocked out.
The T cell according to any one of claims 1 to 3, wherein the T cell is a CAR-T cell.
The T cell according to claim 4, characterized in that: CAR-T cells include CD133 CAR T, CD19 CAR T, CD20 CAR T, BMSA CAR T, MSLN CAR T, EGFRVIII CAR T, Her2 CAR T, GD2 CAR T , CEA CAR T.
The method for constructing T cells according to claim 1, comprising introducing into the T cells plasmid DNA expressing Cas9 and sgRNA, Cas9 protein or sgRNA-Cas9 protein complex, and knocking out at least part of the sequence of SHP-1 or at least part of the expression control sequence Endogenous silencing of the SHP-1 gene.
The method according to claim 6, characterized in that the sgRNA sequence targets the third exon of the SHP-1 gene.
The method according to claim 6 or 7, characterized in that the CAR-T cells include CD19 CART, CD20 CART, BMSA CART, MSLN CART, EGFRVIIICART, Her2 CART, GD2CART, CEACAR T.
A method for treating a tumor includes the following steps:

1) Isolate T cells from tumor patients;

2) CAR modification of T cells and endogenous silencing of SHP-1 gene in T cells;

3) Transfuse the transformed T cells into the patient.
The method according to claim 9, wherein the SHP-1 gene of T cells or a regulatory gene thereof is at least partially knocked out.
The method according to claim 9, wherein the third exon of the SHP-1 gene of the T cell is knocked out.
The method according to any one of claims 9 to 11, wherein the CAR modification is selected from the group consisting of CD133, CD19, CD20, BMSA, MSLN, EGFRVIII, Her2, GD2, and CEA modifications.