CN116179493A - Immune cells knocked out of two immune checkpoint genes, preparation method and application thereof - Google Patents

Abstract

The invention provides an immune cell expressing TGF-β antibody and simultaneously knocking out two immune checkpoint genes, its preparation method and application, and belongs to the technical field of biogenetic engineering. The immune cells of the present invention knock out the PD-1 gene, and at the same time knock out the CTLA-4, LAG-3, TIM-3 or TIGIT gene. The immune cells provided by the present invention not only knock out the immunosuppressive gene, but also express TGF-β antibody. On the one hand, by secreting the combination of TGF-β antibody and TGF-β, the high concentration of TGF-β in the tumor microenvironment can be released Inhibition of effector T cells; on the other hand, the immune cells knocked out of the two immune checkpoint genes can be free from the inhibitory ligands on the surface of tumor cells, so as to ensure that immune cells are not easily affected by the tumor microenvironment after reaching the tumor site. Multiple mechanisms of immunosuppression, thereby enhancing the immune anti-tumor effect.

Description

Immune cells knocked out of two immune checkpoint genes, preparation method and application thereof

This application claims the priority of the Chinese patent application (application number: 202111150622.9) filed on September 29, 2021, the content of which has been incorporated herein.

technical field

The invention belongs to the technical field of biogenetic engineering, and in particular relates to an immune cell knocked out of two immune checkpoint genes, a preparation method and application thereof, and the immune cell can efficiently express TGF-β antibody.

Background technique

Studies have shown that in the early stage of tumor immune response, circulating immune cells T cells can recognize, infiltrate and then eliminate certain cancer cells without damaging normal cells. However, many tumors form an immunosuppressive microenvironment through mechanisms such as high expression of immune checkpoint ligands and secretion of large amounts of inhibitory cytokines, leading to loss of T cell activity and even induction of T cell apoptosis, resulting in tumor immune escape.

Immune checkpoint inhibitor drugs can block this recognition inhibition, allowing immune cells to be reactivated to achieve the purpose of destroying cancer cells. Studies have shown that immune checkpoint inhibitors have shown significant anti-tumor efficacy in clinical trials. For example, CTLA-4 antibodies can specifically relieve the immune suppression of T cells by CTLA-4 and activate T cells to produce IFN-γ and IL. -2 etc. Although a large number of clinical trials of the combination of two immune checkpoint inhibitor antibodies or bispecific antibodies are also being carried out, antibody drugs will show strong side effects, which restricts their development and application.

The cell preparations that directly knock out the relevant immune checkpoints or their combinations on T cells have shown good clinical effects and become a new hot spot in current anti-tumor therapy. PD-1-edited T cell preparations have demonstrated feasibility, safety and preliminary efficacy in patients with advanced non-small cell lung cancer refractory to multiple lines of therapy. The results were published in "Nature Medicine" on April 28, 2020 The full text was published in the above article, laying a solid foundation for anti-tumor therapy in this field.

The transforming growth factor β superfamily (TGF-β superfamily) is involved in the regulation of diverse biological processes (including but not limited to: inhibition of cell growth, tissue homeostasis, extracellular matrix (ECM) remodeling, endothelial-mesenchymal transition (EMT) ), cell migration and invasion and immunomodulation/suppression, and various signaling cascades of mesenchymal-epithelial transition). In association with ECM remodeling, TGF-β signaling can increase fibroblast populations and ECM deposition (eg, collagen). In the immune system, TGF-β can bind to receptors on immune cells, regulate immune cell function and maintain the growth and homeostasis of immune precursor cells; however, with the occurrence and progression of tumors, TGF-β inhibits immune cells Proliferation and activity-reducing effects. In this context, TGF-β may contribute to tumor development through its ability to stimulate angiogenesis, alter the stromal environment, and induce local and systemic immunosuppression.

Although the literature suggests that TGF-β antibody combined with immune checkpoint antibody can more effectively inhibit tumor growth, but the combination of multiple antibodies often produces very large side effects, which is extremely risky for clinical application; The inhibitory effect of TGF-β on T cells can be blocked by TGF-β receptors on TGF-β, but this effect is limited to the knocked-out T cells themselves, and cannot block the suppression of other immune cells in the tumor microenvironment.

How to eliminate the influence of T cells from the tumor microenvironment and realize the complete inhibitory effect on the immune checkpoint inhibitory pathway by means of gene knockout has become a technical problem to be solved urgently in this field.

Contents of the invention

The purpose of the present invention is to solve the above technical problems, and to provide an immune cell expressing TGF-β antibody and simultaneously knocking out two immune checkpoint genes, as well as its preparation method and application. The immune cells provided by the present invention not only knock out the immunosuppressive genes, but also express TGF-β antibody, and activate the immune cells from the knockout of the immunosuppressive point and the release of the tumor microenvironment, which can make the immune cells express better anti-tumor ability.

One of the objectives of the present invention is to provide an immune cell that expresses TGF-β antibody and simultaneously knocks out two kinds of immune checkpoint genes. 3. TIM-3 or TIGIT gene.

Specifically, the sequence of the PD-1 gene is shown in SEQ No.1. The sequences of the CTLA-4, LAG-3, TIM-3 and TIGIT genes are respectively shown in SEQ No.2, SEQ No.3, SEQ No.4 and SEQ No.5.

Commonly used editing systems for knockout include plasmids and RNP. Most of the editing of immune cells in the prior art is knockout or knockin. It is rare to perform two kinds of editing on cells at the same time, or the editing rate is low. The present invention adopts knockout and knockin The simultaneous typing method ensures editing efficiency in the case of simultaneous editing. In addition, most existing patents only edit immune cells from the perspective of immune checkpoints, while ignoring that there are other mechanisms in the microenvironment of solid tumors that can inhibit immune cells.

Therefore, the immune cells expressing TGF-β antibody and simultaneously knocking out two immune checkpoint genes provided by the present invention, on the one hand, release the high concentration of TGF-β in the tumor microenvironment by secreting the combination of TGF-β antibody and TGF-β. β inhibits effector T cells; on the other hand, immune cells knocked out of the two immune checkpoint genes can be free from the inhibitory ligands on the surface of tumor cells, so as to ensure that immune cells are not easily affected by the tumor microenvironment after reaching the tumor site. Immunosuppression by multiple mechanisms in the tumor, thereby enhancing the immune anti-tumor effect.

The second object of the present invention is to provide a sgRNA, which is used to knock out the two immune checkpoint genes in the above-mentioned immune cells, the gene sequence of which is as shown in SEQ No.6 and SEQ No.7 A pair of sgRNA sequences, or a pair of sgRNA sequences as shown in SEQ No.8 and SEQ No.9.

The third object of the present invention is to provide a method for obtaining the above-mentioned sgRNA, which includes the following steps:

(1) Use the website to design two sgRNAs for each of the two immunosuppressive genes to be knocked out;

(2) Electrotransfect HEK293T cells to screen out sgRNA with high transfection efficiency.

The fourth object of the present invention is to provide a method for preparing immune cells as described above, which includes the following steps:

(1) Design the ssDNA sequence according to the cleavage site of the PD-1 gene, and design the left and right homology arms complementary to the cleavage site at the left and right ends of the ssDNA sequence. The length of the left and right homology arms are both 500bp. Knock in the TGF-β antibody sequence at the position of the first exon of the -1 gene;

(2) Prepare electroporation buffer according to a certain ratio, mix equimolar amounts of sgRNA of two immunosuppressive point genes, and mix with Cas9 protein in proportion, and incubate at 25°C for 10 minutes to form RNP complex;

(3) The TGF-β antibody ssDNA solution and the RNP complex were mixed in proportion to prepare an electroporation solution, and the T cell suspension was electroporated to obtain immune cells expressing the TGF-β antibody and simultaneously knocking out two immune checkpoint genes.

Further, the gene sequence of the ssDNA in step (1) is shown in SEQ No.10.

Further, in step (2), after electroporating T cells, add preheated medium to resuspend the cells, add IFN-γ, and place them in a 37° C., 5% CO ₂ incubator for culture.

The fifth object of the present invention is to provide the application of the above-mentioned immune cells in the preparation of drugs for treating tumors.

The present invention provides an immune cell that highly expresses TGF-β antibody and knocks out two immune checkpoint genes with high efficiency. Compared with the existing immune cells, it has the following differences:

1. The present invention simultaneously knocks out the genes expressing two kinds of immune checkpoints in immune cells, and expresses TGF-β antibody, which has a better effect of relieving immunosuppression.

2. The immune checkpoint pathway is the pathway used by most tumors to escape, so this kind of immune cells is not limited to a certain tumor antigen target, retains or even enhances the diversity of antigen recognition of immune cells, and can be used in a wider spectrum field of cancer treatment.

3. The present invention can not only effectively activate T cells to infiltrate into the tumor, but also release the inhibitory ability of TGF-β to other immune cells (such as NK cells, DC cells and monocyte macrophages, etc.) in the tumor microenvironment, regulate The level of TGF-β reaches the normal level of the human body to maintain immune balance and the growth of immune precursor cells, thereby synergizing with T cells to further enhance the anti-tumor ability.

The beneficial effects of the present invention are as follows:

(1) The present invention provides a brand-new gene-edited immune cell, which knocks out two immunosuppressive point genes at the same time, and enhances its anti-tumor effect.

(2) The present invention inserts the gene expressing TGF-β antibody at the cleavage site of the first exon of PD-1, and the TGF-β antibody binds to TGF-β to maintain TGF-β at a normal physiological level, which can Resisting the immunosuppressive ability of T cells and other immune cells in the tumor microenvironment of TGF-β can also continue to maintain the function of T cells and the growth and dynamic balance of immune precursor cells.

(3) The present invention knocks out the genes expressing immune checkpoints in immune cells, which can improve the activity and proliferation of T cells, prolong the survival time of memory T cells, enhance the recognition of T cells to tumor cells, and activate their attack and killing functions , by mobilizing the body's own immune function to achieve anti-tumor effect.

Description of drawings

Figure 1 is a schematic diagram of TGF-β antibody ssDNA design;

Figure 2 shows the CTLA-4 gene editing rate of T cells detected by TA clone;

Fig. 3 is T7 restriction map;

Fig. 4 is the expression level of TGF-β antibody (effect-to-target ratio=10:1);

Figure 5 shows the anti-tumor effect of gene-edited T cells in vitro;

Figure 6 shows the cytokine secretion level of gene-edited T cells (effect-to-target ratio=10:1).

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be described in detail below in conjunction with the examples. It must be pointed out that the following examples are only used to explain and illustrate the present invention, and are not intended to limit the present invention . Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the invention still belong to the protection scope of the present invention.

Example 1

(1) Primary screening of sgRNA

(1) Design of two immunosuppressive point gene sgRNA

Use the CCTop-CRISPR/Cas9target online predictor website to target knockout of two immunosuppressive point genes (two immune suppression point gene combinations include PD-1 gene and one of CTLA-4, LAG-3, TIM-3 or TIGIT genes) . The following scheme takes the combination of PD-1 and CTLA-4 as an example, and designs two sgRNAs for each; the sgRNAs are obtained by chemical synthesis, and the sequences of the two sgRNAs of PD-1 are shown in SEQ No.6 and SEQ No.7. CTLA- The two sgRNA sequences of 4 are shown in SEQ No.8 and SEQ No.9.

Design a pair of in vitro amplification (PCR) primers for CTLA-4 sgRNA and PD-1 sgRNA; anneal the upstream and downstream of the ordered single-stranded sgRNA to form double strands, and the vector plasmid pGL3-U6-PGK-puromycin contains U6 promoter, BsaⅠ-HF Specific enzyme cutting sites, PM and AMP resistance tags are digested by high-fidelity restriction endonuclease BsaⅠ-HF to form cohesive complementary ends, which are ligated with double-stranded sgRNA to form a new plasmid, which is then transformed into DH5α, Paint the plate, pick a single clone for sequencing the next day, select the bacterial solution containing the successfully constructed plasmid to expand the culture and extract the plasmid.

(2) Electroporation of HEK293T cells to screen high-efficiency sgRNA

After digesting and centrifuging the well-growing HEK293T cells, resuspend the cells in DMEM high-glucose medium containing 10% FBS, and cover a 24-well plate at a density of 2× ¹⁰⁵ cells/ml (500 μL cell suspension per well); According to the ratio of plasmid SpCas9:pGL3-U6-sgRNA-PGK-puromycin=2:1 (1 μg in total), add EP tube (solution A), take Lipofectamine20002 μL to another EP tube (solution B), add 50 μL serum-free medium Opti - Dilute and mix in MEM and let stand at room temperature for 5 minutes, add solution B to solution A, let stand at room temperature for 20 minutes after mixing, slowly drop into HEK293T cells (experimental group), control HEK293T cells without adding the plasmid part containing sgRNA, The rest of the conditions remained the same as the experimental group. After 6 hours, replace with fresh complete medium (DMEM+10% FBS+1% double antibody), add PM (final concentration 2 μg/mL) at 24 hours, collect cells to extract genomic DNA at 72 hours, and apply CTLA-4sg1-2F/R and PD-1sg1-2F/R primers amplify the fragments near the corresponding sgRNA, amplify and send for sequencing.

(2) Separation, sorting and activation of T cells

T cells are isolated from fresh whole blood from healthy blood donors. Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using Ficoll-Paque PLUS, and resuspended in RPMI 1640 medium containing 10% FBS and interleukin-2 The RPMI 1640T cell complete medium was cultured in a sterile incubator (37° C., 5% CO ₂ ) at a density of 1×10 ⁶ cells/ml.

(3) Validation of the efficiency of sgRNA in T cells

T cells in good growth state were counted and grouped (2×10 ⁶ cells/part), centrifuged (1200 rpm, 5 min) and the supernatant was discarded. Prepare the electroporation buffer at the ratio of Nucleofector Solution:Supplement=82:18, resuspend T cells with half of the buffer, and gently mix the half according to the ratio of sgRNA (CTLA-4sgRNA or PD-1sgRNA) and Cas9 protein, and incubate at room temperature for ten minutes , forming the RNP complex. Aspirate the T cell suspension, add RNP complex, mix the mixture with T cells (experimental group) and gently transfer it to the electroporation cup, place it in the electroporation instrument for electroporation, add preheated medium to the electroporation cup, control group Cells were not electroporated, and other conditions were kept the same as the experimental group. The cells were resuspended in the preheated medium, cultured in a sterile incubator (37°C, 5% CO ₂ ) for one week, and the cells of the experimental group and the control group were collected respectively. After extracting genomic DNA and performing PCR, a part of the PCR products were digested with T7, and 2% agarose gel electrophoresis was used to detect and analyze the digestion results; a part of the PCR products were cloned by TA, and the transformants were picked for sequencing.

(4) Design of ssDNA

Choose to knock in the TGF-β antibody sequence at the position of the first exon of PD-1, thereby designing a 1446bps ssDNA (as shown in SEQ No.10) according to the PD-1 cleavage site, and in the ssDNA The left and right homology arms complementary to the cleavage site are designed at the left and right ends, and the length of the left and right homology arms is 500bp. The schematic diagram of ssDNA design is shown in Figure 1.

(5) Construction of gene-edited T cells

100 μL of electroporation buffer (experimental group) was prepared according to a certain ratio; electroporation was not performed in the control group, and other conditions remained the same as the experimental group. Mix the sgRNAs of the two genes (PD-1 and CTLA-4) in equimolar amounts, and mix them with the Cas9 protein in proportion, and incubate at 25°C for 10 minutes to form RNP complexes; TGF-β antibody ssDNA solution and RNP complexes Mix in proportion to prepare the final electrotransfer solution for electroporation. Add culture medium after electroporation of T cells Use preheated 2mL culture medium to resuspend cells, add IFN-γ (2000IU/mL) and place in 37°C, 5% CO ₂ incubator for culture. After culturing for one week, appropriate amount of cells were harvested for TA cloning, flow cytometry, and enzyme digestion to detect the editing rate.

Experimental example 1

(1) TA cloning test results and T7 digestion results

Figure 2 shows the results of TA clone detection of CTLA-4 gene editing rate in T cells, and Figure 3 shows the T7 restriction map. TA cloning results showed that the CTLA-4 gene editing rate was 70%; T7 enzyme digestion showed that both CTLA-4 and PD-1 gene editing were knocked out, and the TGF-β antibody gene was inserted at the PD-1 gene position.

(2) Detection of TGF-β antibody expression in gene-edited T cells

The gene-edited T cells were cultured synchronously with the unedited T cells, and the supernatants of the cells were collected at 12, 24, and 48 hours for Elisa assay, and the results are shown in Figure 4.

The experimental results showed that there was no TGF-β antibody expression in the supernatant of unedited T cells, and the expression of TGF-β antibody could be detected in the culture supernatant of gene-edited T cells, and it increased with the increase of culture time. It shows that gene-edited T cells can express and secrete TGF-β antibody, which meets the requirements of T cell transformation.

(3) Detection of the killing effect of gene-edited T cells

Collect the required number of lung cancer cells, gene-edited T cells, and non-edited T cells in the logarithmic growth phase, and wash them twice with PBS; resuspend the washed lung cancer cells in a 15mL centrifuge tube with PBS, and adjust the density. Lung cancer cells were plated in a 6-well plate, 1× ¹⁰ cells/well. After they adhere to the wall, calculate the number of gene-edited T cells and unedited T cells according to the effect-to-target ratio, and spread them into 6-well plates containing target cells for co-culture; add different concentrations of TGF-β factors, and detect Effect of TGF-β on T cell killing. After shaking gently, culture at 37°C for 48h. After 48 hours, the well plate was taken out, 10 μL CCK8 solution was added to each well, the culture plate was incubated in the incubator for 2 hours, and the absorbance at 450 nm was measured with a microplate reader.

The experimental results are shown in Figure 5. The experimental results in Figure 5 show that the death rate of lung cancer cells in the treatment group added with gene-edited T cells was significantly higher than that in the control group, indicating that gene-edited T cells have better anti-tumor effects.

(4) Detecting the secretion of cytokines during the killing process of gene-edited T cells

Collect the required number of lung cancer cells, gene-edited T cells, and non-edited T cells in the logarithmic growth phase, and wash them twice with PBS; resuspend the washed lung cancer cells in a 15mL centrifuge tube with PBS, and adjust the density. Lung cancer cells were plated in a 6-well plate, 1× ¹⁰ cells/well. After they adhered to the wall, the number of gene-edited T cells and unedited T cells was calculated according to the effect-to-target ratio, and they were spread into 6-well plates containing target cells for co-culture. After shaking gently, culture at 37°C for 48h. After 48 hours, the orifice plate was taken out, the suspension was drawn into a 1.5mL centrifuge tube and centrifuged at 2500rpm/min, and the supernatant was drawn for ELISA detection.

The experimental results are shown in Figure 6. It can be seen from Figure 6 that the cytokines IL-6, IL10, and TNF-α were detected in the supernatant, and the levels of IL-6, IL10, and TNF-α detected in the supernatant of the control group were far lower In the experimental group, it shows that gene-edited cells secrete cytokines when they kill tumor cells, thereby enhancing the immune response.

Claims (9)

1. An immune cell expressing TGF-β antibody and simultaneously knocking out two immune checkpoint genes, characterized in that, the immune cell knocks out the PD-1 gene, and also knocks out CTLA-4, LAG-3, TIM-3 or TIGIT gene. 2. The immune cell according to claim 1, wherein the sequence of the PD-1 gene is shown in SEQ No.1. 3. The immune cell according to claim 1, wherein the sequences of the CTLA-4, LAG-3, TIM-3 and TIGIT genes are respectively as SEQ No.2, SEQ No.3, SEQ No.4 , shown in SEQ No.5. 4. A sgRNA, characterized in that, the sgRNA is used to knock out two kinds of immune checkpoint genes in the immune cell as claimed in claim 1, and the gene sequence of the sgRNA comprises such as SEQ No.6 and SEQ No. A pair of sgRNA sequences shown in .7, or a pair of sgRNA sequences shown in SEQ No.8 and SEQ No.9. 5. the method for obtaining sgRNA as claimed in claim 4, is characterized in that, comprises the following steps: (1) Use the website to design two sgRNAs for each of the two immunosuppressive genes to be knocked out; (2) Electrotransfect HEK293T cells to screen out sgRNA with high transfection efficiency. 6. A method for preparing immune cells according to any one of claims 1-3, characterized in that it comprises the following steps: (1) Design the ssDNA sequence according to the cleavage site of the PD-1 gene, and design the left and right homology arms complementary to the cleavage site at the left and right ends of the ssDNA sequence. The length of the left and right homology arms are both 500bp. Knock in the TGF-β antibody sequence at the position of the first exon of the -1 gene; (2) Prepare electroporation buffer according to a certain ratio, mix equimolar amounts of sgRNA of two immunosuppressive point genes, and mix with Cas9 protein in proportion, and incubate at 25°C for 10 minutes to form RNP complex; (3) The TGF-β antibody ssDNA solution and the RNP complex were mixed in proportion to prepare an electroporation solution, and the T cell suspension was electroporated to obtain immune cells expressing the TGF-β antibody and simultaneously knocking out two immune checkpoint genes. 7. The preparation method according to claim 6, characterized in that the gene sequence of the ssDNA in step (1) is as shown in SEQ No.10. 8. The preparation method according to claim 6, characterized in that, in step (2) after electroporating T cells, add preheated medium to resuspend the cells, add IFN-γ and place at 37°C, 5% CO ₂ to incubate cultured in the box. 9. The use of the immune cells according to any one of claims 1-3 in the preparation of tumor therapeutic drugs.

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