WO2020029171A1 - Method for constructing antigen-presenting cell line without endogenous hla gene background, antigen-presenting cell line and use thereof - Google Patents

Abstract

Provided is a method for constructing an antigen-presenting cell line without an endogenous HLA gene background, comprising: using a CRISPR/Cas9 gene editing system to knock out an endogenous HLA-C gene from a C1R antigen-presenting cell of a single HLA type, wherein the C1R antigen-presenting cells comprise genes HLA-A, HLA-B and HLA-C, the HLA-A not expressing, and the HLA-B basically not expressing. Also provided is an antigen-presenting cell line without an endogenous HLA gene background that is obtained by the described method.

Description

Method for constructing antigen-presenting cell line without endogenous HLA gene background, antigen-presenting cell line and use thereof Technical field

The invention relates to the field of immune technology, in particular to the field of antigen-presenting cells, and particularly to a method for constructing an antigen-presenting cell line without an endogenous HLA gene background, an antigen-presenting cell line, and uses thereof.

Background technique

In today's world, the incidence of cancer is increasing year by year, and immunotherapy is considered to be one of the most likely cures for tumors. Tumor-specific immune targets are the key to safe and effective immunotherapy. The surface proteins of tumor cells targeted by traditional antibodies and CAR-T therapy only account for 20-25% of all possible targets for solid tumors, and HLA molecules on the surface of human cells can present all intracellular antigens for T cells to recognize, so the target The epitope presented to the HLA molecule can expand the targets available for immunotherapy by four times that of traditional methods. Therefore, specific typing of HLA antigen presenting cells is extremely important for the study of new antigens, and how to reduce the interference of other HLA molecules has also become an important issue.

Currently, the commonly used antigen presentation tool cells are K562 cells, which are usually directly overexpressed HLA molecules of the required type in K562. The disadvantage of the prior art is that, because K562 cells contain their own HLA molecules, which are obtained by overexpression directly in K562 cells, when used for antigen presentation, there will be background interference and false positive results.

Summary of the invention

The invention solves the problems of high background and false positive of antigen-presenting cells, and provides a tool cell for background-free HLA molecule expression for research related to antigen presentation.

According to a first aspect, an embodiment provides a method for constructing an antigen-presenting cell line without an endogenous HLA gene background, comprising: using a CRISPR / Cas9 gene editing system to type a single C1R antigen-presenting cell for HLA. Endogenous HLA-C gene knockout was performed, in which the C1R antigen-presenting cells contained HLA-A, HLA-B, and HLA-C genes, in which HLA-A was not expressed and HLA-B was not substantially expressed.

Preferably, the endogenous HLA-C gene knockout is achieved by introducing a Cas9-sgRNA protein complex (RNP) into the C1R antigen-presenting cell, wherein the sgRNA includes a recognition sequence GTGAACCTGCGGAAACTGCG (SEQ ID ID NO: 1).

Preferably, the sgRNA is obtained by in vitro transcription from an in vitro transcription template.

Preferably, the above-mentioned in vitro transcription template is obtained by PCR-amplifying a plasmid containing a gRNA backbone, wherein the primers used for PCR amplification include the above-mentioned recognition sequence SEQ ID NO: 1 and a T7 promoter sequence.

Preferably, the above method includes:

(1) An in vitro transcription template of an sgRNA targeted to the HLA-C gene is obtained by PCR amplification, wherein the primers for the above PCR amplification include:

HLA-C0401 gRNA FW:

TAATACGACTCACTATAGTGAACCTGCGGAAACTGCGGTTTTAGAGCTAGAAATAGC (SEQ ID NO: 2); and

gRNA scafford RV: AGCACCGACTCGGTGCCACT (SEQ ID NO: 3);

The template used for the PCR amplification includes a gRNA backbone;

(2) using T7 RNA polymerase to synthesize sgRNA in vitro from the in vitro transcription template obtained in step (1);

(3) The sgRNA and Cas9 protein obtained in step (2) are embedded into a Cas9-sgRNA protein complex (RNP), and the Cas9-sgRNA protein complex is introduced into the C1R antigen-presenting cells, and cultured and sorted An antigen-presenting cell line with a HLA-C gene knockout and no endogenous HLA gene background was obtained.

Preferably, the template used for the PCR amplification in step (1) above is a pMD19-T plasmid containing a gRNA backbone.

Preferably, in the step (3), the Cas9-sgRNA protein complex is introduced into the C1R antigen-presenting cell by means of electrotransformation.

Preferably, in the above step (3), a flow cytometer is used to sort the antigen-presenting cell lines having no endogenous HLA gene background.

According to a second aspect, an embodiment provides an antigen-presenting cell line constructed without the endogenous HLA gene background and constructed according to the method of the first aspect.

Preferably, the HLA-C gene in the above-mentioned antigen-presenting cell line is caused by a frameshift mutation due to insertion or deletion.

According to a third aspect, an embodiment provides the use of an antigen-presenting cell line without the endogenous HLA gene background of the second aspect as a tool cell in antigen research or immunotherapy.

The antigen-presenting cell line without the endogenous HLA gene background obtained by the method of the present invention removes the interference of endogenous HLA molecules in the antigen-presenting cell C1R, and can effectively reduce the background, improve the accuracy, and obtain Trusted results. In addition, on the basis of the antigen-presenting cell line of the present invention, HLA molecules that need to be typed can be overexpressed to quickly obtain cells of interest.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram of a strategy for constructing an antigen-presenting cell line without an endogenous HLA gene background in an embodiment of the present invention; FIG.

FIG. 2 is a schematic diagram of a template plasmid map and an in vitro transcription template of an sgRNA targeted to HLA-C * 0401 molecule obtained by PCR in an example of the present invention. A specific primer PCR is used to synthesize an in vitro transcription template of the HLA-C gene sgRNA. The upstream product contains T7 promoter, which contains a gRNA scaffold downstream;

FIG. 3 is an electrophoresis result diagram of T7E1 detection and editing efficiency after electroporation of Cas9-sgRNA protein complex (RNP) in C1R cells in accordance with an embodiment of the present invention. The lanes from left to right are DNA markers, and the control group without sgRNA (Add only 3 μg of Cas9 protein), and the experimental group (add 3 μg of Cas9 protein + sgRNA 2 μg);

FIG. 4 is a schematic diagram of sequencing results of Sanger sequencing to detect editing efficiency after electroporation of RNP-knocked C1R cells according to an embodiment of the present invention, showing sequencing results of three clones including control, deletion of 8 bases, and deletion of 11 bases. ;

FIG. 5 is a diagram showing the results of various indels generated after editing the HLA molecular sites of C1R cells of different clones in the embodiment of the present invention; FIG.

FIG. 6 is a diagram showing the results of staining with HLA-A, B, and C antibodies after CLA cells of different clones were knocked out of HLA-C in an example of the present invention; FIG.

FIG. 7 is a diagram showing the results of staining with HLA-C antibodies after HLA-C was knocked out from C1R cells of different clones in the example of the present invention.

detailed description

The present invention will be further described in detail below through specific embodiments in combination with the accompanying drawings. In the following embodiments, many details are described so that the present application can be better understood. However, those skilled in the art can effortlessly realize that some of these features can be omitted in different situations or can be replaced by materials and methods.

Please refer to FIG. 1, a strategy for constructing an antigen-presenting cell line without an endogenous HLA gene background in one embodiment of the present invention. A single C1R antigen-presenting cell with HLA typing includes HLA-A, HLA-B and HLA-C Genes, in which HLA-A is not expressed, HLA-B is basically not expressed, that is, HLA-B expression cannot be detected by serology. In the strategy of the present invention, the CRISPR / Cas9 gene editing system is used to knock out HLA-C * 0401 (where * 0401 represents the specific typing of HLA-C class molecules) molecules in C1R cells, and then perform flow sorting. C1R-H-null (not expressed) monoclonal cells were obtained.

The C1R antigen-presenting cells used in the present invention can be obtained from the American Type Culture Collection (ATCC), and the serial number is CRL-1993. At the same time, existing literature (Storkus, WJ, et al. Reversal of natural, killing, susceptibility, and target cells, expressing transfected classes, HLA genes, Proc. Natl. Acad. Sci. USA 86: 2361-2364, 1989.PubMed: 2784569) reported that cell.

The CRISPR / Cas9 gene editing system has a few different variants that work on the same principle. The present invention verifies that the endogenous HLA-C gene knockout is achieved by introducing a Cas9-sgRNA protein complex (RNP) into a C1R antigen-presenting cell. On the basis of the validity of the present invention, those skilled in the art can make some suitable modifications based on the spirit of the present invention, so that different CRISPR / Cas9 gene editing systems can effectively implement the HLA-C gene in C1R antigen presenting cells. knockout.

The embodiment of the present invention verifies a sgRNA (single guide RNA) which is very effective for HLA-C gene knockout, wherein the sgRNA includes a recognition sequence GTGAACCTGCGGAAACTGCG (SEQ ID NO: 1). Therefore, sgRNA containing SEQ ID NO: 1 is preferred in the present invention.

The sgRNA in the embodiment of the present invention can be used to amplify a plasmid containing a gRNA backbone by PCR to obtain an amplified product as an in vitro transcription template, and then transcribe the in vitro transcription template to obtain sgRNA in vitro. As a preferred embodiment, considering that SEQ ID NO: 1 has a very efficient knock-out effect, the primers used in the process of PCR amplification to generate an in vitro transcription template include the above-mentioned recognition sequence SEQ ID ID NO: 1 and take into account the transcription The required promoter also includes a T7 promoter sequence in the primer.

As a most preferred embodiment, the method of the present invention includes the following steps:

(1) An in vitro transcription template of an sgRNA targeted to the HLA-C gene is obtained by PCR amplification, wherein the primers for the above PCR amplification include:

HLA-C0401 gRNA FW:

TAATACGACTCACTATAGTGAACCTGCGGAAACTGCGGTTTTAGAGCTAGAAATAGC (SEQ ID NO: 2); and

gRNA scafford RV: AGCACCGACTCGGTGCCACT (SEQ ID NO: 3);

The template used for the PCR amplification includes a gRNA backbone;

(2) using T7 RNA polymerase to synthesize sgRNA in vitro from the in vitro transcription template obtained in step (1);

(3) The sgRNA and Cas9 protein obtained in step (2) are embedded into a Cas9-sgRNA protein complex (RNP), and the Cas9-sgRNA protein complex is introduced into the C1R antigen-presenting cells, and cultured and sorted An antigen-presenting cell line with a HLA-C gene knockout and no endogenous HLA gene background was obtained.

It should be noted that the template used for PCR amplification in step (1) can be provided by many kinds of template plasmids, such as the commonly used pMD19-T plasmid (Figure 2), which contains the gRNA backbone. Such plasmids also include P1123 plasmid provided by Fenghui Biological Company.

The technical solution of the present invention is described in detail through the following embodiments. It should be understood that the embodiments are merely exemplary and cannot be understood as limiting the protection scope of the present invention.

Examples

1.In vitro transcription template of sgRNA targeting HLA-C * 0401 molecule obtained by PCR

Design and synthesize primer sequences as follows:

HLA-C0401 gRNA FW:

TAATACGACTCACTATAGTGAACCTGCGGAAACTGCGGTTTTAGAGCTAGAAATAGC (SEQ ID NO: 2);

gRNA scafford RV: AGCACCGACTCGGTGCCACT (SEQ ID NO: 3).

The PCR reaction system is shown in Table 1 below:

Table 1

The reaction procedure is shown in Table 2 below:

Table 2

After the PCR reaction, the PCR product is digested and recovered to obtain an in vitro transcription template.

2.In vitro transcription and synthesis of sgRNA

(1) Prepare the reaction mixture shown in the following Table 3 in the EP tube (reagent MMESSAGE, MMACHINE, T7, KIT, Invitrogen, AM1344):

table 3

(2) After mixing the above reaction system, centrifuge immediately and react at 37 ° C for 3 to 4 hours;

(3) Add 1 μL of TUBRO and DNase, mix thoroughly, centrifuge immediately, and react at 37 ° C for 15 minutes;

(4) Terminate the reaction by adding 115 μL Nuclease-free Water and 15 μL Anmonieum Aoleeate;

(5) Add an equal volume of phenol-chloroform (pH 4.5) and shake vigorously, mix well, and centrifuge at 4 ° C, 14000 rpm for 15 min;

(6) Transfer the upper aqueous solution to a new 1.5mL EP tube, add an equal volume of chloroform, shake vigorously, mix well, centrifuge at 14000rpm at 4 ° C, and centrifuge for 15min;

(7) Transfer the upper aqueous solution to a new 1.5mL EP tube, add an equal volume of isopropanol, shake and mix, and let stand at room temperature for 10min or -20 ° C overnight;

(8) Centrifuge at 4 ° C, 14000rpm for 15min, discard the supernatant (carefully suck to prevent RNA precipitation), add 500μL 75% ethanol, and centrifuge at 4 ° C, 14000rpm for 15min;

(9) Discard the supernatant (carefully suck to prevent the RNA precipitation), centrifuge at 14,000 rpm for 3 min at 4 ° C, and blot the supernatant with a small pipette tip containing no RNase free;

(10) Air-dry at room temperature. After the ethanol is evaporated to dryness, 20 μL of RNase free water is added, and it is left at room temperature for 5 minutes, and then placed on ice for 20 minutes to completely dissolve it.

(11) Take 1 μL of the above mRNA solution, dilute it 3 times, and then take 1 μL of the diluted mRNA to detect the OD 260 / 230nm value and OD 260 / 280nm value, take another 1 μL and add to 15.5 μL RNA denaturing buffer, mix, 65 ℃, 20min. After denaturation, quickly place on ice, then agarose gel electrophoresis, combined with OD value, estimate the concentration and degradation degree of synthesized mRNA, or directly perform electrophoresis without denaturation.

(12) Pack the synthesized mRNA into small tubes as required, mark them, and store them in a -80 ° C refrigerator.

3.Electrotransfer Cas9-RNP knocks out H1 molecules of C1R

(1) C1R cells with good growth conditions were cultured in a 24-well plate at 2 × 10 ⁵ cells, and resuspended with 100 μL of Opti-MEM (Invitrogen, 11058-021).

(2) Prepare RNP composites according to the following components in Table 4:

Table 4

After incubating at room temperature for 15min, the RNP complex formed can be stable for 2 hours at room temperature.

(3) Mix the two components of steps (1) and (2), add them to the electric shock cup, and turn them (710V, 30ms, CELETRIX electrorotator);

(4) C1R cells after electroporation were cultured in IMDM medium of 10% FBS at 37 ° C and 5% CO _{2 for} 48 hours.

4. Detection of C1R cell editing efficiency

After 48 hours of electrotransfection of C1R cells, the cells were harvested to extract genomic DNA, and the target fragment was amplified by PCR. T7 nuclease I (T7E1) and sanger sequencing were performed for detection.

(1) T7E1 analysis

PCR of genomic DNA of edited cells was performed using the following primers:

C0401FW: gcttcatcgcagtgggctac (SEQ ID NO: 4);

C0401RV: cgggagatctacgggagatgg (SEQ ID NO: 5).

After the PCR product is recovered by gel, the reaction is performed according to the following components in Table 5:

table 5

Component	content
PCR recovery products	200ng
10XNEBuffer 2	2 μL
DNase / RNase-free water	Make up to 19 μL

The denaturing annealing reaction was performed in a PCR instrument. The reaction conditions are shown in Table 6 below:

Table 6

To 19 μL of the quenched product, 1 μL of T7E1 enzyme (NEB, MO3O2S) was added for digestion. The reaction was carried out at 37 ° C for 15 minutes, and then detected by 1% agarose electrophoresis.

The results are shown in Figure 3. The experimental group was significantly cleaved by T7 nuclease 1 (T7E1 enzyme), proving that the editing knockout was successful.

(2) Sanger sequencing to detect C1R editing efficiency

After genome PCR of C1R cells, the gel recovery products were subjected to Sanger sequencing analysis. Figure 4 shows the control and two edited clone sequencing results, showing the deletion of 8 bases and the deletion of 11 bases. After sequencing, the target gene locus was knocked out.

Figure 5 shows the results of various indels after editing the HLA molecular sites of C1R cells of different clones, indicating that the target gene site has a frameshift mutation and a stop codon was formed in advance, indicating that the HLA-C0401 gene was knocked out. Except for success.

5.Monoclonal screening

C1R cells after electrotransfection were subjected to flow sorting of monoclonals. The steps were as follows:

(1) Take 2 * 10 ⁶ C1R cells after electroporation and place them in three flow tubes;

(2) Wash twice with 1 mL of PBS, remove the supernatant after centrifugation, and resuspend with 200 μL of PBS;

(3) 5 μL HLA-A, B, and C (w6 / 32) (Biolegend, 311402) antibodies were added to one tube, and 5 μL HLA-C (Biolegend, 373302) antibodies were added to one tube, and one tube was left untreated as a control;

(4) Incubate on ice in the dark for half an hour, wash twice with PBS, remove the supernatant, and resuspend in 200 μL PBS;

(5) 5 minutes before the flow cytometry, add 2 μL DAPI (1mg / mL), and mix and run on the machine;

(6) Flow cytometry, remove dead cells, sort HLA-A, B, C and HLA-C double negative cells, one cell per well, and culture;

(7) Cultivate a monoclonal.

The flow cytometry results are shown in Figures 6 and 7. Figure 6 shows the results of staining with HLA-A, B, and C antibodies after HLA-C was knocked out from C1R cells of different clones. clone1) and clone5 (clone5), flow analysis showed that HLA-A, B, and C were negative, proving that HLA-C0401 was successfully knocked out.

Figure 7 shows the results of HLA-C antibody staining of C1R cells of different clones after knocking out HLA-C. Among them, the selected monoclonal 1 (clone 1) and clone 5 (clone 5), and HLA-C was found by flow analysis. It was negative, which further proved that HLA-C0401 was successfully knocked out.

The above uses specific examples to illustrate the present invention, but is only used to help understand the present invention, and is not intended to limit the present invention. For those skilled in the art to which the present invention pertains, according to the idea of the present invention, several simple deductions, deformations, or replacements can also be made.

Claims (10)

1. A method for constructing an antigen-presenting cell line without an endogenous HLA gene background, characterized in that the method comprises: using CRISPR / Cas9 gene editing system to perform endogenous HLA on a single HLA-type C1R antigen-presenting cell -C gene knockout, wherein the C1R antigen presenting cells include HLA-A, HLA-B, and HLA-C genes, wherein HLA-A is not expressed and HLA-B is substantially not expressed.
2. The method for constructing an antigen-presenting cell line without an endogenous HLA gene background according to claim 1, wherein the method is implemented by introducing a Cas9-sgRNA protein complex (RNP) into the C1R antigen-presenting cell. The endogenous HLA-C gene was knocked out, and the sgRNA contained a recognition sequence GTGAACCTGCGGAAACTGCG (SEQ ID NO: 1).
3. The method for constructing an antigen-presenting cell line without an endogenous HLA gene background according to claim 2, wherein the sgRNA is obtained by in vitro transcription from an in vitro transcription template.
4. The method for constructing an antigen-presenting cell line without an endogenous HLA gene background according to claim 3, wherein the in vitro transcription template is obtained by PCR amplification of a plasmid containing a gRNA backbone, wherein PCR amplification is used The primers include the recognition sequence SEQ ID NO: 1 and the T7 promoter sequence.
5. The method for constructing an antigen-presenting cell line with no endogenous HLA gene background according to any one of claims 1-4, wherein the method comprises:

   (1) An in vitro transcription template of an sgRNA targeted to the HLA-C gene is obtained by PCR amplification, wherein the primers for the PCR amplification include:

   HLA-C0401 gRNA FW:

   TAATACGACTCACTATAGTGAACCTGCGGAAACTGCGGTTTTAGAGCTAGAAATAGC (SEQ ID NO: 2); and

   gRNA scafford RV: AGCACCGACTCGGTGCCACT (SEQ ID NO: 3);

   The template used for the PCR amplification includes a gRNA backbone;

   (2) using T7 RNA polymerase to perform in vitro transcription of the in vitro transcription template obtained in step (1) to synthesize sgRNA;

   (3) The sgRNA and Cas9 protein obtained in step (2) are embedded into a Cas9-sgRNA protein complex (RNP), and the Cas9-sgRNA protein complex is introduced into the C1R antigen-presenting cell. The antigen-presenting cell lines from which the HLA-C gene was knocked out and had no endogenous HLA gene background were sorted.
6. The method for constructing an antigen-presenting cell line without an endogenous HLA gene background according to claim 5, wherein the template used for PCR amplification in step (1) is a pMD 19-T plasmid containing a gRNA backbone In the step (3), the Cas9-sgRNA protein complex is introduced into the C1R antigen-presenting cell by means of electrotransformation.
7. The method for constructing an antigen-presenting cell line with no endogenous HLA gene background according to claim 5, wherein in step (3), a flow cytometer is used to sort the antigen-presenting cells without an endogenous HLA gene background. It was a cell line.
8. An antigen-presenting cell line with no endogenous HLA gene background constructed according to the method of any one of claims 1-7.
9. The antigen-presenting cell line according to claim 8, wherein the HLA-C gene in the antigen-presenting cell line is caused by a frameshift mutation due to insertion or deletion.
10. Use of an antigen-presenting cell line without an endogenous HLA gene background according to claim 8 or 9 as a tool cell in antigen research or immunotherapy.

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