CN111705038B

CN111705038B - Construction method and application of cell strain capable of stably expressing human PDL1/CD73 protein

Info

Publication number: CN111705038B
Application number: CN202010853887.4A
Authority: CN
Inventors: 赵静; 琚存祥; 鞠超
Original assignee: Gempharmatech Co ltd
Current assignee: Jiangsu Jicui Yaokang Biotechnology Co., Ltd
Priority date: 2020-08-24
Filing date: 2020-08-24
Publication date: 2020-11-03
Anticipated expiration: 2040-08-24
Also published as: CN111705038A

Abstract

The invention provides a construction method of a cell line for stably expressing double-source PDL1/CD73 protein, which is characterized in that target genes of CD73 and PD-L1 are humanized on a mouse-source tumor cell, and human hCD73 and hPD-L1 genes on the mouse-source tumor cell are used for substituting mouse mCD73 and mCD-L1 for expression, so that a cell line specially used for evaluating the drug effect of an antibody humanized drug in a well-immunized mouse is constructed, and the cell line can be used as a reliable method for directly evaluating the pre-clinical drug effect experiment of the humanized antibody drug.

Description

Construction method and application of cell strain capable of stably expressing human PDL1/CD73 protein

Technical Field

The invention belongs to the field of animal genetic engineering and genetic modification, and particularly relates to a construction method of a stably expressed double-source PDL1/CD73 protein cell strain and application of the cell strain in biomedicine.

Background

The use of immune checkpoint therapies targeting CTLA-4 and PD-1/PD-L1 has revolutionized the treatment methods and progress of solid tumors. However, the clinical efficacy of immune checkpoint therapy still presents problems of low patient positive response rate, and unclear mechanisms of tumor-specific targeting of immune checkpoints between different tumors. To further increase the positive rate of PD1/PDL1 drugs and to expand tumor indications, clinical trials of multiple combined immune checkpoint strategies are being conducted in large numbers.

Tumor cells may evade anti-tumor immune responses by a variety of mechanisms, such as induction and recruitment of various suppressor immune cells, secretion of immunosuppressive cytokines, and production of immunosuppressive metabolites. Various immunosuppressive cells such as regulatory T cells (tregs), myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), non-natural killer T cells (NKTs), and the like, are accumulated in the tumor microenvironment to help tumor escape by suppressing anti-tumor immune responses. Among the various immunomodulatory factors in the tumor microenvironment, adenosine (adenosine) is an ATP-derived nucleoside, an important factor that helps tumor escape. Adenosine metabolic signaling pathway is one of the key signaling pathways in promoting tumorigenesis and progression, and activation and inhibition of the inflammatory and hypoxic states of the tumor microenvironment, which are mainly regulated by adenosine-producing enzymes, enhance the expression of adenosine-producing enzymes, including CD 73. CD73 hydrolyzes adenosine phosphate (AMP) to adenosine by its own enzymatic activity. There have been several studies to date that CD73 is highly expressed in a variety of solid tumors. Meanwhile, in vitro experimental data show that the high expression of the CD73 can promote the proliferation and invasion of various tumor cells.

It has been found that tumor Treg cells undergo apoptosis, and such apoptotic Treg cells abrogate PDL1 inhibitor-mediated anti-tumor T cell immunity. Experimental results indicate that adenosine, rather than typical suppressors such as PD-L1, CTLA-4, TGF-TGF, IL-35 and IL-10, is a key factor in the immunosuppression of Treg cells mediating apoptosis. Apoptotic Treg cells release and convert large amounts of ATP to adenosine via CD39 and CD73 and mediate immunosuppression via the adenosine and A2A pathways. For tumor patients, the targeted blocking of the activation of the CD73 signaling pathway by the antibody drug will effectively inhibit tumor growth. Blocking the CD73 signaling pathway in combination with immune checkpoint inhibitors such as anti-PDL 1 would be a new promising combined-target therapeutic strategy.

The mouse is the most widely applied and representative preclinical experimental animal at present, and the widely used tumor targeted antibody medicine for research at present is a tumor allograft mouse model, namely a model of human tumor formed after a human tumor cell line or tumor tissue is inoculated to an immunodeficiency mouse. However, the model used for the drug efficacy evaluation system has the defects that the targeting property and the specificity of the drug cannot be embodied and researched, and particularly for the drug which plays the drug efficacy function by regulating the immune system, the model cannot reflect the drug efficacy and the mechanism of the drug playing more truly due to the deletion of B cells, T cells and NK cells.

Since the immune system of mice is highly similar to that of human, the Syngeneic tumor Model (Syngeneic Model) of mice is also widely applied to tumor drug screening and drug effect evaluation. The homologous tumor model is to inoculate mouse tumor cells into homotypic mice, and then screen and evaluate the drug effect after the tumor cells form tumor bodies. However, the model has the problems that the drug efficacy of the antibody targeting the murine antigen or the human-mouse consensus antibody can only be evaluated, and the prediction of the drug specifically targeting the human tumor antigen cannot be well performed. In order to solve the problem in a homologous tumor model, a scheme for carrying out gene humanized transformation on tumor cells in the homologous tumor model is provided, the gene of the mouse-derived tumor cell is replaced by a human-derived gene through a gene editing technology, and the humanized mouse-derived cell is inoculated to a mouse of the same type, so that the verification and screening of the drug effect of a targeted human antigen on a mouse with a sound immune function and the function of exploring the mechanism can be met.

Due to the reliability and stability of the homologous tumor model, the long-term future development trend of the antibody drug can be evaluated. The gene humanized tumor cell is an indispensable part of a homologous tumor mouse model, and is necessary and indispensable for the gene modification of a human antigen targeting a tumor cell.

However, since there are still many differences in immune system functions between mice and human bodies, how to better simulate the immune system function of human bodies on mice to construct animal research models that are more suitable for pre-clinical drug of human immune system is still a big challenge for developers of new drugs and pre-clinical CRO companies.

In order to solve the problems, the invention relates to a method for directly evaluating the drug effect evaluation cell line of an antibody humanized drug in an immune-competent mouse by humanizing CD73 and PD-L1 target genes on a murine tumor cell and substituting human hCD73 and hPD-L1 genes on the murine tumor cell for the expression of mouse mCD73 and mCD-L1, so that the method can be used as a reliable method for directly evaluating the pre-clinical drug effect experiment of the humanized antibody drug.

An optimized specific operation method for preparing a stably-expressed double-source PDL1/CD73 protein cell strain is constructed, the method optimizes the element composition on an expression humanized vector, knocks out the optimal sgRNA of a mouse gene, identifies and screens primers and enzyme digestion schemes used and the like, and ensures the high efficiency of preparing a target cell strain.

Disclosure of Invention

The invention provides a construction method of a cell strain for stably expressing double-source PDL1/CD73 protein, which can directly transfer a target segment into a cell without lentivirus packaging and infection to obtain the cell strain for stably and highly expressing the human protein, and comprises the following steps: the whole CDS sequence of a humanized CD73 gene is randomly inserted into a cell line expressing humanized PDL1, a mouse-derived CD73 is knocked out by adopting a Cas9KO technology, so that stable expression of the human-derived PDL1/CD73 gene replacing the mouse-derived PDL1/CD73 gene in a mouse-derived cell is realized, and the CDS sequence of the human-derived CD73 is shown as SEQ ID NO: 2, and the coded amino acid sequence is shown as SEQ ID NO: 3, respectively.

Preferably, the method specifically comprises the following steps: (1) constructing an expression vector expressing humanized CD73 for insertion of the humanized CD73 gene, the vector comprising the following module CAG promoter-hCD 73-PolyA-Loxp-Puromycin-Loxp; (2) constructing sgRNA for knocking out mouse CD 73; (3) an expression vector expressing humanized CD73 and sgRNA and a Cas9 protein are simultaneously transfected into a cell expressing humanized PDL1, and the cell which highly expresses hPDL1 and hCD73 and hardly expresses mPDL1 and mCD73 is obtained.

Preferably, the vector constructed in step (1) is identified by amplification and electrophoresis using the following primer pairs CAG-tF 1/GPT 000349-tR1 and GPT000349-tF 2/GPT 000349-tR2 (SEQ ID NOS: 4-7):

。

preferably, the vector constructed in step (1) is identified by enzyme digestion and electrophoresis, wherein the enzyme digestion adopts a scheme:

restriction enzyme	Length of fragment
		HindIII	5017\3506
SpeI	4362\2857\1304
		NotI	5823\2700

Preferably, the step (2) is specifically: sgrnas were designed at the 5 'end of exon2 and 3' end of exon3, and cell clones with frame shift mutations were selected. According to the CRISPR Cas9 technology, 2 sgRNAs are respectively designed at the 5 'end of exon2 and the 3' end of exon3, the sgRNAs are used for cell electrotransformation, cells are collected to extract genetic materials, the sgRNAs with high cutting efficiency are judged by PCR and TA cloning, and the sgRNAs with high cutting efficiency are screened out for subsequent cell electrotransformation.

Preferably, the sgRNAs with the highest cleavage efficiency are selected from the group consisting of CD73-E2S1 and CD73-E3S2, and the sequences are as follows:

SEQ ID NO：8: AGTTCTCTCTGTTGGCGGTG

SEQ ID NO：9：CGCTCAGAAAGTTCGAGGTG。

preferably, step (3) is specifically:

(a) reviving and passaging the MC38 tumor cells; (b) performing tumor cell electrotransfer on the fourth day, simultaneously electrotransfering the expression vector for expressing the humanized CD73 and the sgRNA and the Cas9 protein into cells, and performing electrotransfer culture; (c) after 48 hours, Puromycin is added for cell screening, culture solution containing corresponding drug concentration is replaced every day, wild type cells are synchronously screened and used as a control until all the wild type cells of the control die, the cells are continuously cultured to a certain density, monoclonal selection is carried out by using a limiting dilution method, the selected monoclonal cells are expanded to a certain density, a part of the cells are frozen, and a part of the cells are subjected to expression detection of human-derived and mouse-derived CD 73; (d) identifying the deletion of murine CD73 in different cell clones by PCR; (e) the expression levels of hCD73 and hCD73 proteins in different cell clones were analyzed by flow cytometry detection.

Preferably, the primers used for PCR identification in step (d) are as follows:

。

preferably, step (3) further comprises: (f) in vivo cell oncogenicity test.

Preferably, the cell is a tumor cell.

Preferably, the cell line in which the humanized PDL1 is expressed is an hPDL1(Tg) -mPDL1(KO) cell line, and the cell line is obtained by replacing an ectodomain sequence of a murine PDL1 gene with an ectodomain CDS of a human PDL1 gene and knocking out a murine Pdl1 gene by using a CRISPR Cas9 technology.

Preferably, the construction of the cell line of humanized PDL1 specifically comprises:

(1) an expression vector expressing humanized PDL1 was constructed for insertion of the humanized PDL1 gene, the amino acid sequence of the humanized PDL1 was as follows (SEQ ID NO: 1):

MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPATHPPQNRTHWVLLGSILLFLIVVSTVLLFLRKQVRMLDVEKCGVEDTSSKNRNDTQFEET。

the carrier comprises the following components: CAG promoter-hPDL 1 extracellular region CDS-mPDL 1 intracellular region CDS-PolyA-Loxp-Neo-Loxp; the successful construction of the vector is verified by PCR identification and enzyme digestion identification.

(2) Constructing sgRNA for knocking out murine PDL 1; the selected sgrnas with the highest cleavage efficiency were as follows:

Gtatggcagcaacgtcacgatgg（SEQ ID NO：18）；agtgaccaccaacccgtgagtgg（SEQ IDNO：19）；

(3) an expression vector expressing humanized PDL1 and sgRNA and a Cas9 protein are simultaneously transfected into cells to obtain the cells which highly express humanized PDL1 and hardly express murine PDL 1.

The invention also provides application of any one of the methods in evaluating the effectiveness of a medicament targeting the humanized CD73, screening and developing the medicament targeting the humanized CD73, evaluating the anti-tumor effect of the medicament targeting the humanized CD73 in combination with other medicaments, or researching the toxicology of the medicament targeting the humanized CD 73.

Also provided are sgrnas that specifically target the mouse CD73 gene, including primer sequences of CD73-E2S1 and CD73-E3S2, CD73-E2S1, CD73-E3S2 as follows:

SEQ ID NO：7：AGTTCTCTCTGTTGGCGGTG

SEQ ID NO：8：CGCTCAGAAAGTTCGAGGTG。

the invention has the following positive effects:

1. the invention provides a modification method of a humanized tumor cell line, which is simple to operate, does not need lentivirus packaging and infection, and can directly transfer a target segment into a cell to obtain a cell strain with stable and high expression of a humanized protein. The method for obtaining the humanized tumor cell line is not limited by the capacity of a lentiviral vector, and can simultaneously carry out the transfer of a polygene vector to obtain the polygene humanized cell line.

2. The invention establishes a system of a humanized PDL1/CD73 tumor cell line and a humanized mouse, and the system can be used for in vivo efficacy evaluation of a human drug. The medicine evaluation is carried out after the humanized cell line is inoculated in the healthy immune system of the humanized mouse, and the curative effect of the medicine can be reflected more truly. Compared with the method for evaluating the drug effect by using the tumor-bearing mice subjected to immune reconstruction, the humanized mice are inoculated with the same humanized tumor cell line model, so that the drug effect evaluation cost is greatly reduced, and a platform is provided for screening a large number of candidate drugs simultaneously.

3. The invention provides an optimized specific operation method for preparing a cell strain stably expressing double-source PDL1/CD73 protein, wherein the method optimizes the element composition on a vector for expressing humanized CD73, knocks out the optimal sgRNA of mouse-source CD73, identifies and screens primers and enzyme digestion schemes used, and ensures the high efficiency of preparing a target cell strain.

Drawings

FIG. 1 shows the CAG-hCD73PolyA-Loxp-Puro-Loxp vector construction strategy.

FIG. 2 is a gel map of the identification of the GPT000349-huCD73 vector.

FIG. 3 is a photograph of the run of the GPT000349-CAG-huCD73-polyA-Puro assay.

FIG. 4 shows the gel pattern of the enzyme cleavage identification of GPT000349-huCD 73.

FIG. 5 shows the murine CD73 gene knockout strategy.

FIG. 6 shows the identification run chart of MC38-hCD73(Tg) -mCD73 (KO) -hPDL1(Tg) -mPDL1 (KO).

FIG. 7 is a flow chart of different cell lines expressing human and murine PDL1 and CD73 proteins.

FIG. 8 is a test of the tumorigenesis of MC38-hPDL1(Tg) -mPDL1(KO) cells and MC38wt cells in C57BL/6-hPD1/hPDL1/hCD137 mice.

Detailed Description

The present invention is further described in detail below with reference to the attached drawings so that those skilled in the art can implement the invention by referring to the description text.

Example 1: construction of MC38-hCD73(Tg) -mCD73 (KO) -hPDL1(Tg) -mPDL1(KO) cell line

1. Construction of CAG-hCD73PolyA Loxp-Puro-Loxp vector

The construction strategy of the CAG-hCD73PolyA-Loxp-Puro-Loxp vector is shown in figure 1, and the specific construction method is as follows:

1.1, determining a human source fragment replacement region and an inserted human source sequence:

the whole CDS sequence of a humanized CD73 gene is randomly inserted into a cell line expressing humanized PDL1, and meanwhile, a mouse-derived CD73 is knocked out by adopting a Cas9KO technology, so that the stable expression of the human-derived PDL1/CD73 gene replacing the mouse-derived PDL1/CD73 gene in a mouse-derived cell is realized.

The cell line hPDL1(Tg) -mPDL1(KO) expressing humanized PDL1 is obtained by replacing an ectodomain sequence of a murine PDL1 gene with a CDS (human PDL1 gene ectodomain region) and knocking out a murine PDL1 gene by using a CRISPR Cas9 technology, and specifically comprises the following steps:

MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVH GEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNAPYNKINQRILVVDP VTSEHELTCQAEGYPKAEVIWTSSDHQVLSGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAE LVIPELPATHPPQNRTHWVLLGSILLFLIVVSTVLLFLRKQVRMLDVEKCGVEDTSSKNRNDTQFEET

The humanized CD73 CDS sequence is as follows (SEQ ID NO: 2):

ATGTGTCCCCGAGCCGCGCGGGCGCCCGCGACGCTACTCCTCGCCCTGGGCGCGGTGCTGTGGCCTGCGGCTGGCGCCTGGGAGCTTACGATTTTGCACACCAACGACGTGCACAGCCGGCTGGAGCAGACCAGCGAGGACTCCAGCAAGTGCGTCAACGCCAGCCGCTGCATGGGTGGCGTGGCTCGGCTCTTCACCAAGGTTCAGCAGATCCGCCGCGCCGAACCCAACGTGCTGCTGCTGGACGCCGGCGACCAGTACCAGGGCACTATCTGGTTCACCGTGTACAAGGGCGCCGAGGTGGCGCACTTCATGAACGCCCTGCGCTACGATGCCATGGCACTGGGAAATCATGAATTTGATAATGGTGTGGAAGGACTGATCGAGCCACTCCTCAAAGAGGCCAAATTTCCAATTCTGAGTGCAAACATTAAAGCAAAGGGGCCACTAGCATCTCAAATATCAGGACTTTATTTGCCATATAAAGTTCTTCCTGTTGGTGATGAAGTTGTGGGAATCGTTGGATACACTTCCAAAGAAACCCCTTTTCTCTCAAATCCAGGGACAAATTTAGTGTTTGAAGATGAAATCACTGCATTACAACCTGAAGTAGATAAGTTAAAAACTCTAAATGTGAACAAAATTATTGCACTGGGACATTCGGGTTTTGAAATGGATAAACTCATCGCTCAGAAAGTGAGGGGTGTGGACGTCGTGGTGGGAGGACACTCCAACACATTTCTTTACACAGGCAATCCACCTTCCAAAGAGGTGCCTGCTGGGAAGTACCCATTCATAGTCACTTCTGATGATGGGCGGAAGGTTCCTGTAGTCCAGGCCTATGCTTTTGGCAAATACCTAGGCTATCTGAAGATCGAGTTTGATGAAAGAGGAAACGTCATCTCTTCCCATGGAAATCCCATTCTTCTAAACAGCAGCATTCCTGAAGATCCAAGCATAAAAGCAGACATTAACAAATGGAGGATAAAATTGGATAATTATTCTACCCAGGAATTAGGGAAAACAATTGTCTATCTGGATGGCTCCTCTCAATCATGCCGCTTTAGAGAATGCAACATGGGCAACCTGATTTGTGATGCAATGATTAACAACAACCTGAGACACACGGATGAAATGTTCTGGAACCACGTATCCATGTGCATTTTAAATGGAGGTGGTATCCGGTCGCCCATTGATGAACGCAACAATGGCACAATTACCTGGGAGAACCTGGCTGCTGTATTGCCCTTTGGAGGCACATTTGACCTAGTCCAGTTAAAAGGTTCCACCCTGAAGAAGGCCTTTGAGCATAGCGTGCACCGCTACGGCCAGTCCACTGGAGAGTTCCTGCAGGTGGGCGGAATCCATGTGGTGTATGATCTTTCCCGAAAACCTGGAGACAGAGTAGTCAAATTAGATGTTCTTTGCACCAAGTGTCGAGTGCCCAGTTATGACCCTCTCAAAATGGACGAGGTATATAAGGTGATCCTCCCAAACTTCCTGGCCAATGGTGGAGATGGGTTCCAGATGATAAAAGATGAATTATTAAGACATGACTCTGGTGACCAAGATATCAACGTGGTTTCTACATATATCTCCAAAATGAAAGTAATTTATCCAGCAGTTGAAGGTCGGATCAAGTTTTCCACAGGAAGTCACTGCCATGGAAGCTTTTCTTTAATATTTCTTTCACTTTGGGCAGTGATCTTTGTTTTATACCAATAG

the amino acid sequence encoded by the CDS of human CD73 is as follows (SEQ ID NO: 3):

MCPRAARAPATLLLALGAVLWPAAGAWELTILHTNDVHSRLEQTSEDSSKCVNASRCMGGVARLFTKVQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNALRYDAMALGNHEFDNGVEGLIEPLLKEAKFPILSANIKAKGPLASQISGLYLPYKVLPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHTDEMFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKFSTGSHCHGSFSLIFLSLWAVIFVLYQ

1.2 construction of the vector CAG-hCD73-PolyA-Loxp-Puro-Loxp

1) V52# MouseH11-CAG-pMD18T (2713 bp, 1783 bp) was digested with XhoI, and a 1783bp fragment was recovered and named GPT000349-huCD 73. As shown in fig. 2. One well represents a reaction system for enzyme digestion, and in order to ensure the concentration of the recovered fragment, two-tube enzyme digestion reaction system is carried out.

2) GPT000349-huCD73 was amplified by high fidelity enzymatic PCR.

3) Treating by using a V52#, PMD18T-CAG-PURO and ASC1 linear enzyme digestion carrier CIAP for later use;

4) the SLIC ligated amplified huCD73 onto the linearized vector recovered in step 3, coated with AMP plate, and the successfully ligated vector was named GPT000349-CAG-huCD73-polyA-Puro TG. The electrophoresis chart of the amplified product is shown in FIG. 3, and it can be seen that the expected 943bp product is generated by the amplification of the CAG-tF 1/GPT 000349-tR1 primer pair, and the expected 1311bp product is generated by the amplification of the GPT000349-tF 2/GPT 000349-tR2 primer pair. Each well represents the plasmid identification of a single colony picked on an AMP plate.

TABLE 1 GPT000349-CAG-huCD73-polyA-Puro TG vector identification primers

。

The restriction enzymes used for the identification and the length of the fragments after cleavage are shown in Table 2. The gel pattern of the enzyme-cleaved and identified GPT000349-huCD73 is shown in FIG. 4. The enzyme digestion identification result shows that the GPT000349-CAG-huCD73-polyA-Puro TG vector is successfully constructed.

Table 2 GPT000349-CAG-huCD73-polyA-Puro TG enzyme digestion identification scheme

。

2. Construction of sgRNA for murine CD73 knockout

A schematic representation of the murine CD73 knock-out strategy is shown in FIG. 5.

2.1 analysis of the CD73 gene revealed that no frameshift occurred after individual knockout of each exon. In order to improve the knockout efficiency and avoid excision of the CDS sequence of the human CD73, sgRNAs are designed at the 5 'end of exon2 and the 3' end of exon3, and cell clones with frame shift mutation are screened. According to CRISPR Cas9 technology, 2 sgrnas were designed at the 5 'end of exon2 and 3' end of exon3, respectively.

2.2 sgRNA transcription preparation method: PCR is carried out by taking PrimerStar or PrimerStarMax system and sgRNA-F, sgRNA-R as primers and puc57-sgRNA plasmid with correct sequencing as a template, and a PCR product is purified to prepare a template for sgRNA transcription. Transcription of sgRNA was performed using T7-ShortScript in vitro transcription kit (AM 1354).

2.3 cells were electroporated using the sgrnas of table 3, and cells were collected to extract genetic material, and the efficiency of sgRNA cleavage was judged by PCR and TA cloning. According to the result, the sgRNA with higher cutting efficiency is screened out: CD73-E2S1 (SEQ ID NO: 8 AGTTCTCTCTGTTGGCGGTG) and CD73-E3S2 (SEQ ID NO: 9: CGCTCAGAAAGTTCGAGGTG) were used for subsequent MC38 cell electroporation.

TABLE 3 sgRNA sequences for knock-out of murine CD73

。

3. The cells were electroporated to obtain the MC38-hCD73(Tg) -mCD73 (KO) -hPDL1(Tg) -mPDL1(KO) cell line.

3.1 the MC38 tumor cells were revived, plated onto 10cm dishes, changed daily and passaged every other day.

3.2 tumor cells were electroporated on day four, GPT000349-CAG-huCD73-polyA-Puro vector and sgRNA together with Cas9 protein were electroporated into the cells simultaneously. After completing the electrotransformation, the cells were placed in a carbon dioxide incubator, incubated at 37 ℃ with 5% CO2, and the medium was changed every day.

3.3 cells were cultured for 48h, followed by cell selection by addition of Puromycin (200 ul/ml). Changing culture solution containing corresponding drug concentration every day, synchronously sieving wild type cells as control until all the wild type cells die, continuously culturing the cells to a certain density, and performing monoclonal selection by using a limiting dilution method. And (3) expanding the selected monoclonal cells to a certain density, freezing and storing a part of cells, and detecting the expression of human-derived and murine CD73 in a part of cells.

3.4 identification of murine CD73 deletions by PCR in different cell clones.

And extracting corresponding cytogenetic materials from 13 selected monoclonal cell strains (G7, 2G7, H7, D11, F7, E11, 2G7, F11, F10, G11, C11, 2C11 and 2F 9), performing PCR amplification, gel recovery and sequencing, and judging the deletion of the mouse-derived CD73 and the transgenosis of the human-derived CD 73. The results are as follows:

1) the genotypes of clones 2G7, H7, E11, 2G7, F11, C11, 2C11 and 2F9 are transferred into the genotype of hCD 73;

2) clones H7, G11 and C11 are knockout mCD73 genotypes;

based on the above PCR and running gel identification results, we selected H7 and C11 cell clones for subsequent validation and study.

Amplification primers used to identify MC38-hCD73(Tg) -mCD73 (KO) -hPDL1(Tg) -mPDL1(KO) are shown in Table 4. The electrophoretogram of the amplification product is shown in FIG. 6. P is positive control, WT is MC38 wild type, N is negative blank control, M is DNA Marker, and the number represents monoclonal cell. And if the amplification band is consistent with the positive control band in size, the construction is considered to be successful.

Table 4 MC38-hCD73(Tg) -mCD73 (KO) -hPDL1(Tg) -mPDL1(KO) identify amplification primers

。

3.5 analysis of hCD73 and hCD73 protein expression levels in different cell clones by flow cytometry detection. The results show that:

(1) MC38WT cells could only detect mPDL1 and mCD73, where mPDL1 > 99%, mCD73 > 9%;

(2) the expression ratio of mPDL1 and mCD73 of cell strains MC38-C11 and MC38-H7 is less than 1 percent, and the expression ratio of hPDL1 and hCD73 is more than 99 percent.

In combination, the cell strains MC38-C11 and MC38-H7 highly express hPDL1 and hCD73 and hardly express mPDL1 and mCD73, and can be selected for in vitro amplification and in vivo nodulation test.

Table 5 different cell lines express the results of human and murine PDL1 and CD73 protein detection.

。

EXAMPLE 2 in vivo cell neoplasia assay

We used the autonomously developed C57BL/6/hPD1/hPDL1/hCD73 humanized mice (6-8 week old female mice) for in vivo tumorigenicity testing of the humanized cell lines.

The detection method of the tumor-bearing and tumor comprises the following steps:

1) and (3) cell recovery: the desired cells were removed from the liquid nitrogen tank, rapidly thawed in a 37 ℃ water bath, and plated on 15cm petri dishes for culture.

2) Passage: MC38 are adherent cells, usually requiring passage every 2-3 days.

3) Mice were randomly divided into 4 groups and were inoculated with either log phase MC38WT cells or different doses of MC38-C11, MC38-H7 cells.

4) Observation and measurement: mice were observed daily for growth and weighed 2 times per week. After confirming that the mouse has the tumor (the tumor size is 80-100 mm)³Grouping) the tumor sizes are detected.

5) An euthanasia end experiment was performed when the tumor volume of a single mouse reached 3000mm3 after inoculation.

The results show that:

1) the same number of MC38-hPDL1(Tg) -mPDL1(KO) -hCD73(Tg) -mCD73 (KO) -C11, MC38-hPDL1(Tg) -mPDL1(KO) -hCD73(Tg) -mCD73 (KO) -H7 cells are inoculated, and the tumor formation speed in vivo is approximate;

2) inoculating the same number of MC38-hPDL1(Tg) -mPDL1(KO) and MC38-hCD73(Tg) -mCD73 (KO) cells, and ensuring that the in vivo tumor formation speed is close;

3) inoculating the same amount of MC38-hPDL1(Tg) -mPDL1(KO), MC38-hCD73(Tg) -mCD73 (KO), MC38-hPDL1(Tg) -mPDL1(KO) -hCD73(Tg) -mCD73 (KO) -C11, MC38-hPDL1(Tg) -mPDL1(KO) -hCD73(Tg) -mCD73 (KO) -H7, wherein the rate of the PDL1 and CD73 double humanized MC38 cells in the in vivo tumorigenesis of a cell mouse is obviously higher than that of single humanized cells PDL1 and CD 73;

4) mice were inoculated with humanized tumor cells and had no effect on body weight changes.

TABLE 6 nodulation test protocol and results

。

While embodiments of the invention have been disclosed above, it is not intended to be limited to the uses set forth in the specification and examples. It can be applied to all kinds of fields suitable for the present invention. Additional modifications will readily occur to those skilled in the art. It is therefore intended that the invention not be limited to the exact details and illustrations described and illustrated herein, but fall within the scope of the appended claims and equivalents thereof.

Sequence listing

<110> Jiangsu Jiejiaokang Biotech limited

<120> construction method for stably expressing human PDL1/CD73 protein cell strain and application thereof

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atgtgtcccc gagccgcgcg ggcgcccgcg acgctactcc tcgccctggg cgcggtgctg 60

tggcctgcgg ctggcgcctg ggagcttacg attttgcaca ccaacgacgt gcacagccgg 120

ctggagcaga ccagcgagga ctccagcaag tgcgtcaacg ccagccgctg catgggtggc 180

gtggctcggc tcttcaccaa ggttcagcag atccgccgcg ccgaacccaa cgtgctgctg 240

ctggacgccg gcgaccagta ccagggcact atctggttca ccgtgtacaa gggcgccgag 300

gtggcgcact tcatgaacgc cctgcgctac gatgccatgg cactgggaaa tcatgaattt 360

gataatggtg tggaaggact gatcgagcca ctcctcaaag aggccaaatt tccaattctg 420

agtgcaaaca ttaaagcaaa ggggccacta gcatctcaaa tatcaggact ttatttgcca 480

tataaagttc ttcctgttgg tgatgaagtt gtgggaatcg ttggatacac ttccaaagaa 540

accccttttc tctcaaatcc agggacaaat ttagtgtttg aagatgaaat cactgcatta 600

caacctgaag tagataagtt aaaaactcta aatgtgaaca aaattattgc actgggacat 660

tcgggttttg aaatggataa actcatcgct cagaaagtga ggggtgtgga cgtcgtggtg 720

ggaggacact ccaacacatt tctttacaca ggcaatccac cttccaaaga ggtgcctgct 780

gggaagtacc cattcatagt cacttctgat gatgggcgga aggttcctgt agtccaggcc 840

tatgcttttg gcaaatacct aggctatctg aagatcgagt ttgatgaaag aggaaacgtc 900

atctcttccc atggaaatcc cattcttcta aacagcagca ttcctgaaga tccaagcata 960

aaagcagaca ttaacaaatg gaggataaaa ttggataatt attctaccca ggaattaggg 1020

aaaacaattg tctatctgga tggctcctct caatcatgcc gctttagaga atgcaacatg 1080

ggcaacctga tttgtgatgc aatgattaac aacaacctga gacacacgga tgaaatgttc 1140

tggaaccacg tatccatgtg cattttaaat ggaggtggta tccggtcgcc cattgatgaa 1200

cgcaacaatg gcacaattac ctgggagaac ctggctgctg tattgccctt tggaggcaca 1260

tttgacctag tccagttaaa aggttccacc ctgaagaagg cctttgagca tagcgtgcac 1320

cgctacggcc agtccactgg agagttcctg caggtgggcg gaatccatgt ggtgtatgat 1380

ctttcccgaa aacctggaga cagagtagtc aaattagatg ttctttgcac caagtgtcga 1440

gtgcccagtt atgaccctct caaaatggac gaggtatata aggtgatcct cccaaacttc 1500

ctggccaatg gtggagatgg gttccagatg ataaaagatg aattattaag acatgactct 1560

ggtgaccaag atatcaacgt ggtttctaca tatatctcca aaatgaaagt aatttatcca 1620

gcagttgaag gtcggatcaa gttttccaca ggaagtcact gccatggaag cttttcttta 1680

atatttcttt cactttgggc agtgatcttt gttttatacc aatag 1725

<210>2

<211>574

<212>PRT

<213>Homo sapiens

<400>2

Met Cys Pro Arg Ala Ala Arg Ala Pro Ala Thr Leu Leu Leu Ala Leu

1 5 10 15

Gly Ala Val Leu Trp Pro Ala Ala Gly Ala Trp Glu Leu Thr Ile Leu

20 25 30

His Thr Asn Asp Val His Ser Arg Leu Glu Gln Thr Ser Glu Asp Ser

35 40 45

Ser Lys Cys Val Asn Ala Ser Arg Cys Met Gly Gly Val Ala Arg Leu

50 55 60

Phe Thr Lys Val Gln Gln Ile Arg Arg Ala Glu Pro Asn Val Leu Leu

65 70 75 80

Leu Asp Ala Gly Asp Gln Tyr Gln Gly Thr Ile Trp Phe Thr Val Tyr

85 90 95

Lys Gly Ala Glu Val Ala His Phe Met Asn Ala Leu Arg Tyr Asp Ala

100 105 110

Met Ala Leu Gly Asn His Glu Phe Asp Asn Gly Val Glu Gly Leu Ile

115 120 125

Glu Pro Leu Leu Lys Glu Ala Lys Phe Pro Ile Leu Ser Ala Asn Ile

130 135 140

Lys Ala Lys Gly Pro Leu Ala Ser Gln Ile Ser Gly Leu Tyr Leu Pro

145 150 155 160

Tyr Lys Val Leu Pro Val Gly Asp Glu Val Val Gly Ile Val Gly Tyr

165 170 175

Thr Ser Lys Glu Thr Pro Phe Leu Ser Asn Pro Gly Thr Asn Leu Val

180 185 190

Phe Glu Asp Glu Ile Thr Ala Leu Gln Pro Glu Val Asp Lys Leu Lys

195 200 205

Thr Leu Asn Val Asn Lys Ile Ile Ala Leu Gly His Ser Gly Phe Glu

210 215 220

Met Asp Lys Leu Ile Ala Gln Lys Val Arg Gly Val Asp Val Val Val

225 230 235 240

Gly Gly His Ser Asn Thr Phe Leu Tyr Thr Gly Asn Pro Pro Ser Lys

245 250 255

Glu Val Pro Ala Gly Lys Tyr Pro Phe Ile Val Thr Ser Asp Asp Gly

260 265 270

Arg Lys Val Pro Val Val Gln Ala Tyr Ala Phe Gly Lys Tyr Leu Gly

275 280 285

Tyr Leu Lys Ile Glu Phe Asp Glu Arg Gly Asn Val Ile Ser Ser His

290 295 300

Gly Asn Pro Ile Leu Leu Asn Ser Ser Ile Pro Glu Asp Pro Ser Ile

305 310 315 320

Lys Ala Asp Ile Asn Lys Trp Arg Ile Lys Leu Asp Asn Tyr Ser Thr

325 330 335

Gln Glu Leu Gly Lys Thr Ile Val Tyr Leu Asp Gly Ser Ser Gln Ser

340 345 350

Cys Arg Phe Arg Glu Cys Asn Met Gly Asn Leu Ile Cys Asp Ala Met

355 360 365

Ile Asn Asn Asn Leu Arg His Thr Asp Glu Met Phe Trp Asn His Val

370 375 380

Ser Met Cys Ile Leu Asn Gly Gly Gly Ile Arg Ser Pro Ile Asp Glu

385 390 395 400

Arg Asn Asn Gly Thr Ile Thr Trp Glu Asn Leu Ala Ala Val Leu Pro

405 410 415

Phe Gly Gly Thr Phe Asp Leu Val Gln Leu Lys Gly Ser Thr Leu Lys

420 425 430

Lys Ala Phe Glu His Ser Val His Arg Tyr Gly Gln Ser Thr Gly Glu

435 440 445

Phe Leu Gln Val Gly Gly Ile His Val Val Tyr Asp Leu Ser Arg Lys

450 455 460

Pro Gly Asp Arg Val Val Lys Leu Asp Val Leu Cys Thr Lys Cys Arg

465 470 475 480

Val Pro Ser Tyr Asp Pro Leu Lys Met Asp Glu Val Tyr Lys Val Ile

485 490 495

Leu Pro Asn Phe Leu Ala Asn Gly Gly Asp Gly Phe Gln Met Ile Lys

500 505 510

Asp Glu Leu Leu Arg His Asp Ser Gly Asp Gln Asp Ile Asn Val Val

515 520 525

Ser Thr Tyr Ile Ser Lys Met Lys Val Ile Tyr Pro Ala Val Glu Gly

530 535 540

Arg Ile Lys Phe Ser Thr Gly Ser His Cys His Gly Ser Phe Ser Leu

545 550 555 560

Ile Phe Leu Ser Leu Trp Ala Val Ile Phe Val Leu Tyr Gln

565 570

<210>3

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

ctagagcctc tgctaaccat gttc 24

<210>4

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

actacaggaa ccttccgccc 20

<210>5

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>5

ctgttggtga tgaagttgtg 20

<210>6

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>6

gatctcagtg gtatttgtga g 21

<210>7

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>7

atagagttct ctctgttggc ggtg 24

<210>8

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>8

aaaccaccgc caacagagag aact 24

<210>9

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>9

atagcgctca gaaagttcga ggtg 24

<210>10

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>10

aaaccacctc gaactttctg agcg 24

<210>11

<211>24

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>11

ctagagcctc tgctaaccat gttc 24

<210>12

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>12

ttgacgcact tgctggagtc 20

<210>13

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>13

aatggtggag atgggttcca g 21

<210>14

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>14

ccaacctttg ttcatggcag 20

<210>15

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>15

atgtggtcag gtgtcataga ggc 23

<210>16

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>16

attcttggaa cgtctcccag c 21

<210>17

<211>23

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>17

tctgctttct aaccgtggat gtc 23

<210>18

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>18

agacacagcc atcgaccatt c 21

Claims

1. A construction method for stably expressing a cell strain of double PDL1/CD73 protein is characterized in that a target fragment is directly transferred into a cell without lentivirus packaging and infection to obtain the cell strain of the stably high-expression human protein, and the construction method comprises the following steps: the method comprises the steps of randomly inserting the whole CDS sequence of a humanized CD73 gene into a cell line expressing humanized PDL1, knocking out mouse-derived CD73 by adopting a Cas9KO technology, and realizing the stable expression of the human-derived PDL1/CD73 gene replacing mouse-derived PDL1/CD73 gene in mouse-derived cells, wherein the CDS sequence of the human-derived CD73 is shown as SEQ ID NO: 2, the coded amino acid sequence is shown as SEQ ID NO: 3 is shown in the specification; the cell line for expressing humanized PDL1 is obtained by replacing an ectodomain sequence of a murine PDL1 gene with a CDS (human PDL1 gene ectodomain), and knocking out a murine Pdl1 gene by using a CRISPR Cas9 technology;

wherein the sgRNA used for the knockout of murine PDL1 is SEQ ID NO: 18 and SEQ ID NO: 19: gtatggcagcaacgtcacgatgg (SEQ ID NO: 18); agtgaccaccaacccgtgagtgg (SEQ ID NO: 19);

sgrnas for murine CD73 knockouts are CD73-E2S1 and CD73-E3S2, the sequences are set forth in SEQ ID NO: 8-9 show:

SEQ ID NO：8: AGTTCTCTCTGTTGGCGGTG

SEQ ID NO：9：CGCTCAGAAAGTTCGAGGTG。

2. the method of claim 1, wherein: the method specifically comprises the following steps: (1) constructing an expression vector expressing humanized CD73 for insertion of the humanized CD73 gene, the vector comprising the following module CAG promoter-hCD 73-PolyA-Loxp-Puromycin-Loxp; (2) constructing sgRNA for knocking out mouse CD 73; (3) the expression vector expressing humanized CD73 and sgRNA as well as Cas9 protein are simultaneously transfected into cells to obtain the cells which highly express hPDL1 and hCD73 and hardly express mPDL1 and mCD 73.

3. The method of claim 2, wherein the vector constructed in step (1) is identified by amplification and electrophoresis using the following primer pairs CAG-tF 1/GPT 000349-tR1 and GPT000349-tF 2/GPT 000349-tR 2:

CAG-tF1：CTAGAGCCTCTGCTAACCATGTTC

GPT000349-tR1：ACTACAGGAACCTTCCGCCC

GPT000349-tF2：CTGTTGGTGATGAAGTTGTG

GPT000349-tR2：GATCTCAGTGGTATTTGTGAG。

4. the method of claim 2, wherein the vector constructed in the step (1) is identified by enzyme digestion and electrophoresis, wherein the enzyme digestion adopts the following scheme: the length of the HindIII fragment after enzyme digestion is 5017\3506bp, the length of the SpeI fragment after enzyme digestion is 4362\2857\1304bp, and the length of the NotI fragment after enzyme digestion is 5823\2700 bp.

5. The method according to claim 2, wherein the step (2) is specifically: designing sgrnas at the 5 'end of exon2 and the 3' end of exon3, screening cell clones with frame shift mutation, respectively designing 2 sgrnas at the 5 'end of exon2 and the 3' end of exon3 according to CRISPR Cas9 technology, performing cell electrotransformation by using the sgrnas, collecting cells to extract genetic materials, judging the sgRNA cleavage efficiency by PCR and TA cloning, and screening the sgrnas with high cleavage efficiency for subsequent cell electrotransformation.

6. The method according to claim 2, wherein the step (3) is specifically:

(a) reviving and passaging the MC38 tumor cells; (b) performing tumor cell electrotransfer on the fourth day, simultaneously electrotransfering the expression vector for expressing the humanized CD73 and the sgRNA and the Cas9 protein into cells, and performing electrotransfer culture; (c) after 48 hours, Puromycin is added for cell screening, culture solution containing corresponding drug concentration is replaced every day, wild type cells of a synchronous drug screen are used as a control until all the wild type cells of the control die, the cells are continuously cultured, a limiting dilution method is used for monoclonal selection, the selected monoclonal cells are amplified, half of the cells are frozen, and the other half of the cells are subjected to expression detection of human-derived and mouse-derived CD 73; (d) identifying the deletion of murine CD73 in different cell clones by PCR; (e) the expression levels of mCD73 and hCD73 proteins in different cell clones were analyzed by flow cytometry detection.

7. The method of claim 6, wherein: the primers used for PCR identification in step (d) are shown below: primer pair 1: CAG-tF1 and GPT000349-hCD73-5tR 1; and (3) primer pair 2: GPT000349-hCD73-3tF1 and Rabbit-PolyA-tR5, primer set 3: GPT000349-mCD73-KO-tF1 and GPT000241-mCD73-KO-tR1, primer set 4: GPT000241-mCD73-wt-tF1 and GPT000241-mCD73-wt-tR1, wherein

CAG-tF1：CTAGAGCCTCTGCTAACCATGTTC（SEQ ID NO：10）

GPT000349-hCD73-5tR1：TTGACGCACTTGCTGGAGTC（SEQ ID NO：11）

GPT000349-hCD73-3tF1：AATGGTGGAGATGGGTTCCAG（SEQ ID NO：12）

Rabbit-PolyA-tR5：CCAACCTTTGTTCATGGCAG（SEQ ID NO：13）

GPT000349-mCD73-KO-tF1：ATGTGGTCAGGTGTCATAGAGGC（SEQ ID NO：14）

GPT000241-mCD73-KO-tR1：ATTCTTGGAACGTCTCCCAGC（SEQ ID NO：15）

GPT000241-mCD73-wt-tF1：TCTGCTTTCTAACCGTGGATGTC（SEQ ID NO：16）

GPT000241-mCD73-wt-tR1：AGACACAGCCATCGACCATTC（SEQ ID NO：17）。

8. The method of claim 6, wherein step (3) further comprises: (f) in vivo cell oncogenicity test.

9. The method of any one of claims 1-8, wherein the cell is a tumor cell.

10. The sgRNA specifically targeting mouse CD73 gene comprises the sequences of CD73-E2S1 and CD73-E3S2, CD73-E2S1 and CD73-E3S2 as follows:

SEQ ID NO：8：AGTTCTCTCTGTTGGCGGTG

SEQ ID NO：9：CGCTCAGAAAGTTCGAGGTG。