CN112251463B

CN112251463B - Construction method of CD73 humanized mouse model

Info

Publication number: CN112251463B
Application number: CN202011062364.4A
Authority: CN
Inventors: 琚存祥; 王韬; 杨笑柳; 于薇薇
Original assignee: Guangdong Yaokang Biotechnology Co ltd
Current assignee: Guangdong Yaokang Biotechnology Co ltd
Priority date: 2020-09-30
Filing date: 2020-09-30
Publication date: 2023-06-06
Anticipated expiration: 2040-09-30
Also published as: CN112251463A

Abstract

The invention discloses a construction method of a CD73 humanized mouse model, which is used for obtaining a CD73 humanized animal model efficiently and successfully by optimizing a targeting vector, sgRNA and verification steps used in the preparation of the model. The model of the invention has significant advantages in screening application of CD73 targeted drugs and other immune check point or small molecule drug combination.

Description

Construction method of CD73 humanized mouse model

Technical Field

The invention relates to the technical field of animal genetic engineering, in particular to a construction method of a CD73 humanized mouse model.

Background

First generation immune checkpoint inhibitors, including PD1, PDL1, CTLA4, etc., have become the mainstay of tumor immunotherapy. Although these immune checkpoints have not been successful in tumor immunotherapy, only a small fraction of patients exhibit a durable response to a single immune checkpoint, even a fraction of patients do not respond to checkpoint inhibitors that are currently on the market. The important aspect of overcoming the drug resistance of solid tumor treatment is to regulate the tumor microenvironment, and the immunosuppression in the microenvironment can obviously improve the tumor immunotherapy effect.

CD73 is an extracellular-5' -nucleotidase, a glycoprotein anchored to the plasma membrane by Glycosyl Phosphatidylinositol (GPI). CD73 is widely distributed on the surface of human tissue cells. CD73 molecule presents high expression in various tumor tissues, not only participates in purine nucleotide metabolism and salvage synthesis, but also is used as an important immune signal negative regulation molecule, and participates in immune escape of tumors by catalyzing the formation of immunosuppressive mediator Adenosine (ADO). Targeting CD73 may have good anti-tumor effects, and the synergy shown by anti-CD 73 in combination with certain immune molecule modulators has also become a recent research hotspot. Thus, the anti-CD 73 targeted therapy is expected to become a novel combined immunotherapy.

Research on pathogenesis of human diseases, screening for effective therapeutic drugs, requires a large number of preclinical trials. Because of ethical limitations in preclinical studies using human cells and tissues directly, animal models are an alternative to human biological studies. Mice are small in size, easy to maintain and operate, short in breeding cycle, similar to human in aspects of genome, physiology and the like, and have correspondingly mature gene modification technology, so that the mice become widely applied mammalian model biological systems. However, since the physiological characteristics of mice are much different from those of humans, experimental results obtained by using animal models are sometimes not applicable to humans. However, by using the method of gene modification or cell and tissue transplantation, the humanized mouse model prepared by placing human genes or cell tissues on the mouse model can simulate the related activities of the human genes or cell tissues to a great extent, and the effectiveness of the mouse model as a model for simulating certain human diseases is greatly improved. This is what we refer to as a humanized mouse model, i.e., a mouse model with human functional genes, human cells or tissues.

The CD73 humanized mouse is characterized in that a mouse with a sound immune system is modified by utilizing a gene modification method, a mouse model capable of interacting with anti-humanized CD73 monoclonal antibody is constructed by replacing a mouse-derived CD73 gene with a humanized gene, and a more economical and easily-obtained animal evaluation system is provided for preclinical anti-humanized CD73 monoclonal antibody screening. We constructed gRNA for mouse CD73 gene and vector carrying human CD73 gene fragment, and replaced murine CD73 with human CD73 gene by CRISPR/Cas9 technology and embryo injection technology. Meanwhile, various elements used in the CRISPR/Cas9 technology, including gRNA and the like, are fully optimized and adjusted, so that the success rate and the accuracy of the humanized CD73 gene animal model prepared by the technology are ensured.

Disclosure of Invention

The invention aims to provide a construction method of a CD73 humanized mouse model, which has the advantage of high-efficiency screening application of combined medication of a CD73 targeting drug and other immune check points or small molecular drugs, solves the problems that pathogenesis research of human diseases, screening of effective therapeutic drugs, a large number of preclinical tests are needed, and preclinical related research is limited in ethical aspects due to direct utilization of human cells and tissues.

In order to achieve the above purpose, the present invention provides the following technical solutions:

the method for constructing the CD73 humanized mouse model is characterized in that a human CD73 coding amino acid sequence is inserted into the rear of the 5'UTR of a mouse CD73 gene, a 3XployA termination sequence is added at the tail end, so that the mouse CD73 protein cannot be expressed, and the human CD73 protein is regulated and controlled by the promoter and the 5' UTR of the mouse CD73 gene, and the method comprises the following steps: (1) Constructing an expression vector for expressing the humanized CD73, inserting the humanized CD73 gene, and connecting a plurality of fragments together by adopting an SLIC mode to construct a targeting vector; (2) determining the sgRNA sequence for model preparation; (3) Injecting an expression vector expressing humanized CD73 and sgRNA together with Cas9 protein into fertilized eggs, and transplanting to pseudopregnant mice, wherein the sequence of the humanized CD73 gene is shown in SEQ ID NO: 2.

Preferably, after inserting the human sequence into the murine 5' UTR, the sgRNA with the lowest off-target rate is selected for the target fragment, and the sequence of the sgRNA is shown as SEQ ID NO: 3.

Preferably, the targeting fragment vector is prepared as follows:

1) Obtaining linearized pl253-DTA linearization by cutting pl253-DTA plasmid with NOTI+SPEI;

2) Amplifying CD73-5arm and CD73-3arm by using a C57BL/6 genome PCR to obtain huCD73-CDS fragments, obtaining 3xployA fragments, and recycling for later use;

3) SLIC ligation of CD73-5arm, CD73-3arm, linearized pl253-DTA, huCD73-CDS and 3xployA was performed using PCR to identify vector integrity;

4) After the final vector was constructed successfully, the band of 3839bp was recovered by PACI/AGEI cleavage, and sequencing was completed for injection.

Preferably, the primers for amplifying the CD73-5arm, CD73-3arm, huCD73-CDS and 3xployA sequences are as follows:

preferably, the method further comprises genotyping pseudopregnant mice, screening positive mice F0 mice successfully inserted with correct human fragments, breeding F0 and B6 background mice to obtain F1, and performing genetic identification on the tail of the F1 generation.

Preferably, the primers used for genotyping are as follows:

preferably, the method further comprises detection of CD73 protein expression, immune system validation and/or validation of anti-tumor efficacy of CD73 inhibitors.

Preferably, wherein the immune system validation step is: selecting thymus or spleen of a main immune organ of a mouse, cutting thymus or spleen of a B6-hCD73 homozygous mouse and a C57BL/6 background mouse, grinding and digesting tissues into single cells, staining extracellular proteins of tissue cells by using a murine T\B\NK surface antibody, washing the cells by using PBS, and detecting the cell numbers of T (CD4+, CD8+), B, NK cells by using flow cytometry.

Preferably, the step of verifying the anti-tumor efficacy of the CD73 inhibitor comprises the following steps: b6 background PD1 and PDL1 double humanized mice and hCD73 humanized mice are bred to obtain B6-hPD1/hPDL1/hCD73 three humanized mice, and the B6-hPD1/hPDL1/hCD73 humanized mice are adopted for drug efficacy evaluation; selecting 6-7wB6-hPD1/hPDL1/hCD73 homozygous mice, subcutaneously inoculating the colon cancer cells MC38-hPDL1 (Tg) -mPDL1 (KO) -hCD73 (Tg) -mCD73 (KO) of the mice, wherein the cell line is tumor cells obtained by knocking out both murine PDL1 and CD73 together with CD73 humanization and PDL1 humanization, and the tumor volume is about 100+/-50 ^mm3 Then, the cells are divided into 4 groups, namely a control group, a PDL1 single drug group, a PDL1 and CD73-1 antibody combined group and a PDL1 and CD73-2 antibody combined group; the dosing frequency was 2 times per week, dosing was continued for 3 weeks, dosing endpoint, mice were sacrificed.

Preferably, wherein the CD73 protein is detected by a flow detection method:

(1) Drawing materials: collecting 100ul to 1.5mL of peripheral blood of a mouse by using an eye socket, lightly stirring to avoid coagulation, transferring the blood into a 10mL centrifuge tube, adding 1mL of 1 XRBC into each tube, uniformly mixing, cracking red for 10min at room temperature and avoiding light, centrifuging for 400g at 8 ℃, removing supernatant, cracking red for 2 times if required, washing for 1 time by using a FACS buffer, and placing on ice for later use;

(2) Closing: according to the experimental requirements, 100uL (about 10 ⁶ Dividing the cells into different flow tubes, adding Fc block (CD 16/32 antibody) and 1ul of CD16/32 antibody (1:100 dilution) into each tube, mixing well, and incubating on ice for 5min;

(3) Antibody incubation: preparing antibody mixed solution (hPD-1, mPD-1, hCD73, mCD73 and CD3 antibodies) according to the number of the sample tubes, adding 50uL of antibody mixed solution into each sample according to the optimal dosage of the antibodies, and uniformly mixing by vortex; adding 0.5ul of antibody into each single-dyeing tube, and uniformly mixing by vortex; incubating for 1h on ice in dark place;

(4) Cleaning: FACS buffer washing, FACS buffer addition, on-press detection, sytox blue addition 5min before loading (final concentration 1:10000 dilution), and dead living cells differentiation.

Preferably, the immune cells are detected by flow-through methods as follows:

drawing materials: cutting spleens of B6-hCD73 homozygous mice and C57BL/6 background mice, weighing, and placing the spleens in a C-shaped tube;

digestion: the spleen was digested with enzyme digestion solution at 37℃for 30min. Stopping digestion of the digested spleen cells by adding 300ul of 0.1M EDTA, filtering 1mL of the spleen cells with a filter membrane to remove undigested tissue blocks, and adding 2mL of FACS buffer to neutralize EDTA in each tube; centrifuging at 8deg.C for 400g for 5min, removing supernatant, adding 3ml of 1×RBC into each tube of spleen, mixing, lysing red blood cells at 8deg.C for 5min at room temperature in dark place, centrifuging at 400deg.C for 5min, removing supernatant, adding 1ml of FACS buffer, resuspending, and filtering; centrifuging at 8deg.C for 5min at 400g, removing supernatant, adding FACS buffer, and re-suspending to adjust cell concentration to 1×10 ⁷ mu.L/mL, split into flow tubes, ready to incubate antibodies;

(1) Closing: according to the experimental requirements, 100uL (about 10 ⁶ Dividing the cells into different flow tubes, adding Fc block (CD 16/32 antibody) and 1ul of CD16/32 antibody (1:100 dilution) into each tube, mixing well, and incubating on ice for 5min;

(2) Antibody incubation: preparing an antibody mixed solution according to the number of the sample tubes: adding antibodies of CD19, CD4, CD8, CD335 and CD3 according to the optimal dosage of the antibodies, adding 50uL of antibody mixed solution into each sample, mixing by vortex, adding 0.5uL of antibody into each single-dyeing tube, mixing by vortex, and incubating for 1h on ice in a dark place;

(3) Cleaning: FACS buffer washing, FACS buffer addition, on-press detection, sytox blue addition 5min before loading (final concentration 1:10000 dilution), and dead living cells differentiation.

Compared with the prior art, the invention has the beneficial effects that:

1. the mouse CD73 protein was humanized to obtain a mouse model that successfully expressed human CD73.

2. The three-target humanized mouse model can be obtained by breeding a mouse model of human CD73 and a PD1/PDL1 mouse, and can simultaneously express human PD1, PDL1 and CD73 proteins, so that perfect identification of PD1 and PDL1 channels is realized, and the model is a high-quality model for evaluating immune checkpoint therapy. The method can be used for single drug evaluation of PD1, PDL1 and CD73 antibodies, can be used for anti-tumor drug effect evaluation of the combination of the CD73 antibodies and the PDL1 antibodies, has profound guiding significance for preclinical drug effect evaluation, and fills the blank of the three-target mouse model in the market due to the established B6-hPD1/hPDL1/hCD73 humanized mouse model, and provides a CD73 inhibitor evaluation platform. And obvious benefits are brought from the economic and social aspects.

3. The invention provides a specific operation method for preparing an animal model for stably expressing humanized CD73, wherein a targeting strategy, element constitution on a carrier for expressing the humanized CD73, the optimal sgRNA for knocking out mouse-derived CD73, and a primer and an enzyme digestion scheme used for identification and screening are optimized, and the high efficiency of preparing a target animal model is ensured by combining subsequent protein expression detection, immune system verification, inhibitor evaluation and the like.

Drawings

FIG. 1 is a schematic representation of the targeting strategy of the present invention.

FIG. 2 shows the results of PCR amplification of the identified vector.

FIG. 3 shows the F1 generation identification strategy.

FIG. 4 is a graph of F1 generation identification results.

FIG. 5 is a graph of F1 generation identification results.

FIG. 6 shows the results of the flow protein assay.

FIG. 7 is a graph of immune system validation results.

FIG. 8 is a graph showing the change in body weight of each group of mice.

Fig. 9 shows tumor change curves of mice in each group.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

EXAMPLE 1 construction of a CD73 humanized mouse model

The construction method of the CD73 humanized mouse model is completed according to the following steps:

1. determining a human fragment substitution region and an inserted human sequence: the CRISPR Cas9 mode is used to replace the Cd73 encoding gene of the mouse with the human CD73 encoding gene, so as to construct a mouse model capable of expressing the human CD73. The amino acid sequence (Aa: 1-574) encoded by the selected human CD73 gene is the following sequence SEQ ID NO:1 is shown in the specification; the human CD73 coding amino acid sequence is inserted into the rear of the 5' UTR of the mouse CD73 gene, and a 3XployA termination sequence is added at the tail end, so that the mouse CD73 protein cannot be expressed, and the strategy diagram is as follows. The human CD73 protein is regulated by the promoter and 5' UTR of mouse CD73 gene, so that the expression of human CD73 protein is regulated and maintained in the mouse sequence to maintain the similarity with the expression space and expression amount of original mouse CD73 protein, and the original stability of mouse body is not destroyed. A targeting strategy diagram is shown in fig. 1.

Human CD73 amino acid sequence SEQ ID NO:1:

MCPRAARAPATLLLALGAVLWPAAGAWELTILHTNDVHSRLEQTSEDSSKCVNASRCMGGVARLFTKVQQIRRAEPNVLLLDAGDQYQGTIWFTVYKGAEVAHFMNALRYDAMALGNHEFDNGVEGLIEPLLKEAKFPILSANIKAKGPLASQISGLYLPYKVLPVGDEVVGIVGYTSKETPFLSNPGTNLVFEDEITALQPEVDKLKTLNVNKIIALGHSGFEMDKLIAQKVRGVDVVVGGHSNTFLYTGNPPSKEVPAGKYPFIVTSDDGRKVPVVQAYAFGKYLGYLKIEFDERGNVISSHGNPILLNSSIPEDPSIKADINKWRIKLDNYSTQELGKTIVYLDGSSQSCRFRECNMGNLICDAMINNNLRHTDEMFWNHVSMCILNGGGIRSPIDERNNGTITWENLAAVLPFGGTFDLVQLKGSTLKKAFEHSVHRYGQSTGEFLQVGGIHVVYDLSRKPGDRVVKLDVLCTKCRVPSYDPLKMDEVYKVILPNFLANGGDGFQMIKDELLRHDSGDQDINVVSTYISKMKVIYPAVEGRIKFSTGSHCHGSFSLIFLSLWAVIFVLYQ

selecting human gene sequences as SEQ ID NO:2 is shown as follows:

atgtgtccccgagccgcgcgggcgcccgcgacgctactcctcgccctgggcgcggtgctgtggcctgcggctggcgcctgggagcttacgattttgcacaccaacgacgtgcacagccggctggagcagaccagcgaggactccagcaagtgcgtcaacgccagccgctgcatgggtggcgtggctcggctcttcaccaaggttcagcagatccgccgcgccgaacccaacgtgctgctgctggacgccggcgaccagtaccagggcactatctggttcaccgtgtacaagggcgccgaggtggcgcacttcatgaacgccctgcgctacgatgccatggcactgggaaatcatgaatttgataatggtgtggaaggactgatcgagccactcctcaaagaggccaaatttccaattctgagtgcaaacattaaagcaaaggggccactagcatctcaaatatcaggactttatttgccatataaagttcttcctgttggtgatgaagttgtgggaatcgttggatacacttccaaagaaaccccttttctctcaaatccagggacaaatttagtgtttgaagatgaaatcactgcattacaacctgaagtagataagttaaaaactctaaatgtgaacaaaattattgcactgggacattcgggttttgaaatggataaactcatcgctcagaaagtgaggggtgtggacgtcgtggtgggaggacactccaacacatttctttacacaggcaatccaccttccaaagaggtgcctgctgggaagtacccattcatagtcacttctgatgatgggcggaaggttcctgtagtccaggcctatgcttttggcaaatacctaggctatctgaagatcgagtttgatgaaagaggaaacgtcatctcttcccatggaaatcccattcttctaaacagcagcattcctgaagatccaagcataaaagcagacattaacaaatggaggataaaattggataattattctacccaggaattagggaaaacaattgtctatctggatggctcctctcaatcatgccgctttagagaatgcaacatgggcaacctgatttgtgatgcaatgattaacaacaacctgagacacacggatgaaatgttctggaaccacgtatccatgtgcattttaaatggaggtggtatccggtcgcccattgatgaacgcaacaatggcacaattacctgggagaacctggctgctgtattgccctttggaggcacatttgacctagtccagttaaaaggttccaccctgaagaaggcctttgagcatagcgtgcaccgctacggccagtccactggagagttcctgcaggtgggcggaatccatgtggtgtatgatctttcccgaaaacctggagacagagtagtcaaattagatgttctttgcaccaagtgtcgagtgcccagttatgaccctctcaaaatggacgaggtatataaggtgatcctcccaaacttcctggccaatggtggagatgggttccagatgataaaagatgaattattaagacatgactctggtgaccaagatatcaacgtggtttctacatatatctccaaaatgaaagtaatttatccagcagttgaaggtcggatcaagttttccacaggaagtcactgccatggaagcttttctttaatatttctttcactttgggcagtgatctttgttttataccaatag

2. determining the sgRNA sequence for model preparation: determining the general region of the sgRNA according to the replacement fragment, selecting to insert a human sequence into a murine 5' UTR in S2 for ensuring that the mouse sequence is not expressed, selecting the target fragment with the lowest off-target rate by using sgRNA evaluation software, and performing a sgRNA transcription preparation method: and (3) performing PCR (polymerase Star or Primer Star Max system, using sgRNA-F, sgRNA-R as a Primer, sequencing correct puc57-sgRNA plasmid (1:30 dilution) as a template, and purifying PCR products to prepare the sgRNA transcription preparation template. Transcription of sgrnas was performed using the T7-ShortScript in vitro transcription kit (AM 1354); the sgRNA sequences used were as follows, targeting efficiencies of this sgRNA were as high as 88%.

3. Vector and graft product construction: the preparation method of the targeting fragment carrier comprises the steps of connecting a plurality of fragments together by adopting a SLIC mode to construct the targeting fragment carrier, wherein the required fragments comprise pl253-DTA framework, huCD73-CDS, 3x stop, CD73-5arm and CD73-3arm, and the preparation steps of the targeting fragment carrier are as follows:

(1) Linearized pl253-DTA linearization was obtained by cleavage of the pl253-DTA plasmid by NOTI+SPEI.

(2) The C57BL/6 genome is used for PCR amplification of CD73-5arm (homologous arm at 5 'end of sgRNA), CD73-3arm (homologous arm at 3' end of sgRNA) is used for obtaining 3xployA sequence, huCD73-CDS fragment is synthesized by the sequence, meanwhile, the fragment with required dosage is obtained by a PCR amplification mode, electrophoresis is carried out, and kit gel is used for recovery for standby. Primers for amplification of CD73-5arm, CD73-3arm, huCD73-CDS and 3xployA sequences are shown in Table 1 below.

TABLE 1 primers for amplification of CD73-5arm, CD73-3arm, huCD73-CDS and 3xployA sequences

(3) CD73-5arm, CD73-3arm, pl253-DTA (linearization), huCD73-CDS and 3xployA were SLIC ligated and vector integrity was verified using PCR. The vector was amplified using the primers shown in Table 2.

TABLE 2 PCR amplification of primers used to identify vectors

The results of the identification are shown in FIG. 2.

(4) After successful construction of the final vector, the band of 3839bp was recovered using PACI/AGEI cleavage (6451 bp, 38399 bp), the correct vector was sequenced and linearized for injection. The sequencing protocol is shown in table 4.

TABLE 4 vector sequencing protocol

4. Positive mice were obtained by transplantation injection: injecting the transplanted product, target sgRNA and cas9 protein into fertilized eggs, transplanting to pseudopregnant mice, carrying out genotype identification on pseudopregnant mice, screening positive mice F0 mice successfully inserted with correct human fragments, carrying out mating propagation on F0 and B6 background mice to obtain F1, carrying out gene identification on the tail of the F1 generation, and carrying out an identification strategy shown in a table 3, wherein the primers are shown in the table 5. See 3 PCR reactions of fig. 3, wherein wild type: (1) (2) PCR did not yield a positive band; (3) the PCR reaction can obtain a single 206bp band; heterozygotes: (1) (2) PCR reaction can obtain a positive band; (3) the PCR reaction can obtain a single 206bp band; homozygote: (1) (2) PCR reaction can obtain a positive band; (3) the PCR reaction did not yield a single 206bp band. The PCR experimental results of the F1 generation mice are shown in fig. 4 and 5, and in fig. 4-5, the negative control is B6 genome DNA; n is a blank control, no template control; the TRANS 2K PLUS II band is 5000bp, 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp. The F1 positive mouse rat tail DNA can be amplified into corresponding bands by PCR, the 5-end band shows 2135bp, and the 3-end band shows 2074bp. The PCR positive products were subjected to second generation sequencing, the sequencing primers are shown in Table 7, and the mice with correct sequencing were positive mice. The positive mice were numbered as follows: and 7#, 12#, 13#, and F1 are subjected to mass propagation and then are matched to obtain the CD73 humanized pure mice, namely B6-hCD73.

TABLE 5 genotyping primers

TABLE 6 genotyping conditions

TABLE 7 genotyping primers

5. Protein expression detection: the CD73 expression is rich, the peripheral blood is selected in the experiment, the expression of the CD73 protein in the blood is detected, and the CD73 protein flow type detection method comprises the following steps:

(1) Drawing materials: collecting 100ul to 1.5mLEP tube (EDTA.2K is added in advance for anticoagulation) of mouse peripheral blood by using an eye socket, lightly stirring to avoid coagulation, transferring the blood into a 10mL centrifuge tube, adding 1mL of 1 xRBC (10 xstock solution is diluted by distilled water) into each tube, uniformly mixing, cracking red at room temperature and avoiding light for 10min, at 8 ℃,400g, centrifuging for 5min, removing supernatant, and if required, cracking red for 2 times, washing with FACSbuffer for 1 time, and standing on ice for later use;

(4) Cleaning: washing FACS buffer, adding FACS buffer, detecting on machine, adding Sytox blue 5min before loading (final concentration 1:10000 dilution), and distinguishing dead and alive cells;

the results of the flow assay for protein expression are shown in FIG. 6, where human CD73 was successfully detected in both naive and mice, and where murine CD73 was not detected.

6. Immune system validation: selecting thymus or spleen of a main immune organ of a mouse, shearing thymus or spleen of a B6-hCD73 homozygous mouse and a C57BL/6 background mouse, grinding and digesting tissues into single cells, staining extracellular proteins of tissue cells by using a murine T\B\NK surface antibody, washing the cells by using PBS, and performing flow cell detection on the number of T (CD4+, CD8+), B, NK cells, wherein the flow detection method of immune cells comprises the following steps:

(1) Drawing materials: cutting spleens of B6-hCD73 homozygous mice and C57BL/6 background mice, weighing, and placing the spleens in a C-shaped tube;

(2) Digestion: the spleen was digested with enzyme digest (PBS containing Ca, mg+2%CS+10mM HEPES+30ug DNase+1.75mg collagenase D) at 37℃for 30min. Digestion was terminated by adding 300ul of 0.1M EDTA to the digested spleen cells, 1mL was filtered through a filter membrane to remove undigested complete tissue pieces, and 2mL of FACS buffer was added to neutralize EDTA per tube. Centrifuging at 8deg.C for 400g for 5min, removing supernatant, adding 3ml of 1×RBC into each tube of spleen, mixing, lysing red blood cells at 8deg.C for 5min at room temperature in dark place, centrifuging at 400deg.C for 5min, removing supernatant, adding 1ml of FACS buffer, resuspending, and filtering; centrifuging at 8deg.C for 5min at 400g, removing supernatant, adding FACS buffer, and re-suspending to adjust cell concentration to 1×10 ⁷ Per mL, 100uL divided into flow tubes, ready to incubate antibodies;

(3) Closing: according to the experimental requirements, 100uL (about 10 ⁶ Individual) cells were separated into different flow tubes, mixed with 1ul of antibody CD16/32 (1:100 dilution) per tube with Fc block (antibody CD 16/32) and incubated on ice for 5min.

(4) Antibody incubation: preparing antibody mixed solution (CD 19, CD4, CD8, CD335 and CD3 antibodies) according to the number of sample tubes, adding 50uL of antibody mixed solution into each sample according to the optimal dosage of the antibodies, mixing uniformly by vortex, adding 0.5uL of antibody into each tube of a single dye tube, mixing uniformly by vortex, and incubating for 1h in the dark on ice;

(5) Cleaning: washing FACS buffer, adding FACS buffer, detecting on machine, adding Sytox blue 5min before loading (final concentration 1:10000 dilution), and distinguishing dead and alive cells;

the results of immune system validation are shown in figure 7, where the relative proportions of T cells, B cells, cd4+ T cells, cd8+ T cells and NK cells in naive mice are substantially similar as compared to naive mice. Indicating that the immune system of the humanized CD73 mouse is normal, and the modeling of the mouse is successful.

7. Evaluation of CD73 inhibitor by PD1, PDL1 and CD73 humanized mice anti-tumor efficacy verification: in vitro data prove that a CD73 humanized mouse can normally express a CD73 protein, and immune response of the protein in the mouse body is normal, but whether the mouse can be used for evaluating the actual curative effect of a CD73 anticancer drug is yet to be verified, in order to adapt to the clinical existing CD73 administration scheme, a combined administration scheme of PD1/PDL1 and CD73 is adopted, B6 background PD1 and PDL1 double humanized mice and hCD73 humanized mice are bred to obtain B6-hPD1/hPDL1/hCD73 three humanized mice, and drug effect evaluation is carried out by adopting B6-hPD1/hPDL1/hCD73 humanized mice;

CD73 inhibitor evaluation B6-hPD1/hPDL1/hCD73 tumor-bearing mouse model: selecting 6-7w B6-hPD1/hPDL1/hCD73 homozygous mice, subcutaneously inoculating the colon cancer cells MC38-hPDL1 (Tg) -mPDL1 (KO) -hCD73 (Tg) -mCD73 (KO) of the mice, wherein the cell line is tumor cells obtained by knocking out both murine PDL1 and CD73 together with CD73 humanization and PDL1 humanization, and the tumor volume is about 100+/-50 ^mm3 Then, the cells are divided into 4 groups, namely a control group, a PDL1 single drug group, a PDL1 and CD73-1 antibody combined group and a PDL1 and CD73-2 antibody combined group; the dosing frequency was 2 times per week, dosing was continued for 3 weeks, dosing endpoint, mice were sacrificed. The dosing regimen is shown in the following table:

results: the change in body weight of mice in each group is shown in fig. 8. The tumor growth curves for each group are shown in figure 9.

The results show that: compared with the control group, the anti-hPDL1 antibody has obvious cancer inhibiting effect (TGI=48.02%) in the single administration group, the tumor inhibiting effect is improved after the anti-hPDL1 antibody is combined with the CD73 antibody, and the TGI reaches 63.97% and 61%. The results show that B6-hPD1/hPDL1/hCD73 mice can be used for evaluation of the combination administration of PDL1 and CD73 human antibodies, and B6-hPD1/hPDL1/hCD73 mice vaccinated with PDL1 and CD73 humanized cell lines are the preferred model for the combination administration of PD1/PDL1 and CD73 inhibitors.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Sequence listing

<110> Guangdong Kangyaokang biotechnology Co., ltd

<120> method for constructing CD73 humanized mouse model

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

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

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

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

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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> 2

<211> 1725

<212> DNA

<213> homo sapiens

<400> 2

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> 3

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

gcggccgcgg gacgcatggc 20

<210> 4

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

gcggccgcgg gacgcatggc 20

<210> 5

<211> 30

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

ggggacacat ggctggctag agcgcgttga 30

<210> 6

<211> 56

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

ctagccagcc atgtgtcccc gagccgcgcg ggcgcccgcg acgctactcc tcgccc 56

<210> 7

<211> 46

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

tattcatcgc gctattggta taaaacaaag atcactgccc aaagtg 46

<210> 8

<211> 33

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

taccaatagc gcgatgaata aatgaaagct tgc 33

<210> 9

<211> 31

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

gccgcgggac gggttccgga tcagcttgat g 31

<210> 10

<211> 36

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

tccggaaccc gtcccgcggc cgctaaggta cccaag 36

<210> 11

<211> 38

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

ggaccggttt aattaaagag gtggaggaga ctgggatg 38

<210> 12

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

tagctgtcag gtttacccgc tag 23

<210> 13

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

ataactgggc actcgacact tg 22

<210> 14

<211> 23

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

ttggaggcac atttgaccta gtc 23

<210> 15

<211> 22

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

tcattgtgct cggacttcag ag 22

<210> 16

<211> 25

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

tcctctactc cgcagtttag tagag 25

<210> 17

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

acgtcgtttg tgtgcaggat c 21

Claims

1. A construction method of a CD73 humanized mouse model is characterized in that a human CD73 coding amino acid sequence is inserted into the rear of the 5'UTR of a mouse CD73 gene, a 3XployA termination sequence is added at the tail end, so that the mouse CD73 protein cannot be expressed, and the human CD73 protein is regulated and controlled by the promoter and the 5' UTR of the mouse CD73 gene, and the construction method comprises the following steps: (1) Constructing an expression vector for expressing the humanized CD73, inserting the humanized CD73 gene, and specifically connecting a plurality of fragments together by adopting an sLIC mode to construct a targeting vector; (2) determining the sgRNA sequence for model preparation: the sgRNA with the lowest off-target rate is selected for the target fragment, and the sequence is shown as SEQ ID NO:3 is shown in the figure; (3) Injecting an expression vector expressing humanized CD73 and sgRNA together with Cas9 protein into fertilized eggs, and transplanting to pseudopregnant mice, wherein the sequence of the humanized CD73 gene is shown in SEQ ID NO: as shown in figure 2, the number of the parts is two,

wherein the targeting vector comprises CD73-5arm, CD73-3arm, huCD73-CDS and 3xployA,

primers for amplifying the CD73-5arm sequence were as follows:

HUCD73-5ARMF: tgcaccggtttaattaacaccgctgagggtcccaaag

HUCD73-5ARMR: ggggacacatggctggctagagcgcgttga；

primers for amplifying huCD73-CDS sequence were as follows:

HUCD73-CDSF:

ctagccagccatgtgtccccgagccgcgcgggcgcccgcgacgctactcctcgccc

HUCD73-CDSR:

Tattcatcgcgctattggtataaaacaaagatcactgcccaaagtg；

primers for amplifying the 3xployA sequence are as follows:

STOPF: taccaatagcgcgatgaataaatgaaagcttgc

STOPR: gccgcgggacgggttccggatcagcttgatg；

primers for amplifying the CD73-3arm sequence were as follows:

HUCD73-3ARMF: tccggaacccgtcccgcggccgctaaggtacccaag

HUCD73-3ARMR: ggaccggtttaattaaagaggtggaggagactgggatg；

and the method also comprises the steps of carrying out genotype identification on the pseudopregnant mice, screening positive mice F0 mice successfully inserted with the correct humanized fragments, carrying out mating propagation on F0 and B6 background mice to obtain F1, and carrying out gene identification on the tail of the F1 generation.

2. The method according to claim 1, characterized in that: wherein the preparation steps of the targeting fragment vector in the step (1) are as follows:

1) Obtaining linearized pl253-DTA by cleavage of pl253-DTA plasmid by NOTI+SPEI;

2) Amplifying CD73-5arm and CD73-3arm by using C57BL/6 genome PCR to obtain huCD73-CDS fragment and 3xployA sequence connected after the CDS fragment, and recovering for standby;

3. The method of claim 1, wherein the primers used to identify the genotype are as follows:

primers for amplifying the 5' arm sequence are as follows:

mCD73-5tF1：tagctgtcaggtttacccgctag

hCD73-5tR1：ataactgggcactcgacacttg；

primers for amplifying the 3' arm sequence were as follows:

hCD73-3tF1：ttggaggcacatttgacctagtc

mCD73-3tR1：tcattgtgctcggacttcagag；

the primers for amplifying the WT sequences were as follows:

mCD73-wt-tF1：tcctctactccgcagtttagtagag

mCD73-wt-tR1：acgtcgtttgtgtgcaggatc。

4. a method according to any one of claims 1-3, characterized in that: also comprises the detection of CD73 protein expression, immune system verification and anti-tumor drug effect verification of the CD73 inhibitor.

5. The method of claim 4, wherein the immune system validation step is: the method comprises the steps of selecting a thymus or spleen of a main immune organ of a mouse, cutting the thymus or spleen of a B6-hCD73 homozygous mouse and a C57BL/6 background mouse, grinding and digesting tissues into single cells, staining extracellular proteins of tissue cells by using a mouse-derived TBNK surface antibody, washing the cells by using PBS, and detecting the cell number of CD4+ T, CD8+ T, B, NK by using flow cytometry.

6. The method of claim 4, wherein the step of verifying the anti-tumor efficacy of the CD73 inhibitor is: b6 background PD1 and PDL1 double humanized mice and hCD73 humanized mice are bred to obtain B6-hPD1/hPDL1/hCD73 three humanized mice, and the B6-hPD1/hPDL1/hCD73 humanized mice are adopted for drug efficacy evaluation; selecting 6-7w B6-hPD1/hPDL1/hCD73 homozygous mice, subcutaneously inoculating the colon cancer cells MC38-hPDL1 (Tg) -Mpdl1-CD37 (TG/KO) of the mice, wherein the cell line is tumor cells obtained by knocking out both PDL1 and CD73 of murine sources simultaneously with CD73 humanization and PDL1 humanization, and the tumor volume is about 100+/-50 mm ³ Then, the cells are divided into 4 groups, namely a control group, a PDL1 single drug group, a PDL1 and CD73-1 antibody combined group and a PDL1 and CD73-2 antibody combined group; the dosing frequency was 2 times per week, dosing was continued for 3 weeks, dosing endpoint, mice were sacrificed.

7. The method of claim 4, wherein CD73 protein is detected by a flow detection method:

(2) Closing: according to the experimental requirement, dividing 100uL cells into different flow tubes, adding Fcblock CD16/32 antibody, adding 1uL of CD16/32 antibody diluted by 1:100 into each tube, uniformly mixing, and incubating on ice for 5min;

(3) Antibody incubation: preparing an antibody mixed solution of hPD-1, mPD-1, hCD73, mCD73 and CD3 antibodies according to the number of the sample tubes, adding 50uL of the antibody mixed solution into each sample according to the optimal dosage of the antibodies, and uniformly mixing by vortex; adding 0.5ul of antibody into each single-dyeing tube, and uniformly mixing by vortex; incubating for 1h on ice in dark place;

(4) Cleaning: FACS buffer washing, adding FACS buffer, loading for detection, and adding Sytoxbue with final concentration of 1:10000 5min before loading to distinguish dead living cells.

8. The method according to claim 5, wherein: the immune cell flow detection method comprises the following steps:

digestion: digestion of spleen with enzyme digestion solution at 37deg.C for 30min, adding 300ul of 0.1M EDTA to the digested spleen cells, filtering with 1mL of filter membrane, removing undigested tissue blocks, and adding 2mL of FACS buffer to neutralize EDTA; centrifuging at 8deg.C for 400g for 5min, removing supernatant, adding 3ml of 1×RBC into each tube of spleen, mixing, lysing red blood cells at 8deg.C for 5min at room temperature in dark place, centrifuging at 400deg.C for 5min, removing supernatant, adding 1ml of F ACSbuffer, resuspension, and filtering; centrifuging at 8deg.C for 5min at 400g, removing supernatant, adding FACSbuffer, and re-suspending to adjust cell concentration to 1×10 ⁷ Per mL, 100uL divided into flow tubes, ready to incubate antibodies;

(1) Closing: according to the experimental requirement, dividing 100uL cells into different flow tubes, adding Fcblock CD16/32 antibody, adding 1uL of CD16/32 antibody diluted by 1:100 into each tube, uniformly mixing, and incubating on ice for 5min;

(2) Antibody incubation: preparing an antibody mixed solution according to the number of the sample tubes: adding the antibodies of CD19, CD4, CD8, CD335 and CD3 according to the optimal dosage of the antibodies, adding 50uL of antibody mixed solution into each sample, mixing by vortex, adding 0.5uL of antibody into each single-dyeing tube, mixing by vortex, and incubating for 1h on ice in a dark place;

(3) Cleaning: FACS buffer washing, adding FACS buffer, loading for detection, and adding Sytoxbue with final concentration of 1:10000 5min before loading to distinguish dead living cells.