CN111647623A

CN111647623A - Construction method and application of SIRPA humanized animal model

Info

Publication number: CN111647623A
Application number: CN202010364829.5A
Authority: CN
Inventors: 琚存祥; 李松; 张明坤; 侯欢欢; 赵静
Original assignee: Gempharmatech Co ltd
Current assignee: Gempharmatech Co ltd
Priority date: 2020-07-14
Filing date: 2020-07-14
Publication date: 2020-09-11
Anticipated expiration: 2040-07-14
Also published as: CN111647623B

Abstract

The invention provides a method for preparing a SIRPA humanized animal model, which utilizes a gene editing technology to replace an extracellular region coded by a murine Sirpa gene with an extracellular region coded by a humanized SIRPA gene, detects the model through immune system index evaluation and tumor pharmacodynamic test, and proves that the obtained mouse model is successfully constructed. The model is used for screening and evaluating the assisted SIRPA targeted drug.

Description

Construction method and application of SIRPA humanized animal model

Technical Field

The invention belongs to the field of animal genetic engineering and genetic modification, and particularly relates to a construction method of a SIRPA gene modification humanized animal model based on a gene editing technology.

Background

Cancer immunotherapy is a therapeutic method for attacking cancer cells by means of the human body's own immune system. The balance between the immune system and cancer cells is a dynamic process of long-term gambling, both positive and interwoven. Immune cells of a healthy body can discover and kill most of cancerated cells, but under the induction of various innate or acquired factors, the immune system loses absolute advantages and is even 'countered' by cancer cells, so that the immune cells become a key member in the occurrence and development of cancers.

Since many tumor-opsonizing antibodies and checkpoint inhibitors are approved and even more drugs are being evaluated for clinical use, antibody-based cancer therapy has become one of the most successful and important strategies for treating patients with malignant tumors. However, one of the major challenges in the field of cancer immunotherapy is that only a small fraction of cancer patients can achieve a fully durable response. Combination immunotherapy can target not only different regulatory pathways in immune and cancer cells, but also different types of tumor infiltrating immune cells, and may be a promising approach to induce rapid and durable anti-tumor responses and prevent therapy-induced resistance, spread, and metastasis of cancer cells.

An inhibitory receptor signal regulatory protein alpha (SIRPA) is a typical inhibitory immunoreceptor in the SIRP family, and it binds to CD47 to form a CD47-SIRPA signal complex, which has the functions of regulating immune response and mediating bidirectional signal regulation, is also involved in various physiological activities, such as neutrophil chemotaxis, macrophage phagocytosis, etc., and plays a regulatory role in inducing immune tolerance, activation, etc. of T cells.

Cancer cells highly express CD47 on their surface, which in combination with SIRPA inhibits macrophage-mediated phagocytosis. In one study, high affinity variants of SIRPA were used to antagonize increased phagocytosis of cancer cells by CD47 on cancer cells. Another study in mice found that anti-SIRPA antibodies, alone or in combination with other drugs, were able to inhibit cancer growth and metastasis. The monoclonal antibody of SIRPA is used for blocking the signal channel of CD47-SIRPA, so that chemotactic phagocytosis of tumor cells by macrophages can be promoted, and a novel method is provided for targeted therapy of tumors.

Screening of immune checkpoint drugs and preclinical testing require evaluation on animal models, and rodent animals, which are the most widely used experimental small animal models, have become indispensable replacement models in human tumor therapy research. However, due to differences in species properties, immune checkpoint antibodies screened on mice are not fully tried for humans. The human coding gene is substituted for the corresponding gene of a mouse by a gene modification method, and the prepared humanized mouse model of the immune checkpoint has human functional genes and can be used for screening and evaluating human drugs of the immune checkpoint.

The SIRPA humanized mouse is a mouse model which is constructed by replacing a mouse-derived SIRPA gene with a human-derived gene on a mouse with a sound immune system by using a gene modification method and can interact with an anti-human SIRPA monoclonal antibody. Compared with the common mouse, the model realizes the humanized modification of key target molecules, reserves the complete immune system, can be used for screening and evaluating drugs aiming at human genes, and is a highly ideal preclinical drug test model. Meanwhile, a PD1 humanized mouse and a SIRPA humanized mouse which are independently developed are bred to obtain a PD1 and SIRPA humanized mouse model, and the model can be used for evaluating the human PD1 antibody, the SIRPA antibody, the tumor inhibition effect of the combined use of the PD1 antibody and the SIRPA antibody and the safety evaluation of drugs, and provides a more effective medication strategy for clinical experiments. In the process of the SIRPA humanized mouse, all elements and steps used in the gene editing technology, including sgRNA, targeting vectors and the like, are fully optimized and adjusted, and the high success rate and the high accuracy rate of the SIRPA humanized animal model prepared by the technology are ensured.

Disclosure of Invention

The invention provides a method for preparing a SIRPA humanized animal model, which is characterized in that an extracellular region coded by a murine Sirpa gene is replaced by an extracellular region coded by a humanized SIRPA gene by using a Crispr/Cas9 technology, the intracellular region of a murine is reserved, and the amino acid sequence of a SIRPA humanized product which is successfully targeted is shown as SEQ ID No. 2.

Preferably, the method comprises the steps of:

1) selecting two ends of an extracellular region of a murine Sirpa gene as targeting sites, and designing a homologous DNA donor containing an extracellular region of a human SIRPA gene;

2) preparing Cas9 or an expression vector thereof; sgRNA against murine sequences were designed in the humanized replacement region, the sgRNA sequences are given below:

sgRNA name	sgRNA sequence (5 '→ 3')
		hSIRPA-5S3	CGTGAATTCCCCACAGA(SEQNo.3)
hSIRPA-3S3	GCTGGCGATCTGGCTCA(SEQNo.4)

3) The Cas9/sgRNA system and the targeting vector are co-injected or co-electroporated into mouse fertilized eggs, and the fertilized eggs are transplanted into recipient mice to produce SIRPA humanized mice.

Preferably, the sequence of the extracellular region of the human SIRPA gene is shown in SEQ ID NO: 1 is shown.

Preferably, wherein the mouse is a C57BL/6 background or BALB/C background mouse.

Preferably, the method further comprises the steps of screening out a target F0 mouse by gene identification, breeding the target F0 mouse with a background mouse, breeding a progeny mouse of the target F0 mouse with F1, and carrying out PCR identification on the obtained F1 mouse.

Preferably, the primers used for PCR identification are:

preferably, when PCR identification is carried out, the primers 003657-msirpa-5tF1/003657-hSIRPA-5tR1 are respectively positioned outside the 5 'homology arm and in the human fragment of the targeting vector, and if the pair of primers is amplified to generate a PCR product, the target vector is effectively inserted in the 5' of the murine genome; 003657-hSIRPA-3tF1/003657-hSIRPA-3tR1 are respectively positioned in the human fragment and outside the 3 'homologous arm of the targeting vector, and if the pair of primers is amplified to generate PCR products, the target vector is effectively inserted in the 3' of the mouse genome.

Preferably, the method further comprises detecting the expression of the human SIRPA and/or evaluating an immune system indicator of the murine model.

The invention also provides application of the method in preparing an animal model for screening the SIRPA targeted drug.

The invention also provides application of the method in preparing an animal model for evaluating the curative effect of the SIRPA targeted drug or the combination of the SIRPA targeted drug and other drugs.

Further, the invention provides sgRNA specifically targeting the extracellular region of the murine Sirpa gene, the sequence of which is shown in seq id NO: 3-4.

The invention has the following positive effects:

1. the invention adopts Crispr/Cas9 technology, uses the extracellular region of human SIRPA to pertinently replace the extracellular region of murine SIRPA, and is different from the means of preparing SIRPA humanized animal model in the prior art. Realizes that the SIRPA gene of a mouse source is replaced by the gene of a human source on a mouse with a sound immune system, and constructs a mouse model which can interact with the SIRPA monoclonal antibody of the human source. Compared with the common mouse, the model realizes the humanized modification of key target molecules, reserves the complete immune system, can be used for screening and evaluating drugs aiming at human genes, and is a highly ideal preclinical drug test model.

2. The invention provides an optimized specific operation method for preparing a humanized SIRPA animal model, and each step and parameter are optimized in the method. Particularly, through a great deal of creative work, the hSIRPA-5S3 and hSIRPA-3S3 pair with optimal effect is obtained, and under the condition that the design difficulty of the sgRNA is large and the effect is unpredictable at present, the selection of the sgRNA has a remarkable significance. In addition, the gene identification means, the immune system evaluation means and the like in the preparation process are determined by the creative work of the applicant, and the high success rate of the animal model preparation is ensured.

Drawings

FIG. 1 is a 5-end and 3-end identification electrophoretograms of SIRPA-KI-target: wherein the B6 negative control is genomic DNA; n is blank control, no template control; TRANS 2K PLUS II band: 8000bp \5000bp \3000bp \2000bp \1000bp \750bp \500bp \250bp \100 bp.

FIG. 2 is a 5-terminal and 3-terminal identification electrophoretograms of SIRPA-KI-target: WT negative control was genomic DNA; n is blank control, no template control; p is a positive control; m is a TRANS 2K PLUS II band: 8000bp \5000bp \3000bp \2000bp \1000bp \750bp \500bp \250bp \100 bp.

FIG. 3 shows the SIRPA expression results in BALB/c-hPD1/hSIRPA homozygous mice and BALB/c background mice.

FIG. 4 shows the results of SIRPA expression in B6-hSIRPA homozygous mice and B6 background mice.

FIG. 5 is a lymphocyte population test in B6-hSIRPA homozygous mice and B6 background mice.

FIG. 6 shows lymphocyte population detection in BALB/c-hPD1/hSIRPA homozygous mice and BALB/c background mice.

FIG. 7 is a graph showing the change in body weight of each group of tumor-bearing mice.

FIG. 8 is a graph of tumor volume change for different groups.

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.

Example 1: establishment of SIRPA humanized mouse model

And (3) replacing the Sirpa gene of the mouse with human SIRPA on the C57BL/6 background and BALB/C background mice by using a CRISPR Cas9 mode, thereby constructing a mouse model capable of expressing the human SIRPA. And the double-source mouse model capable of expressing the human-source SIRPA and the PD1 is obtained by breeding with a PD1 humanized mouse which belongs to the independent property.

1. Determining human source fragment replacement region and inserted human source sequence

According to the structure and function of the human SIRPA, an extracellular region coded by the human SIRPA gene is selected to replace an extracellular region coded by a murine Sirpa gene, an intracellular region of a mouse is reserved, and an extracellular region amino acid sequence (Aa: 28-362) coded by the selected human SIRPA gene is shown as SEQ No. 1.

GVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQWFRGAGPGRELIYNQKEGHFP RVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVS GPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTRE DVHSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQ LTWLENGNVSRTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHD LKVSAHPKEQGSNTAA

2. Splice sequence determination of humanized SIRPA protein

Homologous recombination is utilized to replace the extracellular region coded by the human SIRPA gene with the extracellular region coded by BALB/c and B6 murine Sirpa genes, the intracellular region sequences of BALB/c and B6 mice are reserved, and the amino acid sequence of the SIRPA humanized mouse which successfully targets is shown as SEQ No.2 (the underlined part is the human amino acid sequence, and the bold italic font is the murine amino acid sequence).

SEQ No.2：

MEPAGPAPGRLGPLLLCLLLSASCFCTGVAGEEELQVIQPDKSVLVAAGETATLRCTATSLIPVGPIQW FRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGTYYCVKFRKGSPDDVEFKSGAGTELSVR AKPSAPVVSGPAARATPQHTVSFTCESHGFSPRDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDV HSQVICEVAHVTLQGDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVSRTETA STVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVSAHPKEQGSNTAADNNATHN

3. Positive mouse obtained by carrier injection transplantation

1) Selecting two ends of an extracellular region of a mouse Sirpa gene as targeting sites, and designing a homologous DNA donor containing an extracellular region of a human SIRPA gene and an identification scheme;

2) preparing Cas9 or an expression vector thereof based on CRISPR/Cas9 technology; sgrnas against murine sequences were designed in the humanized replacement regions. Designing and synthesizing to recognize a 5 'end target site and a 3' end target site, and constructing a sgRNA expression vector. The recognition sites of the two sgRNAs are respectively positioned at the two ends of the extracellular region of the mouse Sirpa gene, and the target site sequence of each sgRNA on the Sirpa is shown in Table 1. Each sgRNA sequence was cloned into a pUC57kan-T7-delG vector to construct a pUC57-sgRNA plasmid.

Table 1sgRNA sequences

sgRNA name	sgRNA sequence (5 '→ 3')	PAM
			hSIRPA-5S1	ACAAATTCAGGCCGGGCG	TGG
hSIRPA-5S2	GAGCGTGAATTCCCCACAG	AGG
			hSIRPA-5S3	CGTGAATTCCCCACAGA	AGG
hSIRPA-3S1	CCTCTATGAGACATTAA	AGG
			hSIRPA-3S2	AGGCTGGCGATCTGGCTC	AGG
hSIRPA-3S3	GCTGGCGATCTGGCTCA	GGG

The sgRNA transcription preparation method comprises the following steps: PCR is carried out by taking PrimerStar or PrimerStar Max 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).

sgRNA screening: after the sgrnas at the 5 'end and the 3' end are respectively incubated with Cas9 protein, the mixed solution is injected into a fertilized egg for 0.5 day, and after the fertilized egg is cultured to a blastocyst stage, the ko positive rate of mouse Sirpa genes is identified, so that the sgrnas with high cleavage activity are screened, and the sgrnas with high cleavage activity at the 5 'end and the 3' end are respectively selected to pair into a sgRNA pair.

The sgRNA cleavage identification method comprises the following steps: the collected blastocysts were subjected to PCR amplification (the PCR protocol is shown In Table 2 below), the amplified bands were subjected to second-generation sequencing (5S1-5S3 sequencing primer: Sirpa-sgRNA-In5F1, 3S1-3S3 sequencing primer: Sirpa-sgRNA-In3R1), the probability of mutation was counted by comparison with the wt bands (the identification result is shown In Table 3 below), and finally hSIRPA-5S3 and hSIRPA-3S3 were selected.

Table 2sgRNA cleavage PCR identification protocol

Table 3sgRNA cleavage activity

sgRNA name	Cutting efficiency
		hSIRPA-5S1	19/20＝95％
hSIRPA-5S2	15/16＝93.8％
		hSIRPA-5S3	18/19＝95％
hSIRPA-3S1	20/20＝100％
		hSIRPA-3S2	17/20＝85％
hSIRPA-3S3	18/19＝95％

Based on the results in Table 3, hSIRPA-5S3 and hSIRPA-3S3 with high cleavage efficiency were selected for the next experiment.

3) The Cas9/sgRNA system and the targeting vector are injected into a mouse fertilized egg of 0.5 day (the extracellular region of a mouse Sirpa gene is knocked out and the extracellular region of a human SIRPA gene is inserted into a genome), the mouse fertilized egg is transplanted into a pseudopregnant female mouse of 0.5 day, and after the mouse is born, a middle-target mouse is screened out through gene identification (F0).

4) Genotyping of humanized mouse F1 generation: breeding a targeted F0 mouse and a background mouse through gene identification, wherein the mouse of the descendant is F1, carrying out PCR identification on the obtained rat tail genomic DNA of the F1 mouse at two ends after the targeted by using two pairs of primers respectively, and the primers 003657-msirpa-5tF1/003657-hSIRPA-5tR1 are respectively positioned outside a 5 'homology arm and in a human fragment of a targeting vector, so that if the pair of primers is amplified to generate a PCR product, the target vector is effectively inserted in the 5' of the mouse genome;

003657-hSIRPA-3tF1/003657-hSIRPA-3tR1 are respectively positioned in the human fragment and outside the 3 'homologous arm of the targeting vector, and if the pair of primers is amplified to generate PCR products, the effective insertion of the target vector in the 3' of the mouse genome is demonstrated. And (3) carrying out sequencing verification on the mouse clone with positive PCR identification at two ends, and identifying the clone with correct sequencing as a positive mouse after the homologous DNA donor replaces the corresponding sequence of the mouse in the mouse genome.

TABLE 4F1 identifying primers

Note: KI is an on-target genotype; WT is wild type; KI/KI is homozygote; KI/WT is heterozygote

The PCR reaction system and reaction conditions are shown in the following table:

TABLE 5PCR reaction System

Reagent (TakaraR045)	Volume (μ l)	Specification of
			PrimeSTAR Max Premix(2×)	12.5	\
ddH₂O	9.5	\
			Primer
	1	10μM
			Primer
	1	10μM
			Template
	1

TABLE 6PCR reaction conditions

As a result: BALB/c background 10 positive F1 mice were obtained. As shown in fig. 1, hsrpa F1 rat tail DNA identified the electrophoretogram, and both 5 'and 3' identified the purposive bands as positive, indicating that the mice were positive for correct targeting; the rest identified no band were all negative, mice were treated. 11 positive F0 mice were obtained against B6 background. As shown in fig. 2, hsrpa F1 rat tail DNA identification electropherograms, 5 'and 3' identified that the target bands were positive, indicating that the mice were positive for correct gene recombination; the rest identified no band were all negative, mice were treated.

BALB/c background SIRPA gene identification is carried out on F1 mouse tails, PCR experimental results of F1 mice are shown in figure 1, and identification of 5 'and 3' of humanized SIRPA genes of 92#, 96#, 98-103#, 105#, and 111# mice is positive, which indicates that the mice are positive mice correctly carrying out gene recombination. F1 can be bred with autogenetic PD1 humanized mouse after being largely propagated to obtain SIRPA and PD1 double humanized mouse homozygote mouse, which is called BALB/c-hPD1/hSIRPA for short.

B6 background F1 mouse tail carries out SIRPA gene identification, F1 mouse PCR experiment results are shown in figure 2, 5 'and 3' identifications of humanized SIRPA genes of 71-73#, 77#, 83# mice are positive, and the mice are positive mice which correctly carry out gene recombination. F1 can be bred with humanized mouse PD1 after being largely propagated to obtain SIRPA and PD1 double humanized mouse homozygote mouse, B6-hPD1/hSIRPA for short.

Example 2B6-hSIRPA and BALB/c-hPD1/hSIRPA expression in mice with humanized SIRPA and verification of the immune system

The expression condition of the SIRPA homozygous mouse is analyzed through flow cytometry, immune cell groups are checked, humanized genes can be successfully expressed through analysis, and the mouse without obvious abnormality of an immune system can be used for a tumor pharmacodynamic experiment.

1. Protein expression assay

And (3) selecting a SIRPA main expression tissue thymus or spleen, and detecting the expression of the SIRPA protein in the tissue.

The SIRPA protein flow detection method comprises the following steps:

a) material taking: spleens of humanized mice and background mice were excised, weighed, and placed in a C-tube.

b) Digestion: spleen and thymus were digested with enzyme digests (PBS containing Ca, Mg + 2% CS +10mM HEPES + 30ug DNase +1.75Mg collagenase D) at 37 ℃ for 30 min. Digestion was stopped by adding 300ul of 0.1M EDTA to the spleen cells that had been digested. 1mL of the mixture was filtered through a filter to remove the undigested tissue mass, and 2mL of FACS buffer was added to each tube to neutralize the EDTA. Centrifuging for 5min at 8 deg.C and 400g, removing supernatant, adding 3mL 1 × RBC per tube of spleen, mixing, lysing erythrocytes for 5min at 8 deg.C and 400g in dark at room temperature, centrifuging for 5min, removing supernatant, adding 1mL FACS buffer, resuspending, and filtering; centrifuging at 8 deg.C for 5min at 400g, removing supernatant, adding FACS buffer, resuspending, adjusting cell concentration to 1 × 107/mL, and separating into 100uL in flow tube to prepare for incubation antibody.

c) And (3) sealing: according to the experimental requirements, 100uL (about 106) cells were divided into different flow tubes, mixed with Fcblock (CD16/32 antibody) and 1uL CD16/32 antibody (1:100 dilution) per tube, and incubated on ice for 5 min.

d) Antibody incubation: preparing antibody mixed liquor (hSIRPA, mSRIPA, CD3 antibody) according to the number of sample tubes, adding 50uL of antibody mixed liquor into each sample according to the optimal dosage of the antibody, and uniformly mixing by vortex; adding 0.5ul antibody into each tube of the single staining tube, and uniformly mixing by vortex; incubating for 1h on ice in a dark place;

e) cleaning: and (4) washing by using the FACS buffer, adding the FACS buffer, and detecting on a machine. Sytoxblue (final concentration 1:10000 dilution) was added 5min before loading to differentiate dead and live cells.

And (3) detection results: using human-derived anti-SIRPA antibodies and murine anti-SIRPA antibodies, the expression of human-derived SIRPA by macrophages in B6-hSIRPA homozygous mice and C57BL/6 background mice and BALB/C-hPD1/hSIRPA pure mice and BALB/C background mice was detected by flow cytometry, whereas the expression of human-derived SIRPA was not detected in background mice, while the expression of human-derived SIRPA was detected in B6-hSIRPA homozygous mice and BALB/C-hPD1/hSIRPA homozygous mice. (see FIG. 3, FIG. 4, infra)

2. Immune system validation

Selecting the spleen of a main immune organ of a mouse, shearing the spleen of a SIRPA humanized homozygous mouse and a corresponding background mouse, grinding and digesting tissues into single cells, staining extracellular proteins of the tissue cells by using murine T \ B \ NK surface antibodies, washing the cells by using PBS, and then carrying out flow cytometry to detect the number of T (CD4+, CD8+) and B, NK cells.

The immune cell flow detection method comprises the following steps:

a) material taking: the spleen of a SIRPA humanized homozygous mouse and a corresponding background mouse are cut and placed in a C-shaped tube.

b) 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 30 min. Digestion was stopped by adding 300ul of 0.1M EDTA to the spleen cells that had been digested. 1mL of the mixture was filtered through a filter to remove the undigested tissue mass, and 2mL of FACS buffer was added to each tube to neutralize the EDTA. Centrifuging for 5min at 8 deg.C and 400g, removing supernatant, adding 3mL 1 × RBC per tube of spleen, mixing, lysing erythrocytes for 5min at 8 deg.C and 400g in dark at room temperature, centrifuging for 5min, removing supernatant, adding 1mL FACS buffer, resuspending, and filtering; centrifuging at 8 deg.C for 5min at 400g, removing supernatant, adding FACSBuffer, resuspending, adjusting cell concentration to 1 × 107/mL, and separating into 100uL in flow tube to prepare for incubation of antibody.

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

And (3) detection results: as shown in FIG. 5, the number of each T, B, NK immune cells of the B6-hSIRPA homozygous mice is not obviously different from that of B6 background mice; as shown in FIG. 6, the number of immune cells of T, B, NK of BALB/c-hPD1/hSIRPA homozygous mice was not significantly different from that of BALB/c background mice. The data show that the human SIRPA participates in the immune response process of the mice, the immune system of the humanized mice is normal, and the humanized mice have no difference compared with common background mice and can be used for SIRPA targeted drug evaluation.

Example 3B6-hSIRPA humanized mouse evaluation of tumor pharmacodynamic validation of anti-SIRPA antibodies

Since the in vitro level validation data is not enough to reflect the actual situation of hsrpa in the humanized mouse, the designed experiment of the invention collects the following in vivo experimental data: the tumor-bearing B6-hSIRPA humanized mouse has antitumor drug effect reactivity against the hSIRPA antibody and antitumor drug effect reactivity against the combination of the hSIRPA antibody and the mPDL1 antibody.

The detection method comprises the following steps: MC38-hCD47 (murine CD47 humanized MC38 tumor cell line) cells. ResuscitationThe cells are taken out of a liquid nitrogen tank, quickly thawed in a water bath at 37 ℃, inoculated in 15cm dish for culture, the cells are collected, centrifuged at 1000rpm for 5min, supernatant is discarded, 10ml of PBS without calcium and magnesium ions is used for washing for 2 times, the PBS is used for resuspending the cells, the final concentration of the cells is adjusted to be 5 ×/ml, subcutaneous injection is carried out, 100 mu l of MC38-hCD47 cells are injected to the right back of a B6-hSIRPA humanized mouse, the cells are arranged above the thigh, the growth condition of the mouse is observed every day before grouping, the mouse is weighed for 2 times every week, the tumor size is detected after confirming the tumor bearing of the mouse, the average tumor volume is 80-120mm after connecting the MC38-hCD47 cells for 11 days, the mouse weight is randomly divided into Vehicle weight, the SIRI-SIRPA (30 mg) is obtained after the mice are inoculated with the single drug, the single drug is used for the following administration, the SIRPA 1-1 kg, the SIRPA 1 kg dose is obtained after the mice are inoculated with the single drug, the single drug is used for the following administration, the combined administration of the mice (24 mg) and the single dose of the SIRPA 361 kg) is measured after the mice is carried out, the administration of the mice, the combined dose of the mice is carried out for the combined dose of the injection, the mice for the injection, the injection is carried out, the injection, the average tumor volume is carried out for the measurement, the measurement³An euthanasia end experiment was performed.

Table 7 grouping and dosing regimens

1. Body weight changes in mice

All animals were weighed twice a week after dosing, and the body weight change curves for each group of tumor-bearing mice are shown in fig. 7. There was no significant difference in body weight change between the different groups of mice, indicating that the mice tolerated well at the antibody test dose without significant toxic side effects.

2. Change in tumor volume

FIG. 8 is a graph of the tumor volume change of different groups, and the combined drug has certain tumor growth inhibition effect in the model.

3. Relative tumor inhibition ratio TGI (%)

In D25, group 4 (G4: anti-hSIRPA + anti-mPDL1) had a lower tumor volume than group 1 control (G1: Vehicle control (PBS)), and a TGI (%) of 48.60%.

TABLE 7 statistical analysis of the efficacy of the test articles after treatment

And (4) conclusion:

the result of a pharmacodynamic experiment shows that the combined drug combination of the anti-human SIRPA inhibitor and the anti-mouse PDL1 inhibitor has more obvious tumor inhibition effect compared with a single drug combination and a control group, and the SIRPA humanized mouse is proved to be a powerful tool for evaluating the in-vivo efficacy of the SIRPA inhibitor or the combined drug combination with other inhibitors.

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.

Claims

1. A method for preparing a SIRPA humanized animal model is characterized in that an extracellular region coded by a murine Sirpa gene is replaced by an extracellular region coded by a human SIRPA gene, the intracellular region of a murine Sirpa is reserved, and the amino acid sequence of a SIRPA humanized product which is successfully targeted is shown as SEQ ID No. 2.

2. The method of claim 1, comprising the steps of:

2) preparing a Cas9 protein or an expression vector thereof; sgRNA against murine sequences were designed in the humanized replacement region, the sgRNA sequences are given below:

3) And co-injecting or co-electrically transferring the Cas9/sgRNA system and the targeting vector into mouse fertilized eggs, and transplanting the fertilized eggs into a recipient mother mouse to produce the SIRPA humanized mouse.

3. The method of claim 1 or 2, wherein the sequence of the extracellular region of the human SIRPA gene is as shown in SEQ ID NO: 1 is shown.

4. The method of any one of claims 1-3, wherein the mouse is a C57BL/6 background or BALB/C background mouse.

5. The method as claimed in claim 1 or 2, further comprising the steps of screening out the target F0 mouse by gene identification, breeding it with the background mouse, breeding its offspring mouse to F1, and carrying out PCR identification on the obtained F1 mouse.

6. The method of claim 5, wherein the primers used for PCR identification are:

7. the method of claim 6, wherein the primers 003657-msirpa-5tF1/003657-hSIRPA-5tR1 are located outside the 5 'homology arm and in the human fragment of the targeting vector when PCR identification is performed, and PCR products are generated by amplification of the primers, so that the target vector is effectively inserted into the 5' murine genome; 003657-hSIRPA-3tF1/003657-hSIRPA-3tR1 are respectively positioned in the human fragment and outside the 3 'homologous arm of the targeting vector, and if the pair of primers is amplified to generate PCR products, the target vector is effectively inserted in the 3' of the mouse genome.

8. The method of any one of claims 1-3, further comprising detecting expression of human SIRPA and/or assessing immune system parameters in a murine model.

9. Use of the method of any one of claims 1-8 in the manufacture of an animal model for screening for a SIRPA-targeted drug.

10. Use of the method of any one of claims 1-8 in the preparation of an animal model for the evaluation of the efficacy of a SIRPA-targeted drug or the evaluation of the combination of a SIRPA-targeted drug and another drug.

11. The sequence of sgRNA specifically targeting the extracellular region of the murine Sirpa gene is shown in SEQ ID NO: 3-4.