CN114470241A - Application of liver targeting gamma-interferon expression vector in preparation of anti-liver tumor drugs - Google Patents

Abstract

The invention discloses application of a liver targeting gamma-interferon expression vector in preparation of a medicine for treating and/or preventing liver tumors, wherein the gamma-interferon is expressed in a liver region in a targeting manner through the liver targeting gamma-interferon expression vector, NK cells are activated, and M1 type polarization treatment and/or prevention of macrophages are promoted. The test data of the invention prove that: the liver-targeted expression IFN gamma can activate macrophages and NK cells, obviously inhibit the growth of liver tumors, and is a novel anti-liver-tumor medicament with great potential.

Description

Application of liver targeting gamma-interferon expression vector in preparation of anti-liver tumor drugs

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of a liver targeting gamma-interferon expression vector in preparation of a medicine for treating liver tumor.

Background

Liver tumors refer to tumor lesions occurring in liver parts, including primary tumors and metastatic tumors, wherein benign tumors are rare, malignant tumors are more, the targeted therapy effect is limited at present, and new therapies need to be developed urgently.

Immunotherapy is one of the important anti-tumor strategies, originally established by William B Coley physicians as a bacterially-mediated anti-tumor therapy, and is now evolving into a variety of immune cell-mediated therapeutic modalities. In recent years, with the development of gene editing technology, genetically engineered bacteria have been transformed into new anti-tumor weapons. The genetically modified salmonella YB1 prepared by the hong Kong university by using a gene editing method can effectively inhibit the growth of breast cancer, lung cancer, liver cell and liver cancer and liver metastases.

In conclusion, the therapeutic methods and drugs for liver tumor therapy have very limited effects, and there is a need to develop a new drug for liver tumor therapy.

Disclosure of Invention

Therefore, the invention provides the application of the interferon in preparing the medicine for treating the liver tumor.

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

the embodiment of the invention provides application of a liver targeting gamma-interferon expression vector in preparing a medicament for treating and/or preventing liver tumor, wherein the gamma-interferon is targeted to express in a liver region through the liver targeting gamma-interferon expression vector, NK cells are activated, and M1 type polarization of macrophages is promoted to treat and/or prevent the liver tumor.

In one embodiment of the invention, the liver targeting interferon-gamma expression vector is AAV-IFNr.

In one embodiment of the present invention, the AAV-IFNr is constructed by the following process:

constructing an expression vector pAAV-AAT containing an apoE enhancer-AAT promoter;

constructing a mouse IFNG coding sequence to the expression vector pAAV-AAT to obtain pAAV-IFNr;

pHelper, pAAV2/8 and pAAV-IFNr were co-transfected into 293T cells to obtain AAV-IFNr.

In another aspect, the invention also provides a composition for treating or preventing tumor, which comprises the liver-targeting interferon gamma expression vector.

In one embodiment of the invention, the liver targeting interferon-gamma expression vector is AAV-IFNr.

The invention also provides a product, the active component of which is a liver targeting gamma-interferon expression vector, and the application of the product is any one of the following:

(a) activating NK cells;

(b) promoting M1 type polarization of macrophages;

(c) treating tumor.

In one embodiment of the invention, the tumor is a liver tumor.

In one embodiment of the invention, the liver-targeting interferon-gamma expression vector is used as a delivery vector of IFN gamma, and the liver targetedly expresses the IFN gamma.

The invention has the following advantages:

the test data of the invention prove that: the liver-targeted expression of IFN gamma can activate macrophages and NK cells, obviously inhibit the growth of liver tumor, and is a novel drug with great potential for resisting liver tumor.

YB1 can inhibit the growth of mouse liver tumor by inducing IFN gamma generation, and the liver target expression IFN gamma can reproduce the anti-liver tumor efficacy of YB1, and has great potential as a novel therapy for treating liver tumor.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It should be apparent that the drawings in the following description are merely exemplary, and that other embodiments can be derived from the drawings provided by those of ordinary skill in the art without inventive effort.

The structures, ratios, sizes, and the like shown in the present specification are only used for matching with the contents disclosed in the specification, so as to be understood and read by those skilled in the art, and are not used to limit the conditions that the present invention can be implemented, so that the present invention has no technical significance, and any structural modifications, changes in the ratio relationship, or adjustments of the sizes, without affecting the effects and the achievable by the present invention, should still fall within the range that the technical contents disclosed in the present invention can cover.

Fig. 1 is a graph of the inhibitory effect of YB1 injected into the tail vein in a single injection on liver metastasis of INR1G9 cells (n 10) in an example of the invention, data expressed as mean ± sd, × p < 0.001;

fig. 2 is an analysis of the levels of various inflammatory factors in liver tissue (n-3-5) according to an example of the present invention, data expressed as mean ± sd, × p < 0.001; p < 0.01;

fig. 3 is a graph showing the effect of neutralizing antibodies on the anti-tumor effect of YB1 by inhibiting IFN γ or CCL2 function, in an example of the invention, paraffin sections from liver stained with H & E for tumor growth (n-5);

FIG. 4 shows the design of AAV vectors for liver-targeted expression of IFN γ in accordance with the present invention, adapted from TaKaRa (#6230) specification;

FIG. 5 shows the expression of AAV-EGFP mediated EGFP in multiple organs of mice by a liver targeting expression vector according to an embodiment of the present invention;

fig. 6 shows the expression of AAV-IFNr mediated IFN γ in mouse liver (n-3) using liver targeting expression vector of the present invention, data expressed as mean ± sd, × p < 0.01;

fig. 7 shows the inhibitory effect of the liver targeting expression vector AAV-IFNr of the present example on liver metastasis of INR1G9 cells, the left panel shows 3 representative liver photographs per group, the right panel shows statistics of tumor foci number (n ═ 8) for mice per group, the data are expressed as mean ± standard deviation, × p < 0.001;

FIG. 8 shows the activation of macrophages and NK cells by the liver targeting expression vector AAV-IFNr, in an embodiment of the present invention, NK 1.1: a marker of NK cell activation; iNOS: macrophage M1 type polarization marker; f4/80: a macrophage marker. A scale: 100 μm.

Detailed Description

The present invention is described in terms of particular embodiments, other advantages and features of the invention will become apparent to those skilled in the art from the following disclosure, and it is to be understood that the described embodiments are merely exemplary of the invention and that it is not intended to limit the invention to the particular embodiments disclosed. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

1. Test materials

The golden hamster INR1G9 cells were donated by professor Wang haiyan, university of Rinewa, Switzerland. In the presence of 10% bovine fetal serum⁵U/L penicillin and 100mg/L streptomycin in RPMI 1640(GIBCO) medium (11.2mM glucose). 293T cells (ATCC) were cultured in high-glucose DMEM (GIBCO) medium supplemented with 10% fetal bovine serum.

Bacterial strain YB1 was provided by professor yellow jiangdong, hong kong university. YB1 was grown in LB medium with the addition of 25. mu.g/ml chloramphenicol, 50. mu.g/ml streptomycin, and 100. mu.g/ml 2, 6-Diaminoacrylic Acid (DAP) (Sigma) and shaken at 220rpm at 37 ℃ overnight for about 16 hours. The concentration of the culture was determined by serial dilution of the plates.

Animal experiments were approved by the animal ethics committee of the central friendly hospital and were performed according to the principles of laboratory animal care. Female nu/nu nude mice were used for the experiment at 6-8 weeks. The liver tumor model is prepared as follows: mice were anesthetized with 1% sodium pentobarbital (45mg/kg) by intraperitoneal injection. The mouse skin was disinfected with 75% alcohol and the spleen was removed from the oblique left incision. 25 using a micro BD insulin syringeMu.l of resuspended 5X10 in FBS-free Medium⁵INR1G9 cells were injected into the spleen until spleen envelope swelling was seen. After injection and needle withdrawal, hemostasis and cell leakage inhibition were compressed with a dry cotton swab. The spleen was then returned to the abdominal cavity and the skin was sutured closed.

2. Treatment of INR1G9 liver tumors

Collection of 5X10 from overnight cultures⁷The bacteria were centrifuged, resuspended in 100 μ l PBS and injected splenically one week later with INR1G9 cells into the mouse tail vein. The mice were reared for another 3 weeks, and then the mice were euthanized to obtain tumors. INR1G9 cells were able to form multiple tumor foci in mouse liver following splenic injection. After a single tail vein treatment with YB1, tumor formation in the liver was significantly inhibited, as indicated by a reduced number of tumor foci, smaller size and lack of blood supply, as shown in fig. 1.

Example 2

1. Histological analysis

INR1G9 tumors were fixed in 4% PFA/PBS overnight, and paraffin sections were prepared for H&And E, dyeing. For immunofluorescence on tissue sections, INR1G9 tumors were fixed in 4% PFA/PBS for 2 hours, equilibrated in 30% sucrose/PBS overnight, then OCT embedded, prepared into frozen sections and stained. The main antibodies used were: mouse resistance to iNOS (abcam), mouse resistance to NK1.1(Novus), rat resistance to F4/80 (R)&D) In that respect The secondary antibody used was Alex

555 donkey anti-mouse IgG (H + L) and Alex

Goat anti-rat IgG 555 (H + L) (both from Invitrogen). Mounting (assist in san) was carried out using a mounting solution containing DAPI.

2. Detection of liver inflammatory factors

Weighing about 100mg of mouse liver, homogenizing in PBS + 0.1% Triton X-100 (containing protease inhibitor), centrifuging to remove precipitate, and detecting 6 inflammatory factors simultaneously by CBA flow cytometry method with inflammatory factor detection kit of BD Biosciences company; or taking the supernatant, and verifying the content of the single factor by using an ELISA kit (four-positive cypress) of the IFN gamma factor. Since YB1 has been reported in the literature to proliferate well in the liver and bacterial infection may activate the immune system and promote the anti-tumor effect of the body, it is hypothesized that the local inflammatory factor level in the liver may change after YB1 infection, thereby activating the innate immune system. The mouse liver infected by YB1 for 1 week is taken to detect inflammatory factors such as IL12p70, TNF alpha, IFN gamma, CCL2, IL-10, IL-6 and the like. As shown in figure 2, levels of IFN γ and CCL2 were significantly upregulated in YB1 infected versus uninfected groups.

Example 3

Antibody neutralization experiments: mice vaccinated intraperitoneally with tumors and treated with YB1 were injected intraperitoneally with rat anti-IFN γ or CCL2 antibody (BioXCell) (2mg/kg), twice a week for three weeks. To verify whether IFN γ and CCL2 play a role in YB 1-mediated antitumor processes, neutralizing antibodies to IFN γ and/or CCL2 were administered twice weekly for intervention after tail vein injection of YB 1. As shown in fig. 3, it was found that the neutralizing antibody to CCL2 administered alone did not impair the antitumor effect of YB1, whereas the antitumor effect of YB1 was significantly impaired in the group to which IFN γ or IFN γ + CCL2 was administered.

Example 4

Construction of AAV packaging plasmids:

the Helper Free System (AAV2) System was purchased from TaKaRa. In order to construct a liver specific promoter, two copies of apoE enhancer-AAT promoter sequences are synthesized, the nucleotide sequence of the apoE enhancer-AAT promoter is shown as SEQ ID NO.1, and a CMV promoter sequence in a HindIII/NruI enzyme cutting site of a pAAV-CMV vector is replaced to obtain pAAV-AAT.

To follow the distribution of AAV in vivo, the EGFP gene (sequence from pEGFP-C1, Clontech) was synthesized and constructed into the EcoRI/BamHI site of pAAV-AAT, resulting in pAAV-EGFP.

To express IFN γ, the mouse IFNG coding sequence (NM-008337.4) was synthesized and constructed into the EcoRI/BamHI sites of pAAV-AAT to give pAAV-IFNr.

To obtain AAV2/8 for liver-specific infection, VP1 gene (https:// www.ncbi.nlm.nih.gov/nuccore/af513852) of AAV8 was synthesized in place of VP1 of AAV2 between SwaI/NdeI sites of the original pRC2-mi342 vector, resulting in pAAV 2/8.

Packaging of AAV 2/8: for packaging chimeric AAV2/8, 6X 10 was used⁶293T cells were seeded 24 hours earlier on T75 plates, 9. mu.g each of pHelper, pAAV2/8 and vector (pAAV-AAT, pAAV-EGFP or pAAV-IFNr) were co-transfected into 293T cells, and the cells were changed 12 hours later. After 60 hours, 15ml of culture supernatant was collected. Digesting the cells and collecting the cell precipitate. Using 3ml of lysine buffer (50mM Tris,150mM NaCl,2mM MgCl)₂pH 8.0), repeated freeze-thawing three times. Cell debris was removed by centrifugation at 3000rpm x 5 min. Nuclease benzonase (100U/ml) was added and incubated at 37 ℃ for 1 hour. 15ml of the culture supernatant and 3ml of the cell lysate were mixed, and 4.5ml of PEG8000 solution (40% PEG8000, 2.5N NaCl) was added. Ice-bath for 2 hours, and centrifugation at 2500g × 30min at 4 ℃. The pellet was resuspended in 13ml DMEM and filtered through a 0.45 μm filter to remove impurities. Ultracentrifugation was carried out at 4 ℃ for 150,000g x 3 hours, and the pellet was resuspended in 2ml of serum-free 1640 medium.

Mouse in vivo experiment with AAV2/8 Virus: to test the liver-specific targeting of AAV2/8, 10 was selected⁷Injecting 100 μ l AAV-EGFP virus into mouse via tail vein, taking core, liver, pancreas, stomach, intestine, spleen 7 days later, and detecting green fluorescence signal in each organ under body type fluorescence microscope (Leica).

For tumor treatment with AAV vectors, tumor cells were injected splenically into mice and tail vein injected 10⁷100 μ l AAV-IFNr, and four weeks later, liver was taken to examine tumor growth.

The invention prepares chimeric AAV2/8 specifically targeting liver, and the carrier for IFN gamma delivery is shown in figure 4. Meanwhile, AAV2/8 capable of expressing Enhanced Green Fluorescent Protein (EGFP), namely AAV-GFP, is prepared, and the specific targeting of the virus vector to the liver is observed. The invention discovers that after AAV-GFP is injected into tail vein for one week, the liver of mouse can see obvious green fluorescence, and other organs have no obvious fluorescence signal as shown in figure 5.

As shown in FIG. 6, the present example examined IFN γ levels in the liver of AAV-IFNr-infected mice one week later, and found that IFN γ levels were significantly increased in the liver of AAV-IFNr-infected mice as compared to the control group.

The present example evaluates the effect of AAV-IFNr on liver tumorigenesis and growth. The invention discovers that the AAV empty vector injected alone can not obviously influence the tumor formation, but the liver of the mouse injected with AAV-IFNr has no macroscopic tumor formation as shown in figure 7, which proves that the expression of the liver targeting IFN gamma has strong capacity of inhibiting the tumor formation in the liver.

The invention detects the activation condition of NK cells and macrophages in the liver after AAV-IFNr infection. The invention discovers that after AAV-IFNr infection, the expression of a marker NK1.1 for NK cell activation and a marker iNOS for macrophage M1 type polarization are obviously up-regulated compared with an empty vector control group and an uninfected group, as shown in figure 8, the expression of IFN gamma activates NK cells and promotes M1 type polarization of macrophages, and the mechanism is probably an IFN gamma mediated anti-tumor mechanism.

The invention discovers that YB1 can inhibit the growth of tumor in mouse liver by inducing IFN gamma generation, and the liver target expression IFN gamma can activate NK cell, promote macrophage M1 type polarization, reproduce and even generate anti-tumor effect better than YB1, and has great potential as a novel therapy for treating tumor generation and growth in liver.

Although the invention has been described in detail above with reference to a general description and specific examples, it will be apparent to one skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Zhongri friendly Hospital (Zhongri friendly institute of clinical medicine), Shanghai City eastern Hospital (affiliated eastern Hospital of Tongji university)

Application of liver targeting gamma-interferon expression vector in preparation of anti-liver tumor drugs

<130> GG211040016A

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1039

<212> DNA

<213> Artificial Sequence

<400> 1

ggctcagagg cacacaggag tttctgggct caccctgccc ccttccaacc cctcagttcc 60

catcctccag cagctgtttg tgtgctgcct ctgaagtcca cactgaacaa acttcagcct 120

actcatgtcc ctaaaatggg caaacattgc aagcagcaaa cagcaaacac acagccctcc 180

ctgcctgctg accttggagc tggggcagag gtcagagacc tctctgggcc catgccacct 240

ccaacatcca ctcgacccct tggaatttcg gtggagagga gcagaggttg tcctggcgtg 300

gtttaggtag tgtgagaggg ggctcagagg cacacaggag tttctgggct caccctgccc 360

ccttccaacc cctcagttcc catcctccag cagctgtttg tgtgctgcct ctgaagtcca 420

cactgaacaa acttcagcct actcatgtcc ctaaaatggg caaacattgc aagcagcaaa 480

cagcaaacac acagccctcc ctgcctgctg accttggagc tggggcagag gtcagagacc 540

tctctgggcc catgccacct ccaacatcca ctcgacccct tggaatttcg gtggagagga 600

gcagaggttg tcctggcgtg gtttaggtag tgtgagaggg agatcttgct accagtggaa 660

cagccactaa ggattctgca gtgagagcag agggccagct aagtggtact ctcccagaga 720

ctgtctgact cacgccaccc cctccacctt ggacacagga cgctgtggtt tctgagccag 780

gtacaatgac tcctttcggt aagtgcagtg gaagctgtac actgcccagg caaagcgtcc 840

gggcagcgta ggcgggcgac tcagatccca gccagtggac ttagcccctg tttgctcctc 900

cgataactgg ggtgaccttg gttaatattc accagcagcc tcccccgttg cccctctgga 960

tcgactgctt aaatacggac gaggacaggg ccctgtctcc tcagcttcag gcaccaccac 1020

tgacctggga cagtgaatc 1039

Claims (8)

1. The application of the liver targeting interferon-gamma expression vector in preparing the medicine for treating and/or preventing liver tumor is provided, wherein the interferon-gamma is expressed in a liver region in a targeting way through the liver targeting interferon-gamma expression vector, NK cells are activated, and M1 type polarization of macrophages is promoted to treat and/or prevent the liver tumor.

2. The use according to claim 1,

the liver targeting gamma-interferon expression vector is AAV-IFNr.

3. The use according to claim 2,

the construction process of the AAV-IFNr comprises the following steps:

constructing an expression vector pAAV-AAT containing an apoE enhancer-AAT promoter;

constructing a mouse IFNG coding sequence to the expression vector pAAV-AAT to obtain pAAV-IFNr;

pHelper, pAAV2/8 and pAAV-IFNr were co-transfected into 293T cells to obtain AAV-IFNr.

4. A composition for treating or preventing a tumor comprising the liver-targeting interferon-gamma expression vector of claim 1 or 2.

5. The composition of claim 4,

the liver targeting gamma-interferon expression vector is AAV-IFNr.

6. A product, the active component of which is a liver targeting gamma-interferon expression vector, the product is used by any one of the following:

(a) activating NK cells;

(b) promoting M1 type polarization of macrophages;

(c) treating tumor.

7. The product of claim 6,

the tumor is liver tumor.

8. The product of claim 6,

the liver targeting interferon-gamma expression vector is used as a delivery vector of IFN gamma, and the liver targeting expression of IFN gamma.

Images