Non-human mammal model expressing IL-8 and application thereof

Abstract

The invention relates to a non-human mammal model expressing IL-8 (interleukin-8) and application of the non-human mammal model. In particular, the present invention provides a non-human mammalian model that expresses IL-8. The non-human mammal model expressing IL-8 can be better used for constructing a tumor animal model, the tumor animal model is a non-human mammal tumor model expressing IL-8, and the tumor animal model can be used for researching the influence of IL-8 on tumors. And can be used for screening or identifying potential therapeutic agents for preventing and / or treating tumors.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a non-human mammal model expressing IL-8 and uses thereof. Background Art

[0002] Cancer is a disease that poses a serious threat to human health, and the research and development of anti-tumor drugs has always been a hot topic in drug research and development.

[0003] Tumor animal models can be used to effectively accelerate the development of anticancer drugs, and tumor animal models can reduce the cost of anticancer drug development. However, existing tumor animal models still have many disadvantages, such as the slow growth of constructed tumors and tumor blood vessels, which increases the cost of constructing tumor animal models and makes it difficult to effectively use them for anticancer drug screening, thus limiting the development of tumor drugs.

[0004] Therefore, there is a need in the art to prepare an animal model for promoting the development of anti-tumor drugs. Summary of the invention

[0005] The object of the present invention is to provide a non-human mammal model expressing IL-8, which can be better used to construct a tumor animal model, and the tumor animal model can be used to study the effect of IL-8 on tumors and to screen or identify potential therapeutic agents for preventing and / or treating tumors.

[0006] Another object of the present invention is to provide a use of an IL-8 inhibitor for enhancing the anti-tumor effect of a PD-1 inhibitor.

[0007] A first aspect of the present invention provides a non-human mammal model expressing IL-8.

[0008] In another preferred embodiment, the IL-8 includes human IL-8 or primate IL-8.

[0009] In another preferred embodiment, the non-human mammal is a rodent.

[0010] In another preferred embodiment, the non-human mammals include mice, rats, rabbits and / or monkeys.

[0011] In another preferred embodiment, the mice include C57BL / 6 mice.

[0012] In another preferred embodiment, the expression of IL-8 includes expression of IL-8 in serum or expression of IL-8 in tissue sites.

[0013] In another preferred embodiment, the non-human mammal model expressing IL-8 is heterozygous or homozygous.

[0014] The second aspect of the present invention provides a method for preparing a non-human mammal model expressing IL-8 as described in the first aspect of the present invention, comprising the steps of:

[0015] The non-human mammal is caused to express IL-8, thereby obtaining a non-human mammal model expressing IL-8.

[0016] In another preferred embodiment, the method for preparing the non-human mammal model expressing IL-8 comprises the steps of:

[0017] A non-human mammal model expressing IL-8 is obtained by making the non-human mammal contain a gene expressing IL-8.

[0018] In another preferred embodiment, the method for preparing the non-human mammal model expressing IL-8 comprises the steps of:

[0019] The gene expressing IL-8 is inserted into a non-human mammal so that the non-human mammal expresses IL-8, thereby obtaining a non-human mammal model expressing IL-8.

[0020] In another preferred embodiment, the gene expressing IL-8 is located in the genome of a non-human mammal.

[0021] In another preferred embodiment, the gene expressing IL-8 is located at positions 3023490-3024411 of chromosome 9 of non-human mammalian cells.

[0022] In another preferred embodiment, the method for preparing the non-human mammal model expressing IL-8 comprises the steps of:

[0023] (a) providing non-human mammalian cells, and causing the cells to express IL-8, thereby obtaining non-human mammalian cells expressing IL-8;

[0024] (b) Using the non-human mammal cells expressing IL-8 obtained in step (a), a non-human mammal model expressing IL-8 is prepared.

[0025] In another preferred embodiment, in step (a), the genome of the cell contains a gene expressing IL-8.

[0026] In another preferred embodiment, the step (a) comprises:

[0027] Non-human mammalian cells are provided, and a gene expressing IL-8 is inserted into the cells, thereby obtaining non-human mammalian cells expressing IL-8.

[0028] In another preferred embodiment, the step (a) comprises:

[0029] By using gene insertion technology, the gene expressing IL-8 is inserted into the genome of non-human mammalian cells, thereby obtaining non-human mammalian cells expressing IL-8.

[0030] In another preferred embodiment, the step (b) comprises:

[0031] (b1) preparing a chimeric non-human mammal using the non-human mammal cells expressing IL-8 obtained in step (a);

[0032] (b2) mating and breeding the chimeric non-human mammal obtained in step (b1) with a normal wild-type non-human mammal, and screening the offspring to obtain heterozygous non-human mammals expressing IL-8;

[0033] (b3) obtaining a non-human mammal model expressing IL-8 (homozygous non-human mammal) by mating the heterozygous non-human mammals obtained in step (b2) with each other.

[0034] The third aspect of the present invention provides a use of the non-human mammal model expressing IL-8 as described in the first aspect of the present invention in preparing a tumor model of a non-human mammal expressing IL-8.

[0035] In another preferred embodiment, the non-human mammal tumor model expressing IL-8 carries a tumor and expresses IL-8.

[0036] In another preferred embodiment, the non-human mammal tumor model expressing IL-8 is a non-human mammal model expressing IL-8 and bearing a tumor.

[0037] In another preferred embodiment, the non-human mammal model expressing IL-8 is as described in the first aspect of the present invention.

[0038] In another preferred embodiment, the tumor includes a human tumor or a non-human mammal tumor.

[0039] In another preferred embodiment, the non-human mammal is a rodent.

[0040] In another preferred embodiment, the non-human mammals include mice, rats, rabbits and / or monkeys.

[0041] In another preferred embodiment, the mice include C57BL / 6 mice.

[0042] In another preferred embodiment, the tumor includes one or more of colon cancer, lung cancer, breast cancer, pancreatic cancer, brain tumor and leukemia.

[0043] In another preferred embodiment, the leukemia includes acute myeloid leukemia M5.

[0044] In another preferred embodiment, the tumor cells of the tumor include one or more of glioma cells, GL261 cells, U373 cells and GBM5 cells.

[0045] In another preferred embodiment, the tumor includes one or more of glioblastoma, glioma, and astrocytoma.

[0046] In another preferred embodiment, the tumor is a fluorescently labeled tumor.

[0047] In another preferred embodiment, the tumor-bearing site includes one or more of intracranial and subcutaneous sites.

[0048] In another preferred embodiment, the tumor-bearing site includes the intracranial brain site.

[0049] A fourth aspect of the present invention provides a tumor model of a non-human mammal expressing IL-8.

[0050] In another preferred embodiment, the non-human mammal tumor model expressing IL-8 carries a tumor and expresses IL-8.

[0051] In another preferred embodiment, the non-human mammal tumor model expressing IL-8 is a non-human mammal model expressing IL-8 and bearing a tumor.

[0052] In another preferred embodiment, the non-human mammal model expressing IL-8 is as described in the first aspect of the present invention.

[0053] In another preferred embodiment, the non-human mammal tumor model expressing IL-8 is heterozygous or homozygous.

[0054] In another preferred embodiment, the tumor includes a human tumor or a non-human mammal tumor.

[0055] In another preferred embodiment, the non-human mammal is a rodent.

[0056] In another preferred embodiment, the non-human mammals include mice, rats, rabbits and / or monkeys.

[0057] In another preferred embodiment, the mice include C57BL / 6 mice.

[0058] In another preferred embodiment, the tumor includes one or more of colon cancer, lung cancer, breast cancer, pancreatic cancer, brain tumor and leukemia.

[0059] In another preferred embodiment, the leukemia includes acute myeloid leukemia M5.

[0060] In another preferred embodiment, the tumor cells of the tumor include one or more of glioma cells, GL261 cells, U373 cells and GBM5 cells.

[0061] In another preferred embodiment, the tumor includes one or more of glioblastoma, glioma and astrocytoma.

[0062] In another preferred embodiment, the tumor is a fluorescently labeled tumor.

[0063] In another preferred embodiment, the tumor-bearing site includes one or more of intracranial and subcutaneous sites.

[0064] In another preferred embodiment, the tumor-bearing site includes the intracranial brain site.

[0065] The fifth aspect of the present invention provides a method for preparing a tumor model of a non-human mammal expressing IL-8 as described in the fourth aspect of the present invention, comprising the steps of:

[0066] (i) preparing a non-human mammal model expressing IL-8;

[0067] (ii) The non-human mammal model expressing IL-8 is allowed to bear a tumor, thereby obtaining a non-human mammal tumor model expressing IL-8.

[0068] In another preferred embodiment, in the step (i), the preparation of the non-human mammal model expressing IL-8 is as described in the second aspect of the present invention.

[0069] In another preferred embodiment, the step (ii) comprises:

[0070] Tumor cells are inoculated into a non-human mammal model expressing IL-8, thereby obtaining a non-human mammal tumor model expressing IL-8.

[0071] In another preferred embodiment, compared with a tumor model of a non-human mammal that does not express IL-8, the tumor model of the non-human mammal that expresses IL-8 comprises one or more features selected from the following group:

[0072] (t1) expression of IL-8;

[0073] (t2) accelerated tumor growth;

[0074] (t3) tumor size increases;

[0075] (t4) increased tumor vascular density;

[0076] (t5) increased expression of IL-8 in CD4+T cells;

[0077] (t6) increased expression of IL-8 in CD45+CD3- myeloid cells; and / or

[0078] (t7) Tumor-infiltrating CD4+ and CD8+ T cells carry more exhaustion markers PD-1 and TIM-3.

[0079] In another preferred embodiment, the expression of IL-8 includes expression of IL-8 in serum or expression of IL-8 in tissue sites.

[0080] The sixth aspect of the present invention provides a use of a non-human mammal tumor model expressing IL-8 as described in the fourth aspect of the present invention as an animal model in studying tumor diseases.

[0081] The seventh aspect of the present invention provides a use of the non-human mammal tumor model expressing IL-8 as described in the fourth aspect of the present invention as an animal model for studying the effect of IL-8 on tumors.

[0082] In another preferred embodiment, the influence includes growth influence.

[0083] The eighth aspect of the present invention provides a non-human mammal tumor model expressing IL-8 as described in the fourth aspect of the present invention, for use as an animal model in screening or identifying therapeutic agents for preventing and / or treating tumors.

[0084] In another preferred embodiment, the therapeutic agent comprises a drug.

[0085] The ninth aspect of the present invention provides a method for screening or identifying a potential therapeutic agent for preventing and / or treating tumors, comprising the steps of:

[0086] (1) In a test group, in the presence of a test substance, the test substance is administered to the tumor model of a non-human mammal expressing IL-8 as described in the fourth aspect of the present invention, and the growth condition Q1 of the tumor in the tumor model of a non-human mammal expressing IL-8 in the test group is detected; and in a control group in which the test substance is not administered and other conditions are the same, the growth condition Q2 of the tumor in the tumor model of a non-human mammal expressing IL-8 in the control group is detected;

[0087] (2) comparing the growth status Q1 and the growth status Q2 of the tumor detected in step (1) to determine whether the test substance is a potential therapeutic agent for preventing and / or treating tumors;

[0088] If the growth condition Q1 of the tumor is lower than the growth condition Q2, it indicates that the test substance is a potential therapeutic agent for preventing and / or treating tumors.

[0089] In another preferred example, the growth condition of the tumor includes changes in one or more indicators selected from the following group: changes in tumor morphology, changes in tumor volume, changes in tumor weight, or a combination thereof.

[0090] In another preferred example, the "lower than" includes significantly lower than.

[0091] Preferably, the "lower than" means that the tumor growth condition Q1 of the test group with biological replicates after administration of the test substance is lower than the tumor growth condition Q2 of the control group with biological replicates after administration of the test substance, and the P value after t-test is less than 0.05.

[0092] In another preferred embodiment, the "lower than" refers to that the ratio of the tumor growth state Q1 to the tumor growth state Q2 is less than 1.0, preferably ≤0.9, more preferably ≤0.8, more preferably ≤0.7, more preferably ≤0.6, more preferably ≤0.5, more preferably ≤0.4, more preferably ≤0.3, more preferably ≤0.2, more preferably ≤0.1, more preferably ≤0.05, more preferably ≤0.01.

[0093] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0094] In another preferred embodiment, the test substance is selected from the following group: antibodies, small molecule compounds, nucleic acids, or a combination thereof.

[0095] In another preferred embodiment, the test substance includes one or more of an IL-8 inhibitor and a PD-1 inhibitor.

[0096] In another preferred embodiment, the potential therapeutic agent is selected from the following group: antibodies, small molecule compounds, nucleic acids, or a combination thereof.

[0097] In another preferred embodiment, the potential therapeutic agent includes one or more of an IL-8 inhibitor and a PD-1 inhibitor.

[0098] In another preferred embodiment, the inhibitor is selected from the following group: antibodies, small molecule compounds, nucleic acids, or a combination thereof.

[0099] In another preferred embodiment, the IL-8 inhibitor comprises an anti-IL-8 antibody.

[0100] In another preferred embodiment, the PD-1 inhibitor includes an anti-PD-1 antibody.

[0101] In another preferred embodiment, the inhibitor includes a specific inhibitor or a non-specific inhibitor.

[0102] The tenth aspect of the present invention provides a pharmaceutical composition comprising an IL-8 inhibitor and a PD-1 inhibitor; and a pharmaceutically acceptable carrier.

[0103] In another preferred embodiment, the dosage form of the pharmaceutical composition is a solid preparation, a liquid preparation or a semisolid preparation.

[0104] In another preferred embodiment, the dosage form of the pharmaceutical composition is an oral preparation, an external preparation or an injection preparation.

[0105] In another preferred embodiment, the injection preparation is an intravenous injection preparation.

[0106] In another preferred embodiment, the dosage form of the pharmaceutical composition is tablets, injections, infusions, ointments, gels, solutions, microspheres or films.

[0107] In another preferred embodiment, based on the weight of the pharmaceutical composition, the content of the IL-8 inhibitor is 0.001-99.9 wt %.

[0108] In another preferred embodiment, based on the weight of the pharmaceutical composition, the content of the PD-1 inhibitor is 0.001-99.9 wt %.

[0109] The eleventh aspect of the present invention provides a use of the pharmaceutical composition according to the tenth aspect of the present invention in the preparation of a medicament for preventing and / or treating tumors.

[0110] In another preferred embodiment, the tumor includes a human tumor or a non-human mammal tumor.

[0111] In another preferred embodiment, the non-human mammal is a rodent.

[0112] In another preferred embodiment, the non-human mammals include mice, rats, rabbits and / or monkeys.

[0113] In another preferred embodiment, the mice include C57BL / 6 mice.

[0114] In another preferred embodiment, the tumor includes one or more of colon cancer, lung cancer, breast cancer, pancreatic cancer, brain tumor and leukemia.

[0115] In another preferred embodiment, the leukemia includes acute myeloid leukemia M5.

[0116] In another preferred embodiment, the tumor cells of the tumor include one or more of glioma cells, GL261 cells, U373 cells and GBM5 cells.

[0117] In another preferred embodiment, the tumor includes one or more of glioblastoma, glioma and astrocytoma.

[0118] In another preferred embodiment, the tumor is a fluorescently labeled tumor.

[0119] The twelfth aspect of the present invention provides a method for preventing and / or treating tumors, comprising the step of administering the pharmaceutical composition as described in the tenth aspect of the present invention to a subject in need thereof.

[0120] In another preferred embodiment, the subject includes a human or a non-human mammal.

[0121] In another preferred embodiment, the non-human mammals include rodents, rabbits, monkeys, livestock, dogs, cats, etc.

[0122] In another preferred embodiment, the administration is injection or oral administration.

[0123] In another preferred embodiment, the injection administration is intravenous injection administration.

[0124] The thirteenth aspect of the present invention provides a use of an IL-8 inhibitor in the preparation of a pharmaceutical composition for enhancing the anti-tumor effect of a PD-1 inhibitor.

[0125] In another preferred embodiment, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0126] In another preferred embodiment, the dosage form of the pharmaceutical composition is a solid preparation, a liquid preparation or a semisolid preparation.

[0127] In another preferred embodiment, the dosage form of the pharmaceutical composition is an oral preparation, an external preparation or an injection preparation.

[0128] In another preferred embodiment, the injection preparation is an intravenous injection preparation.

[0129] In another preferred embodiment, the dosage form of the pharmaceutical composition is tablets, injections, infusions, ointments, gels, solutions, microspheres or films.

[0130] In another preferred embodiment, the tumor includes a human tumor or a non-human mammal tumor.

[0131] In another preferred embodiment, the non-human mammal is a rodent.

[0132] In another preferred embodiment, the non-human mammals include mice, rats, rabbits and / or monkeys.

[0133] In another preferred embodiment, the mice include C57BL / 6 mice.

[0134] In another preferred embodiment, the tumor includes one or more of colon cancer, lung cancer, breast cancer, pancreatic cancer, brain tumor and leukemia.

[0135] In another preferred embodiment, the leukemia includes acute myeloid leukemia M5.

[0136] In another preferred embodiment, the tumor cells of the tumor include one or more of glioma cells, GL261 cells, U373 cells and GBM5 cells.

[0137] In another preferred embodiment, the tumor includes one or more of glioblastoma, glioma and astrocytoma.

[0138] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0139] Figure 1The preparation of humanized mice carrying the human IL-8 gene locus (IL8-Hu) is shown; wherein, (1A) shows a human BAC clone (PiggyBac-on-BAC RP11-997L11) containing the human IL-8 gene, wherein the human IL-8 gene contains the entire human IL-8 gene locus and its upstream and downstream regulatory elements, and the human IL-8 gene is inserted into the C57BL / 6 mouse genome; 4 pairs of BAC-specific primers that amplify different parts of the BAC plasmid to 300-800 bp fragments are used to perform PCR analysis and identification on BAC-positive pups; the offspring carrying the complete human IL-8 gene locus are backcrossed with C57BL / 6 mice for 5 generations; (1B) shows the insertion site of IL-8 in the mouse genome; as described above, reverse transcription PCR is used to determine the IL-8 insertion site; briefly, genomic DNA is extracted from the tail, digested with HaeIII (NEB, Cat#R0108S), and then incubated with T4 DNA ligase (NEB, Cat#M0202L) was used for ligation; the primers used to restore the left flanking sequence of the piggyBac transposon were PB-5IF (5'-CTTGACCTTGCCACAGAGGACTATTAGAGG-3' (SEQ ID NO: 1)) and PB-5IR (5'-CAGTGACACTTACCGCATTGACAAGCACGC-3' (SEQ ID NO: 2)); the primers used to restore the right flanking sequence of the piggyBac transposon were PB-3IF (5'-CCTCGATATACAGACCGATAAAACACATGC-3' (SEQ ID NO: 3)) and PB-3IR (5'-AGTCAGTCAGAAACAACTTTGGCACATATC-3' (SEQ ID NO: 4)); the PCR product was cloned into pMD19-T (SinoBiological) for subsequent sequencing; the sequencing results were analyzed by NCBI BLAST search and human and mouse genome integrated database were used for analysis; the human IL-8 gene locus was inserted into the mouse chromosome 9 at position 3023490-3024411.

[0140] Figure 2 The percentage of immune cells in IL8-Hu mice is shown; wherein, (2A), (2B), and (2C) flow cytometry analysis of the percentage of immune cells in IL8-Hu mice; macrophages: CD11b+F4 / 80+; granulocytes: CD11b+Ly6G+; regulatory T cells: CD4+Foxp3+; B cells: B220+; conventional dendritic cells (cDCs): MHCII+CD11c+; spleen cDC1: MHCII+CD11c+CD8+; spleen cDC2: MHCII+CD11c+CD11b+; T cells (Tn): CD62L+CD44-; effector T cells (Teff): CD62L-CD44+; central memory T cells (Tcm): CD62L+CD44+; immature cDC: IA / I-Emid CD11c+; mature cDC: IA / I-Ehi CD11c+; SP: single positive cells; DP: CD4, CD8 double positive cells.

[0141] Figure 3 The tumor growth in IL8-Hu mice is shown; wherein, (3A) in vivo bioluminescence imaging of tumor growth: 10,000 GL261-Luc cells were implanted into the brain of WT or IL8-Hu mice; 25 days later, tumor growth was measured; the scale indicates that the luminescence intensity is p / s / cm2 / sr; (3B) is the survival curve of WT and IL8-Hu mice bearing GL261 tumors: 10,000 GL261-Luc cells were injected into the corpus callosum and their survival was monitored; WT, n=8, IL8-Hu, n=7; ***P<0.001, log-rank test; (3C) is the survival curve of GL261 tumor in IL8-Hu mice Figure 3 Flow cytometric analysis of IL-8 producing cells in GL261 tumors: WT or IL8-Hu mice were implanted with GL261 tumors; mice were sacrificed on day 20; Percoll-enriched cells were stimulated with PMA / ionomycin in the presence of Golgi plug and analyzed by flow cytometry staining; red indicates CXCR5+CD4+ T cells; graphical data are presented as mean ± SD; ****P < 0.0001, two-tailed t-test; (3D) IHC staining of CD31 in GL261 tumors: CD31+ area in each view was analyzed and quantified using ImageJ software; graphical data are presented as mean ± SD; ****P < 0.0001, two-tailed t-test.

[0142] Figure 4 It shows that T cells in IL8-Hu mouse tumors are in a more exhausted state; wherein, (4A) and (4B) show flow cytometry analysis of PD-1 and TIM-3 expression on T cells; graphical data are expressed as mean ± SD; *P < 0.05, **P < 0.01, two-tailed t-test.

[0143] Figure 5Figure 5 shows that systemic expression of IL-8 is increased after anti-PD-1 treatment; wherein, (5A) in vivo bioluminescence imaging of tumor growth: 10,000 GL261-Luc-IL8 cells were implanted into the brain of IL8-Hu mice; starting from day 7, anti-PD-1 (RMP1-14) antibody or isotype control antibody was intraperitoneally injected at a dose of 200 μg / mouse every 3 days, for a total of 4 injections; on day 26, imaging showed tumor growth; the scale indicates that the luminescence intensity is p / s / cm2 / sr; (5B) flow cytometry analysis of PD-1 and TIM-3 expression in tumor T cells; graphical data are expressed as mean ± SD; **P < 0.01, two-tailed t-test; (5C) loading of GL261-Luc after anti-PD-1 treatment or without treatment IL-8 immunohistochemical staining of IL8-Hu mice with tumors: 10,000 tumor cells were implanted into the brains of IL8-Hu mice; anti-PD-1 antibody was intraperitoneally injected at a dose of 200 μg / mouse on day 18; IL-8 expression was detected by IHC staining 5 days later; the number of IL-8+ dots and IL-8+ area in each view were analyzed using ImageJ software; graphical data are presented as mean ± SD; ****P < 0.0001, two-tailed t-test; (5D) Serum was collected on day 5 after antibody treatment, and IL-8 levels were detected by ELISA; graphical data are presented as mean ± SD; ***P < 0.001, two-tailed t-test; (5E) Flow cytometric analysis of tumor MDSCs: Percoll density gradient centrifugation was used to enrich the IL-8+ cells from Immune cells from the brains of WT or IL8-Hu mice (top) or tumor-bearing IL8-Hu mice treated with anti-PD-1 antibody or isotype antibody (bottom) were stained by flow cytometry, and the statistical analysis results are on the right; graphical data are expressed as mean ± SD; *P < 0.05, **P < 0.01, two-tailed t test; (5F) Representative patterns of Ly6C and Ly6G staining in the indicated tissues; (5G) Flow cytometric analysis of MDSCs in the indicated tissues of tumor-bearing mice with or without anti-PD-1 treatment; statistical analysis is as follows: graphical data are expressed as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed t test.

[0144] Figure 6It shows that blockade of the IL-8-CXCR1 / CXCR2 axis enhances the efficacy of anti-PD-1; wherein, (6A) in vivo bioluminescence imaging of tumor growth: 20,000 GL261-Luc cells were implanted into the brains of WT or IL8-Hu mice; starting from the 7th day, anti-PD-1 (clone RMP1-14) antibody or isotype IgG was intraperitoneally injected at a dose of 200 μg / mouse every 3 days, for a total of 4 injections; at the same time, another part of the mice was intraperitoneally injected with 50 μg of Reparixin every other day until death; on the 20th day, tumor growth imaging was performed; the scale indicates that the luminescence intensity is p / s / cm2 / sr; (6B) survival curves of mice treated with different methods in part A; *P<0.05, **P<0.01, ***P<0.001, log-rank test; (6C) flow cytometry was used to detect cells expressing Gr1 and CD11b in the tumor on the 21st day after tumor injection. (6D) Statistical analysis of the percentage and number of CD11bhi Gr1mid, CD11bhi Gr1hi, and CD11bhiGr1 cells in tumors: 95% CIs are shown in translucent shades (brown for WT mice and cyan for IL8-Hu mice); brown stars indicate the corresponding P values ​​between the WT group, and cyan stars indicate the corresponding P values ​​between the IL8-Hu group; dark stars show the corresponding P values ​​between the WT group and the IL8-Hu group; graphical data are expressed as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, t two-tailed test.

[0145] Figure 7 It shows that neutralizing IL-8 enhances the efficacy of anti-PD-1; wherein, (7A) Schematic diagram of the experimental method: 500 GL261-Luc-IL8 cells were implanted into the brain of IL8-Hu mice; starting from the 7th day, each mouse was intraperitoneally injected with 200μg anti-PD-1 antibody (clone RMP1-14) or 200μg anti-IL-8 antibody (clone 6217); on the 26th day, tumor growth was shown; (7B) as Figure 6 In vivo bioluminescence imaging of tumor growth on day 26 of mice treated as shown in part F of FIG. ; the scale indicates the luminescence intensity in p / s / cm2 / sr; (7C) as Figure 6 Survival curves of mice treated with different methods are shown in Part F; ****P<0.0001, log-rank test; (7D) Immunohistochemical staining of tumor tissue with anti-CD31 antibody: The CD31+ area (vascular area) of each view was counted using ImageJ software; graphical data are expressed as mean ± SD; *P<0.05, ***P<0.001, ****P<0.0001, t two-tailed test. DETAILED DESCRIPTION

[0146] The present invention provides a non-human mammal model expressing IL-8, which can be better used to construct a tumor animal model, and the tumor animal model can be used to study the effect of IL-8 on tumors and to screen or identify potential therapeutic agents for preventing and / or treating tumors. The present invention also provides a use of an IL-8 inhibitor in enhancing the anti-tumor effect of a PD-1 inhibitor.

[0147] the term

[0148] As used herein, the terms "include", "comprises", and "comprising" are used interchangeably, and include not only open definitions, but also semi-closed and closed definitions. In other words, the terms include "consisting of", "consisting essentially of".

[0149] As used herein, "interleukin 8" is abbreviated as IL-8.

[0150] As used herein, "PD-1" refers to programmed death-1, also known as programmed cell death protein 1.

[0151] IL-8

[0152] In the present invention, IL-8 refers to interleukin-8.

[0153] In a preferred embodiment, IL-8 refers to human IL-8 or primate IL-8.

[0154] In another preferred embodiment, the expression of IL-8 includes serum expression of IL-8 or tissue expression of IL-8I. In the present invention, it was unexpectedly found that IL-8 inhibitors can significantly enhance the anti-tumor effect of PD-1 inhibitors, and the combined administration of PD-1 inhibitors and IL-8 inhibitors can significantly improve the anti-tumor effect.

[0155] Non-human mammals

[0156] The non-human mammal described in the present invention may be a rodent.

[0157] In a preferred embodiment, the non-human mammals include mice, rats, rabbits and / or monkeys.

[0158] In another preferred embodiment, the mice include C57BL / 6 mice.

[0159] Tumor

[0160] In the present invention, the tumor may be a human tumor or a non-human animal tumor.

[0161] In a preferred example, the tumor includes one or more of colon cancer, lung cancer, breast cancer, pancreatic cancer, brain tumor and leukemia.

[0162] Specifically, the leukemia includes acute myeloid leukemia M5.

[0163] In another preferred embodiment, the tumor cells of the tumor include one or more of glioma cells, GL261 cells, U373 cells and GBM5 cells.

[0164] In another preferred embodiment, the tumor includes one or more of glioblastoma, glioma and astrocytoma.

[0165] In another preferred embodiment, the tumor is a fluorescently labeled tumor.

[0166] Non-human mammal model expressing IL-8, preparation method and use thereof

[0167] The present invention provides a non-human mammal model expressing IL-8.

[0168] In a preferred example, the expression of IL-8 includes expression of IL-8 in serum or expression of IL-8 in tissues.

[0169] The present invention provides a method for preparing a non-human mammal model expressing IL-8, comprising the steps of:

[0170] The non-human mammal is caused to express IL-8, thereby obtaining a non-human mammal model expressing IL-8.

[0171] In a preferred embodiment, the method for preparing a non-human mammal model expressing IL-8 comprises the steps of:

[0172] The non-human mammal is made to contain a gene expressing IL-8, thereby obtaining a non-human mammal model expressing IL-8.

[0173] In another preferred embodiment, the method for preparing a non-human mammal model expressing IL-8 comprises the steps of:

[0174] The gene expressing IL-8 is inserted into a non-human mammal so that the non-human mammal expresses IL-8, thereby obtaining a non-human mammal model expressing IL-8.

[0175] In another preferred embodiment, the gene expressing IL-8 is located in the genome of a non-human mammal.

[0176] In another preferred embodiment, the gene expressing IL-8 is located at positions 3023490-3024411 of chromosome 9 of non-human mammalian cells.

[0177] In another preferred embodiment, the method for preparing a non-human mammal model expressing IL-8 comprises the following steps:

[0178] (a) providing non-human mammalian cells, and causing the cells to express IL-8, thereby obtaining non-human mammalian cells expressing IL-8;

[0179] (b) Using the non-human mammal cells expressing IL-8 obtained in step (a), a non-human mammal model expressing IL-8 is prepared.

[0180] In another preferred embodiment, in step (a), the genome of the cell contains a gene expressing IL-8.

[0181] In another preferred embodiment, the step (a) comprises:

[0182] Non-human mammalian cells are provided, and a gene expressing IL-8 is inserted into the cells, thereby obtaining non-human mammalian cells expressing IL-8.

[0183] In another preferred embodiment, the step (a) comprises:

[0184] By using gene insertion technology, the gene expressing IL-8 is inserted into the genome of non-human mammalian cells, thereby obtaining non-human mammalian cells expressing IL-8.

[0185] In another preferred embodiment, the step (b) comprises:

[0186] (b1) preparing a chimeric non-human mammal using the non-human mammal cells expressing IL-8 obtained in step (a);

[0187] (b2) mating and breeding the chimeric non-human mammal obtained in step (b1) with a normal wild-type non-human mammal, and screening the offspring to obtain heterozygous non-human mammals expressing IL-8;

[0188] (b3) obtaining a non-human mammal model expressing IL-8 (homozygous non-human mammal) by mating the heterozygous non-human mammals obtained in step (b2) with each other.

[0189] The present invention provides a use of a non-human mammal model expressing IL-8 in preparing a tumor model of a non-human mammal expressing IL-8.

[0190] Tumor model of non-human mammal expressing IL-8, preparation method and use thereof

[0191] The present invention provides a tumor model of a non-human mammal expressing IL-8.

[0192] In another preferred embodiment, the non-human mammal tumor model expressing IL-8 is a non-human mammal model expressing IL-8 and bearing a tumor.

[0193] In another preferred embodiment, the tumor-bearing site includes one or more of intracranial and subcutaneous sites.

[0194] In another preferred embodiment, the tumor-bearing site includes the intracranial brain site.

[0195] The present invention provides a method for preparing a tumor model of a non-human mammal expressing IL-8, comprising the steps of:

[0196] (i) preparing a non-human mammal model expressing IL-8;

[0197] (ii) The non-human mammal model expressing IL-8 is allowed to bear a tumor, thereby obtaining a non-human mammal tumor model expressing IL-8.

[0198] In another preferred embodiment, the step (ii) comprises:

[0199] Tumor cells are inoculated into a non-human mammal model expressing IL-8, thereby obtaining a non-human mammal tumor model expressing IL-8.

[0200] In another preferred embodiment, compared with a tumor model of a non-human mammal that does not express IL-8, the tumor model of the non-human mammal that expresses IL-8 comprises one or more features selected from the following group:

[0201] (t1) expression of IL-8;

[0202] (t2) accelerated tumor growth;

[0203] (t3) tumor size increases;

[0204] (t4) increased tumor vascular density;

[0205] (t5) increased expression of IL-8 in CD4+T cells;

[0206] (t6) increased expression of IL-8 in CD45+CD3- myeloid cells; and / or

[0207] (t7) Tumor-infiltrating CD4+ and CD8+ T cells carry more exhaustion markers PD-1 and TIM-3.

[0208] In another preferred embodiment, the expression of IL-8 includes expression of IL-8 in serum or expression of IL-8 in tissue sites.

[0209] The present invention provides a tumor model of a non-human mammal expressing IL-8, which is used as an animal model in studying tumor diseases.

[0210] The present invention provides a tumor model of a non-human mammal expressing IL-8, which is used as an animal model in studying the effect of IL-8 on tumors.

[0211] In another preferred embodiment, the influence includes growth influence.

[0212] The present invention provides a tumor model of a non-human mammal expressing IL-8, which is used as an animal model in screening or identifying therapeutic agents that can prevent and / or treat tumors.

[0213] In another preferred embodiment, the therapeutic agent comprises a drug.

[0214] Screening or identification of potential therapeutic agents for the prevention and / or treatment of tumors

[0215] The present invention provides a method for screening or identifying a potential therapeutic agent for preventing and / or treating tumors, comprising the steps of:

[0216] (1) In a test group, in the presence of a test substance, the test substance is administered to the tumor model of a non-human mammal expressing IL-8 as described in the fourth aspect of the present invention, and the growth condition Q1 of the tumor in the tumor model of a non-human mammal expressing IL-8 in the test group is detected; and in a control group in which the test substance is not administered and other conditions are the same, the growth condition Q2 of the tumor in the tumor model of a non-human mammal expressing IL-8 in the control group is detected;

[0217] (2) comparing the growth status Q1 and the growth status Q2 of the tumor detected in step (1) to determine whether the test substance is a potential therapeutic agent for preventing and / or treating tumors;

[0218] If the growth condition Q1 of the tumor is lower than the growth condition Q2, it indicates that the test substance is a potential therapeutic agent for preventing and / or treating tumors.

[0219] In another preferred example, the growth condition of the tumor includes changes in one or more indicators selected from the following group: changes in tumor morphology, changes in tumor volume, changes in tumor weight, or a combination thereof.

[0220] In another preferred example, the "lower than" includes significantly lower than.

[0221] Preferably, the "lower than" means that the tumor growth condition Q1 of the test group with biological replicates after administration of the test substance is lower than the tumor growth condition Q2 of the control group with biological replicates after administration of the test substance, and the P value after t-test is less than 0.05.

[0222] Preferably, the “lower than” refers to that the ratio of the tumor growth state Q1 to the tumor growth state Q2 is less than 1.0, preferably ≤0.9, more preferably ≤0.8, more preferably ≤0.7, more preferably ≤0.6, more preferably ≤0.5, more preferably ≤0.4, more preferably ≤0.3, more preferably ≤0.2, more preferably ≤0.1, more preferably ≤0.05, more preferably ≤0.01.

[0223] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0224] In another preferred embodiment, the test substance is selected from the following group: antibodies, small molecule compounds, nucleic acids, or a combination thereof.

[0225] In another preferred embodiment, the test substance includes one or more of an IL-8 inhibitor and a PD-1 inhibitor.

[0226] In another preferred embodiment, the potential therapeutic agent is selected from the following group: antibodies, small molecule compounds, nucleic acids, or a combination thereof.

[0227] In another preferred embodiment, the potential therapeutic agent includes one or more of an IL-8 inhibitor and a PD-1 inhibitor.

[0228] In another preferred embodiment, the inhibitor is selected from the following group: antibodies, small molecule compounds, nucleic acids, or a combination thereof.

[0229] In another preferred embodiment, the IL-8 inhibitor comprises an anti-IL-8 antibody.

[0230] In another preferred embodiment, the PD-1 inhibitor includes an anti-PD-1 antibody.

[0231] In another preferred embodiment, the inhibitor includes a specific inhibitor or a non-specific inhibitor.

[0232] Pharmaceutical composition

[0233] The present invention provides a composition, preferably a pharmaceutical composition, comprising an IL-8 inhibitor and a PD-1 inhibitor.

[0234] In a preferred example, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0235] In another preferred embodiment, the dosage form of the pharmaceutical composition is a solid preparation, a liquid preparation or a semisolid preparation.

[0236] In another preferred embodiment, the dosage form of the pharmaceutical composition is an oral preparation, an external preparation or an injection preparation.

[0237] In another preferred embodiment, the injection preparation is an intravenous injection preparation.

[0238] In another preferred embodiment, the dosage form of the pharmaceutical composition is tablets, injections, infusions, ointments, gels, solutions, microspheres or films.

[0239] The term "pharmaceutically acceptable carrier" refers to one or more compatible solid, semisolid, liquid or gel fillers that are suitable for human or animal use and must have sufficient purity and sufficiently low toxicity. "Compatibility" refers to the components of the pharmaceutical composition and the active ingredients of the drug and their mutual blending without significantly reducing the efficacy of the drug.

[0240] It should be understood that in the present invention, the carrier is not particularly limited, and can be selected from commonly used materials in the art, or prepared by conventional methods, or purchased from the market. Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as methylcellulose, ethylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate, etc.), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as Tween), wetting agents (such as sodium lauryl sulfate), buffers, integrators, thickeners, pH regulators, transdermal enhancers, colorants, flavoring agents, stabilizers, antioxidants, preservatives, antibacterial agents, pyrogen-free water, etc.

[0241] Typically, in addition to the active pharmaceutical ingredient, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide, and oil, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or a mixture of these substances, etc. In addition to these inert diluents, the composition may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, etc.

[0242] The pharmaceutical preparation should match the mode of administration. The pharmaceutical preparations involved in the present invention can also be used with other synergistic therapeutic agents (including before, during or after use). When using a pharmaceutical composition or preparation, a safe and effective amount of the drug is administered to the desired subject (such as a human or non-human mammal), and the safe and effective amount is usually at least about 10 μg / kg body weight, and in most cases does not exceed about 8 μg / kg body weight. Preferably, the dose is about 10 μg / kg body weight to 1 mg / kg body weight. It is understandable that the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of professional physicians.

[0243] The main technical effects of the present invention include:

[0244] 1. The present invention prepares a non-human mammal model expressing IL-8, which can be better used to construct a tumor animal model, and the tumor animal model can be used to study the effect of IL-8 on tumors and to screen or identify potential therapeutic agents for preventing and / or treating tumors.

[0245] 2. The present invention also provides a use of an IL-8 inhibitor for enhancing the anti-tumor effect of a PD-1 inhibitor. The combined administration of a PD-1 inhibitor and an IL-8 inhibitor can significantly improve the anti-tumor effect.

[0246] The present invention is further described below in conjunction with specific examples. It should be understood that the following specific examples are based on the technical solution, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to these examples. The experimental methods without specific conditions described in the following examples are generally carried out under conventional conditions or according to the manufacturer's instructions.

[0247] Example 1

[0248] 1. Methods

[0249] Generation of IL-8 humanized (IL8-Hu) mouse strain

[0250] The piggyBac-on-BAC RP11-997L11 containing the human IL-8 locus was inserted into the genome of C57BL / 6 mice. Four pairs of BAC-specific primers that amplified different parts of the BAC plasmid to 300-800 bp fragments were used for PCR analysis and identification of BAC-positive pups. The offspring carrying the complete human IL-8 gene locus were backcrossed with C57BL / 6 mice for five generations. The IL-8 insertion site was determined by reverse transcription polymerase chain reaction (Inverse PCR). Briefly, genomic DNA was extracted from mouse tails, digested with HaeIII (NEB, Cat#R0108S), and then ligated with T4 DNA ligase (NEB, Cat#M0202L). The primers used to restore the left flanking sequence of the piggyBac transposon were PB-5IF (5'-CTTGACCTTGCCACAGAGGACTATTAGAGG-3' (SEQ ID NO: 1)) and PB-5IR (5'-CAGTGACAC TTACCGCATTGACAAGCACGC-3' (SEQ ID NO: 2)). The primers used to restore the right flanking sequence of the piggyBac transposon were PB-3IF (5'-CCTCGATATACAGACCGATAAAACACATGC-3' (SEQ ID NO: 3)) and PB-3IR (5'-AGTCAGTCAGAAACAACTTTGGCACATATC-3' (SEQ ID NO: 4)). The PCR product was cloned into pMD19-T (Sino Biological) for subsequent sequencing. The sequencing results were analyzed using NCBI BLAST search (www.ncbi.nlm.nih.gov), human and mouse genome integrated database (www.ensembl.org). The human IL-8 gene locus was inserted into the mouse chromosome 9 at position 3023490-3024411. All mice used in this example were housed in individually ventilated cages under specific pathogen-free conditions, and all mouse protocols were approved by the TMMU Ethics Committee.

[0251] Glioma cell lines

[0252] GL261 (ATCC, Cat#CRL-1887) and U373 MG cells (ECACC, Cat#08061901) were transfected with lentivirus carrying Luc-GFP, and GFP-positive cells were sorted by flow cytometry to generate a fluorescent stable cell line. To construct a cell line producing IL-8, IL-8 was overexpressed in GL261-Luc cells using a lentivirus carrying IL8-RFP, and RFP-positive cells were sorted by flow cytometry. In vitro cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco, Cat#10099), 2mM glutamine, and 100U / mL penicillin / streptomycin at 37°C and 5% CO 2 nourish.

[0253] Glioma Mouse Model

[0254] For the GL261 tumor model, C57BL / 6 or IL8-Hu mice (8-12 weeks, 3-10 mice per group) were anesthetized with a mixture of ketamine (50 mg / kg) and thiazide (5 mg / kg) injected intraperitoneally. The mice were shaved and then placed in a stereotaxic frame. After disinfecting the scalp with alcohol and iodophor, a 1 cm incision was made in the midline of the scalp to expose the skull. Then, 4% H 2 O 2 The periosteum was removed and a single hole was drilled 2 mm lateral and 1 mm posterior to the bregma. Injections were made with a 10 μL syringe (Hamilton, 1701RN no NDL) and a 33G needle at a depth of 2 millimeters (mm) and retracted 0.5 mm to form a reservoir. 3 μL of tumor cells were injected at a rate of 1 μL / min using a micropump. The syringe was left in place for 1 min and then removed. The skull burr hole was filled with bone wax and the skin was adhered and cleaned. Mice were placed in a heated cage until they fully recovered. GBM5, U373-Luc, or U373-Luc+T cells were injected into 6- to 8-week-old B-NDG mice (NOD.CB17-Prkdc scid Iq tm1 / Bcgen, Biocytogen, China), 5-7 mice per group. To measure tumor growth, tumor-bearing mice were anesthetized with isoflurane and intraperitoneally injected with D-luciferin (150 mg / kg body weight; Promega, Cat# P1043). After 10 min, the animals were transferred to the IVIS100 imaging system (Caliper Life Sciences) and luminescence was detected. The data were then analyzed using Living Image2.5 software (Caliper Life Sciences).

[0255] Preparation of mouse tumor tissue cell suspension

[0256] The tumor tissue was cut into pieces, incubated with a digestion cocktail containing 1 mg / mL collagenase D (Roche, Cat#11088882001) and 30 μg / mL DNase I (Sigma-Aldrich, Cat#7291-2mg) at 37°C for 15 minutes in a C tube (Miltenyi Biotec, Cat#130-096-334), and then separated on a gentleMACS using the m_brain_01 program and incubated at 37°C for another 15 minutes. The homogenate was then filtered with a 70 μm filter and centrifuged at 500 x g for 10 minutes at 4°C. Immune cell enrichment was then performed using 30% and 70% Percoll (Fisher Scientific, Cat#10607095) density gradient centrifugation in RPMI 1640 medium containing 10% fetal bovine serum. Centrifuge at 2000 x g for 20 minutes at 20°C (without brakes during acceleration and deceleration) to collect the middle transparent layer containing immune cells. After washing with PBS containing 1.5% FBS and 20 mM EDTA, the cells are subjected to flow cytometric analysis or sorting.

[0257] Cell surface and intracellular cytokine staining in flow cytometry

[0258] Cells from different experiments were stained with fluorescently labeled antibody cocktails at 4°C for 30 minutes and then analyzed with a BDLSRFortessa instrument. To detect cytokines in lymphocytes, cells were stimulated for 5 hours with 100 ng / mL of phorbol-12-myristate 13-acetate (PMA) and 1 μg / mL of ionomycin in the presence of Golgi plug (BD Biosciences, Cat#555028). Intracellular staining was performed using a fixation / permeabilization kit (BD Biosciences, Cat#555028). Data were analyzed using FlowJo.

[0259] Immunohistochemistry (IHC) staining

[0260] Formalin-fixed paraffin-embedded tumor tissue blocks or frozen tumor tissue were cut into 3 μm thick serial sections. After blocking with streptavidin peroxidase, heat-induced antigen epitope retrieval was performed in citrate buffer (pH: 6.0). Sections were incubated with primary antibodies Cit-H3 (Abcam, Cat#ab5103, 1:300 dilution), CD31 (Abcam, Cat#ab28364, 1:200 dilution), IL-8 (Bio-Rad, Cat#AHP781, 1:100 dilution) or human CD4 (Abcam, Cat#ab133616, 1:200 dilution) at 4°C overnight and then counterstained with hematoxylin. Staining was performed by Dako REAL TM EnVision TM The slides were displayed using a detection system (Dako, Cat#K5007) and scanned with an Axio Scan.Z1 (ZEISS). Quantitative analysis of CD31- and IL-8-positive areas or IL-8-positive spots was performed using ImageJ 1.46r as described previously. Images were converted to RGB stacks, and the pixel areas or pixel particles stained with CD31 and IL-8 were measured by a blinded examiner using a thresholding method. In this case, 3 to 10 tumor fields (magnification 200 times) were randomly selected for analysis.

[0261] ELISA

[0262] 5 μg / mL IL-8 capture antibody (Biolegend, Cat#514602) was coated on a 96-well plate (Thermo, Cat#442404) overnight at 4°C. After 1% BSA blocking for 1 hour, mouse serum or standards (Biolegend, Cat#570909) were incubated overnight at 4°C. After washing, 2 μg / mL biotin-labeled detection antibody (Biolegend, Cat#514704) was added and incubated at room temperature for 1 hour. After incubation with streptavidin-HRP (R&D, Cat#DY998) for another 1 hour, IL-8 concentration was quantified by HRP-catalyzed oxidation of o-phenylenediamine (Sigma, Cat#P8936) and measured with a MULTISKAN GO instrument (Thermo Scientific).

[0263] Statistics

[0264] The comparison between the two groups was performed using an unpaired two-tailed Student's t-test. Survival analysis was performed using the Kaplan-Meier method, and comparison was performed using the log-rank test. HRs and their 95% CIs were calculated using a stratified Cox proportional hazard regression model. The relevant P values ​​were calculated using a stratified two-tailed test. The threshold point optimization for patients in the TCGA database was calculated using SPSS statistical software. Statistical analysis was performed using GraphPadPrism 9. All quantitative data are expressed as mean ± SD. All P values ​​< 0.05 were defined as significant (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

[0265] 2. Results

[0266] Malignant gliomas have a very poor prognosis and remain challenging due to limited standard treatment options, including surgery and radiotherapy / chemotherapy. Although immunotherapy targeting inhibitory receptors PD-1 and CTLA-4 can reactivate tumor-specific T cells and produce durable anti-tumor responses in various cancer types, the clinical benefit of ICB in the treatment of malignant gliomas remains low. Glioblastoma is the most aggressive type of glioma, and a recent phase III clinical trial of anti-PD-1 treatment failed to confirm a survival benefit in recurrent glioblastoma (GBM). Possible reasons include the intrinsic properties of glioma cells, such as huge genetic heterogeneity and low mutation load, as well as some external features: a highly immunosuppressive microenvironment mediated by ubiquitous tumor-associated myeloid cells and a multivesicular microenvironment that undermines T cell-mediated anti-tumor immunity.

[0267] Increasing evidence suggests that the heterogeneity and functional status of tumor-infiltrating T cells play a key role in shaping antitumor immunity and clinical characteristics such as response to immunotherapy. + and CD8 +T cells, although they are present at a lower frequency than myeloid cells, and their number increases with increasing tumor grade, indicating that they have functional diversity. In fact, recent research focused on the evaluation of T cell exhaustion at the single-cell level has shown that glioma-infiltrating T cells contain a variety of different subpopulations, of which less than 20% of T cells undergo clonal expansion, which can be mainly classified as exhausted, effector or memory subpopulations, while the remaining 80%-90% of tumor-infiltrating T cells (the so-called bystander T cells) do not undergo clonal expansion, and their functions remain to be explored. In addition, the individual developmental origin of T cells in gliomas is still unclear. Here, this example uses single-cell RNA sequencing (scRNA-seq), CyTOF, and flow cytometry to perform a panoramic map of T cells from adapted human blood and tumor tissues. Through comparative analysis, the unique characteristics of tumor-infiltrating T cells compared to the corresponding T cells in peripheral blood were revealed. Specifically, a unique CD4 that produces interleukin-8 (IL-8) was identified. + T cell subsets that exhibit innate immune-like characteristics and are associated with poor prognosis. Given the absence of interleukin-8 (IL-8) in rodents, further studies of IL-8-mediated immune responses in vivo and its role in attenuating immune checkpoint blockade (ICB) therapy have been hampered. This example adapts the strategy shown in previous studies to develop an IL-8 humanized mouse strain that carries a bacterial artificial chromosome (BAC) containing the entire IL-8 gene locus and upstream and downstream regulatory elements. In a glioma transplant mouse model, IL-8 + CD4 + T cells can act alone or in conjunction with IL-8-producing myeloid cells and stromal cells to enhance the immunosuppressive properties of the glioma microenvironment by promoting angiogenesis and recruiting MDSCs. Surprisingly, anti-PD-1 immunotherapy increases systemic IL-8 expression and weakens the efficacy of immune checkpoint blockade therapy by enhancing immunosuppression. Therefore, the study of this example enhances the understanding of bystander T cells in tumors and emphasizes that the IL-8-CXCR1 / CXCR2 axis can be used as a target for combined immunotherapy.

[0268] Due to the lack of IL-8 gene in rodents, the function of IL-8 in vivo, especially its function as a key immune regulator in tumorigenesis, remains unclear. This example follows the previous strategy to develop an IL-8 humanized (IL8-Hu) mouse strain, in which an artificial BAC clone (RPL11-997L11) containing the complete human IL-8 gene locus and upstream and downstream regulatory elements with a length of about 160 kbs was inserted into the intergenic region of mouse chromosome 9 (see Figure 1Compared with wild-type (WT) control mice, There were no differences in appearance, body weight, lifespan, or fertility between IL8-Hu mice (data not shown). Further evaluation of T cells, B cells, dendritic cells (DCs), macrophages, and granulocytes in the thymus, spleen, and peripheral lymph nodes (LNs) also revealed no significant differences (see Figure 2 A, B, and C in Figure 2), which is consistent with the observations of a previous study. Subsequently, a human glioblastoma model was established by intracranial injection of luciferase-expressing GL261 cells into mice. Compared with the wild-type group, IL8-Hu mice transplanted with GL261 cells showed significantly larger tumor volume and shorter lifespan (see Figure 3 Histological examination of tumor tissues from IL8-Hu mice by IHC and H&E staining showed an increase in tumor vascular density (see Figure 3 More importantly, flow cytometric analysis of IL8-Hu mouse tumors showed that CD4 + The expression of IL-8 in T cells was significantly increased, while in CD45 + CD - The expression was more significantly increased in the myeloid cell compartment, confirming the results obtained from human samples (see Figure 3 Of note, tumor-infiltrating CD4 + and CD8 + T cells carry more exhaustion markers PD-1 and TIM-3 (see Figure 4 Therefore, compared with WT mice, the newly constructed IL8-Hu mouse strain in this example is more suitable for constructing a human glioma model, in which the physiological expression of human IL-8 can reproduce the accelerated tumor growth and angiogenesis.

[0269] It is increasingly recognized that the immunosuppressive effect enhanced by IL-8 remains an obstacle to the good efficacy of ICB therapy in several tumor types. Taking advantage of the newly developed IL8-Hu mouse model, this example attempts to explore whether IL-8 is a potential factor leading to the poor efficacy of anti-PD-1 therapy in malignant gliomas. This example conducted a tumor implantation experiment, the method is similar to Figure 5Similar to part C of , IL8-Hu mice were injected intracranially with luciferase-expressing GL261 cells. After tumor formation, tumor-bearing mice received intraperitoneal (ip) injections of anti-PD-1 antibody or isotype IgG. Consistent with previous observations as monotherapy, anti-PD-1 treatment did not show a good antitumor response, although anti-PD-1 treatment significantly alleviated T cell exhaustion in tumors (see Figure 5 Unexpectedly, evaluation of IL-8 levels in these mice revealed that anti-PD-1 treatment significantly increased serum and tumor-site IL-8 expression compared with controls (see Figure 5 Given that human IL-8 can attract mouse CD11b + Myeloid cells, this example analyzes CD11b from tumor sites + Myeloid cell populations were found to be significantly higher in mice treated with anti-PD-1 compared with controls. + CD11b hi Gr1 mid and CD45 + CD11b hi Gr1 hi The numbers of these two cell populations, MDSCs, increased significantly, while CD11b hi Gr1 - There was no significant change in cell number (see Figure 5 The phenotypic characteristics of these two types of MDSCs in the tumor site showed that CD11b hi Gr1 mid MDSCs are monocytic (M)-MDSCs, which are characterized by Ly6C + , while CD11b hi Gr1 hi Cells contain both Ly6G + G-MDSCs, also contain Ly6G + M-MDSCs, which is consistent with previous studies (see Figure 5 Unlike the tumor site, only Ly6G + G-MDSCs consist of CD11b hi Gr1 hi MDSCs, and CD11b in the bone marrow and blood of mice treated with anti-PD-1 compared with control mice hi Gr1 hi MDSCs increased significantly, but there was no significant change in the spleen (see Figure 5F and G in the Figure). Since MDSC populations were greatly increased in both peripheral blood and tumor sites, when MDSC production was assessed, it was found that MDSCs from the spleen, blood, and tumor sites (but not the bone marrow) produced a large amount of IL-8, suggesting that MDSCs and IL-8 may interact in promoting systemic immunosuppression in tumor-bearing mice and cancer patients. Overall, these findings suggest that in mouse gliomas, anti-PD-1 treatment systemically increases IL-8 expression and recruitment of MDSCs, which in turn impairs the efficacy of anti-PD-1 therapy.

[0270] This example investigates whether blocking the IL-8-CXCR1 / CXCR2 axis can enhance the efficacy of anti-PD-1 therapy in malignant gliomas. Reparixin, a reported pharmacological inhibitor of CXCR1 and CXCR2, was used to test the above hypothesis. As previously described, luciferase-expressing GL261 cells were implanted intracranially into WT mice and IL8-Hu mice for seven days, followed by intraperitoneal (ip) injection of isotype IgG, anti-PD-1 antibody, reparixin, and reparixin combined with anti-PD-1. It was further verified that anti-PD-1 monotherapy did not inhibit tumor growth in both IL8-Hu mice and WT mice (see Figure 6 Treatment with reparixin significantly slowed tumor growth in both IL8-Hu and WT mice, and the improvement in survival was almost identical in both (see Figure 6 More strikingly, both IL8-Hu mice and WT mice that received dual administration of Reparixin and anti-PD-1 showed the best effect in reducing tumor burden and prolonging survival (see Figure 6 Flow cytometry evaluation showed that reparixin treatment not only abolished anti-PD-1-induced CD45 + CD11b hi Gr1 hi MDSCs and CD45 + CD11b hi Gr1 mid , and also reduced the basal levels of both, which were relatively small in WT mice (see Figure 6 Therefore, these results provide evidence to support the view that inhibiting the trafficking of MDSCs by targeting CXCR1 and CXCR2 can improve the therapeutic efficacy of anti-PD-1 therapy in glioma.

[0271] This example further studies whether neutralizing IL-8 has a similar effect. Since human primary glioblastoma cells also express IL-8 at high levels, this example rigorously constructed a human glioblastoma model by injecting GL261 cells expressing both IL-8 and luciferase into IL8-Hu mice (sequencing was performed later). Figure 6 As shown in part F of Figure 2, mice were treated with control isotype antibody, PD-1 blockade alone, IL-8 blockade alone, or both PD-1 and IL-8 blockade. Mice receiving anti-IL-8 antibody had significantly smaller tumors and prolonged survival compared with mice receiving isotype antibody (see Figure 7 Notably, simultaneous blockade of PD-1 and IL-8 significantly prolonged median survival by approximately 18 days compared with a control group that received isotype antibodies (see Figure 7 In view of the aforementioned data showing that administration of anti-PD-1 antibodies increased systemic IL-8 levels in IL8-Hu mice, and the fact that IL-8 can promote tumor angiogenesis, this example performed a histological assessment of microvessels in glioma tumors by IHC staining for CD31. Compared with control mice, tumor-bearing IL8-Hu mice treated with anti-PD-1 antibodies showed significantly higher microvessel density, which was significantly reduced by anti-IL-8 treatment (see Figure 7 In summary, the above results indicate that neutralization of IL-8 (equivalent to blocking its receptors CXCR1 and CXCR2) can enhance T cell-mediated anti-tumor immunity by eliminating suppressive MDSCs.

[0272] The specific embodiments of the present invention are described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions made to the present invention are also within the scope of the present invention. Therefore, the equalization changes and modifications made without departing from the spirit and scope of the present invention should be included in the scope of the present invention.

Claims

1. A non-human mammalian model expressing IL-8.

2. A method for preparing a non-human mammal model expressing IL-8 as claimed in claim 1, characterized in that: The preparation method comprises the steps of: The non-human mammal is caused to express IL-8, thereby obtaining a non-human mammal model expressing IL-8.

3. Use of the non-human mammal model expressing IL-8 as claimed in claim 1 in preparing a tumor model of a non-human mammal expressing IL-8.

4. A tumor model of a non-human mammal expressing IL-8.

5. A method for preparing a tumor model of a non-human mammal expressing IL-8 as claimed in claim 4, characterized in that: The preparation method comprises the steps of: (i) preparing a non-human mammal model expressing IL-8; (ii) The non-human mammal model expressing IL-8 is allowed to bear a tumor, thereby obtaining a non-human mammal tumor model expressing IL-8.

6. A use of a non-human mammal tumor model expressing IL-8 as claimed in claim 4, characterized in that: The use is to use it as an animal model in studying tumor diseases, studying the effects of IL-8 on tumors, and / or screening or identifying therapeutic agents for preventing and / or treating tumors.

7. A method for screening or identifying potential therapeutic agents for preventing and / or treating tumors, characterized in that: The method comprises the steps of: (1) In a test group, in the presence of a test substance, the test substance is applied to the tumor model of a non-human mammal expressing IL-8 as described in claim 4, and the growth condition Q1 of the tumor in the tumor model of a non-human mammal expressing IL-8 in the test group is detected; and in a control group in which the test substance is not applied and other conditions are the same, the growth condition Q2 of the tumor in the tumor model of a non-human mammal expressing IL-8 in the control group is detected; (2) comparing the growth status Q1 and the growth status Q2 of the tumor detected in step (1) to determine whether the test substance is a potential therapeutic agent for preventing and / or treating tumors; If the growth condition Q1 of the tumor is lower than the growth condition Q2, it indicates that the test substance is a potential therapeutic agent for preventing and / or treating tumors.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises an IL-8 inhibitor and a PD-1 inhibitor; and a pharmaceutically acceptable carrier.

9. Use of the pharmaceutical composition according to claim 8 in the preparation of a medicament for preventing and / or treating tumors.

10. Use of an IL-8 inhibitor in the preparation of a pharmaceutical composition for enhancing the anti-tumor effect of a PD-1 inhibitor.

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