CN113274502A - Compositions for specific type three-negative breast cancer immunotherapy - Google Patents

Compositions for specific type three-negative breast cancer immunotherapy Download PDF

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CN113274502A
CN113274502A CN202110487230.5A CN202110487230A CN113274502A CN 113274502 A CN113274502 A CN 113274502A CN 202110487230 A CN202110487230 A CN 202110487230A CN 113274502 A CN113274502 A CN 113274502A
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breast cancer
glycosylation
fut8
negative breast
cells
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CN113274502B (en
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邓蓉
朱孝峰
唐军
黄韵
杜天
李智玲
张海亮
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Sun Yat Sen University Cancer Center
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract

The invention discloses a composition for immunotherapy of specific type three-negative breast cancer. The inventors identified aberrant B7-H3 glycosylation and showed that N-glycosylation of B7-H3 on the NXT motif is associated with its protein stability and immunosuppression in TNBC tumors. Fucosyltransferase FUT8 catalyzes the N-glycan core fucosylation of B7-H3 to maintain its high expression. The FUT8 gene knockout can save the B7-H3 mediated immunosuppression function of TNBC cell glycosylation. The B7-H3 glycosylation abnormality mediated by FUT8 overexpression has important physiological significance and clinical significance in TNBC patients. Notably, the combined use of the core fucosylation inhibitor 2F-Fuc and anti-PDL1 enhanced the therapeutic effect on B7-H3-positive TNBC tumors. These findings suggest that targeting the FUT8-B7-H3 axis may be a promising strategy for improving anti-tumor immune responses in TNBC patients.

Description

Compositions for specific type three-negative breast cancer immunotherapy
Technical Field
The invention relates to treatment of breast cancer, in particular to a composition for improving curative effect of triple negative breast cancer immunotherapy with abnormal B7-H3 protein N-glycosylation modification and application thereof.
Background
Triple Negative Breast Cancer (TNBC) refers to a subtype of Breast Cancer that lacks ER, PR and HER-2 protein expression. Clinically, TNBC is an aggressive subtype that accounts for 15% to 20% of all diagnosed breast cancer cases, and is more prevalent in younger women and women of african or african america. TNBC is mainly used for invasive ductal carcinoma and is characterized by poor differentiation, strong proliferation capacity and large tumor volume. TNBC is prone to metastatic spread to the lung and brain compared to other breast cancer subtypes migrating to bone and soft tissue. Furthermore, TNBC has a 5-year survival rate of approximately 70%, which is lower than 80% for the other subtypes. TNBCs can be subdivided into 7 subclasses. These subclasses include basal-like BL1 and BL2, mesenchymal cell-like M, mesenchymal stem cell-like MSL, intracavity androgen receptor-expressing LAR and immunomodulatory IM. TNBC has heterogeneity between different subtypes, with differences in morphology, mutant phenotype, and signal transduction profiles between tumors.
TNBC lacks specific targets and anthracycline and paclitaxel based chemotherapy remains the mainstay of treatment for early and late stage TNBC patients, and effective treatment of such malignant invasive breast cancer would be a significant challenge. The results of recent clinical trials prove that the addition of platinum and ruthenium drugs in a new adjuvant chemotherapy regimen can improve the surgical treatment effect of chemotherapy-sensitive TNBC patients. Despite comprehensive and aggressive treatment, over 50% of TNBC patients experience relapse, with over 37% dying within 5 years. This is probably due to the presence of an undefined multi-drug resistance molecular mechanism in relapsed patients, impairing the therapeutic efficacy of chemotherapeutic drugs on malignant tumors. Therefore, the search for new effective methods for treating TNBC has become one of the major hotspots in the study of breast cancer.
Only a small fraction of patients with triple negative breast cancer are effective in the treatment with existing immune checkpoint inhibitors, and the response rate of the treatment is far less than that of other tumors, so that a new effective immunotherapy scheme needs to be explored urgently.
B7-H3 is also named as CD276 or B7RP-2, and plays an important role in non-immunization as a tumor-specific related antigen. B7-H3 plays an important role in regulating glycolysis, migration, proliferation and chemotherapy resistance of tumor cells. Although there is prior evidence that B7-H3 may promote tumor immune responses, there is increasing evidence suggesting that B7-H3 play a negative regulatory role in tumors. Roth et al performed cohort analysis on 823 patients after radical prostatectomy, and found that B7-H3 is highly expressed in patients with prostatic intraepithelial neoplasia, but B7-H3 is expressed at a lower level in normal prostate tissues, the staining intensity of the B7-H3 is positively correlated with tumor metastasis, recurrence and tumor-specific death, and the correlation between the expression level of B7-H3 and the immunosuppressive efficiency of the patients is stronger. These data suggest that the inventors B7-H3 play an inhibitory role in prostate cancer immune response. Also, B7-H3 expression in renal, endometrial, breast, colon, ovarian, pancreatic, and non-small cell lung cancer patients can be used as an indicator of poor prognosis. In neuroblastoma and glioma, 4Ig-B7-H3 on the surface of tumor cells inhibits the NK cell regulated cell killing ability after binding to NK cell surface inhibitory receptors. Recent studies by Chen et al have found that macrophages, when co-cultured with lung cancer cells, induce the expression of macrophages B7-H3, and B7-H3 associated tumor-specific macrophages strongly suppress T cell mediated immune responses. In addition, B7-H3 can up-regulate IL-10 and down-regulate secretion of cytokines such as IL-12, so that immune escape of tumors is generated, and generation and development of the tumors are promoted. At present, the role of B7-H3 in tumor resistance is controversial, and the specific functions and the regulated molecular mechanism of the B7-H3 are clear to provide a new idea for tumor immunotherapy. B7-H3 is a highly glycosylated protein. However, the molecular mechanisms that regulate the expression of glycosylated B7-H3 in cancer cells and the molecular mechanisms that glycosylated B7-H3 affect the immune response remain unclear.
Fucosylation, particularly core fucosylation, is one of the most common cancerous changes in the N-sugar chain. Alpha-1, 6-fucosyltransferase (alpha-1, 6-fucosyltransferase, FUT-8) is the only enzyme currently known that produces an alpha-1, 6-fucosylation structure in the core of an N-sugar chain. FUT8 has been reported to be up-regulated in various cancers, such as breast cancer, lung cancer, prostate cancer, hepatocellular carcinoma, colorectal cancer and melanoma, indicating that FUT8 is associated with tumor biological characteristics and patient prognosis. The specific role of FUT8 in tumors is still not clear enough, and the prior art does not treat tumors by regulating FUT-8. Since it is a glycosyltransferase and does not affect the intrinsic properties of the protein, it is considered difficult to target tumor therapy.
Disclosure of Invention
The invention aims to overcome at least one defect of the prior art and provide a technology capable of remarkably improving the curative effect of treating triple negative breast cancer.
Most Triple Negative Breast Cancer (TNBC) patients were non-responsive to anti-PD 1/PDL1 immunotherapy, suggesting a need to explore immune checkpoint targets. B7-H3 is a highly glycosylated protein. However, the mechanism by which B7-H3 glycosylation is regulated and whether the glycosyl group is involved in immunosuppression are not known. The inventors identified aberrant B7-H3 glycosylation and showed that N-glycosylation of B7-H3 on the NXT motif is associated with its protein stability and immunosuppression in TNBC tumors. Fucosyltransferase FUT8 catalyzes the N-glycan core fucosylation of B7-H3 to maintain its high expression. The FUT8 gene knockout can save the B7-H3 mediated immunosuppression function of TNBC cell glycosylation. The B7-H3 glycosylation abnormality mediated by FUT8 overexpression has important physiological significance and clinical significance in TNBC patients. Notably, the combined use of the core fucosylation inhibitor 2F-Fuc and anti-PDL1 enhanced the therapeutic effect on B7-H3-positive TNBC tumors. These findings suggest that targeting the FUT8-B7-H3 axis may be a promising strategy for improving anti-tumor immune responses in TNBC patients.
The technical scheme adopted by the invention is as follows:
in a first aspect of the present invention, there is provided:
application of B7-H3 protein core fucosylation modification intervention agent in preparation of triple negative breast cancer immunotherapy synergist.
In some examples, the triple negative breast cancer is a glycosylation B7-H3 positive triple negative breast cancer.
In some examples, the glycosylation is N-glycan core fucosylation.
In some examples, the core fucosylation-modifying intervention agent is selected from at least one of an inhibitor of glycosyltransferase FUT8 expression, an inhibitor of glycosyltransferase FUT8 activity, a core fucose analog.
In some examples, the core fucose analog is selected from at least one of 2-fluoro-L-fucose, 6-alkynyl fucose.
In some examples, the inhibitor of glycosyltransferase FUT8 expression is an siRNA or sgRNA of FUT 8.
In some examples, the siRNA has a sequence of siFUT8#1: 5'-CUGCAGUGUGGUGGGUGUCTT-3' or siFUT8# 2: 5'-AGGUCUGUCGAGUUGCUUATT-3'; the sequence of the sgRNA is sgRNA2: 5'-CACCGACAGCCAAGGGTAAATATGG-3' or sgRNA 75 '-CACCGTGAAGCAGTAGACCACATGA-3'.
In some examples, the immunotherapy is anti-PDL1 immunotherapy.
In a second aspect of the present invention, there is provided:
use of a composition in the preparation of a formulation for the treatment of triple negative breast cancer that is glycosylated B7-H3 positive, the composition comprising:
at least one B7-H3 protein core fucosylation modification intervention agent; and
at least one immunotherapeutic agent.
In some examples, the triple negative breast cancer is a glycosylation B7-H3 positive triple negative breast cancer.
In some examples, the glycosylation is N-glycan core fucosylation.
In some examples, the core fucosylation-modifying intervention agent is selected from an inhibitor of the expression of the glycosyltransferase FUT8, an inhibitor of the activity of the glycosyltransferase FUT8, a core fucose analog.
In some examples, the core fucose analog is selected from at least one of 2-fluoro-L-fucose, 6-alkynyl fucose.
In some examples, the inhibitor of glycosyltransferase FUT8 expression is an siRNA or sgRNA of FUT 8.
In some examples, the siRNA has a sequence of siFUT8#1: 5'-CUGCAGUGUGGUGGGUGUCTT-3' or siFUT8# 2: 5'-AGGUCUGUCGAGUUGCUUATT-3'; the sequence of the sgRNA is sgRNA2: 5'-CACCGACAGCCAAGGGTAAATATGG-3' or sgRNA 75 '-CACCGTGAAGCAGTAGACCACATGA-3'.
In some examples, the immunotherapeutic agent is selected from the group consisting of anti-PDL1 immunotherapeutic agents.
The invention has the beneficial effects that:
some examples of the invention provide a promising strategy for improving the anti-tumor immune response of TNBC patients, break through the defect that the existing triple negative breast cancer lacks an effective treatment strategy, and provide a new strategy for prolonging the survival time of triple negative breast cancer patients.
Drawings
FIG. 1: B7-H3 protein expression in triple negative breast cancer tissue samples; (A, B) expression of B7-H3 protein in a sample of a patient with primary breast cancer. Western blot analysis of expression of glycosylation B7-H3 in representative samples of breast cancer patients. Immunohistochemical analysis of B7-H3 expression in breast cancer patient samples in cancer and paracarcinoma. (C) A plot of B7-H3 expression versus overall patient survival in immunohistochemical data.
FIG. 2: bioinformatics analysis of B7-H3 mRNA expression in breast cancer; (A) the BC GenExMiner website analyzes B7-H3 expression of breast cancer in the TCGA dataset versus the overall survival curve of the patient. Patients were stratified according to the two algorithms of Hu' S SSP and SCMGENE. (B) The Kaplan-Meier website analyzes the B7-H3 expression versus Relapse Free Survival (RFS) and disease free survival (DMFS) curves of breast cancer in the TCGA dataset. It has statistical significance by student's t-test. All error bars are expressed as mean ± SD of 3 independent experiments.
FIG. 3: analysis of glycosylation modification types of B7-H3 in triple negative breast cancer cell lines; (A) a glycosylation modification type of B7-H3 protein. Cell lysates were treated with peptide-N-glycosidase F (PNGase F), endoglycosidase (Endo H) and disaccharide O-glycanase (O-glycanase) and analyzed by Western blot analysis. (B) cells were treated with N-linked or O-linked glycosylation inhibitors and analyzed for B7-H3 expression by Western blotting. Filled circles, glycosylated B7-H3; star, non-glycosylated B7-H3. (C) treating cells with tunicamycin, and analyzing the expression of B7-H3 protein on the cell surface by using a flow cytometer. .
FIG. 4: constructing glycosylated and non-glycosylated B7-H3 triple negative breast cancer cell strain; (A) n-glycosylation motif structure, humanized and murine B7-H3 amino acid sequence diagram. (B, D) schematic representation of the B7-H3 NQ mutant used in this study. Numbers indicate amino acid positions (C, E) on B7-H3 knock-out, glycosylated and non-glycosylated B7-H3 cell lysates were detected by western blot analysis.
FIG. 5: glycosylation modification enhances the stability of B7-H3 protein; (A, B, C, D) MDA-MB-231 cells (A), HCC1806 cells (B) and HEK293T cells (C), MDA-MB-231 cells expressing B7-H3-Flag (D) were treated with 20mM Cycloheximide (CHX) at the indicated time intervals and the B7-H3 protein was detected by Western blot analysis. The protein levels of glycosylated or non-glycosylated B7-H3 were quantified by ImageJ and cells were treated with 20mM Cycloheximide (CHX) at intervals specified by GAPDH normalization (E), treated with or without MG132 (100 mM) for 6 hours, and analyzed by Western blot analysis (F) inhibition of glycosylation B7-H3 to enhance ubiquitination modification. B7-H3-8NQ transfected HEK293T cells were treated with or without MG132 and subjected to B7-H3 Immunoprecipitation (IP) and Western blot analysis. (G) Cell surface B7-H3 proteins were analyzed using flow cytometry.
FIG. 6: glycosylation B7-H3 inhibited T cell-mediated triple negative breast cancer cell death. (A, B, C) knockout, glycosylated and non-glycosylated B7-H3 triple negative breast cancer cells were co-cultured with or without activated T cells and the percentage of T cells killing tumor cells was examined at 6 hours.
FIG. 7: glycosylation B7-H3 inhibited T cell proliferation in vitro. (A, B) CD4 stimulated with anti-CD 3 and anti-CD 28 in the presence of knockout, glycosylated and non-glycosylated B7-H3 triple negative breast cancer cells+ T cells and CD8+Proliferation (A) and activity (B) of T cells.
FIG. 8: in vitro glycosylation B7-H3 had no effect on the proliferation and invasion capacity of triple negative breast cancer cells. (A) Glycosylated (B7-H3-WT) and non-glycosylated (B7-H3-8NQ) B7-H3 triple negative breast cancer cell proliferation assays were analyzed by CellTiter Glo, with three replicates per condition. (B) Plate colony formation assay for glycosylated (B7-H3-WT) and non-glycosylated (B7-H3-8NQ) B7-H3 triple negative breast cancer cells. (C) glycosylated (B7-H3-WT) and non-glycosylated (B7-H3-8NQ) B7-H3 triple negative breast cancer cells were subjected to a transwell experiment and migrated cells were quantified by counting the number of cells that migrated to the substrate side of the transwell insert after 26 hours.
FIG. 9: glycosylated B7-H3 inhibited infiltration and activity of immune cells in the transplanted tumors. (A) Murine B7-H3, which overexpresses glycosylation (B7-H3-WT) and non-glycosylation (B7-H3-4NQ), knockouts triple negative breast cancer cell line 4T1, was allogenously inoculated into Balb/c mice, tumor volume was measured at the indicated time points, and tumor weight was measured (n = 6). (B) TIL was isolated from 4T1 tumors and tested for CD4+CD8+T cell and NK cell population ratio, at CD8+ Frequency of GzmB and IFN-positive T cells in T cells.
FIG. 10: glycosylated B7-H3 did not affect the growth of transplanted tumors in Balb/c SCID mice. (A) Murine B7-H3, which overexpresses glycosylation (B7-H3-WT) and non-glycosylation (B7-H3-4NQ), knockouts triple negative breast cancer cell line 4T1, was allogeneously inoculated to Balb/c SCID mice and tumor growth was observed. Tumor volume was measured at the indicated time points and tumor weight was measured (n = 11). (B) human B7-H3 with over-expression of glycosylation (B7-H3-WT) and non-glycosylation (B7-H3-4NQ) knockouts of triple negative breast cancer cell line MDA-MB-231, which was allografted to Balb/c SCID mice and observed for tumor growth. Tumor volume was measured at the indicated time points and tumor weight was measured (n = 7).
FIG. 11: FUT8 catalyzes glycosylation modification of B7-H3. (A) FUT8 was knocked out in MDA-MB-231 and HCC1806 cell lines and detected by Western blot analysis for B7-H3 protein. (B) real-time fluorescent quantitative PCR showed no effect of knocking down FUT8 on B7-H3 mRNA expression (C) Whole cell lysates of MDA-MB-231-WT/8NQ cells knocked out with negative control or FUT8 sgRNA were subjected to LcH affinity chromatography, and the effect of FUT8 knocking down on B7-H3 expression was analyzed by Western blotting method. (D) MDA-MB-231-WT/8NQ cells transduced with negative control or gRNA of Fut8 gene alone were analyzed by flow cytometry to detect the expression of core fucose (Lens collinaris agglutinin [ LCA ]) and B7-H3 on the cell surface.
FIG. 12: silencing FUT8 restores the immunosuppressive effects of glycosylation B7-H3 on immunity. (A) Western blot analysis of siRNA FUT8 (B) knockdown of FUT8 in human B7-H3 knockout triple negative breast cancer cell line MDA-MB-231 overexpressing both glycosylation (B7-H3-WT) and non-glycosylation (B7-H3-4NQ), followed by co-culture with or without activated T cells and percentage of T cell mediated tumor cell killing by flow assay after 6 hours of co-culture. (C) Flow cytometry was used to analyze CD4+ T cell proliferation stimulated with anti-CD 3 and anti-CD 28 in the presence of triple negative breast cancer cells with FUT8 silencing glycosylation B7-H3. (D, E, F, G) CD4+ T cells and CD8+ T cells stimulated with anti-CD 3 and anti-CD 28 express IL-2 and IFN gamma index ratios in the presence of triple negative breast cancer cells with FUT8 silencing glycosylated B7-H3.
FIG. 13: blocking core fucosylation down-regulates B7-H3 expression and enhances T cytotoxicity. (A, C) MDA-MB-231 cells (A) and 4T1 cells (C) were treated with DMSO or 200. mu.M or 300. mu.M 2F-Fuc and cell surface B7-H3 expression was analyzed by flow cytometry. (B) Glycosylated and non-glycosylated B7-H3 triple negative breast cancer cells pretreated with DMSO or 400 μ M2F-Fuc, were then co-cultured with or without activated T cells and the percentage of T cell mediated tumor cell killing was measured by flow after 6 hours of co-culture.
FIG. 14: blocking the B7-H3 core fucosylation enhances the anti-tumor immunity sensitivity of the PD-L1 antibody. (A) Schematic representation of the treated groups (control, anti-PD-L1, 2F-Fuc, anti-PD-L1 and 2F-Fuc). (B) Xenograft 4T1-B7-H3KO-B7-H3-WT tumors were treated according to treatment protocols, observed for tumor growth in Balb/C mice, and tumor volume and final tumor weight (n = 5.) were measured at indicated time points (C) immunohistochemistry to examine the expression of tumors B7-H3 in the different treatment groups. (D) Frequency of IFN γ positive cells in CD8+ T cells, CD4+ T cells and NK cells (E) Tunel staining detected apoptosis in tumors of different treatment groups.
Detailed Description
The technical scheme of the invention is further explained by combining experiments.
Identification of glycosylation patterns and sites of B7-H3 in triple negative breast cancer
1.1B 7-H3 is highly expressed in most breast cancer tissues, mainly in glycosylated form
In order to determine the expression condition of the B7-H3 protein in human breast cancer tumor tissues, the inventor randomly selects 15 to carry out WB detection analysis on breast cancer and matched tissue protein beside the breast cancer, and the WB result indicates that the B7-H3 protein is mainly expressed in the molecular weight range of 90-110KD (indicated by a black circle), while the relative molecular weight of the B7-H3 is 45-66KD, and the inventor guesses that the B7-H3 in the tumor tissues can exist in a glycosylated form according to the previous report that glycosylation modification exists in B7-H3; in addition, the inventors found that the expression of B7-H3 protein was higher in tumor tissue compared to normal breast tissue, indicating that the glycosylated form B7-H3 is highly expressed in breast cancer tissue (fig. 1A); to further clarify whether high expression of the B7-H3 protein was associated with poor prognosis in the triple negative breast, the inventors performed sectioning and immunohistochemical staining of triple negative breast cancer samples from the 1999-2005 pathology department of the university of Zhongshan tumor prevention center and counted the staining scores of normal tissues and tumor tissues in each sample. Immunohistochemistry was performed on median basis, and the results of histochemistry suggested that B7-H3 was significantly higher in tumor tissues than in normal tissues (fig. 1B), and the patients in B7-H3 high expression group had shorter survival time than in B7-H3 low expression group and the results were statistically different (P =0.033, fig. 1C).
High expression of B7-H3 mRNA water in triple negative breast cancer alone was associated with poor prognosis
To observe the correlation between the difference in B7-H3 mRNA levels and the prognosis of triple negative breast cancer, the inventors analyzed the relationship between the level of B7-H3 mRNA and the overall survival time (OS) of patients in the TCGA database using the bc-GenExMiner website, and compared the correlation between B7-H3 mRNA expression and the prognosis of each molecular subtype of breast cancer using the SCMG and Hu's SSP algorithms. The results suggest that both of the above calculations are statistically different in Basal-like triple negative breast cancer alone, with shorter Overall Survival (OS) for patients with high B7-H3 mRNA expression (P =0.0116 and P =0.0046, fig. 2A). Meanwhile, the inventors also further analyzed the correlation between B7-H3 mRNA expression difference in TCGA database and patient recurrence-free survival (RFS) and distant metastasis-free survival (DMFS) using KM-Plot website, and found that B7-H3 mRNA expression was negatively correlated with RFS and DMFS and statistically significant (P =0.0043, P =0.047) only in Basal-like patients, while other breast cancer subtypes did not have this feature (fig. 2B).
B7-H3 is mainly modified by N-glycosylation in triple negative breast cancer
In 2015 Min-Huey Chen et al identified that B7-H3 in oral cancer cells was mainly N-glycosylated and 8N-glycosylation sites were identified by means of glycosylation mass spectrometry, to further determine whether other glycosylation modification forms existed in triple negative breast cancer B7-H3, MDA-MB-231 and HCC1806 triple negative breast cancer cell lines were selected, and two cell line proteins were treated with peptide-N-glycosidase F (PNGase F) which removed all N-sugar chain structures, endoglycosidase (Endo H) which removed high mannose and part of oligosaccharide, and O-linked disaccharide O-glycase (O-glycanase), respectively, and the results showed that B7-H3 significantly decreased from 110KD (black circle) to 70KD (black asterisk) without significant changes in Endo H and O-glycanase, this suggests that B7-H3 is mainly modified by N-glycosylation (FIG. 3A). Using the N-linked glycosylation inhibitor Tunicamycin (TM), and the O-linked glycosylation inhibitors Thiamet G and PUGNAc to treat MDA-MB-231 and Hcc1806 triple negative breast cancer cell lines, the inventors found that the N-glycosylation inhibitor TM was able to significantly inhibit the expression of glycosylation B7-H3 to lower its molecular weight to 70KD (FIG. 3B). In addition, the inventor treated MDA-MB-231 and HCC1806 cells with different concentrations of TM and detected the expression of B7-H3 protein on the cell membrane surface in 24 hours of flow-type detection, and the flow-type result suggests that B7-H3 on the cell membrane surface is obviously reduced after the treatment of the N-linked glycosylation inhibitor and is related to the concentration of TM and the treatment time (FIG. 3C).
Construction of three-negative breast cancer cell model of glycosylation B7-H3
N-glycosylation modification mainly occurs in a section of conserved amino acid sequence Asn-X-Thr/Ser (X is not equal to P), a glycan chain synthesized in a cell is connected to the amide nitrogen of asparagine in a peptide chain through related transferase (figure 4A), mutation modification is carried out on gene sequences of human source (8 sites: N91, N104, N189, N215, N309, N322, N407, N433) and mouse source (4 sites: N91, N104, N189, N215) according to the structure of the motif, asparagine in the conserved sequence is mutated into glutamine (figures 4B and 4D), and human-derived glycosylation B7-H3-WT and mutation B7-H3-8NQ plasmids, and mouse-derived glycosylation B7-H3-WT and mutation B7-H3-4NQ plasmids are successfully constructed; meanwhile, a Flag tag sequence is connected to the N end and is constructed on a vector containing a CMV promoter to promote the expression of the marker in cells. B7-H3 in human triple negative breast cancer MDA-MB-231 and HCC1806 cells and murine triple negative breast cancer 4T1 cell strains are knocked Out in a targeted mode through a CRISPR-Cas9 gene knockout technology, monoclonal B7-H3-Knock Out cell strains are screened, meanwhile, glycosylated forms and non-glycosylated forms of plasmids are expressed in a reversion mode on the basis of the knocked Out cell strains, and human sources (MDA-MB-231-B7-H3KO-WT and MDA-MB-231-B7-H3KO-8NQ, HCC1806-B7-H3KO-WT and HCC1806-B7-H3KO-8NQ) and murine triple negative breast cancer cell models (4T1-B7-H3KO-WT and 584T 5-B7-B KO-NQ 5734) are constructed; WB results showed that human glycosylation B7-H3 had a molecular weight of about 110KD and about 70KD after mutation, while murine glycosylation was 55KD and 40KD respectively (FIGS. 4C, 4E).
The protein is glycosylated, the stability is enhanced, and the expression on the cell membrane is increased
To determine whether N-glycosylation modification affects the stability of B7-H3 protein, MDA-MB-231, HCC1806 and HEK293T cell lines were treated with the protein synthesis inhibitor Cycloheximide (CHX), and cells were collected at different treatment time points for WB assay, showing that non-glycosylated B7-H3 (black asterisk) degrades faster than glycosylated protein (FIGS. 5A-C). Flag-tagged glycosylation and non-glycosylation B7-H3 were overexpressed in MDA-MB-231-B7-H3KO cells, and a consistent conclusion was also drawn by comparing their degradation rates (fig. 5D). Using MG132 to block both the proteasomal pathway and the autophagosomal pathway while inhibiting protein synthesis by CHX, the non-glycosylated forms B7-H3 of the MG 132-treated group did not show a significant decrease with treatment time, suggesting that the non-glycosylated forms B7-H3 are degraded mainly by the proteasomal pathway (fig. 5E). To explore whether non-glycosylated B7-H3 could undergo ubiquitination modification, we turned B7-H3-8NQ and wild-type ubiquitin (Ub) out of HEK293T cells, and B7-H3-8NQ could undergo ubiquitination modification when both plasmids were co-transfected and its ubiquitination level increased after MG132 blocking degradation (fig. 5F). The expression condition of B7-H3 on cell membranes in the constructed cell model is detected, and the expression of the protein on the cell membranes is obviously increased only when B7-H3 is glycosylated, which indicates that glycosylation modification can not only increase the stability of the B7-H3 protein, but also promote the expression of the protein on the cell membranes (FIG. 5G).
Glycosylation B7-H3 mediates immune escape from triple negative breast cancer
2.1 killing ability of T lymphocytes to glycosylated B7-H3 triple-negative breast cancer cells in vitro is significantly reduced
In order to search the difference of tumor immunity caused by glycosylation and non-glycosylation B7-H3, the inventor uses CD28 and CD3 antibodies to non-specifically activate T lymphocytes of healthy donors in vitro for three to six days, and successfully constructed MDA-MB-231 overexpressed by B7-H3-WT and B7-H3-8NQ and HCC1806 triple-negative breast cancer cell strain and effector T cells are co-cultured for 6 to 12 hours according to the ratio of 15: 1; collecting a cell flow cytometry to detect the proportion of the tumor cells with Caspase-3 positive apoptosis index. Flow cytometry results showed a significant reduction in the apoptotic capacity of T-cell induced glycosylation of triple negative breast cancer cells highly expressed in B7-H3 (fig. 6A, 6C). After the MDA-MB-231 cell strain is cultured, sucking part of the supernatant, and detecting the tumor killing efficiency of the T cells by using a lactate dehydrogenase kit; the results of lactate dehydrogenase detection show that the killing rate of T cells to glycosylated B7-H3 cells is obviously reduced, but the killing capacity to non-glycosylated B7-H3 is strong (FIG. 6B).
In vitro glycosylation B7-H3 triple negative breast cancer cells inhibit proliferation of T lymphocytes
In order to further determine the influence of glycosylation B7-H3 and non-glycosylation on T cell functions, live cell fluorescence labeling dye hydroxyl fluorescein diacetate succinimide ester (CFSE) is used for staining T cells, after the successfully constructed triple negative breast cancer cell line is irradiated by 80Gy dose, the triple negative breast cancer cell line and the staining labeled T cells are co-planted into a pore plate coated with CD28 and CD3 antibodies, and after co-culture for 4-7 days, the proliferation condition of the T cells is detected by flow cytometry. Experimental results show that the glycosylated B7-H3 cell strain can obviously inhibit CD4+And CD8+Proliferation of T cells but not glycosylated B7-H3-8NQ had no significant inhibitory effect on T cell proliferation, suggesting that glycosylated B7-H3 had the ability and activity to inhibit T cell proliferation (fig. 7A, 7B).
The glycosylation modification of the B7-H3 protein in vitro has no significant influence on the proliferation and migration of the triple-negative breast cancer cells
Next, in order to clarify the influence of other glycosylated and non-glycosylated B7-H3 on tumors except tumor immunity, the inventors studied whether the glycosylated and non-glycosylated B7-H3 have difference on tumor cell proliferation and clone formation by using in vitro MTT experiment and clone formation experiment, and observed and recorded the cell growth condition for 7 consecutive days, wherein the growth curve indicates that the proliferation capacity of the glycosylated and non-glycosylated B7-H3 triple negative breast cancer cell line has no significant difference; a similar phenomenon was observed with the clonogenic capacity of glycosylated versus non-glycosylated B7-H3 cells (FIGS. 8A, 8B). The successfully constructed tumor cell line is placed in a Transwell chamber and cultured for 16-24 hours, the number of different tumor cells penetrating through a cell permeable membrane is observed to evaluate the migration capacity of the tumor cells, and the experimental result proves that the influence of glycosylation and non-glycosylation B7-H3 on the migration capacity of the triple negative breast cancer cells is not different (FIG. 8C).
Glycosylation B7-H3 significantly promoted the growth of transplanted tumors in immune normal mice and inhibited the infiltration and activity of T lymphocytes in tumors
Inoculating two groups of cell strains, namely 4T1-B7-H3KO-WT and 4T1-B7-H3KO-4NQ, on abdominal fat pads of immune normal Balb/c mice to construct a three-negative breast cancer animal model of glycosylated and non-glycosylated B7-H3; regular measurement and recording of mouse tumor bodyTaking out the mouse tumor and weighing after the experiment is finished; experimental results show that glycosylation B7-H3 can obviously promote the growth of transplanted tumors of mice, and statistical analysis shows that the tumor volume and the tumor weight of the mice are obviously increased compared with those of 4T1-B7-H3KO-4NQ groups (figure 9A). Dissociating the mouse tumor, performing flow type analysis, and analyzing infiltration immune cells in the mouse tumor; comparing the flow results of 4T1-B7-H3KO-WT group with those of 4T1-B7-H3KO-4NQ, it was found that the group of glycosylated B7-H3 infiltrated CD4+T cell CD8+The number of T cells and NK cells is obviously reduced, and CD8 infiltrates+The activity indexes of IFN-gamma and Granzyme in T cells are also obviously reduced (figure 9B), the evidence suggests that glycosylation B7-H3 can inhibit tumor immunity by inhibiting infiltration and activity of immune cells in tumors, thereby promoting the generation and development of tumors.
2.5 glycosylation B7-H3 had no significant effect on the growth of immunodeficient mouse transplantable tumors
In order to further verify that glycosylation B7-H3 mainly plays a role of promoting tumor by regulating an immune system, the inventor respectively inoculates two cell strains of 4T1-B7-H3KO-WT and 4T1-B7-H3KO-4NQ on bilateral abdominal breast fat pads of immunodeficient Balb/c Nude mice, regularly measures and records the tumor volume of the mice, and takes out and weighs the tumors of the mice after the experiment is finished; the mouse tumor growth curve suggests that glycosylation B7-H3 has no effect on tumor growth in immunodeficient mice, and statistical analysis of tumor weights of mice revealed no difference between the 4T1-B7-H3KO-WT group and the 4T1-B7-H3KO-4NQ group (FIG. 10A). In addition, the inventor also inoculates MDA-MB-231-B7-H3KO-WT and MDA-MB-231-B7-H3KO-8NQ cell strains of human origin to the abdominal bilateral breast fat pad of the immunodeficient Balb/c Nude mouse, and records the fat pad by regular measurement. The results of the experiments were consistent with the above conclusions for murine cell lines, and the nodulation capacity and effect on tumor volume and tumor weight for humanized glycosylated and non-glycosylated B7-H3 cell lines were not significantly different (FIG. 10B). The above results further show that glycosylation B7-H3 affects tumor growth mainly through the immune system in triple negative breast cancer animal models.
B7-H3 glycosylation modification molecular mechanism
To explore the relationship between FUT8 and B7-H3, the inventors select different sgRNAs to knock down FUT8 in MDA-MB-231 and HCC1806 cell strains, and detect the expression of B7-H3, and the experimental result shows that when FUT8 is knocked down, glycosylated B7-H3 protein is significantly reduced, but mRNA of B7-H3 is not significantly changed, which suggests that FUT8 can influence the expression of B7-H3 by regulating glycosylation modification of B7-H3, but has no influence on the mRNA of B7-H3 (FIGS. 11A and 11B). To further clarify whether core fucose modification occurred in B7-H3, the inventors performed lectin enrichment experiments (mainly to reflect the expression of FUT 8-regulated core fucoidin) using antibodies to lectin lch (lca) that specifically binds to core fucose, and as a result, showed that B7-H3 of core fucose type was significantly reduced after FUT8 knockout, and that reduction of FUT8 also had some effect on total glycosylation B7-H3 (fig. 11C). The expression of B7-H3 on MDA-MB-231 cell membranes after FUT8 knockout is detected in a flow mode, and as a result, FUT8 can obviously reduce the expression of core fucose on the cell membrane surface (FIG. 11D), and the expression of B7-H3 on the membranes is also obviously reduced, which indicates that the expression of B7-H3 inhibiting core fucose on the cell membranes after FUT8 is also reduced (FIG. 11D).
Negative regulation of killing capacity and activity of T cells through B7-H3
Early experiments prove that the high-expression glycosylated B7-H3 triple-negative breast cancer cell can obviously inhibit the T lymphocyte from killing the breast cancer cell and obviously inhibit the proliferation of the T lymphocyte. The inventor uses RNA interference technology to knock down FUT8(siRNA is siFUT8#1: 5'-CUGCAGUGUGGUGGGUGUCTT-3' (SEQ ID NO: 1); siFUT8# 2: 5'-AGGUCUGUCGAGUUGCUUATT-3' (SEQ ID NO: 2); sgRNA2: 5'-CACCGACAGCCAAGGGTAAATATGG-3' (SEQ ID NO: 3) or sgRNA 75 '-CACCGTGAAGCAGTAGACCACATGA-3' (SEQ ID NO: 4)) on the basis of MDA-MB-231-B7-H3KO-WT and MDA-MB-231-B7-H3KO-8NQ cell strains, cocultivation of the treated tumor cells and T lymphocytes is carried out after 48 hours, a positive proportion of an apoptosis index Caspase-3 is detected in a flow mode, flow results indicate that after knocking down FUT8, the inhibition effect of glycosylation B7-H3 on the T lymphocyte killing tumor cells can be obviously relieved, but did not alter the killing ability of T lymphocytes against non-glycosylated B7-H3 cells (fig. 12A, 12B). In addition, the inventor adopts the RNA interference technology to knock down FUT8 in MDA-MB-231-B7-H3KO-WT cell strain, and treated tumor cells are subjected to 80Gy dosageAfter dose irradiation, the cells are cultured for 4 to 7 days together with T lymphocytes stained by hydroxyl fluorescein diacetate succinimide ester (CFSE) of a fluorescent labeling dye of living cells, the proliferation and activity indexes IL-2 and IFN-gamma of the T lymphocytes are detected in a flow mode, and from the flow analysis result, the inventor finds that FUT8 in a B7-H3 cell strain can obviously enhance CD4 when the low-glycosylation is carried out+Proliferation of T cells (FIG. 12C), and enhancement of CD4+T cells and CD8+The ratio of IL-2 and IFN- γ expression in T (FIGS. 12D,12E,12F, 12G). The above experiment results suggest that the killing of the T cell on the glycosylation B7-H3 cell strain is enhanced after the knocking-down of FUT8, and the inhibition effect of the glycosylation B7-H3 on the activity of the T cell is relieved.
Targeted intervention FUT8 reduces B7-H3 expression to enhance sensitivity of triple negative breast cancer to immunotherapy
4.1 inhibition of FUT8 enhances the killing ability of T cells to triple negative breast cancer cells in vitro by reducing B7-H3
2-Fluoro-L-Fucose (2F-Fuc) is a fucosylation inhibitor, and 2F-Fuc enters cells to compete with a fucosylation raw material GDP-Fuc for GDP, so that GDP-2F-Fuc is formed and the synthesis of natural GDP-Fuc is inhibited, and the Fucose structure is reduced. To find whether 2F-Fuc can reduce the expression of glycosylated B7-H3, the inventors used different concentrations of 2F-Fuc to treat MDA-MB-231-B7-H3KO-WT, MDA-MB-231-B7-H3KO-8NQ and 4T1-B7-H3KO-WT cell strains, and after four days, the expression of B7-H3 protein on the surface of the cell membrane was detected by a flow method, and the flow result suggests that B7-H3 on the surface of the cell membrane is significantly reduced after 2F-Fuc is added to treat human and murine breast cancer cell strains (fig. 13A,13C), which suggests that 2F-Fuc can reduce the expression of glycosylated B7-H3 on the cell membrane. In order to determine whether the 2F-Fuc can reduce the expression of glycosylation B7-H3 and restore the killing capacity of T cells to tumor cells, MDA-MB-231-B7-H3KO-WT and MDA-MB-231-B7-H3KO-8NQ cell strains are pretreated by the 2F-Fuc, co-cultured with the T lymphocytes after four days, and the positive ratio of Caspase-3 serving as an apoptosis index is detected by flow; the results found that the killing ability of glycosylated B7-H3 cell line pretreated with 2F-Fuc was significantly enhanced compared to that of the untreated group, while 2F-Fuc had no significant effect on the ability of T lymphocytes to kill non-glycosylated tumor cells (fig. 13B), which indicates that 2F-Fuc enhances the killing of T lymphocytes by mainly reducing the expression of glycosylated B7-H3.
The targeted intervention FUT8 combined with the PD-L1 monoclonal antibody can obviously inhibit the growth of mouse transplanted tumor and enhance the activity of T lymphocyte
The above results indicate that 2F-Fuc can enhance the immune killing effect by reducing the expression of glycosylation B7-H3. Respectively pretreating 4T1-B7-H3-KO-WT cell strains by using 2F-Fuc and DMSO for 7 days, and then inoculating two differently treated 4T1-B7-H3-KO-WT cell strains to abdominal fat pads of two groups of Balb/c mice which are immunized normally; after one week of inoculation, the two groups of mice are respectively divided into two groups, and PBS + Isotype, PBS + Anti-PD-L1, 2F-Fuc + Isotype and 2F-Fuc + Anti-
PD-L1 treated mice (fig. 14A); the inoculated mice are treated by the cell strain through 2F-Fuc, and the 2F-Fuc intragastric treatment is carried out three times per week after inoculation; Anti-PD-L1 and Isotype were given as intraperitoneal injections for a total of three times every 3 days. And (3) regularly observing the tumor volume of the mouse, recording and drawing a growth curve, and dissociating the tumor of the mouse after the experiment is finished to perform flow detection on the number and activity of the infiltrated immune cells in different groups. According to the growth curve, the inventor finds that the tumor sizes of the control group and the single-drug treatment group are not obviously different, but the tumor volume and the tumor weight of the mice can be obviously reduced when the 2F-Fuc and the Anti-PD-L1 are used in combination (figure 14B), the B7H3 expression in the tumor tissues is obviously reduced after the 2F-Fuc treatment by immunohistochemical analysis (figure 14C), and the CD4 can be obviously increased by the combination of the 2F-Fuc and the Anti-PD-L1 by flow analysis+T cell, CD8+Activity of T cells and NK cells was indicative of IFN- γ expression (FIG. 14D), and Tunel staining analysis showed that 2F-Fuc in combination with Anti-PD-L1 enhanced the apoptotic rate of tumors. Therefore, the inventor can find that the 2F-Fuc and Anti-PD-L1 can achieve the Anti-tumor effect by enhancing the function of immune cells in the tumor in combination.
And (4) conclusion:
in triple negative breast cancer, B7-H3 protein is mainly subjected to N-glycosylation modification, and the glycosylation modification enhances the stability and increases the expression on the surface of a membrane; the glycosylated B7-H3 triple negative breast cancer cells are not tolerant to T cell killing, and can inhibit proliferation, infiltration and activity of T cells, so that the triple negative breast cancer is mediated to have immune escape. Fucosyltransferase FUT8 positively modulates core fucosylation of B7-H3 and affects its immunosuppressive effects. Increased expression of B7-H3 and FUT8 was associated with poor prognosis in triple negative breast cancer patients, and B7-H3 was positively correlated with FUT8 in triple negative breast cancer tissues. Targeted intervention FUT8 may improve the sensitivity of triple negative breast cancer to PD-L1 antibody therapy by reducing the level of B7-H3 glycosylation.
The foregoing is a more detailed description of the invention and is not to be taken in a limiting sense. It will be apparent to those skilled in the art that simple deductions or substitutions without departing from the spirit of the invention are within the scope of the invention.
<110> Zhongshan university tumor prevention and treatment center (Zhongshan university affiliated tumor hospital, Zhongshan university tumor research institute)
<120> composition for immunotherapy of specific type of triple negative breast cancer
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Claims (10)

  1. The application of B7-H3 protein core fucosylation modification intervention agent in preparing triple negative breast cancer immunotherapy synergist.
  2. 2. Use according to claim 1, characterized in that: the triple-negative breast cancer is glycosylated B7-H3 positive triple-negative breast cancer.
  3. 3. Use according to claim 2, characterized in that: the glycosylation is an N-glycan core fucosylation.
  4. 4. Use according to any one of claims 1 to 3, characterized in that: the core fucosylation modification intervention agent is at least one selected from glycosyltransferase FUT8 expression inhibitor, glycosyltransferase FUT8 activity inhibitor and core fucose analogue.
  5. 5. Use according to claim 1, characterized in that:
    the core fucose analogue is at least one of 2-fluoro-L-fucose and 6-alkynyl fucose;
    the glycosyltransferase FUT8 expression inhibitor is siRNA or sgRNA of FUT 8.
  6. 6. Use according to claim 5, characterized in that: the sequence of the siRNA is siFUT8#1: 5'-CUGCAGUGUGGUGGGUGUCTT-3' or siFUT8# 2: 5'-AGGUCUGUCGAGUUGCUUATT-3'; the sequence of the sgRNA is sgRNA2: 5'-CACCGACAGCCAAGGGTAAATATGG-3' or sgRNA 75 '-CACCGTGAAGCAGTAGACCACATGA-3'.
  7. 7. Use according to any one of claims 1 to 3, characterized in that: the immunotherapy is anti-PDL1 immunotherapy.
  8. 8. Use of a composition in the preparation of a formulation for the treatment of triple negative breast cancer that is glycosylated B7-H3 positive, the composition comprising:
    at least one B7-H3 protein core fucosylation modification intervention agent; and
    at least one immunotherapeutic agent.
  9. 9. Use according to claim 8, characterized in that: the core fucosylation modification intervention agent is at least one selected from glycosyltransferase FUT8 expression inhibitor, glycosyltransferase FUT8 activity inhibitor and core fucose analogue.
  10. 10. Use according to claim 8 or 9, characterized in that: the immunotherapy preparation is selected from anti-PDL1 immunotherapy preparation.
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