CN113274502B - 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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CN113274502B
CN113274502B CN202110487230.5A CN202110487230A CN113274502B CN 113274502 B CN113274502 B CN 113274502B CN 202110487230 A CN202110487230 A CN 202110487230A CN 113274502 B CN113274502 B CN 113274502B
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breast cancer
glycosylation
glycosylated
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negative breast
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CN113274502A (en
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邓蓉
朱孝峰
唐军
黄昀
杜天
李智玲
张海亮
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Sun Yat Sen University Cancer Center
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Abstract

The invention discloses a composition for immunotherapy of specific type three-negative breast cancer. The inventors have identified aberrant B7-H3 glycosylation and have shown 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 abnormal B7-H3 glycosylation 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 immunotherapy of specific triple negative breast cancer
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 the expression of ER, PR, and human epidermal growth factor receptor 2 (HER-2) proteins. 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 morphological, mutant phenotype and signal transduction profiles varying 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, more than 50% of TNBC patients experience relapse, with more than 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, also known as CD276 or B7RP-2, is a tumor specific associated antigen and also plays an important role in non-immunization. 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 that B7-H3 plays 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 prostate intraepithelial neoplasia patients, but B7-H3 is expressed at a lower level in normal prostate tissues, the staining intensity of which is positively correlated with tumor metastasis, recurrence and tumor-specific death, and the correlation between B7-H3 expression level and immunosuppressive efficiency in these patients is stronger. These data suggest that the inventors B7-H3 play an inhibitory role in the immune response to prostate cancer. 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 that B7-H3-associated tumor-specific macrophages strongly suppress T cell mediated immune responses. In addition, B7-H3 can enable the tumor to generate immune escape by up-regulating IL-10 and down-regulating secretion of cytokines such as IL-12 and the like, thereby promoting the generation and development of the tumor. At present, the function of B7-H3 in tumor resistance is controversial, and the specific function 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 that glycosylated B7-H3 affects 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 is reported to be up-regulated in various cancers such as breast cancer, lung cancer, prostate cancer, hepatocellular carcinoma, colorectal cancer and melanoma, and the expression of FUT8 is related to the biological characteristics of tumors and the prognosis of patients. The specific function of FUT8 in tumors is still not clear enough, and the prior art does not treat the 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 do not respond to anti-PD 1/PDL1 immunotherapy, suggesting that it is necessary 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 have identified aberrant B7-H3 glycosylation and have shown that N-glycosylation of B7-H3 on the NXT motif is associated with its protein stability and immunosuppression in TNBC tumors. Fucosyltransferase FUT8 catalyzes 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 abnormalities mediated by FUT8 overexpression have important physiological and clinical significance in TNBC patients. Notably, the combination 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 glycosylated B7-H3 positive triple-negative breast cancer.
In some examples, the glycosylation is N-glycan core fucosylation.
In some examples, the core fucosylation-modifying interfering 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 glycosyltransferase FUT8 expression inhibitor is an siRNA or sgRNA of FUT 8.
In some examples, the siRNA has the sequence of siFUT8#1: 5; the sequence of the sgRNA is sgRNA2: 5-.
In some examples, the immunotherapy is anti-PDL1 immunotherapy.
In a second aspect of the present invention, there is provided:
use of a composition for the preparation of a formulation for the treatment of triple negative breast cancer, said triple negative breast cancer being glycosylated B7-H3 positive triple negative breast cancer, said composition comprising:
at least one B7-H3 protein core fucosylation modification agent; and
at least one immunotherapeutic agent.
In some examples, the triple negative breast cancer is a glycosylated 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 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 glycosyltransferase FUT8 expression inhibitor is an siRNA or sgRNA of FUT 8.
In some examples, the sequence of the siRNA is siFUT8#1: 5-; the sequence of the sgRNA is sgRNA2: 5-.
In some examples, the immunotherapeutic agent is selected from anti-PDL1 immunotherapeutic agents.
The beneficial effects of the invention are:
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.
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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 glycosylated B7-H3 expression in representative samples from breast cancer patients. Immunohistochemical analysis of B7-H3 expression in samples from breast cancer patients 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 TCGA data sets versus overall patient survival curves. Patients were stratified according to the two algorithms of Hu' S SSP and SCMGENE. (B) The Kaplan-Meier website analyzes B7-H3 expression versus Relapse Free Survival (RFS) and disease free survival (DMFS) curves for breast cancer in the TCGA dataset. It is statistically significant by student's t-test. All error bars are expressed as mean ± SD of 3 independent experiments.
FIG. 3: analyzing the glycosylation modification type of B7-H3 in the triple-negative breast cancer cell line; (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) Cells were treated with tunicamycin and cell surface B7-H3 protein expression was analyzed using flow cytometry. .
FIG. 4: constructing glycosylated and non-glycosylated B7-H3 triple-negative breast cancer cell strains; (A) The N-glycosylation motif structure, the human source and the mouse source B7-H3 amino acid sequence diagram. (B, D) schematic representation of the B7-H3 NQ mutants 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 the 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 B7-H3 protein was detected by Western blot analysis. 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), with or without MG132 (100 mM) for 6 hours and analyzed by Western blot analysis (F) for inhibition of glycosylated B7-H3 enhancing 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 of B7-H3 inhibits 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 is a schematic view of: glycosylation B7-H3 inhibits 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: glycosylation B7-H3 in vitro had no effect on the proliferation and invasion capacity of triple negative breast cancer cells. (A) Glycosylated (B7-H3-WT) and non-glycosylated (B7-H3-8 NQ) B7-H3 triple negative breast cancer cell proliferation assays were analyzed by CellTiter Glo, with three replicates per condition. (B) (ii) plate colony formation assay for glycosylated (B7-H3-WT) and non-glycosylated (B7-H3-8 NQ) B7-H3 triple negative breast cancer cells. (C) Glycosylated (B7-H3-WT) and non-glycosylated (B7-H3-8 NQ) B7-H3 triple negative breast cancer cells were subjected to a transwell experiment and the number of migrating cells 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 inhibits infiltration and activity of immune cells in the transplanted tumor. (A) Murine B7-H3 knockout triple negative breast cancer cell line 4T1, overexpressing both glycosylation (B7-H3-WT) and non-glycosylation (B7-H3-4 NQ), was allo-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 CD4 was detected therein + 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 knockout triple negative breast cancer cell lines 4T1 with over-expression of glycosylation (B7-H3-WT) and non-glycosylation (B7-H3-4 NQ) were 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 knockout triple negative breast cancer cell lines MDA-MB-231 with over-expression of glycosylation (B7-H3-WT) and non-glycosylation (B7-H3-4 NQ) were allogeneously inoculated into Balb/c SCID mice and tumor growth was observed. 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 B7-H3 protein was detected by Western blot analysis. (B) Real-time fluorescent quantitative PCR showed that knocking down FUT8 had no effect on B7-H3 mRNA expression (C) whole cell lysates of MDA-MB-231-WT/8NQ cells knocked out with negative controls or FUT8 sgrnas were subjected to LcH affinity chromatography, and the effect of FUT8 knocking down on B7-H3 expression was analyzed by western blot method. (D) MDA-MB-231-WT/8NQ cells transduced with a negative control or gRNA of the Fut8 gene alone were analyzed by flow cytometry to detect the expression of core fucose (Lens collinaris lectin [ LCA ]) and B7-H3 on the cell surface.
FIG. 12: silencing FUT8 restores the inhibitory effect of glycosylated B7-H3 on immunity. (A) Western blot analysis (B) of siRNA FUT8 was knocked down in human B7-H3 knockout triple negative breast cancer cell line MDA-MB-231 overexpressing both glycosylated (B7-H3-WT) and non-glycosylated (B7-H3-4 NQ), then co-cultured with or without activated T cells, and the percentage of T cell-mediated tumor cell killing was detected by flow after 6 hours of co-culture. (C) Flow cytometry analyses 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 glycosylated 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-cell toxicity. (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 analyzed for cell surface B7-H3 expression by flow cytometry. (B) Glycosylated and non-glycosylated B7-H3 triple negative breast cancer cells pretreated with DMSO or 400 μ M2F-Fuc, and then co-cultured with or without activated T cells, and the percentage of T cell-mediated tumor cell killing was determined by flow-based assay after 6 hours of co-culture.
FIG. 14: blocking B7-H3 core fucosylation enhances the anti-tumor immunity sensitivity of the PD-L1 antibody. (A) Schematic representation of the treatment 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, tumor volume and final tumor weight were measured at indicated time points (n = 5.) (C) immunohistochemistry to detect expression of tumor B7-H3 in 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 the different treatment groups.
Detailed Description
The technical scheme of the invention is further explained by combining experiments.
Identification of B7-H3 glycosylation patterns and sites in triple negative breast cancer
1.1 B7-H3 is highly expressed in most breast cancer tissues mainly in a 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 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 glycosylation form according to the previous report that the B7-H3 has glycosylation modification; 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 of B7-H3 is highly expressed in breast cancer tissue (FIG. 1A); to further clarify whether B7-H3 protein overexpression is associated with poor prognosis in triple negative breast, we performed sectioning and immunohistochemical staining of triple negative breast cancer samples from 1999-2005 pathologists, university of zhongshan, tumor prevention and treatment center, and counted staining scores for normal tissues and tumor tissues in each sample. Immunohistochemistry was performed on median basis, and the results of histochemistry suggested significantly higher B7-H3 in tumor tissues than normal tissues (fig. 1B), and the patients were found to have shorter survival time in B7-H3 high expression group than in B7-H3 low expression group and statistically different results (P =0.033, fig. 1C).
High expression of B7-H3 mRNA water in triple negative breast cancer alone is 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 B7-H3 mRNA levels and overall patient survival (OS) 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 two algorithms, SCMG and Hu's SSP. 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). While the inventors also further analyzed the correlation of B7-H3 mRNA expression differences in TCGA databases with patient relapse-free survival (RFS) and distant metastasis-free survival (DMFS) using the KM-Plot website, it was found that B7-H3 mRNA expression was negatively correlated with RFS and DMFS and statistically significant (P =0.0043, P = 0.047) only in patients of Basal-like type, whereas other breast cancer subtypes did not have this feature (fig. 2B).
B7-H3 is mainly modified by N-glycosylation in triple-negative breast cancer
To further determine whether B7-H3 has other glycosylation modification forms in triple negative breast cancer, the inventors selected MDA-MB-231 and HCC1806 triple negative breast cancer cell lines, treated the two cell line proteins with peptide-N-glycosidase F (PNGase F) that removes all N-sugar chain structures, endoglycosidase (Endo H) that removes high mannose and part of oligosaccharides, and disaccharide O-glycase (O-glycanase) that removes all O-linked disaccharides respectively, and the results showed that B7-H3 significantly decreased from 110KD (black circle) to 70KD (black asterisk) when PNGase F was used, while Endo H and O-glycanase did not significantly change, suggesting that B7-H3 has mainly N-glycosylation modification (fig. 3A). Treatment of MDA-MB-231 and Hcc1806 triple negative breast cancer cell lines with the N-linked glycosylation inhibitor Tunicamycin (TM), and the O-linked glycosylation inhibitors Thiamet G and PUGNAc, the inventors found that the N-glycosylation inhibitor TM was able to significantly inhibit the expression of glycosylated 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 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 glycosylated 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 motif structure, asparagine in the conserved sequence is mutated into glutamine (figures B, 4D), and plasmids of human glycosylation B7-H3-WT and mutation B7-H3-8NQ, and plasmids of mouse glycosylation B7-H3-WT and mutation B7-H3-4NQ 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 manner by a CRISPR-Cas9 gene Knock-Out technology, a monoclonal B7-H3-Knock Out cell strain is screened, meanwhile, plasmids of glycosylated forms and non-glycosylated forms are expressed in a reversion manner on the basis of the knocked-Out cell strains, and human source (MDA-MB-231-B7-H3 KO-WT and MDA-MB-231-B7-H3KO-8NQ, HCC1806-B7-H3KO-WT and HCC1806-B7-H3KO-8 NQ) and murine triple negative breast cancer cell models (4T 1-B7-H3KO-WT and 4T1-B7-H3KO-4 NQ) of the glycosylated forms and the non-glycosylated forms B7-H3KO-8 NQ) are constructed; WB experimental results showed that human-derived glycosylated B7-H3 has a molecular weight of about 110KD and about 70KD after mutation, while murine-derived glycosylated B7-H3 has a molecular weight of 55KD and 40KD respectively (FIG. 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 Cycloheximide (CHX), which is a protein synthesis inhibitor, 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 could also be 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 form of B7-H3 in the MG 132-treated group did not show a significant decrease with treatment time, suggesting that the non-glycosylated form of B7-H3 is degraded mainly by the proteasomal pathway (fig. 5E). To explore whether non-glycosylated B7-H3 could be ubiquitinated, the inventors exotransformed B7-H3-8NQ and wild-type ubiquitin (Ub) in HEK293T cells, which was able to ubiquitinate B7-H3-8NQ when both plasmids were co-transfected and increased its ubiquitination level after MG132 blocking degradation (fig. 5F). The expression of B7-H3 on cell membranes in the cell model constructed above was detected, and the expression of the protein on cell membranes was significantly increased only when the glycosylation of B7-H3 occurred, which suggests that the glycosylation modification not only can increase the stability of the B7-H3 protein, but also can promote its expression on cell membranes (FIG. 5G).
Glycosylation B7-H3 mediated immune escape from triple negative breast cancer
2.1 The killing ability of T lymphocyte to glycosylation B7-H3 triple negative breast cancer cell in vitro is obviously reduced
In order to explore the tumor immunity difference of glycosylated and non-glycosylated B7-H3, the inventor uses CD28 and CD3 antibodies to non-specifically activate T lymphocytes of healthy blood donors in vitro for three to six days, and the 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 following ratio of 15; 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 cells to induce glycosylated B7-H3 highly expressed triple negative breast cancer cells (fig. 6A, 6C). After MDA-MB-231 cell line is cultured, sucking part of supernatant, and detecting the tumor killing efficiency of 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 of B7-H3 triple-negative breast cancer cells inhibits 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 strain is irradiated by 80Gy dosage, the triple negative breast cancer cell strain and the staining labeled T cells are co-planted into a pore plate coated with CD28 and CD3 antibodies, and after co-culture is carried out 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 T cell proliferation inhibitory effect, suggesting that glycosylated B7-H3 has the ability and activity to inhibit T cell proliferation (7 a, 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 clonal formation by using in vitro MTT experiment and clonal formation experiment, and observed and recorded the cell growth condition for 7 days continuously, 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 for clonogenic, with no difference in clonogenic capacity between glycosylated and non-glycosylated B7-H3 cells (FIGS. 8A, 8B). The successfully constructed tumor cell strain is placed in a Transwell chamber and cultured for 16-24 hours to observe the number of different tumor cells penetrating through a cell permeable membrane so as to evaluate the migration capability of the tumor cells, and the experimental result proves that the influence of glycosylation and non-glycosylation B7-H3 on the migration capability of the triple negative breast cancer cells is not different (FIG. 8C).
Glycosylated B7-H3 significantly promotes the growth of transplanted tumors in immunocompromised mice and inhibits infiltration and activity of T lymphocytes in the 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; measuring and recording the tumor volume of the mouse periodically, taking out the tumor of the mouse after the experiment is finished, and weighing; experimental results show that glycosylation B7-H3 can obviously promote the growth of mouse transplantation tumor, and statistical analysis shows that the tumor volume and tumor weight of the mouse are obviously increased compared with those of 4T1-B7-H3KO-4NQ (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 and 4T1-B7-H3KO-4NQ, it was found that the group of B7-H3 was glycosylated and the CD4 infiltrated therein was + T cell CD8 + The number of T cells and NK cells is obviously reduced, and CD8 infiltrated + Activity index IFN-gamma in T cellsAnd the Granzyme content was also significantly reduced (fig. 9B), the above evidence suggests that glycosylation B7-H3 can inhibit tumor immunity by inhibiting infiltration and activity of immune cells in tumors, thereby promoting tumor development.
2.5 The glycosylation B7-H3 has no obvious influence on the growth of the transplantation tumor of the immunodeficient mouse
In order to further verify that the glycosylation B7-H3 mainly plays a role of promoting tumors by regulating an immune system, the inventor respectively inoculates two cell strains, namely 4T1-B7-H3KO-WT and 4T1-B7-H3KO-4NQ, on abdominal bilateral breast fat pads of a Balb/c Nude mouse with immunodeficiency, regularly measures and records the tumor volume of the mouse, and takes out the tumor of the mouse and weighs the tumor after the experiment is finished; the tumor growth curves in mice suggest that glycosylated B7-H3 had no effect on tumor growth in immunodeficient mice, and statistical analysis of tumor weights in 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 bilateral abdominal breast fat pad of the immunodeficient Balb/c Nude mice, and records the fat pad by regular measurement. The results of the experiments were consistent with the above conclusions for murine cell lines, and there was no significant difference in the tumorigenic capacity and effect on tumor volume and tumor weight for humanized glycosylated and non-glycosylated B7-H3 cell lines (FIG. 10B). The above results further show that glycosylated B7-H3 mainly affects tumor growth through an immune system in a triple negative breast cancer animal model.
And the molecular mechanism of glycosylation modification of B7-H3
To explore the relationship between FUT8 and B7-H3, the inventors selected different sgRNAs to knock out FUT8 in MDA-MB-231 and HCC1806 cell strains, and detected the expression of B7-H3, and the experimental results found that the glycosylated B7-H3 protein is significantly reduced but mRNA of B7-H3 is not significantly changed when FUT8 is knocked out, 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, 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 lectin LCH (LCA) antibodies that specifically bind to core fucose), and as a result, it was shown that B7-H3 of core fucose type was significantly reduced after FUT8 knockout, and that reduction of FUT8 had some effect on total glycosylation of 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, the expression of core fucoidin on the cell membrane surface can be obviously reduced by FUT8 (FIG. 11D), and the expression of B7-H3 on the membranes is also obviously reduced, which indicates that the expression of B7-H3 of core fucose on the cell membranes is also reduced after the FUT8 is inhibited (FIG. 11D).
Negative regulation of T cell killing ability and activity through B7-H3
Early experiments prove that the B7-H3 triple-negative breast cancer cells with high expression glycosylation can obviously inhibit the T lymphocytes from killing the breast cancer cells and obviously inhibit the proliferation of the T lymphocytes. The inventors used RNA interference technology to knock down FUT8 (siRNA is siFUT8#1: 5- -. In addition, the inventor discovers that the inventor can obviously enhance CD4 when knocking down FUT8 in the B7-H3 cell line by the low glycosylation of FUT8 in the MDA-MB-231-B7-H3KO-WT cell line through flow analysis, after irradiating the treated tumor cells with 80Gy dosage, co-culturing the tumor cells and T lymphocytes stained by hydroxyl fluorescein diacetate succinimide ester (CFSE) of living cell fluorescent labeling dye for 4-7 days, and detecting the proliferation and activity indexes IL-2 and IFN-gamma of the T lymphocytes in a flow mode + 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 experimental results suggest enhancement after knocking down FUT8Killing of the glycosylated B7-H3 cell strain by the T cell is realized, and the inhibiting effect of the glycosylated B7-H3 on the activity of the T cell is relieved.
Targeted intervention of FUT8 to reduce B7-H3 expression to enhance sensitivity of triple negative breast cancer to immunotherapy
4.1 Inhibition of FUT8 enhances 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 explore whether 2F-Fuc can reduce the expression of glycosylated B7-H3, the inventors treated MDA-MB-231-B7-H3KO-WT, MDA-MB-231-B7-H3KO-8NQ and 4T1-B7-H3KO-WT cell strains with different concentrations of 2F-Fuc, and after four days, the expression of surface B7-H3 protein on the surface of the cell membrane was detected by a flow method, and the flow result suggests that the B7-H3 on the surface of the cell membrane is significantly reduced after the 2F-Fuc is added to treat breast cancer cell strains of human and mouse sources (FIG. 13A, 13C), which suggests that the 2F-Fuc can reduce the expression of glycosylated B7-H3 on the cell membrane. In order to determine whether the 2F-Fuc can restore the killing capacity of T cells to tumor cells by reducing the expression of glycosylated B7-H3, MDA-MB-231-B7-H3KO-WT and MDA-MB-231-B7-H3KO-8NQ cell strains are pretreated by the 2F-Fuc, co-cultured with T lymphocytes after four days, and the positive ratio of a Caspase-3 index is detected by flow; the results found that the T cell killing ability of glycosylated B7-H3 cell line was significantly enhanced by pretreatment with 2F-Fuc compared to the untreated group, while 2F-Fuc had no significant effect on the T lymphocyte killing ability of non-glycosylated tumor cells (fig. 13B), indicating that 2F-Fuc enhances the T lymphocyte killing of tumor cells mainly by 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 killing effect by reducing the expression of glycosylated 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 two groups of belly breast fat pads of Balb/c mice with normal immunity; 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 gastric lavage treatment is carried out three times per week after inoculation; anti-PD-L1 and Isotype were treated by intraperitoneal injection at intervals of 3 days for a total of three times. 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 detect the number and activity of the infiltrated immune cells in different groups in a flow manner. 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 combined (figure 14B), the B7H3 expression in the tumor tissues is obviously reduced after the 2F-Fuc treatment by the immunohistochemical analysis (figure 14C), and the CD4 can be obviously increased by the combination of the 2F-Fuc and the Anti-PD-L1 by the flow analysis + T cell, CD8 + Expression of IFN- γ as an indicator of T-cell and NK-cell activity (FIG. 14D), tunel staining analysis showed that 2F-Fuc in combination with Anti-PD-L1 enhanced the apoptotic rate of tumors. Therefore, the inventor can obtain that the 2F-Fuc and Anti-PD-L1 can achieve the Anti-tumor effect by enhancing the function of immune cells in the tumor.
And (4) conclusion:
B7-H3 protein mainly generates N-glycosylation modification in triple-negative breast cancer, 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 the core fucosylation of B7-H3 and affects its immunosuppressive effects. The increased expression of B7-H3 and FUT8 is related to the poor prognosis of the three-negative breast cancer patients, and B7-H3 and FUT8 are positively correlated in the three-negative breast cancer tissues. Targeted intervention FUT8 may increase the sensitivity of triple negative breast cancer to PD-L1 antibody therapy by decreasing the level of B7-H3 glycosylation.
The foregoing is a further detailed description of the invention and is not to be taken in a limiting sense as the invention is defined by the appended claims. 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 (1)

  1. Application of a B7-H3 protein core fucosylation modification intervention agent in preparation of a triple-negative breast cancer immunotherapy synergist, wherein the triple-negative breast cancer is glycosylated B7-H3 positive triple-negative breast cancer, the glycosylation is N-glycan core fucosylation, the core fucosylation modification intervention agent is siRNA of FUT8, the sequence of the siRNA is siFUT8#1: 5.
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