Application of IGSF3 inhibitor in preparation of medicine for treating cervical cancer

Abstract

The invention discloses application of an IGSF3 inhibitor in preparation of a medicine for treating cervical cancer, and belongs to the field of biological medicine. Based on high expression of IGSF3 in cervical cancer tissues, the invention finds that IGSF3 weakens anti-tumor immune response in a cervical cancer tumor immune microenvironment and inhibits anti-tumor immunity by regulating and controlling regulatory T cells. According to the invention, the red phenol is further screened and can be used as a novel IGSF3 inhibitor, and experimental results show that the red phenol can significantly inhibit tumor growth and can effectively improve the tumor immune microenvironment; rhodol and anti-PD1 or anti-CTLA4 are combined for use, combined treatment or sequential treatment is carried out on cervical cancer, and a remarkable anti-tumor effect is shown. The invention provides a new medicine and a treatment strategy for treating cervical cancer, and has outstanding practical application value and wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of an IGSF3 inhibitor in the preparation of a drug for treating cervical cancer. Background Technology

[0002] Cervical cancer ranks fourth among the most common malignant tumors in women. In recent decades, immune checkpoint blockade (ICB) therapy—especially inhibitors targeting programmed cell death protein 1 (PD-1)—has revolutionized cancer treatment. According to the latest NCCN guidelines, ICBs are now an integral part of the treatment system as a first-line treatment for recurrent, metastatic, and locally advanced cervical cancer. However, their efficacy remains limited, with an objective response rate (ORR) of less than 30%. This ongoing clinical challenge underscores the need to elucidate other immune escape mechanisms employed by tumor cells in order to develop more effective treatment strategies.

[0003] Immunoglobulin superfamily member 3 (IGSF3) possesses eight immunoglobulin-like domains and is overexpressed in various human malignancies. It mediates cell-cell adhesion and promotes tumor metastasis and invasion. Notably, recent evidence suggests that IGSF family members can interact with components of the tumor immune microenvironment (TIME). Although IGSF3 is a classic member of this family, its role in tumor immunity remains unclear, and further research is needed to investigate its specific impact on immune escape and ICB response in cervical cancer.

[0004] Purpurogallin is a natural polyphenolic compound isolated from plants and is generally used for anti-inflammatory or antioxidant purposes. Studies have shown that purpurogallin also has significant xanthine oxidase inhibitory activity. However, there are no reports on the application of purpurogallin in cancer treatment, especially as an IGS-SF3 inhibitor. Summary of the Invention

[0005] The purpose of this invention is to provide an application of an IGSF3 inhibitor in the preparation of a drug for treating cervical cancer, in order to solve the problems existing in the prior art. This invention screened red betaine as a novel IGSF3 inhibitor. Red betaine can significantly inhibit tumor growth. Red betaine, when used in combination with anti-PD1 or anti-CTLA4, has a significant anti-tumor effect.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the application of an IGSF3 inhibitor in the preparation of a drug for treating cervical cancer.

[0008] Furthermore, the IGSF3 inhibitor is erythrophenol.

[0009] The present invention also provides a composition comprising erythrophenol and a PD1 inhibitor.

[0010] The present invention also provides the use of the above-described composition in the preparation of a medicament for treating cervical cancer.

[0011] The present invention also provides a medicament for treating cervical cancer, wherein the medicament is based on the above-described composition as the main active ingredient.

[0012] Furthermore, it also includes pharmaceutically acceptable excipients.

[0013] The present invention also provides a composition comprising erythrophenol and a CTLA4 inhibitor.

[0014] The present invention also provides the use of the above-described composition in the preparation of a medicament for treating cervical cancer.

[0015] The present invention also provides a drug for treating cervical cancer, wherein the above-described composition is the main active ingredient of the drug.

[0016] Furthermore, it also includes pharmaceutically acceptable excipients.

[0017] The present invention discloses the following technical effects:

[0018] This invention, based on the high expression of IGSF3 in cervical cancer tissues, discovered that IGSF3 weakens the anti-tumor immune response in the tumor immune microenvironment of cervical cancer by regulating regulatory T cells, thereby inhibiting anti-tumor immunity. Further screening revealed that erythrophenone can serve as a novel IGSF3 inhibitor. Experimental results show that erythrophenone can significantly inhibit tumor growth and effectively improve the tumor immune microenvironment. Combining erythrophenone with anti-PD1 or anti-CTLA4 agents for combination or sequential therapy against cervical cancer also demonstrated significant anti-tumor effects. This invention provides new drugs and treatment strategies for cervical cancer, possessing outstanding practical application value and broad application prospects. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1The image shows the results of the experiment on the upregulation of IGSF3 expression in cervical cancer epithelial cells; where A is a t-SNE dimensionality reduction plot of 77,252 cells from 10 patients with normal cervix, HSIL, and cervical cancer, colored by cell type (left) and tissue group (right); B is the GO enrichment analysis of genes that were significantly upregulated in cervical cancer epithelial cells; the horizontal axis (Gene) (percent) represents the percentage of upregulated genes associated with each GO entry out of all annotated genes in that category; C is the t-SNE plot of 18,199 epithelial cells from normal cervix, HSIL, and cervical cancer in 10 patients, colored by subgroup (left) and tissue type (right); D is the distribution of the proportion of each epithelial subgroup in normal cervix, HSIL, and cervical cancer; E is a heatmap of differentially expressed genes between normal cervix / HSIL and cervical cancer (left) and a schematic diagram of the IGSF3 structure predicted by AlphaFold3 (right); the heatmap color represents the Z-value of gene expression in each cluster, and the number in parentheses indicates the number of immunoglobulin (Ig) domains contained in the protein; F is the representative immunohistochemistry of IGSF3 protein in normal cervix, early and late cervical cancer. Chromosomal staining; scale bars are 200 μm (left) and 50 μm (right); G is a box plot of IGSF3 expression levels in normal cervix, HSIL, and cervical cancer based on scRNA-seq; H is a pseudo-temporal analysis trajectory plot of epithelial cells based on scRNA-seq (showing two lineages), with color intensity representing gene-normalized expression levels; I is the overall survival curve of squamous cervical cancer patients in the TCGA database with high / low IGSF3 expression (n=231, p=0.0224, HR=2.174); J is a Spearman scatter plot showing the negative correlation between IGSF3 expression and immune scores calculated by the ESTIMATE algorithm; *P<0.05, ***P<0.0001; CC indicates cervical cancer;

[0021] Figure 2 Figure 1 shows the results of experiments relating IGSF3 expression to regulatory T cell (Treg) infiltration; where A is a tumor growth curve showing the effect of IGSF3 expression on tumor growth; and B is subcutaneous IGSF3 in C57 mice. KD With IGS F3 NC CD45 in TC1 tumors + Percentage of immune cells; C is the volcano plot showing IGSF3. KD With IGS F3 NC Differentially expressed genes in tumor tissues of TC1 tumor-bearing mice; D is a GO enrichment plot showing pathways significantly enriched in TIME after IGSF3 knockdown; the horizontal axis (Gene percent) represents the percentage of upregulated genes associated with each GO item out of all annotated genes in that category; E is the immune score of each immune cell subtype in the control group and the IGSF3 knockdown group; F is the GSEA enrichment analysis showing IGSF3... KDAlterations in Treg differentiation-related pathways in tumors; G is a box plot showing the negative correlation between IGSF3 expression and Treg immunosuppressive function calculated by the Xcell algorithm in cervical cancer samples from the TCGA database; H is immunofluorescence staining of human cervical cancer tissue showing the spatial distribution of Tregs in epithelial cell regions with high IGSF3 expression (left) and low expression (right); linear regression analysis showed a significant positive correlation between IGSF3 expression level and Treg infiltration density (R0). 2 =0.7776, P<0.0001); scale bars are 200μm (left) and 50μm (right); I represents IGSF3. NC / IGSF3 KD CD4 in tumor tissue of tumor-bearing mice + CD25 in T cells + FOXP3 + Tregs percentage; J represents IGSF3. NC / IGSF3 KD CD4 in Siha cell and PBMC co-culture system + CD25 in T cells + FOXP3 + Treg percentage; unless otherwise noted, all data are expressed as mean ± standard error; *p<0.05, ***p<0.0001; NC represents normal control group, KD represents IGSF3 knockdown group, TIME represents tumor immune microenvironment, PBMCs represent peripheral blood mononuclear cells;

[0022] Figure 3 Figure 1 shows the experimental results of IGSF3 enhancing Treg function and inhibiting antitumor immunity; where A represents... Figure 3 Experimental design flowcharts for B and C; B represents IGSF3. KD / IGSF3 NC The expression ratio of PD1 and CTLA4 on the surface of Tregs within TC1 tumor-bearing mice; C represents IGSF3. KD / IGSF3 NC The proportion of TGF-β and IL-10 secreted by Tregs within the tumor of TC1 tumor-bearing mice; D is Figure 3 Experimental design flowcharts for E and F; E represents IGSF3. KD / IGSF3 NC The proportion of MHC-II expression on the surface of intratumoral dendritic cells (DCs) in TC1 tumor-bearing mice; F represents IGSF3. KD / IGSF3 NC The expression ratio of CD80 and CD86 on the surface of DCs within tumors in TC1 tumor-bearing mice; G represents... Figure 3 Experimental design flowchart for HJ; H represents IGSF3. KD / IGSF3NC TC1 tumor-bearing mice with CD8 in tumors + The ratio of GZMB and IFN-γ secreted by T cells; I represents IGSF3. KD / IGSF3 NC TC1 tumor-bearing mice with CD8 in tumors + The percentage of memory T cells (Tmem) in T cells; J represents IGSF3. KD / IGSF3 NC TC1 tumor-bearing mice with CD8 in tumors + The percentage of exhausted precursor T cells (Tpex) in T cells; K is... Figure 3 Experimental design flowcharts for L and M; L represents a co-culture system without Tregs (tumor cells + DCs + CD8). + CD8 in T cells + T cell CD25 expression ratio; M represents a co-culture system containing Tregs (tumor cells + DCs + CD8+). + CD8 in T cells + Tregs + The proportion of IFN-γ expression on T cells; data are expressed as mean ± standard error, and the Student's t-test was used for statistical analysis; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; Tmem represents memory T cells, and Tpex represents exhausted precursor T cells;

[0023] Figure 4 The diagram shows the experimental results of IGSF3 enhancing Treg function by binding to TNFR2. A is the experimental design flowchart for identifying the IGSF3 receptor on the surface of Tregs; B is a volcano plot showing differentially expressed genes in the IGSF3-positive and negative groups; C is a schematic diagram of the immunoprecipitation-mass spectrometry (IP-MS) experimental design; D is a Venn diagram of RNA-seq and IP-MS analysis of common genes; E is a flow cytometry histogram and mean fluorescence intensity (MFI) bar chart showing an increased ΔMFI in the TNFR2 overexpression group compared to the control group; F is a schematic diagram of the IGSF3-TNFR2 binding pattern predicted by AlphaFold3; G is an ELISA binding curve showing increasing TNFR2-Fc coating concentrations at 2 μg / ml. Binding affinity of IGSF3-His protein; H is the sensor map of IGSF3-TNFR2 interaction detected by surface plasmon resonance (SPR); I is the schematic diagram of the pull-down experiment design and the experimental results of IGSF3 binding to TNFR2-His / GFP-His; J is the immunofluorescence microscopy image showing the co-localization of IGSF3 and TNFR2 (scale bar: 4 μm); K is IGSF3 NC / IGSF3 KDThe proportion of TNFR2 expression on the surface of Tregs in tumor tissues of TC1 tumor-bearing mice; L represents the expression of CD4+ in the Siha cell and PBMC co-culture system. + The percentage of Tregs in T cells (divided into three groups: IGSF3) NC Siha+PBMCs, IGSF3 NC Siha+PBMCs+anti-TNFR2 antibody, IGSF3 KD Siha+PBMCs calculation); data are expressed as mean ± standard error; **P<0.01, ***P<0.001; OE indicates overexpression, IF indicates immunofluorescence;

[0024] Figure 5 Figure 1 shows the experimental results of the interaction sequence and downstream signaling pathways between IGSF3 and TNFR2; Figure 2 shows the schematic diagram of the domain composition of IGSF3 and TNFR2; Figure 3 shows the pull-down experimental design: comparing the binding affinity of wild-type and binding sequence deletion mutants; Figure 4 shows the pull-down experimental results of IGSF3-WT-HA / IGSF3-MUT-HA and TNFR2-His protein; Figure 5 shows the pull-down experimental results of TNFR2-WT-HA / TNFR2-MUT-HA and IGSF3-His protein; Figure 6 shows the flow cytometry histogram and bar chart showing the effect of TNFR2-His on the interaction between IGSF3 and TNFR2. Different affinities of IGSF3-WT-HA / MUT-HA; F shows flow cytometry histograms and bars illustrating the different affinities of IGSF3-His for TNFR2-WT-HA / MUT-HA; G shows GSEA enrichment analysis demonstrating the downstream pathway characteristics of IGSF3-TNFR2; H shows Western blotting showing the difference in NF-κB pathway induction in Jurkat cells transfected with TNFR2-WT-HA / MUT-HA plasmids under IGSF3-His stimulation; data are expressed as mean ± standard error; *P<0.05, **P<0.01; WT represents wild-type, MUT represents a binding domain deletion mutant;

[0025] Figure 6 The diagram shows the experimental results of red betaine inhibiting tumor growth by blocking IGSF3; where A is a schematic diagram of the virtual screening process for IGSF3 small molecule inhibitors; B is... Model-predicted potential active binding pocket scores for IGSF3; C is a schematic diagram of the site2 active pocket structure of GSF3; D shows the preliminary binding affinity of the small molecule compound to IGSF3 as shown by SPR at a fixed concentration; E confirms the direct binding of rubrin to IGSF3 by SPR kinetic analysis (KD = 1.32 μM); F shows the binding conformation of rubrin in the Ig-like-C2 type 5 domain of IGSF3 predicted by the AlphaFold3 model; G is... A schematic diagram of the simulated IGSF3 activity pocket and the molecular dynamics (MD) trajectory of erythromycin; H represents the effect of ELISA detection showing that erythromycin significantly reduces the binding affinity of IGSF3-TNFR2; I represents the CD4+ ionotropic protein in the Siha cell and PBMC co-culture system. + The proportion of Tregs in T cells; J represents the in vivo antitumor efficacy curve of erythromycin (20 / 30 / 40 mg / kg dose groups); K represents the intratumoral CD4 count in different erythromycin treatment groups. + The percentage of Tregs in T cells; L represents the tumor infiltration CD8 in different dose groups. + GZMB expression percentage in T cells; data are expressed as mean ± standard error; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001;

[0026] Figure 7 The results of the experimental study on the efficacy of erythromycin in enhancing anti-PD1 / anti-CTLA4 activity are shown in Figure 1. A represents the experimental design, including monotherapy (left), erythromycin combined with αPD1 / αCTLA4 (middle), and erythromycin pretreatment followed by sequential αPD1 / αCTLA4 treatment (right). B shows the tumor volume growth curves in mice of different treatment groups. C shows the CD4+ levels in tumor tissues of different treatment groups. + The proportion of Tregs in T cells; D represents the CD8 infiltrating tumor in different treatment groups. + The proportion of GZMB expression in T cells; E represents the tumor volume growth curves in mice of different treatment groups; F represents the CD4 expression in tumor tissues of different treatment groups. + The proportion of Tregs within T cells; G represents the CD8 infiltrating tumor cells in different treatment groups. + The proportion of GZMB expression in T cells; H represents the comparison of the gross morphology of tumor tissues in different treatment groups (scale bar: 1cm); data are expressed as mean ± standard error; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Detailed Implementation

[0027] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0028] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0029] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0030] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0031] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0032] Purpurogallin of this invention is a phenolic substance with the molecular formula C2. 11 H8O5, CAS number 569-77-7.

[0033] This invention designs and synthesizes shRNA using lentivirus as a vector based on the IGSF3 DNA sequence of mice and humans, and transfects it into TC1 and Siha cells to achieve IGSF3 knockdown. After determining the interaction between IGSF3 and TNFR2, we used AlphaFold3 to predict the structure of their complex, and designed and constructed a series of mutants based on the obtained key binding sequences. Subsequently, we evaluated the effect of each mutation on protein binding ability through pull-down experiments.

[0034] Example 1: IGSF3 is highly expressed in cervical cancer (CC) and is closely related to disease progression and prognosis.

[0035] To investigate the mechanisms by which malignant cells in cancer (CC) remodel the tumor immune microenvironment (TIME) and mediate immune escape, this embodiment analyzed single-cell transcriptome sequencing (scRNA-seq) data previously generated by the inventors' team. This data included 4 normal cervical samples, 2 high-grade squamous intraepithelial lesion (HSIL) samples, and 4 CC samples. Using known lineage markers, all single cells were classified into 12 cell populations: epithelial cells, T cells, neutrophils, macrophages, natural killer (NK) cells, dendritic cells (DCs), plasma cells, B cells, mast cells, fibroblasts, endothelial cells, and smooth muscle cells (SMCs). Figure 1 (A). Notably, there was significant overlap in immune cells and stromal cells among the three tissue groups, while the epithelial cells of tumor, HSIL, and normal samples showed low similarity, suggesting that cervical epithelial cells underwent significant changes during carcinogenesis. GO enrichment analysis showed that upregulated genes in CC tissue epithelial cells were mainly enriched in cell adhesion, DNA replication, and immunosuppression pathways. Figure 1 (B) indicates that CC epithelial cells have strong metastatic and immune escape capabilities.

[0036] After further dividing the epithelial cell subsets into 16 subsets ( Figure 1 From the data (C), it can be observed that Epi_0 and Epi_4 subpopulations contain three types of cell samples, Epi_1 and Epi_2 are mainly derived from HSIL / CC tissues, while Epi_5 is almost entirely derived from tumor samples. Figure 1 The expression of differentially expressed genes (DEGs) in epithelial cells reflects their tissue specificity. Analysis of differentially expressed genes (DEGs) in CC-derived and normal / HSIL-derived epithelial cells revealed that most upregulated DEGs encode adhesion molecules, with significant enrichment of immunoglobulin superfamily (IGSF) members. Notably, IGSF3, a type I transmembrane protein that has not been clearly characterized, has attracted significant attention due to the presence of the largest number of Ig-like domains in its extracellular region (suggesting strong recognition and adhesion capabilities). Figure 1 (E).

[0037] To investigate the role of IGSF3 in the progression of cervical cancer (CC), this study performed immunohistochemical (IHC) staining on 6 normal cervical samples, 5 early-stage CC samples, and 6 late-stage CC samples. Figure 1 In the study, it was found that IGSF3 expression increased with disease progression, consistent with scRNA-seq results. Figure 1 (G). When constructing the developmental trajectory of CC epithelial cells through pseudo-time series analysis, combined with the expression characteristics of malignant markers MKI67 and TOP2A, it was found that IGSF3 levels gradually increased during the malignant transformation of CC epithelial cells. Figure 1 (H). Further data from TCGACESC showed that patients with high IGSF3 expression had worse overall survival (HR = 2.174, P = 0.0224). Figure 1 I). Given that IGSF3 contains multiple Ig-like domains, it is speculated that it may influence the CC microenvironment through immune regulation. ESTIMATE algorithm analysis showed that IGSF3 expression was negatively correlated with CC immune scores. Figure 1 The presence of J in the sample suggests that it may play a negative immunomodulatory role.

[0038] In summary, IGSF3 is significantly overexpressed in CC tissues compared to normal cervix and precancerous lesions, and may participate in disease progression and prognosis by negatively regulating CC TIME.

[0039] Example 2: IGSF3 remodels the cervical cancer immune microenvironment by regulating regulatory T cell (Treg) infiltration.

[0040] To elucidate the effect of IGSF3 on chronological time (TIME) in cervical cancer, this embodiment first constructs an IGSF3 knockdown (IGSF3) assay. KD ) and control (IGSF3) NC The TC1 cell line was implanted into C57 mice and cultured for 3 weeks. Results showed that knockdown of IGSF3 in tumor cells significantly inhibited tumor growth in vivo. Figure 2 (A), accompanied by CD45 + Increased proportion of immune cells Figure 2 The results (B) indicate that IGSF3 knockdown enhances immune infiltration. Batch RNA sequencing of mouse tumor tissues revealed that IGSF3 silencing in tumor cells upregulates adaptive immune-related functional genes such as CCL21, ULBP1, and TNFSF18, while downregulating immune checkpoint molecules such as PDL1 (CD274), CTLA4, and SIGLEC1. Figure 2 In the middle C), a weakened immunosuppressive phenotype was observed. GO analysis showed that after IGSF3 knockdown, upregulated genes were significantly enriched in anti-tumor immune-related pathways ( Figure 2 These results confirm the negative regulatory role of IGSF3 in cervical cancer TIME (d).

[0041] To clarify the main immune cell subsets affected by IGSF3, the inventors analyzed batch RNA sequencing data using the ConsensusTME algorithm and found that Tregs showed a significant decrease in immune scores. Figure 2 This is consistent with the downregulation of Treg marker genes (FOXP3 and CTLA4) after IGSF3 knockdown. Figure 2 (C). GSEA analysis further confirmed that IGSF3 knockdown led to a significant decrease in the enrichment of Treg-related pathways. Figure 2 Analysis using the Xcell algorithm based on the TCGA CESC database showed that high IGSF3 expression was positively correlated with enhanced Treg inhibitory function. Figure 2 The results showed that IGSF3 has a significant effect on Tregs in cervical cancer TIME.

[0042] To verify this association, this embodiment employs multiple techniques for a systematic investigation: immunofluorescence staining of 80 cervical cancer tissues showed that regions with high IGSF3 expression had more Treg infiltration ( Figure 2 (H); flow cytometry analysis revealed IGSF3. KD The proportion of Tregs in TC1 cell tumor-bearing mice during TIME was significantly lower than that in the control group. Figure 2 (I); In vitro experiments were conducted by constructing IGSF3 KD / NC Co-culturing Siha cell lines with peripheral blood mononuclear cells confirmed IGSF3 NC Cellular ratio IGSF3 KD Cells can better promote CD4 + T cells differentiate into Tregs ( Figure 2 These results consistently confirmed the positive correlation between IGSF3 expression and Tregs accumulation at the tissue, animal model, and cell experiment levels.

[0043] In summary, this invention reveals that IGSF3 negatively regulates the cervical cancer immune microenvironment by promoting Tregs infiltration, providing a new molecular perspective for understanding the immune escape mechanism of cervical cancer.

[0044] Example 3: IGSF3 deficiency inhibits Tregs function and enhances anti-tumor immunity

[0045] After elucidating the effect of IGSF3 on the number of Tregs in cervical cancer TIME, this embodiment further explores the regulatory effect of IGSF3 knockdown on Treg function. Figure 3 (A) Compared with the control group, IGSF3 KD In TC1 tumor-bearing mice, Tregs not only showed decreased expression of effector activating molecules (PD1, CTLA4) in tumor tissues, but also... Figure 3 (B) and at the same time, the secretion of lymphokines is reduced ( Figure 3 The results (C) indicate that under IGSF3 deficiency, both contact-dependent and non-contact inhibitory functions of Tregs are impaired. In vitro experiments also confirmed that IGSF3, regardless of the presence or absence of cell contact, [is affected]. KD The inhibitory function of Tregs was impaired in the Siha cell co-culture system. These results collectively indicate that tumor cells expressing high levels of IGSF3 can enhance the immunosuppressive function of Tregs, while IGSF3 deficiency weakens this effect.

[0046] Given that Tregs exert their immunomodulatory effects by inhibiting antitumor responses, we further analyzed DCs and CD8s after IGSF3 silencing. + Changes in the function of T cells, two key cells in anti-tumor immunity. Regarding DC antigen presentation ability ( Figure 3 While IGSF3 deficiency did not significantly restore the primary MHC-II stimulus signal, it did enhance the expression of co-stimulatory molecules such as CD80 / CD86. Figure 3 E and F in the middle stage indicate improved immune surveillance function within TIME. For tumor-infiltrating CD8... + T cells ( Figure 3In G), IGSF3 deficiency leads to increased secretion of granzyme B (GZMB) and IFN-γ. Figure 3 The presence of H+ indicates enhanced cytotoxicity. Furthermore, the proportion of memory T cells (Tmem, CD62LLowCD44High) is increased. Figure 3 (I), exhausted precursor T cells (Tpex, TCF1) + TIM3 - This subset, which can differentiate into highly cytotoxic terminal TILs, mediate long-term tumor control, and respond to PD-1 therapy, also showed a significant increase. Figure 3 (J) indicates that IGSF3 knockdown can improve CD8 + Dysfunctional T-cell phenotype.

[0047] To clarify the role of Tregs in this process, the inventors isolated CD8+ from mouse spleens. + T cells, DCs, and Tregs, with IGSF3 NC / KD Detection of CD8 after co-culturing TC1 cells + T cell activation markers CD25 and effector molecule IFN-γ ( Figure 3 (Middle K). It is worth noting that, under the condition of no Tregs, IGSF3 NC With IGS F3 KD TC1 cells against CD8 + There was no significant difference in the direct activation of T cells; however, the presence of Tregs significantly amplified the effect of IGSF3 on CD8. + The regulatory effect of T cells ( Figure 3 (L and M). These results reveal that IGSF3 weakens the antitumor immune response in cervical cancer TIME through a Treg-dependent pathway.

[0048] Example 4: Interaction between IGSF3 and TNFR2 on the Treg surface

[0049] Given that IGSF3 is highly expressed on the surface of tumor cells and can enhance the immunosuppressive function of Tregs, it is hypothesized that IGSF3 may exert its regulatory role through interaction with specific receptors on the surface of Tregs. To identify the binding partners of IGSF3 on Tregs, this invention employs the following research strategy: firstly, CD4+ receptors are separated into Tregs by flow cytometry... + T cells are divided into two groups: those that can bind IgSF3 protein and those that cannot. Figure 4 In A), CD4 binding to IGSF3 was found. + T cells highly express FOXP3 and immune checkpoint molecules such as PDCD1, CTLA4, and LAG3. Figure 4The results (B) confirmed that this cell group consisted of Tregs with strong immunosuppressive capabilities. Differential gene analysis showed that known immunosuppressive membrane receptor genes, including TNFR2, TNFRSF11B, and CD123, were significantly upregulated in this cell group. Secondly, lysis of IGSF3-binding cells and mass spectrometry analysis identified membrane proteins such as BTN3A3, TNFR2, and ABCB7. Notably, TNFR2 was the only membrane protein co-occurring in both analyses. Figure 4 The presence of D suggests that it may be a receptor on the surface of Tregs that recognizes IGSF3. To verify this hypothesis, the inventors overexpressed TNFR2 in Jurkat cells and found that its binding ability to IGSF3-biotin was significantly enhanced. Figure 4 The study confirmed that IGSF3 specifically binds to TNFR2.

[0050] TNFR2, a member of the TNF receptor superfamily, is known to be highly expressed in immunosuppressive cells such as Tregs and myeloid-derived suppressor cells (MDSCs). Using the AlphaFold3 model, we found that IGSF3 and TNFR2 can form 12 hydrogen bonds at specific sites. Figure 4 The combined ipTM+pTM score of TNFR2 and IgG3 (ECF) reached 0.89, indicating high affinity between the two. ELISA experiments showed that TNFR2 ECD-Fc binds to IgG3 (ECF) in a concentration-dependent manner. 50 =0.22μg / mL)( Figure 4 (G); Surface plasmon resonance (SPR) analysis showed a binding affinity of 4.48 μM (G); Figure 4 (H); pull-down experiments also confirmed the direct interaction between the two (H); Figure 4 (I) Immunofluorescence showed that there was co-localization signal of IGSF3-TNFR2 at the contact site between tumor cells and Tregs (I). Figure 4 (J). In vivo experiments showed that knockdown of IGSF3 reduced the proportion of TNFR2+Tregs in tumor tissue. Figure 4 In an in vitro co-culture system, the anti-TNFR2 antibody could reverse the activation effect of Tregs by TC1 cells that highly expressed IGSF3. Figure 4 These results collectively confirm the specific molecular interaction between IGSF3 on the surface of tumor cells and TNFR2 on the surface of Tregs.

[0051] Example 5: Identification of the minimum binding sequence of IGSF3 and TNFR2

[0052] To further elucidate the direct interaction mechanism between IGSF3 and TNFR2, this embodiment further investigated the binding sequences of this receptor-ligand pair. Based on the AlphaFold3 model prediction, the binding region of IGSF3 is mainly located in the Ig-like-C2-type5 domain, while the binding site of TNFR2 is concentrated in the CRD2 domain. Figure 5 (A). To validate this prediction, four plasmids were constructed: those containing wild-type (IGSF3-WT-HA, TNFR2-WT-HA) and mutants lacking the predicted binding region (IGSF3-MUT-HA, TNFR2-MUT-HA). Pull-down experiments were performed after transfection into Siha or Jurkat cells. Figure 5 The results showed that the deletion of these key sequences significantly weakened the binding ability of the two (B). Figure 5 (C and D). Flow cytometry analysis revealed that when transfected cells were exposed to recombinant TNFR2 or IGSF3 protein, the loss of the predicted domain also led to weakened ligand-receptor interactions. Figure 5 (E and F in the middle). These pieces of evidence collectively indicate that the Ig-like-C2 type 5 domain of IGSF3 and the CRD2 domain of TNFR2 are key sites mediating their interaction.

[0053] To elucidate the Tregs immunosuppressive signaling pathway activated by IGSF3-TNFR2 interaction, this study reanalyzed RNA-Seq data from mouse tumor tissues. The results showed that, compared to the IGSF3 knockdown group, the TNFR2-related signaling pathway in the control group was significantly enriched in the NF-κB pathway. Figure 5 The IgG pathway (G) is known to regulate Treg homeostasis and proliferation. Therefore, we hypothesize that IGSF3-TNFR2 interaction may regulate Treg cell activation and differentiation by activating the NF-κB pathway. Experimental results show that Jurkat cells transfected with TNFR2-WT-HA can induce NF-κB activation upon IGSF3 protein stimulation, while the TNFR2-MUT-HA transfected group has no such effect. Figure 5 These results not only clarified the key structural domains of IGSF3-TNFR2 interaction, but also revealed the molecular mechanism by which its downstream pathway enhances the immunosuppressive function of Tregs through the NF-κB pathway.

[0054] Example 6: Red phenol can restore anti-tumor immunity by interfering with the interaction between IGSF3 and TNFR2.

[0055] Based on the crucial role of IGSF3 in tumor immune escape, it is hypothesized that targeting IGSF3 may offer survival benefits for cervical cancer patients. Since there are currently no small molecule inhibitors or neutralizing antibodies targeting IGSF3, this embodiment uses... Model predictions revealed that the site2 region of IGSF3 had the highest active binding site score. Figure 6 (B and C). Subsequently, virtual docking screening was performed on 25,245 small molecule compounds ( Figure 6 (A) The 30 candidate compounds with the highest binding affinity scores were selected. Surface plasmon resonance (SPR) screening showed that four compounds—red phenol, rhodiosin, atechin gallate, and salvianolic acid A—had significant binding affinity. Figure 6 (Middle D). Concentration gradient SPR experiments further confirmed that erythromycin binds most strongly to IGSF3 (KD = 1.32 μM). Figure 6 The results (E) indicate that this traditional antioxidant can serve as a novel IGSF3 inhibitor.

[0056] AlphaFold3 modeling prediction revealed that erythrophenol can form 5 hydrogen bonds within the IGSF3 active pocket. Figure 6 Molecular dynamics (MD) simulations show that it can stably bind to this region (F). Figure 6 (G). Competitive ELISA experiments confirmed that erythromycin can significantly inhibit the interaction between IGSF3 and TNFR2. Figure 6 In the Siha cell-PBMC co-culture system, erythromycin effectively inhibited tumor cell-induced Treg differentiation (H). Figure 6 (I). Animal experiments showed that after one week of daily intraperitoneal injection of 20 / 30 / 40 mg / kg of erythromycin, the growth of TC1 subcutaneous xenografts was inhibited in a dose-dependent manner. Figure 6 (J), and reduced Treg infiltration in the tumor microenvironment accompanied by CD8 + Enhanced T-cell cytotoxicity ( Figure 6 (K and L). Importantly, no significant toxic reactions were observed in the treated mice. Based on the optimal efficacy response, subsequent experiments were conducted at a dose of 40 mg / kg for further investigation.

[0057] Example 7: Red phenol can sensitize immune checkpoint inhibitors

[0058] This invention ultimately evaluated the therapeutic efficacy of erythromycin as a monotherapy or in combination with anti-PD1 (Bioxcell BE0131) / anti-CTLA4 (Bioxcell BE0146) in cervical cancer. Figure 7(A). Given the crucial role of Tregs in limiting immunotherapy, the emerging concept of sequential immunotherapy suggests that immunosuppressive TIME should be improved first through specific therapies, followed by secondary therapy to enhance anti-tumor immunity. Based on this, the inventors hypothesized that pretreatment of TC1 tumor-bearing mice with erythropoietin (targeting IGSF3) could enhance the subsequent anti-PD1 / anti-CTLA4 efficacy by pre-blocking Treg accumulation. Experimental results confirmed that compared with the IgG control group, all treatment groups showed significant anti-tumor activity, with the combination therapy (erythropoietin + anti-PD1 / CTLA4) and sequential therapy (erythropoietin pretreatment followed by anti-PD1 / CTLA4) showing particularly outstanding effects. Figure 7 B, Figure 7 E, Figure 7 (H). Flow cytometry analysis showed that the combination and sequential groups significantly reduced Tregs infiltration and better restored CD8 compared to the single-drug groups. + T cell function Figure 7 (CG). Notably, the sequential strategy in anti-CTLA4 therapy did not show any additional advantage, while pretreatment with erythromycin followed by anti-PD1, although not statistically significant, resulted in smaller tumor volume and stronger CD8 activity. + T-cell toxicity. This evidence collectively supports the therapeutic value of erythromycin in combination with anti-PD1 / CTLA4 (especially the erythromycin → anti-PD1 sequential regimen).

[0059] This invention elucidates that erythromycin, as an IGSF3 inhibitor, can reshape innate immunity and enhance anti-PD1 / anti-CTLA4 sensitivity by blocking the IGSF3-TNFR2 interaction, with sequential therapy showing the greatest promise. More importantly, this invention proposes a method to restore CD8 by inhibiting Tregs. + Novel treatment strategies that enhance the anti-tumor function of T cells provide a theoretical basis for improving the efficacy of cervical cancer immunotherapy.

[0060] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of an IGSF3 inhibitor in the preparation of a drug for treating cervical cancer.

2. Use according to claim 1, wherein The IGSF3 inhibitor is erythrophenol.

3. A composition characterized in that, Including erythromycin and PD1 inhibitors.

4. The use of the composition as described in claim 3 in the preparation of a medicament for treating cervical cancer.

5. A medicament for treating cervical cancer, characterized by, The drug uses the composition of claim 3 as its main active ingredient.

6. The medicament according to claim 5, wherein It also includes pharmaceutically acceptable excipients.

7. A composition characterized in that, Including erythromycin and CTLA4 inhibitors.

8. Use of the composition as described in claim 7 in the preparation of a medicament for treating cervical cancer.

9. A drug for treating cervical cancer, characterized in that, The drug uses the composition of claim 7 as its main active ingredient.

10. The medicament as claimed in claim 9, characterized in that, It also includes pharmaceutically acceptable excipients.

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