CKS1B as immunotherapy response prediction biomarker and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and provides CKS1B serving as an immunotherapy response prediction biomarker and application of the CKS1B, and according to the application, CKS1B serves as an immunotherapy response marker, and a CKS1B inhibitor is combined with an active component to treat a model mouse. In-vitro cell experiments are adopted to evaluate the immunotherapy prediction effect of the CKS1B as a biomarker on esophageal squamous carcinoma, a Cks1b overexpression tumor mouse model, a homologous mouse model and a human immune reconstruction mouse model are established, and a CKS1B inhibitor is combined with active ingredients to treat the two models; results show that the CKS1B inhibitor combined with the active component can promote removal of esophageal squamous carcinoma cells by CD8 + T cells, inhibit interferon signal channels and antigen presentation, effectively recover immune response, inhibit tumor cell proliferation and significantly reduce tumor volume, so as to achieve the purpose of treating esophageal squamous carcinoma.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to immune response prediction, specifically to CKS1B as a biomarker for predicting immune therapy response and its application. Background Technology

[0002] Currently, cancer immunotherapy, especially immune checkpoint inhibitors (such as PD-1 / PD-L1 inhibitors) and CAR-T cell therapy, has been widely used in clinical practice. However, the efficacy of immunotherapy varies significantly among different patients, with some exhibiting drug resistance or immune escape, leading to treatment failure. This is mainly attributed to the following shortcomings: lack of accurate and effective biomarkers, the complexity of immune escape mechanisms, and inadequate combination strategies for immunotherapy.

[0003] Currently, although there are some biomarkers for predicting immunotherapy response (such as PD-L1 expression and tumor mutational burden (TMB), there is still a lack of biomarkers with sufficient sensitivity and specificity to accurately predict which patients will respond effectively to immunotherapy, especially in the context of tumor immune escape and immune resistance.

[0004] Tumor cells suppress the immune system's response through various immune escape mechanisms, such as T cell depletion and an immunosuppressive microenvironment. However, current immunotherapies often treat patients indiscriminately, lacking personalized assessment and targeted intervention, making it difficult to address the challenges posed by immune escape. Furthermore, genomic data from esophageal squamous cell carcinoma show that the activation and depletion of CD8+ T cells in the tumor microenvironment are not related to tumor microbiome size (TMB).

[0005] While immune checkpoint inhibitors and CAR-T cell therapy have demonstrated promising therapeutic potential, improving the efficacy of immunotherapy in drug-resistant patients remains an unsolved challenge. Strategies for combining immunotherapy with other treatments are not yet well-defined, particularly the combination with immunosuppressants, which is still in the exploratory stage, lacking precise combination regimens.

[0006] In summary, this invention utilizes CKS1B as a novel biomarker to assess the sensitivity of patients to immunotherapy (including immune checkpoint inhibitors and CAR-T cell therapy) for esophageal squamous cell carcinoma by detecting the expression level of CKS1B in esophageal squamous cell carcinoma tumor tissue. It aims to address the technical challenges in existing technologies, such as inaccurate prediction of immunotherapy response in esophageal squamous cell carcinoma, immune escape and immune resistance, and the lack of personalized guidance for combined immunotherapy strategies. Summary of the Invention

[0007] The purpose of this invention is to provide CKS1B as a biomarker for predicting immunotherapy response and its application. The invention applies CKS1B biomarker to the prediction of immunotherapy response in esophageal squamous cell carcinoma treatment, and applies CKS1B in combination with inhibitors to the treatment of esophageal squamous cell carcinoma. This provides a new approach and means for the prediction and treatment of immunotherapy response in esophageal squamous cell carcinoma, in order to solve the problems of inaccurate prediction of immunotherapy response, immune escape and immune resistance, as well as insufficient combination strategies for immunotherapy.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

[0009] This invention provides the application of CKS1B inhibitors in the preparation of drugs that enhance the sensitization of esophageal squamous cell carcinoma immunotherapy. The inhibitors include substances that reduce CKS1B protein expression or CKS1B protein content, or substances that inhibit CKS1B gene expression. The substances include one or more of nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, peptides, proteins, gene editing vectors, lentiviruses, or adeno-associated viruses.

[0010] This invention also provides the application of the above-mentioned CKS1B inhibitor combined with the active ingredient in the preparation of a drug for treating esophageal squamous cell carcinoma.

[0011] Preferably, the drug is used to promote the clearance of esophageal squamous cell carcinoma cells by CD8+ T cells.

[0012] Preferably, the drug is used to improve the interferon signaling pathway and antigen presentation, thereby restoring the immune response.

[0013] Preferably, the drug is used to inhibit the proliferation of tumor cells.

[0014] Preferably, the drug comprises a CKS1B inhibitor and medically approved excipients.

[0015] Preferably, the drug includes various acceptable dosage forms.

[0016] Preferably, the dosage form is an injection, pill, capsule, granule, tablet, or oral liquid.

[0017] The beneficial effects of this invention are:

[0018] This invention evaluated the predictive effect of CKS1B as a biomarker for immunotherapy of esophageal squamous cell carcinoma using in vitro cell experiments. A CKS1B overexpressing tumor mouse model, a homologous mouse model, and a human immune reconstitution mouse model were established. Both models were treated with a CKS1B inhibitor and the active ingredient. Results showed that combined treatment with the inhibitor promoted the clearance of esophageal squamous cell carcinoma cells by CD8+ T cells, improved interferon signaling pathways and antigen presentation, effectively restored the immune response, inhibited tumor cell proliferation while restoring T cell function, and significantly reduced tumor volume, thus achieving the goal of treating esophageal squamous cell carcinoma. Attached Figure Description

[0019] Figure 1 This invention relates to the correlation between tumor CKS1B expression and CD8+T depletion (A is the correlation between CKS1B expression and CD8+T depletion genes; B is the correlation between CKS1B expression and CD8+T terminal depletion).

[0020] Figure 2 This invention validates the susceptibility and mechanism of in vitro CD8+ T cell killing in tumors under CKS1B knockdown and non-knockdown conditions (A is a series of photographs of tumor cells co-cultured with CD8+ T cells under CKS1B knockdown and non-knockdown conditions; orange represents surviving tumor cells, green represents activated CD8+ T cells, and the disappearance of fluorescence represents cell death; B shows the degree of CD8+ T cell depletion and the degree of tumor cell apoptosis after co-culturing tumor cells with CD8+ T cells under CKS1B knockdown and non-knockdown conditions; C shows the changes in the expression of interferon pathway and antigen presentation pathway in tumor cells under CKS1B knockdown and non-knockdown conditions; **P<0.01, ***P<0.001, ****P<0.0001, compared with the control group).

[0021] Figure 3 This invention demonstrates the targeting effect of the CKS1B inhibitor 14i on esophageal squamous cell carcinoma cells (A shows the changes in the expression of interferon and antigen presentation pathways in mouse esophageal squamous cell carcinoma cells after 14i treatment; B shows the changes in the expression of interferon and antigen presentation pathways in human esophageal squamous cell carcinoma cells after 14i treatment; C shows the killing effect of esophageal squamous cell carcinoma cells co-cultured with CD8+ T cells after 14i treatment; **P<0.01, ***P<0.001, ****P<0.0001, compared with the control group).

[0022] Figure 4This invention demonstrates the efficacy of CKS1B in combined immunotherapy (A is a representative image of multiplex immunofluorescence of tumor tissue in esophageal squamous cell carcinoma patients before treatment; B shows the expression of CKS1B in tumor tissue in esophageal squamous cell carcinoma patients before treatment; C shows the relationship between CKS1B expression and survival in the melanoma immunotherapy cohort in public data; D shows the relationship between CKS1B expression and survival in the glioma immunotherapy cohort in public data; E shows the expression of CKS1B in the melanoma immunotherapy RNA-seq sequencing cohort; F shows the expression of CKS1B in the esophageal squamous cell carcinoma immunotherapy RNA-seq sequencing cohort; G shows the expression of CKS1B in the esophageal squamous cell carcinoma immunotherapy single-cell sequencing cohort; *P<0.05, compared with the control group).

[0023] Figure 5 This invention validates the use of the CKS1B inhibitor 14i combined with monoclonal antibody in a mouse subcutaneous tumor transplantation model of esophageal squamous cell carcinoma (A is the result of anti-PD-1 injection in the CKS1B overexpressing tumor model; B is the result of treatment with 14i + anti-PD-1 in the homologous mouse model (mEC25); C is the result of treatment with 14i + Nivolumab in the human immune reconstituted mouse model (KYSE30); *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, compared with the control group). Detailed Implementation

[0024] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0025] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0027] Example: Relationship between CKS1B expression and tumor development

[0028] In vitro cell experiments were used to verify the relationship between CKS1B expression and tumor development.

[0029] 1. Experimental instruments and materials

[0030] 1.1 Experimental Apparatus

[0031] Clean bench / biosafety cabinet, CO2 incubator, centrifuge, microscope, constant temperature shaker (for 37°C digestion), rotary mixer, cell counting chamber / cell counter, refrigerator (4°C, -20°C, -80°C), water bath, flow cytometer, Quantagne q900 qPCR (Kubo Technology, Beijing).

[0032] 1.2 Experimental Materials

[0033] 1.2.1 Consumables related to flow cytometry analysis

[0034] CD8a BB700 antibody (BD Biosciences, catalog number: 566409), CD45 BV421 antibody (BioLegend, catalog number: 109832), PD-1 APC-Cyanine7 antibody (BioLegend, catalog number: 135224), TIM-3 APC antibody (BioLegend, catalog number: 134008), LAG-3 antibody (Invitrogen, catalog number: 12-2231-82), Granzyme B PE-Cyanine7 antibody (BioLegend, catalog number: 372214), TNF-α PE antibody (BioLegend, catalog number: 506306), IFN-γ APC antibody (BioLegend, catalog number: 505810), FIX & PERM™ cell permeation kit (Invitrogen, catalog number: GAS004), Fixable Live / Dead Dye (Invitrogen, catalog number: L23101), APC Annexin V Apoptosis Detection Kit (BioLegend), BD GolgiPlug Protein Transport Inhibitor (BD Pharmingen, catalog number: 550583), Leukocyte Activation Mixture (BD Pharmingen, catalog number: 550583).

[0035] 1.2.2 Consumables related to cell culture and model establishment

[0036] RPMI 1640 medium (Gibco / Cytiva), collagenase IV (Gibco / Sigma), DNase I (Sigma-Aldrich), Percoll separation solution (GE Healthcare, catalog number: 17-0891-09), CCK-8 cell counting kit (DOJINDO, catalog number: CK04), Growth Factor Reduced Matrigel® (Corning, catalog number: 356231).

[0037] 2 Experimental Methods

[0038] 2.1 T cell-mediated tumor cell killing assay

[0039] Activated OT-I T cells were co-cultured with OVA-expressing mEC25 cells in 96-well plates at different ratios, with the culture time and ratios shown in the figure. After culture, T cells and cell debris were removed by washing with PBS, and the remaining surviving tumor cells were counted using a cell counting kit (DOJINDO).

[0040] 2.2 Co-culture of CD8+ T cells and tumor cells in vitro

[0041] OT-I T cells were activated with SIINFEKL peptide for 24 hours, and after removing the peptide, they were cultured in fresh T cell culture medium for one day. Activated OT-I T cells (1×10⁻⁶) 6 ) and OVA-expressing mEC25 cells (1×10 5 Co-cultivation.

[0042] 2.3 Flow cytometry analysis

[0043] For surface staining, OT-I T cells were co-stained with PD-1-TIM-3 antibody at 4°C for 30 minutes. Apoptotic tumor cells were detected using the 7AAD-Annexin-V apoptosis detection kit. Data were analyzed using FlowJo software.

[0044] 2.4 Real-time quantitative PCR (qRT-PCR) analysis

[0045] Total RNA was extracted from mouse and human cells using the RNA-Quick Purification Kit (ES Science), and reverse transcription was performed using PrimeScript™ RT Master Mix (Takara). mRNA levels were detected using a Quantagne q900 qPCR system (Coopers) with TB Green® Premix Ex Taq™ II (Takara). The mRNA expression levels of each target gene were calculated relative to the mRNA expression levels of GAPDH or ACTB as internal controls.

[0046] 2.5 Data Analysis

[0047] Statistical analyses were performed using R software (version 4.1.2) and GraphPad Prism 9. P-values ​​were calculated using either the Wilcoxon rank-sum test or the Student t-test. The Wilcoxon rank-sum test was used to compare whether there were significant differences between two groups of non-normally distributed quantitative data; in functional experiments, the Student t-test was used to compare the two treatment groups. Differences in patient survival time were estimated using the Kaplan-Meier method, and P-values ​​were obtained using the log-rank test. Hazard ratios were calculated using a multivariate Cox proportional hazards model.

[0048] 3. Experimental Results

[0049] 3.1 The expression of tumor CKS1B is highly correlated with CD8+ T depletion markers and the proportion of terminally depleted CD8+ T cells.

[0050] In the publicly available single-cell dataset of esophageal cancer from Shantou University, analysis of tumor cells and CD8+ T cells revealed a significant positive correlation between the expression of CKS1B in tumor cells and terminally exhausted CD8+ T cells, as well as a significant positive correlation with various exhaustion markers of CD8+ T cells. Figure 1 ).

[0051] 3.2 CKS1B expression can inhibit antigen presentation function and interferon pathway in tumor cells.

[0052] OT-I T cells are a specific type of T cell, a type of CD8+ T cell, commonly used as a model system in immunological research. CKS1B expression can inhibit the antigen-presenting function of tumor cells, suppress the clearance of tumor cells by immune cells, and prolong the interaction time between CD8+ T cells and tumor cells, leading to the depletion of CD8+ T cells due to repeated stimulation by tumor antigens.

[0053] Real-time tracking of esophageal squamous cell carcinoma tumor cells and CD8+ T cells in vitro revealed that tumor cells with low CKS1B expression could be rapidly cleared by CD8+ T cells (fluorescence disappearance), reducing the time required for CD8+ T cell antigen-TCR stimulation. Figure 2 A). Flow cytometry analysis of co-cultured tumor cells and CD8+ T cells revealed that the degree of apoptosis in the CKS1B knockdown group was significantly higher than that in the control group, while the degree of exhaustion of CD8+ T cells was significantly lower than that in the control group. Figure 2 B). qPCR results showed that the expression of tumor cell antigen presentation pathway and interferon pathway genes was significantly increased in the CKS1B knockdown group (B). Figure 2 C).

[0054] 3.3 Targeting CKS1B can enhance the ability of tumor antigen presentation and promote its clearance by CD8+ T cells.

[0055] The CKS1B inhibitor 14i significantly increased the expression of antigen presentation and interferon pathway genes in tumor cells, and in vitro culture showed a significant increase in the killing effect on tumor cells after drug administration. Figure 3 ).

[0056] 3.4 In the immunotherapy cohort, patients with low CKS1B expression showed better efficacy with immunotherapy.

[0057] Publicly available data on immunotherapy show that tumor patients with high CKS1B expression have significantly lower survival rates than those with low expression; according to efficacy grouping, non-responder patients have higher CKS1B expression levels ( Figure 4 ).

[0058] Application examples

[0059] Establish allogeneic transplantation and humanized immunotherapy models to verify the effect of CKS1B combined with inhibitors on immunotherapy.

[0060] 1. Experimental Instruments and Materials

[0061] 1.1 Experimental Apparatus

[0062] Image analysis software (inForm) (PerkinElmer).

[0063] 1.2 Experimental Materials

[0064] Growth Factor Reduced Matrigel® (Corning, catalog number: 356231), C57BL / 6J mice (Jackson Laboratory, catalog number: 000664), NOG mice (Jackson Laboratory), mEC25 cells (laboratory strain), KYSE30 cells (ATCC, catalog number: CRL-2115), 14i (CKS1B inhibitor) (TargetMol, catalog number: 2760612-77-7), aPD-1 antibody (Bio X Cell, catalog number: BE0146), Nivolumab (MCE, catalog number: 173-2120-0539), DMSO (Sigma-Aldrich, catalog number: D8418), peripheral blood mononuclear cells (from healthy donors), NSG mice (6 weeks old) (Jackson Laboratory), C57BL / 6J mice (Jackson Laboratory).

[0065] 2 Experimental Methods

[0066] 2.1 Cks1b overexpression tumor mouse model

[0067] mEC25 cells stably overexpressing Cks1b (8 × 10⁻⁶) 6 The drug was suspended in 140 μL of diluted growth factor-depleted Matrigel (Corning) and subcutaneously injected into the back of C57BL / 6J mice. An equal amount of mEC25 cells without Cks1b overexpression were used as a control group to establish a tumor model. Mice inoculated with Cks1b-free and Cks1b-overexpressing mice were randomly assigned to groups:

[0068] Placebo group: intraperitoneal injection of an equal volume of PBS

[0069] aPD-1 treatment group: intraperitoneal injection of aPD-1 (Bio X cell, 10 mg / kg, dissolved in PBS);

[0070] 2.2 Homologous mouse model (mEC25)

[0071] mEC25 cells (8 × 10⁻⁶) 6 A tumor was established on the right side of C57BL / 6J mice via subcutaneous injection. When the tumor volume reached approximately 100 mm³, the mice were randomly assigned to different groups:

[0072] 14i treatment group: Oral administration of 14i (25 mg / kg, dissolved in PBS containing 2% DMSO);

[0073] aPD-1 treatment group: intraperitoneal injection of aPD-1 (Bio X cell, 10 mg / kg, dissolved in PBS);

[0074] Combination therapy group: 14i and aPD-1 were administered simultaneously.

[0075] Each group was given the medication once every 3 days for a total of 4 cycles (12 days).

[0076] Note: The tumor cells and the mice inoculated with subcutaneous tumors were from the same strain, and the transplanted tumors were compatible with the mice's immune system.

[0077] 2.3 Human immune reconstitution mouse model (KYSE30)

[0078] KYSE30 cells (5 × 10⁻⁶) 6 Subcutaneous injection was administered to the right side of 6-week-old NOG mice. Fourteen days after inoculation, peripheral blood mononuclear cells (5 × 10⁶) from healthy donors were injected via the tail vein. 6 When the tumor volume reached approximately 100 mm³, the mice were randomly divided into 4 groups:

[0079] Nivolumab group: Nivolumab (10 mg / kg) was administered intraperitoneally.

[0080] 14i group: Oral administration of 14i (25 mg / kg);

[0081] Combination therapy group: Nivolumab combined with 14i;

[0082] PBS control group: PBS carrier was administered.

[0083] Each group was administered the drug once every 3 days for a total of 5 cycles (15 days). During the experiment, tumor length, width and volume were measured and volume was calculated regularly, and body weight was recorded to assess efficacy and systemic toxicity.

[0084] Note: This is a human immune reconstitution mouse model, which is a heterologous transplantation. The mice were inoculated with human tumor cells and subcutaneous tumor mice, which are from different sources. Human immune cells were reinfused into immunodeficient mice to reconstruct human immunity.

[0085] 2.4 Data Analysis

[0086] Statistical analysis was performed using R software (version 4.1.2) and GraphPad Prism 9. P-values ​​were calculated using either the Wilcoxon rank-sum test or the Student t-test. The Wilcoxon rank-sum test was used to compare whether there were significant differences between two groups of non-normally distributed quantitative data; in the functional experiments, the Student t-test was used to compare the two treatment groups.

[0087] 3. Experimental Results

[0088] 3.1 Targeting CKS1B in combination with immune checkpoint inhibitors can enhance the efficacy of immunotherapy. Figure 5 ).

[0089] Anti-PD-1 targeted drugs are a novel type of tumor immunotherapy that activates the patient's own immune system to attack tumor cells by blocking the binding of PD-1 to its ligand PD-L1. Nivolumab is a monoclonal antibody targeting the PD-1 receptor; it enhances the immune system's anti-tumor response by blocking the interaction between PD-1 and PD-L1 and PD-L2. 14i targets the binding of tumor cell CKS1B-SKP2 substrates, inhibiting the degradation of downstream substrate ubiquitination, thereby promoting downstream interferon and antigen recognition pathways, facilitating the recognition and killing of tumor cells by immune cells, avoiding prolonged stimulation of immune cells, and reducing the degree of immune cell exhaustion.

[0090] In a mouse model of tumors overexpressing Cks1b, the overexpression group showed poorer efficacy with aPD-1 (anti-PD-1) compared to the control group, while the control group showed greater sensitivity to anti-PD-1. Figure 5A). Furthermore, in both homologous mouse models and human immune reconstituted mouse models, the combined use of drugs demonstrated superior therapeutic effects compared to monotherapy, indicating that the combination of CKS1B inhibitors and anti-PD-1 enhances the efficacy of immunotherapy. Figure 5 (BC in the middle).

[0091] In summary, the statistical data demonstrates a high correlation between CKS1B expression and tumor development indicators. By detecting CKS1B expression levels in tumor tissues, the sensitivity of cancer patients to immune checkpoint inhibitors or CAR-T cell therapy can be assessed, thus using CKS1B expression as a biomarker for immunotherapy. Upon detection of high CKS1B expression, CKS1B inhibitors can be used, or CKS1B inhibitors can be combined with active ingredients to inhibit tumor cell proliferation while simultaneously restoring T cell function, increasing the expression of genes related to the interferon signaling pathway and antigen presentation, reducing immune resistance, overcoming immune escape mechanisms, thereby enhancing the efficacy of immunotherapy and promoting immunosensitization.

[0092] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. The use of a CKS1B inhibitor in the preparation of a drug for promoting the sensitization of esophageal squamous cell carcinoma immunotherapy, characterized in that, The inhibitor includes a substance that reduces the expression or content of CKS1B protein, or a substance that inhibits the expression of CKS1B gene, including one or more of nucleic acid molecules, carbohydrates, lipids, small molecule compounds, antibodies, polypeptides, proteins, gene editing vectors, lentivirus or adeno-associated virus.

2. Use of the CKS1B inhibitor combined with the active ingredient in the preparation of a drug for treating esophageal squamous cell carcinoma according to claim 1.

3. Use according to claim 2, characterized in that, The drug is used to promote the clearance of CD8+ T cells on esophageal squamous cell carcinoma cells.

4. Use according to claim 2, characterized in that, The drug is used to improve the interferon signaling pathway and antigen presentation, and restore immune response.

5. Use according to claim 2, characterized in that, The drug is used to inhibit tumor cell proliferation.

6. Use according to claim 2, characterized in that, The drug includes a CKS1B inhibitor and a medically acceptable excipient.

7. Use according to claim 2, characterized in that, The drug includes various acceptable dosage forms.

8. Use according to claim 7, characterized in that, The dosage form is injection, pill, capsule, granule, tablet or oral liquid.

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