Biomarker for evaluating treatment effect of KRAS mutation lung adenocarcinoma immune checkpoint inhibitor and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a biomarker for evaluating the treatment effect of a KRAS mutation lung adenocarcinoma immune checkpoint inhibitor and application of the biomarker. The invention provides a biomarker for evaluating the treatment effect of an immune checkpoint inhibitor of KRAS mutation lung adenocarcinoma, aiming at the problems that the immunotherapy effect of KRAS mutation lung adenocarcinoma is difficult to evaluate due to lack of a biomarker capable of accurately predicting the treatment effect during immunotherapy of KRAS mutation late lung adenocarcinoma. The invention further provides application of the KRAS mutant lung adenocarcinoma immune checkpoint inhibitor in a kit for evaluating the treatment effect of the KRAS mutant lung adenocarcinoma immune checkpoint inhibitor and application of the KRAS mutant lung adenocarcinoma immune checkpoint inhibitor in preparation of drugs for treating KRAS mutant lung adenocarcinoma. The invention provides a new biomarker for predicting the curative effect of the immunotherapy of the KRAS mutation lung adenocarcinoma, and provides an effective combined treatment strategy for overcoming the immunotherapy resistance of the tumors.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a biomarker for evaluating the therapeutic effect of an immune checkpoint inhibitor on KRAS mutant lung adenocarcinoma and use thereof. BACKGROUND

[0002] KRAS (Kirsten rat sarcoma viral oncogene homolog) mutation is one of the most common oncogenic drivers in lung adenocarcinoma (LUAD), accounting for about 30% of the western population and 10% of the Asian population. Although targeted drugs for KRAS G12C mutations have brought hope to some patients, most patients with non-G12C mutant subtypes still lack effective targeted therapy. At present, the immune checkpoint inhibitor represented by PD-1 / PD-L1 inhibitor combined with chemotherapy has become the first-line standard treatment for KRAS mutant advanced lung adenocarcinoma. However, primary or secondary drug resistance leads to a considerable proportion of patients who cannot benefit from it, and its efficacy still faces great challenges.

[0003] Because of the complex resistance mechanism of KRAS mutant lung adenocarcinoma to immunotherapy, current research has not fully elucidated it, which may involve the combined effects of factors such as inhibition of tumor immune microenvironment, up-regulation of co-inhibitory immune checkpoints, defects in tumor antigen presentation, and secretion of immunosuppressive cytokines. At present, the strategy to overcome drug resistance mainly focuses on combination therapy. For example, combined with anti-angiogenic drugs or other immunomodulators. However, the limited efficacy of these strategies is accompanied by increased toxicity.

[0004] Therefore, there is an urgent need in clinical practice for biomarkers that can accurately predict the efficacy of immunotherapy, and new combination therapy targets that can effectively reverse the immunosuppressive microenvironment based on clear molecular mechanisms.

[0005] Receptor activator of nuclear factor kappa B ligand (RANKL) is overexpressed in a variety of malignant tumors and is associated with poor prognosis. Recent studies have suggested that the RANKL / RANK signaling pathway may play a key role in tumor immune regulation, but whether RANKL plays a role in KRAS mutant lung adenocarcinoma immunotherapy resistance remains unclear and needs to be studied. SUMMARY

[0006] The technical problem to be solved by the present application is that there is a lack of biomarkers that can accurately predict the therapeutic effect of immunotherapy for KRAS mutant advanced lung adenocarcinoma, making it difficult to evaluate the efficacy of immunotherapy for KRAS mutant lung adenocarcinoma.

[0007] The technical scheme for solving the above technical problems of the present application is: a biomarker for evaluating the treatment effect of an immune checkpoint inhibitor on KRAS mutant lung adenocarcinoma is provided, and the biomarker is receptor activator of nuclear factor kappa B ligand (RANKL).

[0008] The second aspect of the present application also provides a use of RANKL in a kit for evaluating the treatment effect of an immune checkpoint inhibitor on KRAS mutant lung adenocarcinoma.

[0009] The third aspect of the present application also provides a use of a RANKL inhibitor in the preparation of a pharmaceutical composition for treating KRAS mutant lung adenocarcinoma.

[0010] In the above use, the RANKL inhibitor includes desumab or recombinant OPG-Fc fusion protein.

[0011] In the above use, the KRAS mutant lung adenocarcinoma is a lung adenocarcinoma of a mutant subtype.

[0012] Further, in the above use, the KRAS mutant lung adenocarcinoma is a lung adenocarcinoma that generates primary immune resistance when treated with a PD-1 inhibitor.

[0013] In the above use, the pharmaceutical composition further includes a PD-1 inhibitor.

[0014] The present application also provides a pharmaceutical composition for treating KRAS mutant lung adenocarcinoma, which includes a RANKL inhibitor and a PD-1 inhibitor.

[0015] The present application has the following beneficial effects:

[0016] The present application first predicts the responsiveness and prognosis of a lung adenocarcinoma patient carrying a KRAS mutation to an immune checkpoint inhibitor treatment by detecting the expression level of RANKL in a tumor tissue or a blood sample, and finds that a KRAS mutant lung adenocarcinoma patient with high expression of RANKL shows resistance to PD-1 inhibitor treatment. Based on this, the present application provides a biomarker RANKL for evaluating the treatment effect of an immune checkpoint inhibitor on KRAS mutant lung adenocarcinoma, and also provides a use of a RANKL inhibitor in a drug for treating KRAS mutant lung adenocarcinoma. The RANKL inhibitor combined with the PD-1 inhibitor (i.e., the RLICi regimen) can effectively reverse the immune suppression state during the use of the PD-1 inhibitor and more effectively inhibit tumor growth. The present application provides a new biomarker for predicting the efficacy of immunotherapy for KRAS mutant lung adenocarcinoma, and provides an effective combination therapy strategy for overcoming the immunotherapy resistance of this type of tumor. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1LUAD enriched RANKL expression and accompanied by suppressive TIME. High expression of RANKL in KRAS mutant lung adenocarcinoma was validated by transcriptome data from different public databases (A-C). Protein expression level of RANKL was also confirmed in CICAMS cohort study (D). Heatmap (E) and scatter plots (F, G) showed the correlation of transcriptome data from TCGA database. Comparison of TIDE scores between patients with low and high expression of RANKL in KRAS mutant LUAD subgroup of TCGA database (H). Heatmap (I) and scatter plots (J, K) showed the correlation of transcriptome data from GEO and CPTAC databases. Comparison of TIDE scores between patients with low and high expression of RANKL in KRAS mutant LUAD subgroup of GEO and CPTAC databases (L). Heatmap of correlation of protein expression data in CICAMS cohort (M). Representative immunohistochemical sections of RANKL, PD-L1 and CD8A in KRAS mutant LUAD patients of CICAMS cohort (N). Note: mut, mutant; wt, wild type; TIME, tumor immune microenvironment; LUAD, lung adenocarcinoma; IHC, immunohistochemical staining; PD-L1, programmed death-ligand 1; TIDE, tumor immune dysfunction and exclusion score. Significance of two groups of samples was determined by Wilcoxon test or two-tailed unpaired Student's t-test, and correlation analysis was performed by Pearson correlation coefficient method.

[0018] Figure 2 NCC retrospective cohort study confirmed that KRAS mutant LUAD patients benefited more from RLICi treatment. KRAS mutant LUAD patients who received RLICi treatment had significantly better prognosis (Fig. A and B) and tumor control (Fig. C and D) than those who did not, while this was not observed in KRAS non-mutant lung adenocarcinoma patients. Multivariate analysis confirmed that KRAS mutant lung adenocarcinoma patients benefited more from RLICi treatment (Fig. E). Note: NCC, National Cancer Center; LUAD, lung adenocarcinoma; RLICi, RANKL inhibitor combined with immune checkpoint inhibitor; DCB, durable clinical benefit; ECOG PS, Eastern Cooperative Oncology Group performance status score; PD-L1, programmed death-ligand 1; Neg, negative; Pos, positive; NA, data missing; yrs, years; HR, hazard ratio; CI, confidence interval. ∗, statistically significant.

[0019] Figure 3The DEMAIN prospective phase II clinical trial demonstrated that the RLICi regimen brought survival benefits to patients and was safe. The flowchart of the DEMAIN study (A). Waterfall plot (B). Swim plot (C). The disease status marked represents the initial efficacy evaluation. The arrow indicates the patient who continues to be treated, and no arrow indicates the patient who stops treatment due to subsequent disease progression. Typical cases of the DEMAIN trial (D). The yellow dotted circle marks the tumor lesion. Kaplan-Meier curves of progression-free survival (E) and overall survival (F). ELISA results show that RLICi can effectively reduce the serum soluble RANKL level and the concentration of collagen metabolites CTX and NTX in urine (G-I). Kaplan-Meier curves suggest that both baseline serum soluble RANKL and baseline tissue RANKL levels are associated with poor prognosis. RLICi, RANKL inhibitor combined with immune checkpoint inhibitor; PD-1, programmed death receptor 1; sRANKL, soluble RANKL; uCTX, urine type I collagen C-terminal peptide; uNTX, urine type I collagen N-terminal peptide; ELISA, enzyme-linked immunosorbent assay. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. The significant difference between the two groups of samples was determined by two-tailed unpaired Student's t-test. DETAILED DESCRIPTION

[0020] The present application predicts the responsiveness and prognosis of lung adenocarcinoma patients carrying KRAS mutations to immune checkpoint inhibitor therapy by detecting the expression level of RANKL in tumor tissue or blood samples.

[0021] Through retrospective and prospective clinical cohort analysis, it is found that KRAS mutant lung adenocarcinoma patients with high expression of RANKL show resistance to PD-1 inhibitor therapy and have shorter overall survival.

[0022] Therefore, the present application provides a biomarker for evaluating the therapeutic effect of immune checkpoint inhibitors on KRAS mutant lung adenocarcinoma, and the biomarker is receptor activator of nuclear factor kappa B ligand RANKL. The present application also provides a use of RANKL in a kit for evaluating the therapeutic effect of immune checkpoint inhibitors on KRAS mutant lung adenocarcinoma.

[0023] Further, through mechanism research, it is found that RANKL down-regulates the expression of PD-L1 in tumor cells by activating the PI3K-AKT signaling pathway, and inhibits the secretion of T cell chemotactic factors such as CXCL9 / 10 / 11 by macrophages, resulting in reduced CD8+ T cell infiltration and forming an immunosuppressive microenvironment.

[0024] Therefore, based on this mechanism, the present application further proposes a RLICi regimen for combined treatment of RANKL inhibitors and PD-1 inhibitors. In the animal experiments of the present application, it is shown that the combined treatment of RANKL inhibitors and PD-1 inhibitors significantly inhibits tumor growth and enhances CD8+ T cell and M1 macrophage infiltration in the tumor. At the same time, the prospective DEMAIN clinical trial further verifies the good efficacy and safety of the combined treatment of RANKL inhibitors and PD-1 inhibitors in KRAS mutant lung adenocarcinoma patients.

[0025] Therefore, the present application further provides a use of a RANKL inhibitor in the preparation of a drug for preventing or treating KRAS mutant lung adenocarcinoma.

[0026] Specifically, the RANKL inhibitor of the present application can be a drug such as denosumab, and any substance that can cause RANKL target inhibition can be used as the RANKL inhibitor of the present application and has a corresponding use.

[0027] In particular, the KRAS mutant lung adenocarcinoma of the present application refers especially to non-G12C mutant subtype lung adenocarcinoma. Further, it refers to lung adenocarcinoma that produces immunosuppression after treatment with a PD-1 inhibitor. The present application finds that a RANKL inhibitor can reverse the immunosuppression caused by treatment with a PD-1 inhibitor, and produce better therapeutic effect. When a RANKL inhibitor and a PD-1 inhibitor are combined for treatment, the therapeutic effect is better.

[0028] The present application provides a new biomarker for predicting the efficacy of immunotherapy for KRAS mutant lung adenocarcinoma, and provides an effective combined treatment strategy for overcoming the immunotherapy resistance of this type of tumor.

[0029] The specific embodiments of the present application will be further explained by the following examples, but it does not mean that the protection scope of the present application is limited within the scope described in the examples.

[0030] The equipment and materials used in the examples are all ordinary commercially available products.

[0031] Example 1: Detection of the expression level of RANKL in tumor tissue

[0032] The present application first detects the expression level of RANKL in tumor tissue samples to predict the responsiveness and prognosis of lung adenocarcinoma patients carrying KRAS mutations to immunological checkpoint inhibitor treatment. Specifically as follows:

[0033] Analysis of The Cancer Genome Atlas (TCGA) transcriptome datasets (KRAS-mut: N = 137; KRAS-wt: N = 291) showed that RANKL expression was significantly higher in KRAS-mut LUAD patients compared to KRAS-wt patients (p < 0.001) (Figure 1A). We validated this finding using the Gene Expression Omnibus (GEO) dataset (GEO: GSE31210) (KRAS-mut: N = 20; KRAS-wt: N = 68) (Figure IB) and the Clinical Proteomic Tumor Analysis Consortium (CPTAC)_LUAD_2019 dataset (KRAS-mut: N = 29; KRAS-wt: N = 32) (Figure 1C), obtaining consistent results (p = 0.009 and p = 0.006, respectively). Subsequently, we validated the protein expression level of RANKL using CICAMS cohort (KRAS-mut: N = 98; KRAS-wt: N = 118) tissue microarray, and immunohistochemistry (IHC) staining results confirmed the above transcriptome data findings (p < 0.001) (Figure ID).

[0034] wherein the RNA sequencing raw data and related clinical information of LUAD were manually obtained from TCGA database (https: / / portal.gdc.cancer.gov / ), GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ) and CPTAC database (https: / / www.linkedomics.org / admin.php). According to the median value of RANKL expression, the patients were divided into RANKL low expression group and RANKL high expression group. The processed data were subjected to expression analysis, correlation analysis, immune infiltration analysis (quanTIseq score) and TIDE score calculation on Tumor Immune Dysfunction and Exclusion (TIDE) website (http: / / tide.dfci.harvard.edu / ), respectively.

[0035] Among them, the tissue microarray (TMA) was taken from the resected tissues of 216 NSCLC patients who underwent surgery in China Medical Science Academy Cancer Hospital / Institute (CICAMS cohort) from January 2008 to December 2013. We evaluated the expression of RANKL in 4 μm frozen LUAD tissue sections in the CICAMS cohort to assess the differences in protein levels. The representative unstained sections of the primary tumor of each patient were sent to the pathology laboratory of our hospital for RANKL IHC staining. The primary antibodies used in this study for IHC include RANKL (#NB100-56512; Novus Biologicals, USA), RANK (#ab13918; Abeam, UK), CD68 (#ab213363; Abeam), PD-L1 (#740-4907; Ventana Medical Systems, USA), CD8 (#ZA-0508; Zsbio Tech, China), CXCL9 (#22355-1-AP; Proteintech, USA), CXCL10 (#MAB2662-SP; R&D, USA), and CXCL11 (#MAB672-SP; R&D).

[0036] Example 2 Retrospective clinical cohort analysis experiment

[0037] It was also confirmed by retrospective clinical cohort analysis that KRAS mutant lung adenocarcinoma patients with high expression of RANKL showed resistance to PD-1 inhibitor treatment and shorter overall survival. Specifically as follows:

[0038] From December 2020 to December 2022, 71 LUAD patients with bone metastases who received bone-targeted drugs combined with immune checkpoint inhibitors (ICI) were enrolled in the NCC cohort. Thirty patients (42.3%) received RLICi treatment, and 41 patients (57.7%) received bisphosphonate combined with ICI (non-RLICi) treatment. Kaplan-Meier survival analysis showed that the median progression-free survival (mPFS) of KRAS mutant LUAD patients receiving RLICi treatment was significantly longer than that of patients receiving non-RLICi treatment (338 days vs 143 days, p < 0.001) (Figure 2A), while there was no significant difference in KRAS wild-type (WT) patients (Figure 2B). In KRAS mutant LUAD patients receiving RLICi treatment, the proportion of patients who achieved durable clinical benefit (PFS > 6 months) was significantly higher than that of patients receiving non-RLICi treatment (70.8% vs 26.3%, p = 0.004) (Figure 2C), while there was no significant difference in KRAS wild-type patients (Figure 2D). Multivariate analysis showed that both RLICi regimen and KRAS mutation status could independently predict the improvement of PFS in LUAD patients (hazard ratio [HR]: 4.157; 95% confidence interval [CI]: 1.683-10.264; p = 0.002) (HR: 0.611; 95% CI: 0.400-0.933; p = 0.022) (E). These indicate that KRAS mutant LUAD patients are the main beneficiaries of RLICi. Figure 2 E}). These indicate that KRAS mutant LUAD patients are the main beneficiaries of RLICi.

[0039] Regarding the recruitment of the retrospective cohort (NCC cohort): From December 2020 to December 2022, 71 patients with advanced LUAD treated in our hospital received chemotherapy, immune checkpoint inhibitors (ICI), and bone-targeted drugs such as bisphosphonates (including zoledronic acid, pamidronic acid disodium, or ibandronic acid monosodium) or denosumab. They were identified as the NCC cohort. Concurrent treatment was defined as receiving bone metastasis treatment (BTA) within 30 days before starting systemic treatment or after starting systemic treatment. Demographics, clinicopathological, molecular pathological, detailed treatment history, and survival data were obtained from electronic medical records and telephone follow-up. This study was approved by the CICAMS Ethics Committee, with protocol number #NCC22 / 145-3346.

[0040] Example 3 Prospective clinical cohort analysis experiment

[0041] Finally, a prospective phase II clinical trial analysis confirmed that patients with KRAS-mutant lung adenocarcinoma who highly expressed RANKL benefited more from the RLICi combination therapy, and that tissue / blood RANKL levels effectively predicted patient survival benefits. Details are as follows:

[0042] Our hospital's prospective single-arm phase II clinical trial (DEMAIN) enrolled 20 patients. The overall design of the DEMAIN trial is as follows: Figure 3 As shown in A, in 8 of these patients, the tumors shrank further during RLICi maintenance therapy. Figure 3 B), and the remaining patients all achieved stable conditions. As of March 28, 2024, the lesions in 6 patients remained stable ( Figure 3 C). In patient 2, the primary lesion ceased to change during first-line treatment but continued to shrink upon receiving RLICi treatment; furthermore, in patient 4, the primary lesion completely disappeared after treatment, and the mediastinal lymph nodes in region 4 also shrank. Figure 3 D). To improve the validity of the study, we included 22 KRAS-mutant LUAD patients who received ICI and chemotherapy at our hospital during the same period as a historical control cohort. Regarding efficacy, we found that the PFS in the RLICi maintenance group was not inferior to that in the ICI plus chemotherapy maintenance group. The median PFS1 in the prospective cohort was 347.0 days (95% CI, 268.1–425.9 days), and the median PFS1 in the retrospective cohort was 390.0 days (95% CI, 260.7–519.3 days). Figure 3 E). The median overall survival (mOS) of the prospective cohort was 1,134.0 days, and the median overall survival of the historical cohort was 1,256.0 days (p = 0.792). Figure 3 F).

[0043] ELISA results of biosamples before and after RLICi treatment showed that RLICi effectively reduced serum sRANKL levels and urinary levels of collagen metabolites type I collagen C-terminal peptide and type I collagen N-terminal peptide (Figure 3 G–I). Exploratory analysis of baseline blood and tumor tissue biomarkers indicated that high serum sRANKL levels (n = 16) or positive baseline tissue RANKL expression (n = 11) were associated with poor prognosis. This is consistent with our baseline finding that high RANKL levels are associated with a “cold” time, predicting poor outcomes from immunotherapy.

[0044] Regarding the prospective trial design and recruitment (DEMAIN trial): This study used Simon's two-stage optimal design to evaluate the efficacy of RLICi in maintenance therapy for KRAS-mutant LUAD. Based on historical data from IMpower150 subgroup analysis (mPFS ≈ 8 months with ICI maintenance therapy alone), the intervention aimed to extend mPFS to 9 months. Statistical parameters included a one-sided α of 0.10 and an 80% power. Phase 1 enrolled 20 patients; progression to phase 2 (a total of 40 patients) required mPFS ≥ 9 months. If the mPFS threshold was reached, the regimen would be considered clinically promising, balancing ethical considerations (early futility termination) and statistical rigor at α = 0.10 and β = 0.20. A total of 45 patients were enrolled, taking into account a 10% dropout rate. From July 2021 to March 2023, 20 patients were prospectively enrolled in our hospital for this single-arm phase II clinical trial (DEMAIN) (clinical trial license number: #NCC2022C-545). All KRAS-mutant LUAD patients received maintenance therapy with durvalumab combined with PD-1 checkpoint inhibitors after first-line PD-1 inhibitor-based immunotherapy. The primary endpoint was PFS, and secondary endpoints included OS and safety. Efficacy was evaluated every 3 cycles using the Response Evaluation Criteria in Solid Tumors version 1.1. Considering the lack of parallel controls in single-arm studies, we also introduced a historical control cohort of KRAS-mutant LUAD patients treated with PD-1 checkpoint inhibitors combined with chemotherapy for maintenance therapy in our hospital. This study was approved by the CICAMS Ethics Committee, with protocol number #NCC2022C-545. All subjects have signed written informed consent. All procedures involving human subjects were in accordance with the ethical standards of the institutional and / or national research committee and the Declaration of Helsinki (revised in 2013).

Claims

1. A biomarker for evaluating the efficacy of immune checkpoint inhibitor therapy in KRAS-mutant lung adenocarcinoma, characterized in that, The biomarker is RANKL, the receptor-activated nuclear factor κB ligand.

2. Use of RANKL in kits for evaluating the efficacy of immune checkpoint inhibitor therapy in KRAS-mutant lung adenocarcinoma.

3. Use of RANKL inhibitors in the preparation of pharmaceutical compositions for the treatment of KRAS-mutant lung adenocarcinoma.

4. The use of the RANKL inhibitor according to claim 3 in the preparation of a pharmaceutical composition for the prevention or treatment of KRAS-mutant lung adenocarcinoma, characterized in that: The RANKL inhibitors include denosumab or recombinant OPG-Fc fusion protein.

5. The use of the RANKL inhibitor according to claim 3 in the preparation of a pharmaceutical composition for treating KRAS-mutant lung adenocarcinoma, characterized in that: The KRAS-mutated lung adenocarcinoma is a mutated subtype of lung adenocarcinoma.

6. The use of the RANKL inhibitor according to claim 3 in the preparation of a pharmaceutical composition for treating KRAS-mutant lung adenocarcinoma, characterized in that: The KRAS-mutated lung adenocarcinoma mentioned refers to lung adenocarcinoma that has developed primary immune resistance after treatment with PD-1 inhibitors.

7. The use of the RANKL inhibitor according to claim 3 in the preparation of a pharmaceutical composition for treating KRAS-mutant lung adenocarcinoma, characterized in that: The pharmaceutical composition also includes a PD-1 inhibitor.

8. A pharmaceutical composition for treating KRAS-mutant lung adenocarcinoma, characterized in that: This includes RANKL inhibitors and PD-1 inhibitors.

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