WO2024055179A1 - Pd-1抗体药物继发耐药免疫抑制靶点及应用 - Google Patents

Pd-1抗体药物继发耐药免疫抑制靶点及应用 Download PDF

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WO2024055179A1
WO2024055179A1 PCT/CN2022/118637 CN2022118637W WO2024055179A1 WO 2024055179 A1 WO2024055179 A1 WO 2024055179A1 CN 2022118637 W CN2022118637 W CN 2022118637W WO 2024055179 A1 WO2024055179 A1 WO 2024055179A1
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drug
gpnmb
tumor
resistant
antibody
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房健民
徐小晴
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同济大学苏州研究院
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  • the present invention relates to the field of biomedicine, and in particular to immunosuppressive targets related to secondary drug resistance of PD-1 antibody drugs, and the application of the targets in the treatment of tumors with secondary drug resistance to PD-1 therapy.
  • PD-1 (programmed death 1) programmed death receptor 1 is an important immunosuppressive molecule, a member of the immunoglobulin superfamily, and a membrane protein of 268 amino acid residues. It was originally cloned from the apoptotic mouse T cell hybridoma 2B4.11. Immune regulation targeting PD-1 is of great significance in anti-tumor, anti-infection, anti-autoimmune diseases and organ transplant survival. The combination of PD-1 and PD-L1 initiates programmed death of T cells, allowing tumor cells to obtain immune escape.
  • PD-1 antibody drugs have broad-spectrum anti-cancer effects. Patients with lung cancer, kidney cancer, gastric cancer, colon cancer, ovarian cancer, breast cancer, skin cancer and brain tumors can all benefit from PD-1 immunotherapy. However, there are still some patients who do not respond to PD1 therapy. Taking colorectal cancer as an example, the clinical trial results of Keytruda in the treatment of colorectal cancer showed an ORR of 32% (95% CI, 21-45), including 2 cases of CR and 18 cases of PR.
  • Clinical trial data show that the effective rate of PD-1/PD-L1 monoclonal antibodies in treating tumors is about 20%, and among these patients who benefit, 15%-35% use PD-1/PD for a period of time -L1 monoclonal antibody has caused disease recurrence, that is, secondary drug resistance.
  • the issue of PD-1 antibody drug resistance is one of the current hot topics in academic research.
  • current basic research findings mainly focus on 1 the immune occurrence or initiation stage, 2 the immune effect stage, the process of directly killing tumor cells, and 3 the immune memory stage. Specifically divided into internal causes of the tumor and external causes of the tumor, through the expression or up-regulation of specific genes or pathways on tumor cells, the infiltration and function of immune cells in the tumor microenvironment are inhibited.
  • the current research on the mechanism of drug resistance is still in its infancy, and the understanding of the mechanism of drug resistance is not comprehensive and in-depth enough.
  • the existing solution to PD-1 antibody resistance is mainly combined medication, such as the combination of TIM-3 antibody and PD-1 antibody, and the combination of PD-L1 antibody and PD-1 antibody.
  • target GPNMB is to use GPNMB as the ADC drug target, and use toxic small molecules conjugated with ADC drugs to target and kill tumors.
  • the present invention solves the problem of drug resistance of PD-1 antibody immunotherapy, discovers a new mechanism of drug resistance of PD-1 antibody immunotherapy, discovers new targets related to drug resistance of PD-1 antibody immunotherapy, and discovers new targets in Application in reversing secondary resistance to PD-1 antibody immunotherapy.
  • the present invention provides GPNMB as a key target related to immune monitoring point molecule PD-1 inhibitor immunotherapy resistance, which can overcome the secondary resistance to PD-1/PD-L1 antibody drugs or improve PD-1/PD- Application of L1-targeted drugs in anti-tumor efficacy.
  • GPNMB is highly expressed in tumor tissues under treatment with PD-1 inhibitors, which in turn triggers tumor immunosuppression and triggers tumor resistance to PD-1 inhibitors.
  • the expression level of GPNMB in drug-resistant tumors is significantly higher than that in non-drug-resistant tumors.
  • the anti-tumor efficacy can be improved through combined treatment with PD-1 inhibitors and GPNMB targeted drugs.
  • the PD-1 inhibitor can be a PD-1 or PD-L1 antibody drug, a small molecule drug, or any other drug form
  • the GPNMB targeted drug can be a GPNMB antibody drug, Antibody drug conjugates, small molecule drugs, or any other drug form.
  • the present invention also provides the use of reagents for detecting GPNMB in preparing reagents or kits for screening tumor patients resistant to PD-1/PD-L1 antibody drugs.
  • the present invention also provides a method for screening tumor patients who are resistant to PD-1/PD-L1 antibody drugs, using a reagent for detecting GPNMB to screen tumor patients.
  • the present invention also provides a treatment method for tumor patients who develop secondary drug resistance to PD-1/PD-L1 antibodies.
  • GPNMB-targeted drugs can be administered to tumor patients.
  • GPNMB can be used as a single drug, or in combination with one or more drugs. combination therapy with other drugs.
  • the present invention uses a PD-1 drug resistance model to conduct high-throughput transcriptome sequencing of PD-1 antibody treatment-resistant tumors, draw a map of differentially expressed genes, screen key targets related to PD-1 antibody treatment resistance, and discover immunosuppression
  • the expression of the target GPNMB in drug-resistant tumor cells was significantly higher than that in non-drug-resistant tumors, and the expression of the target GPNMB was verified through experiments.
  • GPNMB Knocking out GPNMB in drug-resistant tumor cells, the proportion of immune cell infiltration in the tumor tissue with GPNMB knockout was significantly increased, and PD-1 antibody treatment was given. It was found that knocking out the target GPNMB reversed the tumor's response to PD-1 antibodies. The resistance to treatment shows that GPNMB is a new PD-1 resistance-related immunosuppression target, and the target GPNMB has strong applicability in treating PD-1-resistant tumors.
  • the applicant used transcriptome high-throughput sequencing analysis to compare the transcriptome characteristics of anti-PD-1 treatment-resistant and sensitive tumors.
  • GPNMB is significantly upregulated in tumor cells resistant to anti-PD-1 therapy, and deletion of GPNMB in resistant cells restores sensitivity to anti-PD-1 therapy in vivo. Tumors may suppress anti-tumor immunity by upregulating the expression of GPNMB, thereby producing resistance to anti-PD-1 immunotherapy.
  • GPNMB is a new immunosuppressive target related to PD-1 resistance. Knocking out the target GPNMB is important for overcoming PD- 1 Resistance has strong applicability.
  • Figure 1 shows the construction and verification of the in vivo anti-PD-1 drug-resistant MC38 model
  • Figure 1A shows the construction process of the in vivo anti-PD-1 drug-resistant MC38 model
  • Figure 1B shows the in vivo anti-PD-1 drug-resistant MC38 model receiving PD- 1 tumor growth curve during therapy
  • Figure 1C shows the tumor growth curve of the anti-PD-1 non-resistant MC38 model in vivo when receiving PD-1 therapy
  • Figure 2 shows the verification that GPNMB is up-regulated in anti-PD-1 treatment-resistant tumor cells;
  • Figure 2A shows the anti-PD-1-resistant MC38 model transcriptome high-throughput sequencing differential gene volcano plot;
  • Figure 2B shows the anti-PD-1 Cluster diagram of differential gene grouping by high-throughput sequencing of the transcriptome of the drug-resistant MC38 model;
  • Figure 2C shows the expression level of GPNMB in the anti-PD-1 drug-resistant MC38 model;
  • Figure 2D shows the expression level of GPNMB in the anti-PD-1 drug-resistant MC38 model.
  • Figure 2E shows the protein expression of GPNMB on anti-PD-1-resistant/non-drug-resistant MC38 cells;
  • Figure 3 shows the correlation between patient survival rate and GPNMB gene expression levels in various cancers;
  • Figure 3A shows BRCA, breast invasive cancer;
  • Figure 3B shows LGG, brain low-grade glioma;
  • Figure 3C shows STAD, gastric gland cancer;
  • Figure 4 shows the infiltration of immune cells in PD-1 antibody-resistant tumor tissues
  • Figure 4A shows the expression of immune-related genes in PD-1 drug-resistant/non-drug-resistant tumor tissues
  • Figure 4B shows the expression of immune cells in PD-1-resistant tumor tissues.
  • Figure 4C shows the infiltration of CD8 T cells in PD-1-resistant/non-drug-resistant tumor tissues
  • Figure 4D shows the infiltration of NK cells in PD-1-resistant/non-resistant tumor tissues.
  • Figure 4E shows the interactive relationship between immune cells and immune factors in PD-1-resistant/non-drug-resistant tumor tissues;
  • Figure 5 shows the application of knocking out the target GPNMB in reversing PD-1 drug resistance
  • Figure 5A shows the expression of GPNMB protein after knocking out GPNMB in drug-resistant tumor cells
  • Figure 5B shows knocking out the drug-resistant target GPNMB Afterwards, the growth of tumors treated with PD-1 antibody
  • Figure 5C shows the infiltration of CD8 T cells in tumor tissues after knocking out the drug-resistant target GPNMB.
  • the invention discloses GPNMB, an immunosuppressive target related to PD-1 antibody treatment resistance, and the application of knocking out GPNMB in reversing PD-1 antibody treatment resistance.
  • the target application of GPNMB in the existing technology is an ADC target, with limited efficacy and insufficient application scope;
  • the drug-resistant target GPNMB discovered by the present invention is an immunosuppressive target and is used in PD-1 drug-resistant tumor types to knock out GPNMB can increase the proportion of immune cell infiltration in tumor tissue and restore the anti-tumor immune response, making it more targeted and more effective.
  • the existing technology targets targets on T cells, and there is a risk of abnormal activation of T cells, which is prone to toxic and side effects; the new PD-1 drug resistance-related immunosuppressive target discovered by the present invention is located on tumor cells, targeting this The target drug has strong specificity and low toxic and side effects.
  • This invention is based on the practical problem of low response rate of existing tumor immune checkpoint inhibitors during clinical use, explores the immune checkpoint therapy-resistant tumor microenvironment from multiple angles, and discovers new immune suppression targets related to PD-1 resistance. point.
  • This project used bioinformatics analysis results of drug-resistant and wild tumor tissue transcriptome sequencing to find GPNMB, a new target for resistance to PD-1 immunotherapy.
  • GPNMB protein in therapy-resistant tumors is significantly higher than that in non-resistant tumors; at the same time, the infiltration levels of various immune cells in PD-1 therapy-resistant tumors are significantly reduced, and they are in an immunosuppressive state; they are resistant to PD-1 Knocking out GPNMB in tumor cells can reverse the resistance to PD-1 antibody treatment and enhance the infiltration of immune cells in tumor tissues.
  • GPNMB is a new target for secondary resistance to PD-1 therapy, and this target is related to the immunosuppression of tumors.
  • This application focuses on protecting the phenomenon of high expression of the immunosuppressive target GPNMB during the process of resistance to PD-1 therapy, and the application of knocking out GPNMB in reversing tumor PD-1 resistance. Any results obtained based on the above experiments are within the scope of the present invention.
  • mice Each of the two groups of mice was injected with 200 ⁇ g of PD-1/PD-L1 antibody drug or PBS twice a week, and the weight and tumor size of the mice were measured and recorded at the same time.
  • mice are sacrificed and the tumor cells are isolated. These are the first-generation PD-1/PD-L1 antibody drug-resistant cells and the first-generation control untreated cells. Cells for treating PD-1/PD-L1 antibody drugs.
  • first-generation PD-1/PD-L1 antibody drug-resistant cell tumor-bearing mice were treated with PD-1/PD-L1 antibody drug (pembrolizumab), each injected with 200 ⁇ g PD-1/PD -L1 antibody drug, twice a week.
  • PD-1/PD-L1 antibody drug pembrolizumab
  • the first generation of tumor-bearing mice that control untreated cells continue to be injected with PBS, and the dosage is the same as that of the initial tumor cells. The number of times is the same as that of the experimental group.
  • Colon cancer PD-1-resistant cell lines were screened for 5 rounds in vivo and continuously treated with anti-PD-1 mAb (Figure 1A). After five rounds of screening, anti-PD-1 immunotherapy failed to effectively control MC38 tumor growth ( Figure 1B).
  • Example 2 Perform high-throughput sequencing analysis of transcriptomes of colon cancer PD-1 therapy secondary drug-resistant/non-drug-resistant tumors, comparatively analyze differentially expressed genes, and draw gene expression maps
  • MC38 tumor cells were inoculated subcutaneously into the right flank of Pdcd1 humanized mice (6 to 8 weeks old) to form tumors. Tumors that grew to approximately 400 mm were dissected, and their total RNA was extracted using TRIzol reagent. The cDNA library was verified using Agilent 2100 Bioanalyzer (Santa Clara, CA, USA). Use IlluminaHiSeqTM 2500 or IlluminaHiSeqX Ten for sequencing to obtain 125bp or 150bp paired-end read data. Analyze the results of transcriptome high-throughput sequencing and draw differential gene expression maps.
  • Example 3 uses the results of high-throughput sequencing of the transcriptome of drug-resistant tumors as a starting point and finds that the expression level of GPNMB molecules on the surface of drug-resistant tumors is significantly higher than that of non-drug-resistant tumors.
  • GPNMB is a new target for secondary resistance to PD-1 therapy in tumors. point
  • Example 4 In various tumors (breast cancer, low-grade brain glioma and gastric adenocarcinoma), the higher the expression level of the new target GPNMB, the worse the patient's survival situation is.
  • the Cancer Genome Atlas was used to analyze the expression of GPNMB gene in breast cancer, brain low-grade glioma and gastric adenocarcinoma (BRCA: breast invasive carcinoma, LGG: brain low-grade glioma, STAD: gastric adenocarcinoma).
  • KMplotter was used to analyze the overall survival (OS) of patients with breast cancer, brain low-grade glioma, and gastric adenocarcinoma.
  • Example 5 In the immune microenvironment of drug-resistant tumor tissues, the infiltration level of various immune cells decreases, and the drug-resistant tumors are in a state of severe immunosuppression.
  • Fluorescence quantitative PCR, flow cytometry and tumor tissue section immunofluorescence technology were used to comparatively analyze the differences in the immune microenvironment of drug-resistant/non-drug-resistant tumor tissues, focusing on the analysis of the infiltration ratio of immune cells after tumor resistance.
  • the infiltration ratio of CD8 T cells, NK cells, SDC cells and other immune cells in tumor tissues is significantly reduced.
  • drug-resistant tumor tissues a variety of important anti-tumor immune cells are in a state of exhaustion, the immune system cannot fully exert its anti-tumor effect, and the tumor escapes the attack of the immune system, thereby developing resistance to PD-1 antibody treatment.
  • Example 6 Knocking out GPNMB in drug-resistant tumor cells can increase the infiltration level of CD8 T cells in the tumor immune microenvironment and reverse tumor resistance to PD-1 antibody treatment.
  • the therapeutic effect of knocking out the target GPNMB against PD-1-resistant tumors was tested in the MC38 colon cancer model.
  • Use drug-resistant MC38 cells with GPNMB knockout and drug-resistant MC38 cells without GPNMB knockout to inoculate human PD-1 transgenic mice by subcutaneous injection, 6 to 8 weeks old, 18 to 22 g, and measure and record daily after inoculation.
  • Tumor size When the tumor volume grows to 100-200mm3, they are randomly divided into two groups for treatment, with 6 to 8 animals in each group.
  • PBS and anti-hPD-1 antibody were injected through the orbital vein. Each mouse in each group was injected with 200 ⁇ g of anti-hPD-1 antibody drug or PBS twice a week. The body weight and tumor size of the mice were measured and recorded at the same time.
  • Tumor growth curves were drawn to verify the therapeutic effect of knocking out the target GPNMB on PD-1-resistant tumors. After knocking out the target GPNMB, anti-hPD-1 antibody drugs can well inhibit the growth of drug-resistant MC38 tumors, while drug-resistant tumors without GPNMB knockout do not respond to anti-hPD-1 antibody drug treatment.
  • CRISPR-Cas9 gene knockout technology was used to knock out the GPNMB gene of drug-resistant tumor cells, so that the tumor cells no longer express GPNMB protein, thus blocking the GPNMB-mediated immunosuppressive signal, and finally successfully reversed the PD- 1 Secondary resistance to antibody drugs.
  • the MC38 drug-resistant strain and the GPNMB-knockout MC38 drug-resistant strain were inoculated into immune-competent mice. After tumor formation, the tumor tissue was peeled off and frozen sections were immunofluorescently stained. Compared with the control MC38 drug-resistant tumors, the GPNMB-knockout The infiltration of CD8 T lymphocytes in MC38-resistant tumor tissues increased significantly. Knocking out the GPNMB target of drug-resistant MC38 cell lines can relieve the immunosuppressive state of drug-resistant tumor tissues.

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Abstract

提供了PD-1抗体疗法耐药靶点GPNMB及耐药靶点在逆转PD-1抗体治疗继发耐药性方面的应用。

Description

PD-1抗体药物继发耐药免疫抑制靶点及应用 技术领域
本发明涉及生物医药领域，特别涉及PD-1抗体药物继发耐药相关免疫抑制靶点，以及靶点在治疗PD-1疗法继发耐药肿瘤中的应用。
背景技术
PD-1(programmed death 1)程序性死亡受体1，是一种重要的免疫抑制分子，为免疫球蛋白超家族，是一个268氨基酸残基的膜蛋白。其最初是从凋亡的小鼠T细胞杂交瘤2B4.11克隆出来。以PD-1为靶点的免疫调节对抗肿瘤、抗感染、抗自身免疫性疾病及器官移植存活等均有重要的意义。PD-1和PD-L1结合启动T细胞的程序性死亡，使肿瘤细胞获得免疫逃逸。
PD-1抗体药物具有广谱的抗癌效果，肺癌、肾癌、胃癌、结肠癌、卵巢癌、乳腺癌、皮肤癌和脑肿瘤等病人均能够从pd-1免疫疗法中获益。然而，仍有部分病人不响应pd1疗法。以结直肠癌为例，Keytruda治疗结直肠癌临床实验结果ORR为32％(95％CI，21-45)，其中2例CR和18例PR。临床试验数据显示，PD-1/PD-L1单抗治疗肿瘤的有效率在20％左右，且在这部分受益的患者中，有15％-35％的患者使用一段时间的PD-1/PD-L1单抗后出现了疾病的复发，即继发耐药现象。
PD-1抗体药物耐药问题是目前学术研究的热点之一。关于免疫耐药机制，目前基础研究所发现的主要集中在①免疫发生或启动阶段②免疫效应阶段，直接杀伤肿瘤细胞的过程③免疫记忆阶段。具体分为肿瘤内部原因以及肿瘤外部原因，通过肿瘤细胞上特定基因或通路的表达或上调，从而导致肿瘤微环境中免疫细胞的浸润以及功能受到抑制。然而，目前对于耐药机理的研究仍处于初级阶段，对耐药机理的认知不够全面和深入。
现有的PD-1抗体继发耐药机理的研究集中在免疫细胞上免疫抑制分子的表达水平变化，比如位于T细胞上的TIM-3分子。
现有的PD-1抗体耐药的解决方法主要是联合用药，比如TIM-3抗体 和PD-1抗体联合用药、PD-L1抗体和PD-1抗体联合用药。
现有的靶点GPNMB的应用是以GPNMB为ADC药物靶标，利用ADC药物偶联的的毒性小分子，靶向杀伤肿瘤。
探究PD-1疗法耐药的新机理，发现PD-1疗法耐药相关新靶点，建立逆转PD-1疗法继发耐药性的有效方法，这些都是目前尚待解决的具体问题。
发明内容
有鉴于此，本发明解决了PD-1抗体免疫疗法耐药性问题，发现PD-1抗体免疫疗法耐药新机理，发现PD-1抗体免疫疗法耐药相关新靶点，发现新靶点在逆转PD-1抗体免疫疗法继发耐药性方面的应用。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了GPNMB作为与免疫监测点分子PD-1抑制剂免疫治疗耐药相关的关键靶点，在克服PD-1/PD-L1抗体药物产生继发耐药或提高PD-1/PD-L1靶向药物抗肿瘤疗效中的应用。
在本发明的一些具体实施方案中，肿瘤组织中GPNMB在PD-1抑制剂治疗下发生高表达，进而引发肿瘤发生免疫抑制，引发肿瘤对PD-1抑制剂发生耐药。
在本发明的一些具体实施方案中，GPNMB在耐药肿瘤中的表达量显著高于非耐药肿瘤。
在本发明的一些具体实施方案中，可以通过PD-1抑制剂和GPNMB靶向药物联合治疗提高抗肿瘤疗效。
在本发明的一些具体实施方案中，所述PD-1抑制剂可以是PD-1或PD-L1抗体药物、小分子药物、或其他任何药物形式；所述GPNMB靶向药物可以GPNMB抗体药物、抗体药物偶联药物、小分子药物、或其他任何药物形式。
本发明还提供了检测GPNMB的试剂在制备筛选PD-1/PD-L1抗体药物耐药的肿瘤患者的试剂或试剂盒中的应用。
本发明还提供了PD-1/PD-L1抗体药物耐药的肿瘤患者的筛选方法，采用检测GPNMB的试剂对肿瘤患者进行筛选。
本发明还提供了对PD-1/PD-L1抗体产生继发耐药性的肿瘤患者的治疗方法，对肿瘤患者施用GPNMB靶向的药物，可以用GPNMB单药治疗，或联合一种或多种其他药物的联合治疗。
本发明利用PD-1耐药模型，对PD-1抗体治疗耐药肿瘤进行转录组高通量测序，绘制差异表达基因图谱，筛选PD-1抗体治疗耐药相关的关键靶点，发现免疫抑制靶点GPNMB在耐药肿瘤细胞中的表达显著高于非耐药肿瘤，并通过实验验证了靶点GPNMB的表达情况。
实验结果表明：耐药肿瘤与非耐药肿瘤相比，抗肿瘤免疫反应相关基因的表达显著下调，对应的抗肿瘤免疫细胞的浸润比例显著减少，耐药肿瘤组织处于严重的免疫抑制状态，该免疫抑制状态与靶点GPNMB的过表达密切相关。除此之外，在多种癌症中病人生存率和GPNMB基因表达水平有很强的相关性，GPNMB基因表达水平越高，病人生存情况越差。
对耐药肿瘤细胞的GPNMB进行敲除，敲除GPNMB的肿瘤组织中免疫细胞的浸润比例显著增多，给与PD-1抗体治疗，发现敲除靶点GPNMB后，逆转了肿瘤对PD-1抗体治疗的耐药性，说明GPNMB是一种新型PD-1耐药相关免疫抑制靶点，靶点GPNMB对于治疗PD-1耐药的肿瘤具有很强的应用性。
综上，在本发明中，本申请人使用转录组高通量测序分析比较了抗PD-1治疗耐药和敏感肿瘤的转录组特征。GPNMB在抗PD-1治疗耐药的肿瘤细胞中显著上调，耐药细胞中GPNMB的缺失恢复了体内抗PD-1治疗的敏感性。肿瘤可能通过上调GPNMB的表达来抑制抗肿瘤免疫，从而产生抗PD-1免疫治疗耐药性，GPNMB是一种新型PD-1耐药相关免疫抑制靶点，敲除靶点GPNMB对于克服PD-1耐药有很强的应用性。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1示体内抗PD-1耐药MC38模型的构建和验证；其中，图1A示体内抗PD-1耐药MC38模型的构建过程；图1B示体内抗PD-1耐药 MC38模型接受PD-1疗法时的肿瘤生长曲线；图1C示体内抗PD-1非耐药MC38模型接受PD-1疗法时的肿瘤生长曲线；
图2示GPNMB在抗PD-1治疗耐药肿瘤细胞中上调的验证；其中，图2A示抗PD-1耐药MC38模型转录组高通量测序差异基因火山图；图2B示抗PD-1耐药MC38模型转录组高通量测序差异基因分组聚类图；图2C示GPNMB在抗PD-1耐药MC38模型中的表达水平；图2D示GPNMB在抗PD-1耐药MC38模型中的相对表达量；图2E示GPNMB在抗PD-1耐药/非耐药MC38细胞上的蛋白表达情况；
图3示在多种癌症中病人生存率和GPNMB基因表达水平的相关性；其中，图3A示BRCA，乳腺浸润性癌；图3B示LGG，脑低级别胶质瘤；图3C示STAD，胃腺癌；
图4示PD-1抗体治疗耐药肿瘤组织免疫细胞浸润情况；其中，图4A示免疫相关基因在PD-1耐药/非耐药肿瘤组织中的表达情况；图4B示免疫细胞在PD-1耐药/非耐药肿瘤组织中的浸润比例；图4C示CD8T细胞在PD-1耐药/非耐药肿瘤组织中的浸润情况；图4D示NK细胞在PD-1耐药/非耐药肿瘤组织中的浸润情况；图4E示PD-1耐药/非耐药肿瘤组织中免疫细胞和免疫因子的交互关系；
图5示敲除靶点GPNMB在逆转PD-1耐药性方面的应用；其中，图5A示耐药肿瘤细胞敲除GPNMB后，GPNMB蛋白的表达情况；图5B示敲除耐药靶点GPNMB后，PD-1抗体治疗的肿瘤生长情况；图5C示敲除耐药靶点GPNMB后，肿瘤组织中CD8T细胞的浸润情况。
具体实施方式
本发明公开了PD-1抗体治疗耐药相关的免疫抑制靶点GPNMB，以及敲除GPNMB在逆转PD-1抗体治疗耐药性方面的应用。本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用 进行改动或适当变更与组合，来实现和应用本发明技术。
本发明的有益效果包括但不限于：
①现有技术针对GPNMB的靶点应用为ADC靶标，疗效有限，应用范围不够精确；本发明发现的耐药靶点GPNMB为免疫抑制靶点，应用于PD-1耐药肿瘤类型中，敲除GPNMB能够提高肿瘤组织中免疫细胞浸润比例，恢复抗肿瘤免疫反应，针对性更强，疗效更佳。
②现有技术针对的为T细胞上的靶点，存在T细胞异常激活的风险，容易发生毒副作用；本发明发现的全新PD-1耐药相关免疫抑制靶点，位于肿瘤细胞上，针对该靶点的药物特异性强，毒副作用小。
③现有技术针对的是肺癌中的PD-1疗法继发耐药问题，癌种有一定的局限性，适应范围小；本发明发现的PD-1免疫疗法继发耐药新靶点，不仅针对结肠癌，在乳腺癌、脑低级别胶质瘤和胃腺癌中都有较高水平的表达，适应范围广。
本发明立足于现有肿瘤免疫检查点抑制剂在临床使用过程中的应答率低这一实际问题，多角度探究免疫检查点疗法耐药肿瘤微环境，发现PD-1耐药相关免疫抑制新靶点。本项目利用耐药与野生肿瘤组织转录组测序生物信息学分析结果，找到了针对PD-1免疫疗法耐药的新靶点GPNMB。分析PD-1免疫疗法耐药/非耐药肿瘤转录组高通量测序的结果，本申请人在差异基因的列表中发现GPNMB在耐药肿瘤中的表达量显著高于非耐药肿瘤；使用实时荧光定量PCR技术验证检测GPNMB分子在耐药肿瘤和非耐药肿瘤中的表达量，发现GPNMB基因转录的RNA水平在耐药的肿瘤中显著提高；使用流式细胞术检测发现，PD-1疗法耐药肿瘤中GPNMB蛋白的表达量显著高于非耐药的肿瘤；同时，PD-1疗法耐药肿瘤中多种免疫细胞的浸润水平显著降低，处于免疫抑制状态；对PD-1耐药肿瘤细胞的GPNMB进行敲除，能够逆转PD-1抗体治疗的耐药性，并且能够增强免疫细胞在肿瘤组织中的浸润。
综上所述，本申请人的研究发现，GPNMB是PD-1疗法继发耐药的新靶点，并且该靶点和肿瘤的免疫抑制相关。本申请着重保护在PD-1疗法耐药的过程中会出现免疫抑制靶点GPNMB高表达这一现象，以及敲 除GPNMB在逆转肿瘤PD-1耐药性方面的应用。任何在上述实验基础上获得的结果均在本发明的保护范围之内。
本发明提供的PD-1/PD-L1抗体药物耐药模型的构建方法以及PD-1疗法耐药靶点，所用原料及试剂均可有市场购得。下面结合实施例，进一步阐述本发明：
实施例1结肠癌PD-1疗法继发耐药模型的构建
a)体外培养结肠癌MC38肿瘤细胞，以皮下注射的方式接种人PD-1转基因小鼠，6至8周龄，18至22g，接种后每日测量记录肿瘤大小。待肿瘤体积长至100～200mm3时，随机分成两组进行治疗，每组6至8只。
b)两组小鼠每只分别注射200μgPD-1/PD-L1抗体药物或PBS，每周两次，同时测量记录小鼠的体重和肿瘤大小。
c)待治疗组和对照组生长最快的肿瘤分别达到1500mm 3时，处死小鼠并分离肿瘤细胞，此为第一代PD-1/PD-L1抗体药物耐药细胞和第一代对照未治疗PD-1/PD-L1抗体药物的细胞。
d)将第一代PD-1/PD-L1抗体药物耐药细胞和第一代对照未治疗PD-1/PD-L1抗体药物的细胞按同样的方法接种到小鼠皮下。
e)成瘤后，第一代PD-1/PD-L1抗体药物耐药细胞荷瘤小鼠给与PD-1/PD-L1抗体药物(pembrolizumab)治疗，每只注射200μg PD-1/PD-L1抗体药物，每周两次，为了排除肿瘤在小鼠体内多次传代可能会出现与起始肿瘤细胞不同的性状，第一代对照未治疗细胞的荷瘤小鼠继续注射PBS，计量与次数同实验组。
f)待治疗组和对照组生长最快的肿瘤分别达到1500mm 3时，使用同样方法分别剥离生长速度最快的细胞,并获得第二代PD-1/PD-L1抗体药物耐药细胞和第二代对照未治疗PD-1/PD-L1抗体药物的细胞。
g)以此方法反复在小鼠皮下接种肿瘤细胞，并进行耐药肿瘤细胞的筛选，经过多轮接种与筛选后，持续使用PD-1/PD-L1抗体药物治疗的肿瘤细胞表现出耐药性状，即使在使用PD-1/PD-L1抗体药物治疗的条件下， 癌细胞的生长速度越来越快，相应的半数生存时间也越来越短。
h)当持续使用PD-1/PD-L1抗体药物治疗的荷瘤小鼠的半数死亡时间接近于持续传代而未使用PD-1/PD-L1抗体药物治疗的荷瘤小鼠的半数死亡时间时，将此代的耐药细胞系分离，培养扩增，液氮冻存，即建成PD-1/PD-L1抗体药物耐药模型，如图1所示。
结肠癌PD-1耐药细胞株在体内进行了5轮筛选，并持续使用抗pd-1mAb治疗(图1A)。经过五轮筛选后，抗pd-1免疫治疗未能有效控制MC38肿瘤的生长(图1B)。
实施例2进行结肠癌PD-1疗法继发耐药/非耐药肿瘤转录组高通量测序分析，对比分析差异表达的基因，绘制基因表达图谱
将MC38肿瘤细胞皮下接种于Pdcd1人源化小鼠(6～8周龄)右侧侧翼形成肿瘤。将长至约400mm 3的肿瘤解剖，并使用TRIzol试剂提取其总RNA。cDNA文库使用Agilent 2100Bioanalyzer(Santa Clara,CA,USA)进行验证。采用IlluminaHiSeqTM 2500或IlluminaHiSeqX Ten进行测序，获得125bp或150bp双端读数据。分析转录组高通量测序的结果，绘制差异基因表达图谱。
MC38-R细胞的转录组测序分析显示，在转录水平上有多种变化(图2A,B)。在MC38-R肿瘤细胞系中，相对于MC38-U，免疫抑制分子GPNMB显著上调(p＝0.016)(图2C)。为验证GPNMB在耐药和非耐药肿瘤细胞株中的差异表达，采用RT-qPCR检测GPNMB mRNA的相对丰度。与MC38-U细胞株相比，MC38-R肿瘤细胞株表达的GPNMB水平显著升高(p＝0.0004)(图2D)。GPNMB在MC38-R肿瘤细胞细胞膜上的蛋白表达量也高于MC38-U(图2E)。
实施例3以耐药肿瘤转录组高通量测序结果为切入点，发现耐药肿瘤表面的GPNMB分子表达量显著高于非耐药肿瘤，GPNMB是肿瘤PD-1疗法继发耐药的新靶点
分析PD-1免疫疗法耐药/非耐药肿瘤转录组高通量测序的结果(如图 2C～图2E所示)，我们在差异基因的列表中发现GPNMB在耐药肿瘤中的表达量显著高于非耐药肿瘤；使用实时荧光定量PCR技术验证检测GPNMB分子在耐药肿瘤和非耐药肿瘤中的表达量，我们发现GPNMB基因转录的RNA水平在耐药的肿瘤中显著提高；接下来我们使用流式细胞术这一项技术，检测发现，PD-1疗法耐药肿瘤中GPNMB蛋白的表达量显著高于非耐药的肿瘤。综上所述，我们的研究发现耐药肿瘤表面的GPNMB分子表达量显著高于非耐药肿瘤，GPNMB是肿瘤PD-1疗法继发耐药的新靶点。
实施例4在多种肿瘤(乳腺癌、脑低级别胶质瘤和胃腺癌)中，新靶点GPNMB的表达水平越高，病人的生存情况越差
使用肿瘤基因组图谱(The Cancer GenomeAtlas,TCGA)分析GPNMB基因在乳腺癌、脑低级别胶质瘤和胃腺癌中的表达情况(BRCA:乳腺浸润性癌,LGG:脑低级别胶质瘤,STAD:胃腺癌)。使用KMplotter分析乳腺癌、脑低级别胶质瘤和胃腺癌患者的总生存率(OS)。
结果如图3所示，我们发现，在多种肿瘤(乳腺癌、脑低级别胶质瘤和胃腺癌)中，新靶点GPNMB的表达水平越高，病人的生存情况越差。这说明GPNMB作为PD-1疗法耐药新靶点，可用于多种癌症。
实施例5在耐药肿瘤组织免疫微环境中，多种免疫细胞浸润水平下降，耐药肿瘤处于严重免疫抑制状态
使用荧光定量PCR、流式细胞术和肿瘤组织切片免疫荧光技术，对比分析耐药/非耐药肿瘤组织免疫微环境的差异，重点分析肿瘤耐药后免疫细胞的浸润比例。如图4所示，肿瘤发生PD-1抗体治疗耐药后，CD8T细胞、NK细胞、SDC细胞等免疫细胞在肿瘤组织的浸润比例显著下调。耐药肿瘤组织中，多种重要的抗肿瘤免疫细胞处于耗竭状态，免疫系统无法充分发挥抗肿瘤作用，肿瘤逃逸免疫系统的攻击，进而产生PD-1抗体治疗的耐药性。
实施例6敲除耐药肿瘤细胞的GPNMB，能够提高肿瘤免疫微环境中CD8T细胞的浸润水平，逆转肿瘤对于PD-1抗体治疗的耐药性
为了验证靶点GPNMB在治疗PD-1抗体耐药肿瘤方面的应用，我们对PD-1抗体耐药肿瘤细胞MC38的GPNMB基因进行了敲除，从而阻断了GPNMB介导的免疫抑制信号。
在MC38结肠癌模型中测试了敲除靶点GPNMB对PD-1耐药肿瘤的治疗作用。使用敲除GPNMB的耐药MC38细胞和未敲除GPNMB的耐药MC38细胞，以皮下注射的方式接种人PD-1转基因小鼠，6至8周龄，18至22g，接种后每日测量记录肿瘤大小。待肿瘤体积长至100～200mm3时，随机分成两组进行治疗，每组6至8只。
通过眼眶静脉注射PBS和抗hPD-1抗体，各组小鼠每只分别注射200μg抗hPD-1抗体药物或PBS，每周两次，同时测量记录小鼠的体重和肿瘤大小。
绘制肿瘤生长曲线，验证敲除靶点GPNMB对PD-1耐药肿瘤的治疗作用。敲除靶点GPNMB后，抗hPD-1抗体药物能够很好的抑制耐药MC38肿瘤的生长，而未敲除GPNMB的耐药肿瘤则不响应抗hPD-1抗体药物治疗。
结果：
使用CRISPR-Cas9基因敲除技术，敲除耐药肿瘤细胞的GPNMB基因，使得肿瘤细胞不再表达GPNMB蛋白，进而阻断GPNMB介导的免疫抑制信号，最终成功逆转MC38耐药细胞株对PD-1抗体药物的继发耐药性。
将MC38耐药株和敲除GPNMB的MC38耐药株接种于免疫健全的小鼠体内，成瘤后剥离肿瘤组织进行组织冷冻切片免疫荧光染色，与对照MC38耐药肿瘤相比，敲除GPNMB的MC38耐药肿瘤组织中CD8T淋巴细胞的浸润明显增多，敲除耐药MC38细胞株的GPNMB靶点，能够解除耐药肿瘤组织的免疫抑制状态。
如图5所示，敲除免疫抑制靶点GPNMB之后，耐药肿瘤细胞恢复了对PD-1抗体治疗的敏感性，生长速度显著减缓，耐药性成功被逆转。 以上研究说明，免疫抑制分子GPNMB是逆转PD-1免疫检查点疗法继发耐药性的关键靶标，敲除肿瘤细胞GPNMB在破解PD-1耐药性方面有很强的应用性。
以上对本发明所提供的PD-1抗体药物继发耐药免疫抑制靶点及应用进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。

Claims (8)

  1. GPNMB作为与免疫监测点分子PD-1抑制剂免疫治疗耐药相关的关键靶点，在克服PD-1/PD-L1抗体药物产生继发耐药或提高PD-1/PD-L1靶向药物抗肿瘤疗效中的应用。
  2. 如权利要求1所述的应用，其特征在于，肿瘤组织中GPNMB在PD-1抑制剂治疗下发生高表达，进而引发肿瘤发生免疫抑制，引发肿瘤对PD-1抑制剂发生耐药。
  3. 如权利要求1或2所述的应用，其特征在于，GPNMB在耐药肿瘤中的表达量显著高于非耐药肿瘤。
  4. 如权利要求1至3任一项所述的应用，其特征在于，可以通过PD-1抑制剂和GPNMB靶向药物联合治疗提高抗肿瘤疗效。
  5. 如权利要求4所述的应用，其特征在于，所述PD-1抑制剂可以是PD-1或PD-L1抗体药物、小分子药物、或其他任何药物形式；所述GPNMB靶向药物可以GPNMB抗体药物、抗体药物偶联药物、小分子药物、或其他任何药物形式。
  6. 检测GPNMB的试剂在制备筛选PD-1/PD-L1抗体药物耐药的肿瘤患者的试剂或试剂盒中的应用。
  7. PD-1/PD-L1抗体药物耐药的肿瘤患者的筛选方法，其特征在于，采用检测GPNMB的试剂对肿瘤患者进行筛选。
  8. 对PD-1/PD-L1抗体产生继发耐药性的肿瘤患者的治疗方法，其特征在于，对肿瘤患者施用GPNMB靶向的药物，可以用GPNMB单药治疗，或联合一种或多种其他药物的联合治疗。
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WO2021158091A2 (ko) * 2020-02-06 2021-08-12 아주대학교산학협력단 케모카인 억제제, 콜로니 자극 인자 억제제 및 면역항암제를 포함하는 암의 예방 또는 치료용 조성물 및 병용 요법
WO2022052874A1 (zh) * 2020-09-09 2022-03-17 深圳微芯生物科技股份有限公司 西奥罗尼联合免疫检查点抑制剂在抗肿瘤治疗中的应用

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