ChiCTR2400094237 版本V1.0 版本创建时间2024/12/19 09:10:34 中国临床试验注册中心

审核状态：

Project audit state：

通过审核

Successful

注册号：

Registration number：

ChiCTR2400094237 

最近更新日期：

Date of Last Refreshed on：

2024-12-19 09:10:26 

注册时间：

Date of Registration：

2024-12-19 00:00:00 

注册号状态：

预注册

Registration Status：

Prospective registration

注册题目：

MHCⅡ类肿瘤新抗原ITGB2通过激活CD4+T细胞提高PD-1抑制剂疗效的作用及机制研究

Public title：

Comprehensive Analysis of the Mechanisms Underlying the Potentiation of PD-1 Inhibitor Efficacy through Vaccination with ITGB2-Derived Neoantigens

注册题目简写：

English Acronym：

研究课题的正式科学名称：

MHCⅡ类肿瘤新抗原ITGB2通过激活CD4+T细胞提高PD-1抑制剂疗效的作用及机制研究

Scientific title：

Investigation into the Mechanistic Underpinnings and Therapeutic Implications of MHC Class II-Restricted Tumor Neoantigen ITGB2 in Augmenting the Efficacy of PD-1 Blockade by Engaging CD4+ T Cells

研究课题代号(代码)：

Study subject ID：

在二级注册机构或其它机构的注册号：

The registration number of the Partner Registry or other register：

申请注册联系人：

黄孝伦 

研究负责人：

黄孝伦 

Applicant：

Huang Xiaolun 

Study leader：

Huang Xiaolun 

申请注册联系人电话：

Applicant telephone：

+86 18602893677

研究负责人电话：

Study leader's telephone：

+86 28 85420691

申请注册联系人传真 ：

Applicant Fax：

研究负责人传真：

Study leader's fax：

申请注册联系人电子邮件：

Applicant E-mail：

huangxiaolun@med.uestc.edu.cn

研究负责人电子邮件：

Study leader's E-mail：

huangxiaolun@med.uestc.edu.cn

申请单位网址(自愿提供)：

Applicant website(voluntary supply)：

研究负责人网址(自愿提供)：

Study leader's website(voluntary supply)：

申请注册联系人通讯地址：

四川省成都市人民南路

研究负责人通讯地址：

成都市人民南路四段55号

Applicant address：

Renmin South Road, Chengdu, Sichuan Province

Study leader's address：

No.55,Section 4,South Renmin Road,Chengdu,China

申请注册联系人邮政编码：

Applicant postcode：

研究负责人邮政编码：

Study leader's postcode：

申请人所在单位：

四川省肿瘤医院

Applicant's institution：

Sichuan Cancer Hospital

研究负责人所在单位：

四川省肿瘤医院

Affiliation of the Leader：

Sichuan Cancer Hospital

是否获伦理委员会批准：

是/Yes

Approved by ethic committee：

Yes

伦理委员会批件文号：

Approved No. of ethic committee：

SCCHEC-02-2024-222

伦理委员会批件附件：

Approved file of Ethical Committee：

 查看附件View

批准本研究的伦理委员会名称：

四川省肿瘤医院医学科研与医疗新技术伦理委员会

Name of the ethic committee：

Ethics Committee for Medical Research and New Medical Technology of Sichuan Cancer Hospital

伦理委员会批准日期：

Date of approved by ethic committee：

2024-11-19 00:00:00

伦理委员会联系人：

王青青

Contact Name of the ethic committee：

Wang Qingqing

伦理委员会联系地址：

成都市人民南路四段55号

Contact Address of the ethic committee：

No.55,Section 4,South Renmin Road,Chengdu,China

伦理委员会联系人电话：

Contact phone of the ethic committee：

+86 28 85420681

伦理委员会联系人邮箱：

Contact email of the ethic committee：

975095403@qq.com

研究实施负责（组长）单位：

四川省肿瘤医院

Primary sponsor：

Sichuan Cancer Hospital

研究实施负责（组长）单位地址：

成都市人民南路四段55号

Primary sponsor's address：

No.55,Section 4,South Renmin Road,Chengdu,China

试验主办单位(项目批准或申办者)：

Secondary sponsor：

国家：

中国

省(直辖市)：

四川

市(区县)：

Country：

China

Province：

Sichuan

City：

单位(医院)：

四川省肿瘤医院

具体地址：

成都市人民南路四段55号

Institution
hospital：

Sichuan Cancer Hospital

Address：

No.55,Section 4,South Renmin Road,Chengdu,China

经费或物资来源：

自选课题（自筹）

Source(s) of funding：

Self-selected project (self-funded)

Target disease：

Hepatocellular carcinoma

Target disease code：

研究类型：

观察性研究

Study type：

Observational study

研究所处阶段：

 其它 

Study phase：

N/A

研究设计：

连续入组 

Study design：

Sequential 

研究目的：

研究拟通过mRNA纳米脂质颗粒实现ITGB2在患者DC细胞的稳定表达，进一步通过免疫细胞活性检测、肝癌类器官杀伤实验，明确ITGB2对CD4+T细胞活性、PD-1抑制剂应答的作用及分子机制。旨在拓展肿瘤新抗原的作用机制，为肿瘤疫苗的临床应用提供新的依据。  

Objectives of Study：

The study aims to achieve stable expression of ITGB2 in patient dendritic cells (DCs) through mRNA nanolipid particles. Further, the study will determine the role and molecular mechanisms of ITGB2 in CD4+ T cell activity and response to PD-1 inhibitors through immune cell activity assays and liver cancer organoid killing experiments. The goal is to expand the understanding of the mechanisms of action of tumor neoantigens and provide new evidence for the clinical application of tumor vaccines.

药物成份或治疗方案详述：

第一部分：研究 MHC-Ⅱ类抗原对 CD4+T 细胞的作用 1.利用肝癌患者外周血分离获得 PBMC，将具有粘附能力的中性粒细胞诱导培养成DC 细胞，在 DC 细胞培养过程中转染 ITGB2mRNA-LNP 复合物:收集肝癌患者的外周血，应用 Ficoll 分离液离心获得 PBMC 培养 24 小时：将具有粘附能力的单核细胞加入 I 段包含 GM-CSF、IL-4、血清的 RPMI 培养基进行 DC 细胞培养； 2. 在肿瘤新抗原溶液中按照 2ug/mL 加入 II 段包含 TNF-α、IL-6、PGE2、血清的RPMI 培养基, 同时设置空载 mRNA-LNP 组及空白对照组；DC 细胞经过 I 段培养基培养 7 天后更换 II 段培养基 48h 进行成熟诱导； 3.将培养 DC 过程中未粘附的悬浮细胞加入包含 IL-2、血清的 RPMI 细胞培养基，获得混合淋巴细胞。 4. 分别 mRNA-LNP 组、Negative control mRNA-LNP 空载对照组、空白对照组的 DC细胞，按照 1: 100 比例，分别与混合淋巴细胞进行共培养： 5. 应用流式分析、酶联免疫斑点试验比较 CD4+T 细胞的细胞分型、比例及功能变化，明确 ITGB2 对 CD4+T 细胞活性的影响；第二部分：研究 MHC-Ⅱ类抗原 ITGB2对 PD-1 抑制剂免疫应答的影响:应用流式细胞术检测 CD3、CD4、CD28、CD44、CD45R、IFN-γR1 以及 CXCR3、酶联免疫斑点试验及流式多因子检测 IFN-γ、TNFα、GM-CSF、IL-2、LT-α、LT-β、CXCR-3 等，比较不同分组混合免疫细胞T 细胞数量及亚群比例的差异及辅助 T 细胞常见细胞因子的分泌水平差异。 第二部分：研究 MHC-Ⅱ类抗原 ITGB2 对 PD-1 抑制剂免疫应答的影响 1. 利用肝癌患者手术切除的新鲜组织构建肝癌类器官、癌旁类器官:收集以上肝癌患者手术切除的新鲜肝癌组织及配对癌旁正常组织，快速冰冻转移至生物安全柜进行清洗、消化、过滤后，加入基质胶进行预混后，在包含 DMEM/F-1、B-27、N-2、烟酰胺、N-乙酰-L-半胱氨酸、[Leu15]-胃泌素、毛喉素、A83-01、EGF、FGF10、HGF、RSpo1-条件培养基、Wnt3a-条件培养基的完全培养基中进行类器官构建，2-3 天换液 1 次，7 天传代一次； 2. 按照 DC 细胞是否转 mRNA-LNP、DC 细胞混合淋巴细胞共培养阶段是否加入 PD-1抑制剂，分为 PD-1 抑制剂组、mRNA-LNP 组、PD-1 抑制剂+mRNA-LNP 组、空白对照组，分别获得混合淋巴细胞； 3. 将不同分组的混合淋巴细胞与患者自体的肝癌类器官、癌旁类器官共培养，通过凋亡标志物荧光检测比较不同分组类器官杀伤效应差别，明确 ITGB2 是否可以提升 PD-1 抑制剂的疗效:收集肝癌类器官及癌旁类器官，按照 1：10 比例将类 器 官 与 上 述 不 同 分 组 的 混 合 免 疫 细 胞 共 培 养 ， 每 组 加 入 等 体 积 的Casepase3/7 荧光探针细胞活性，后将培养板放入 Incucyte S3 成像设备中，实时荧光拍摄凋亡标志物的荧光水平变化，比较不同分组类器官凋亡标志物荧光强度-时间曲线，明确肿瘤新抗原是否可以提升 PD-1 抑制剂的疗效，增强效应 T 细胞的类器官杀伤作用。 第三部分：阐明 ITGB2 影响 PD-1 抑制剂疗效的作用机制 1. 将 PD-1 抑制剂组、mRNA-LNP 组、PD-1 抑制剂+mRNA-LNP 组、空白对照组的混合淋巴细胞，每组送样 4D lable-free 蛋白组学测序，着重分析 Th1、Th2、Th17、Treg、CD8+T 细胞、DC 细胞等免疫细胞分泌的 IFN-γ、TNF-α、LT-α、IL-4、IL-6、IL-10、IL-17 等炎症因子的差别，筛选与新抗原疫苗显著相关的下游因子； 2. 利用体外试验过表达及干扰 ITGB2，检测混合淋巴细胞中 PD-1、CTLA-4、LAG-3 等经典耗竭标志物的差异，验证 ITGB2 蛋白组学结果:用流式细胞术、Westernblot 检测 PD-1、CTLA-4、LAG-3 等经典耗竭标志物的差异，间接阐明肿瘤新抗原的具体作用机制； 3. 收集 30 例肝癌患者血液建立临床肝癌患者队列，应用免疫组化检测 ITGB2 在肝癌患者中的表达，结合患者的临床病理特征、外周血淋巴亚群分析结果，探究ITGB2 的表达与肝癌患者预后、免疫状态的相关性，阐明其未来的临床应用价值:结合患者的临床病理特征包括肿瘤大小、分期、肝功能储备、微血管侵犯、卫星结节、淋巴结转移及相关血清生化学检验结果、外周血淋巴亚群分析、HLA分群检测结果，并随访肝癌患者获得总生存率、复发率、无病生存期等预后结果。探究肿瘤新抗原的表达与肝癌患者预后、免疫状态的相关性。 

Description for medicine or protocol of treatment in detail：

Part I: Investigating the effect of MHC-II class antigens on CD4+ T cells 1. PBMCs were obtained by peripheral blood separation from liver cancer patients, neutrophils with adhesion ability were induced and cultured into DC cells, and ITGB2mRNA-LNP complexes were transfected during DC cell culture: the peripheral blood of liver cancer patients was collected, and PBMCs were obtained by centrifugation with Ficoll separation solution for 24 hours: monocytes with adhesion ability were added to RPMI medium containing GM-CSF, IL-4, and serum in section I for DC cell culture; 2. Add RPMI medium containing TNF-α, IL-6, PGE2 and serum in section II at 2ug/mL in tumor neoantigen solution, and set up a no-load mRNA-LNP group and a blank control group. DC cells were cultured in section I medium for 7 days and then replaced with section II medium for 48 h for maturation induction; 3. Mixed lymphocytes were obtained by adding non-adherent suspension cells from the process of culturing DCs to RPMI cell culture medium containing IL-2 and serum. 4. The DC cells of the mRNA-LNP group, the negative control mRNA-LNP no-load control group, and the blank control group were co-cultured with mixed lymphocytes at a ratio of 1:100: 5. Flow cytometry analysis and enzyme-linked immunospot assay were used to compare the cell typing, proportion and functional changes of CD4+ T cells, and the effect of ITGB2 on CD4+ T cell activity was clarified. Part II: To study the effect of MHC-II class antigen ITGB2 on the immune response of PD-1 inhibitors: flow cytometry for CD3, CD4, CD28, CD44, CD45R, IFN-γR1 and CXCR3, enzyme-linked immunospot assay and flow cytometry multiplex detection of IFN-γ, TNFα, GM-CSF, IL-2, LT-α, LT-β, CXCR-3, etc., and compare different groups of mixed immune cell T Differences in cell number and subset ratio, and differences in the secretion of common cytokines in helper T cells. Part II: To investigate the effect of MHC-II class antigen ITGB2 on the immune response of PD-1 inhibitors 1. Use the fresh tissues surgically resected by liver cancer patients to construct liver cancer organoids and paracancerous organoids: collect the fresh liver cancer tissues and paired adjacent normal tissues from liver cancer patients, quickly freeze and transfer them to a biological safety cabinet for cleaning, digestion and filtration, add Matrigel for premixing, and then add DMEM/F-1, B-27, N-2, nicotinamide, N-acetyl-L-cysteine, [Leu15]-gastrin, forskolin, A83-01, EGF, FGF10, Organoid construction was carried out in complete medium of HGF, RSpo1-conditioned medium, and Wnt3a-conditioned medium, and the fluid was changed once every 2-3 days, and the passage was passed once every 7 days. 2. According to whether the DC cells were transconverted to mRNA-LNP and whether PD-1 inhibitors were added to the mixed lymphocyte co-culture stage of DC cells, they were divided into PD-1 inhibitor group, mRNA-LNP group, PD-1 inhibitor + mRNA-LNP group, and blank control group, and mixed lymphocytes were obtained respectively. 3. Different groups of mixed lymphocytes were co-cultured with patients' own liver cancer organoids and paracancerous organoids, and the differences in the killing effect of organoids in different groups were compared by apoptosis marker fluorescence detection, and whether ITGB2 could improve the efficacy of PD-1 inhibitors was clarified: liver cancer organoids and paracancerous organoids were collected, and the organoids were co-cultured with mixed immune cells of different groups mentioned above at a ratio of 1:10, and the cell activity of Casepase3/7 fluorescent probes of the same volume was added to each group, and the culture plate was put in In the Incucyte S3 imaging device, the fluorescence level of apoptosis markers was photographed in real time, and the fluorescence intensity-time curves of apoptosis markers in different groups of organoids were compared to determine whether tumor neoantigens could improve the efficacy of PD-1 inhibitors and enhance the organoid killing effect of effector T cells. Part III: Elucidate the mechanism of action by which ITGB2 affects the efficacy of PD-1 inhibitors 1. Mixed lymphocytes from PD-1 inhibitor group, mRNA-LNP group, PD-1 inhibitor+mRNA-LNP group, and blank control group were sent to each group for 4D lable-free proteomic sequencing, focusing on the analysis of the differences in inflammatory factors such as IFN-γ, TNF-α, LT-α, IL-4, IL-6, IL-10, and IL-17 secreted by immune cells such as Th1, Th2, Th17, Treg, CD8+ T cells, and DC cells. Screening for downstream factors significantly associated with neoantigen vaccines; 2. Detect the differences of classical depletion markers such as PD-1, CTLA-4, and LAG-3 in mixed lymphocytes by using in vitro assays to overexpress and interfere with ITGB2, and verify the results of ITGB2 proteomics: Flow cytometry and Westernblot were used to detect the differences in classical depletion markers such as PD-1, CTLA-4, and LAG-3, and indirectly elucidate the specific mechanism of action of tumor neoantigens; 3. The blood of 30 patients with liver cancer was collected to establish a clinical cohort of liver cancer patients, and the expression of ITGB2 in liver cancer patients was detected by immunohistochemistry, combined with the clinicopathological characteristics of the patients and the results of peripheral blood lymphosubset analysis, to explore the correlation between the expression of ITGB2 and the prognosis and immune status of liver cancer patients, and to clarify its future clinical application value: combined with the clinicopathological characteristics of the patients, including tumor size, stage, liver function reserve, microvascular invasion, satellite nodules, lymph node metastasis and related serum biochemical test results, peripheral blood lymphosubset analysis, HLA cluster test results, and follow-up of liver cancer patients to obtain overall survival rate, recurrence rate, disease-free survival and other prognostic results. To explore the correlation between the expression of tumor neoantigens and the prognosis and immune status of liver cancer patients. 

纳入标准：

1.受试者自愿加入本研究，签署知情同意书，依从性好，配合随访； 2.年龄>=18岁(以签署知情同意当日计算)，男女皆可； 3.根据 RECIST1.1标准，至少具有一个可测量病灶； 4.ECOG体力状态评分2分，ASA评分3分； 5.五年内未发生其他恶性肿瘤。

Inclusion criteria

1. Subjects voluntarily joined the study, signed the informed consent form, had good compliance, and cooperated with follow-up; 2. Age>=18 years old (calculated on the day of signing informed consent), both male and female; 3. Have at least one measurable lesion according to RECIST1.1 criteria; 4. ECOG physical state score 2 points, ASA score 3 points; 5. No other malignant tumors have occurred within five years.

排除标准：

1.病检提示为：纤维板层HCC、肉瘤样HCC或混合型胆管癌。肝移植状态或具有活动性自身免疫性疾病史； 2.病人有其他严重的合并症：如合并严重心肺疾病，心功能在临床分级2级以下，肺部感梁，中重度COPD，慢支炎等，合并严重糖尿病和/或肾功能不全，合并严重营养不良等； 3.妊娠或乳期妇女。

Exclusion criteria：

1. The examination showed that fibrolamellar HCC, sarcomatoid HCC or mixed cholangiocarcinoma. Liver transplant status or history of active autoimmune disease; 2. The patient has other serious comorbidities: such as severe cardiopulmonary diseases, cardiac function below clinical grade 2, pulmonary sensibilities, moderate to severe COPD, chronic bronchiitis, etc., severe diabetes and/or renal insufficiency, severe malnutrition, etc.; 3. Pregnant or lactating women.

研究实施时间：

Study execute time：

从 From 2024-12-31 00:00:00至 To 2025-12-31 00:00:00  

征募观察对象时间：

Recruiting time：

从From 2024-12-31 00:00:00 至 To 2025-12-31 00:00:00  

干预措施：

Interventions：

组别：

观察组

样本量：

30

Group：

Observation group

Sample size：

干预措施：

无

干预措施代码：

Intervention：

None

Intervention code：

研究实施地点：

Countries of recruitment and research settings：

国家：

 中国

省(直辖市)：

 四川省 

市(区县)：

  

Country：

China 

Province：

Sichuan Province 

City：

 

单位(医院)：

四川省肿瘤医院 

单位级别：

三级甲等 

Institution
hospital：

Sichuan Cancer Hospital

Level of the institution：

Tertiary A

测量指标：

Outcomes：

指标中文名：

ITGB2表达

指标类型：

主要指标

Outcome：

ITGB2 expression level

Type：

Primary indicator

测量时间点：

入组

测量方法：

免疫组化

Measure time point of outcome：

Enrollment

Measure method：

Immunohistochemistry

指标中文名：

无进展生存期

指标类型：

次要指标

Outcome：

Progression-free survival (PFS)

Type：

Secondary indicator

测量时间点：

三月一次

测量方法：

影像学随访

Measure time point of outcome：

Every three months

Measure method：

Imaging follow-up

指标中文名：

总生存期

指标类型：

次要指标

Outcome：

Overall Survival (OS)

Type：

Secondary indicator

测量时间点：

随访期间

测量方法：

电话随访

Measure time point of outcome：

Clinical follow-up period

Measure method：

Telephone

采集人体标本：

Collecting sample(s)
from participants：

标本中文名：

血液

组织：

Sample Name：

Blood

Tissue：

人体标本去向

 使用后销毁  

说明

Fate of sample：

Destruction after use  

Note：

标本中文名：

肝组织

组织：

Sample Name：

Liver tissue

Tissue：

人体标本去向

 使用后销毁  

说明

Fate of sample：

Destruction after use  

Note：

征募研究对象情况：

Recruiting status：

尚未开始

Not yet recruiting

年龄范围：

Participant age：
最小 Min age 18 岁 years
最大 Max age 岁 years

性别：

男女均可

Gender：

Both

随机方法（请说明由何人用什么方法产生随机序列）：

无

Randomization Procedure (please state who generates the random number sequence and by what method)：

None

是否公开试验完成后的统计结果:

Calculated Results after the Study Completed public access:

不公开/Private

盲法：

无

Blinding：

None

是否共享原始数据：

IPD sharing

No

共享原始数据的方式（说明：请填入公开原始数据日期和方式，如采用网络平台，需填该网络平台名称和网址）：

无

The way of sharing IPD”(include metadata and protocol, If use web-based public database, please provide the url)：

None

数据采集和管理（说明：数据采集和管理由两部分组成，一为病例记录表(Case Record Form, CRF)，二为电子采集和管理系统(Electronic Data Capture, EDC)，如ResMan即为一种基于互联网的EDC：

CRF

Data collection and Management (A standard data collection and management system include a CRF and an electronic data capture：

CRF

数据与安全监察委员会：

Data and Safety Monitoring Committee：

有/Yes

注册人：

Name of Registration：

  2024-12-19 09:10:26