本论文的研究内容分为两个部分：第一部分以我国自主研发的1类新药FCN-411和特瑞普利单抗为研究对象，基于质谱技术分别建立了快速、可靠和灵敏的超高效液相色谱串联质谱方法（ultra-high performance liquid chromatography-tandem mass spectrometry，UPLC-MS/MS），测定人血浆中药物浓度，为这两种药物的临床药代动力学研究以及治疗药物浓度监测、药效学等方面的研究提供了评价方法和合理用药依据。第二部分基于高通量蛋白芯片技术揭示结直肠癌患者血浆中自身抗体表达情况，筛选和验证有潜力成为结直肠癌早期诊断和良恶性结直肠肿瘤鉴别诊断的自身抗体标志物。

第一部分：建立FCN-411和特瑞普利单抗的检测方法

第一章：建立和验证人血浆中FCN-411浓度检测的UPLC-MS/MS方法。FCN-411是新型的二代表皮生长因子受体酪氨酸激酶抑制剂（epithelial growth factor receptor-tyrosine kinase inhibitor，EGFR-TKI），具有部分三代EGFR-TKI的特性。为进一步探索FCN-411对非小细胞肺癌患者的疗效，我们建立了UPLC-MS/MS方法用于临床药代动力学和药效学研究。应用操作简便的蛋白沉淀法对血浆样品进行前处理，并根据生物样品分析方法的指导原则进行全部的方法学验证。结果显示血浆样品中FCN-411的标准曲线在2-500 ng/mL范围内线性较好，三批次分析结果中r²均大于0.98；低定量下限样品的批内和批间的精密度在9.18%～17.46%之间，准确度在-17.50%～7.14%之间；其余三个浓度质控样品的批内和批间的精密度在4.24%～14.35%之间，准确度在2.00%～11.89%之间；低、高浓度质控样品内标归一化基质因子均值分别为101.67%和94.74%，变异系数分别为4.69%和2.27%，不同个体间的基质差异不影响FCN-411的定量；低、高浓度质控样品中FCN-411的相对提取回收率均值在85%～100%之间，变异系数分别为5.78%和1.23%，较为一致；血浆样品在室温放置2 h、-80℃保存7天和冻融循环三次以及样品被处理后在10℃进样器放置26 h后均保持稳定，且FCN-411和内标的储备液在-20℃冰箱放置4.8个月仍稳定。该方法顺利通过全部的方法学验证，并成功用于检测1例FCN-411的Ⅰ期临床试验中接受4 mg 剂量治疗的非小细胞肺癌患者血样，初步验证该方法具有较好的临床适用性。单个样品的检测时间仅需4.5分钟，能在较短时间内实现大批量样本的检测，满足临床样本的检测需求，为后续进行FCN-411的Ⅰ期临床试验的药代动力学研究建立了方法学基础。

第二章：建立和验证人血浆中特瑞普利单抗浓度检测的UPLC-MS/MS方法，并与电化学发光免疫分析法（electrochemiluminescence immunoassay，ECLIA）进行比较。特瑞普利单抗是针对PD-1的人源化IgG4型单克隆抗体，我们建立UPLC-MS/MS法检测特瑞普利单抗的浓度，用于后续临床药物治疗浓度监测。我们应用数据库筛选结合基质样本的上机检测，筛选出特瑞普利单抗的特征性肽段，基于其序列合成延长同位素标记的内标标准品，优化前处理和色谱质谱参数，并完成全部的方法学验证，结果均符合生物样品分析方法的检测要求。我们成功检测了特瑞普利单抗I期临床研究入组患者的77份血浆样本，证实该方法具有较好的临床适用性。同时基于56个样本检测结果，与ECLIA方法进行比较。结果显示两组数据具有较好的相关性（r=0.96），但UPLC-MS/MS方法的检测结果显著高于ECLIA的结果（p<0.0001）。Passing-Bablok线性回归和Bland-Altman分析均表明两组数据存在一定的系统差异和比例差异。这主要是由于两种方法的检测原理不同所致，ECLIA法采用双抗原夹心法测定血浆中游离态的单抗药物，并且血浆中存在的可溶性PD1抗原可能与特瑞普利单抗结合，可能影响药物的定量；而我们建立的UPLC-MS/MS法通过监测单抗酶解后的特征性肽段来间接定量单抗的浓度，血浆中结合和游离态的单抗都能被酶解生成肽段而被检测到，因此我们的方法检测血浆中总的特瑞普利单抗浓度，并且不易受血浆基质的干扰。综上，我们建立的方法可以作为ECLIA方法的补充，用于检测不同形式单抗的浓度。

第二部分：结直肠癌早期检测相关的血浆自身抗体研究

第一章：Anti-p53自身抗体对结直肠癌诊断价值的meta分析。基于在Pubmed、Embase和Cochrane library这三个数据库提取相关文献，最终筛选到80篇文献，囊括145个自身抗体。约70%以上文献结果显示自身抗体的灵敏度低于30%，约80%的结果显示其特异度相对较高，超过90％。我们针对研究最多的anti-p53自身抗体进行meta分析，包括来自24篇文献的27个研究。由于纳入的研究间存在明显的异质性，meta回归和亚组分析显示阈值效应是主要的异质性来源。我们将研究根据阳性界值的定义分为5组，由于其余4组依然存在阈值或非阈值效应，只有group 3组内的研究间较为均一，因此我们将group 3的研究采用固定效应模型进行合并分析。group 3由5篇文章组成，包括733例结直肠癌患者和172例对照（126例健康个体和46例良性疾病）。合并灵敏度、特异性、阳性和阴性似然比，诊断比值比和受试者工作特性曲线下面积分别为0.21、0.99、12.26、0.80、15.46和0.87。基线外周血中抗p53自身抗体可能有助于将结直肠癌与健康对照或良性疾病区分开来，但需进一步探索与其他标志物联合使用以提高检测的灵敏度。

第二章：应用高通量蛋白芯片筛选和验证与结直肠癌早期检测相关的自身抗体研究。基于高通量Huprot蛋白芯片的发现阶段和候选蛋白芯片的验证阶段这两步实验，揭示血浆自身抗体在结直肠癌、良性结直肠肿瘤和健康人群中的表达情况。Huprot蛋白芯片发现阶段，共纳入35例早期结直肠癌患者（TNM分期I-II期）、25例良性结直肠肿瘤患者和20例健康人，经过两两比较后共筛选到198个差异自身抗体；候选蛋白芯片验证阶段，纳入305例早期结直肠癌患者、61例良性结直肠肿瘤患者和240例健康人，我们首先基于早期结直肠癌患者和健康人的数据评价4种定义阳性界值法，发现最大约登指数+至少90%特异度法能确保较高的特异度，同时提高检测的灵敏度，后续采用这种方法进行分析，结果如下：（1）发现11个与结直肠癌早期诊断相关的差异自身抗体，单个自身抗体的检测阳性率在12.46%～20.00%之间，其中p53、GATA6、MAP3K5、SOX9-AS1_frag和TIMM8A这5个自身抗体组合构建Logistic回归模型时，灵敏度达到51.47%，特异度为75.42%；（2）发现13个用于Early CRC和Benign组鉴别诊断的差异自身抗体，单个抗体的检测灵敏度在14.10%～28.20%之间，其中6个自身抗体HIST1H1C、OSGIN1、CRY2、PRR7、REG3A和SLC43A2被纳入构建Logistic回归模型，AUC达到0.78，灵敏度达74.10%，特异度为70.49%，具有较好的潜力用于良恶性结直肠肿瘤的鉴别诊断；（3）发现13个与良性结直肠肿瘤相关的自身抗体，单个抗体的检测阳性率在1.64%～24.59%之间，其中p53、RXFP2和UROS这3个自身抗体组合构建Logistic回归模型，检测的灵敏度为34.43%，特异度为86.67%；（4）与健康人和良性结直肠肿瘤患者相比，早期结直肠癌患者主要表达≥2个阳性自身抗体，因此限定≥2个阳性抗体时能显著提高联合检测的特异度，诊断价值与Logistic回归模型的相当。综上，多个血浆自身抗体在早期结直肠癌和良性肿瘤患者中出现显著升高，具有较大潜力用于结直肠癌的早期诊断和良恶性鉴别诊断，后续可在更大的人群队列中再次验证，确认最佳的联合检测组合，以最大程度识别出早期结直肠癌患者。

关键词：FCN-411；特瑞普利单抗；超高效液相色谱串联质谱；自身抗体；结直肠癌

The present study was constituted of two parts: firstly, ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) methods were developed for quantifying epithelial growth factor receptor-tyrosine kinase inhibitor (FCN-411) and PD1 monoclonal antibody (toripalimab) concentrations in human plasma. The two methods provided a basis for analyzing clinical pharmacokinetic (PK), pharmacodynamic (PD) study and monitoring of therapeutic drug concentration (TDM) of the two drugs; secondly, the high-throughput protein microarray was applied to detect plasma autoantibody levels in colorectal cancer (CRC), benign colorectal tumor and healthy controls, then screen and validate these autoantibodies as promising biomarkers for early diagnosis of colorectal cancer and differential diagnosis of benign and malignant colorectal tumors.

Section 1: Developing methods to detect FCN-411 and toripalimab levels

Chapter 1: Developing and validating a UPLC-MS/MS method to quantify FCN-411 in human plasma. Protein precipitation procedure was used to pretreat plasma samples. The method was validated in accordance with the guidelines for bioanalytical method validation. It yielded a good linearity in the range of 2-500 ng/mL; the intra and inter accuracy and precision were 9.18%～17.46% and -17.50%～7.14% for the low limit of quantification samples (LLOQ), and 4.24%～14.35% and 2.00%～11.89% for the high, medium and low quality controls (QCs), respectively; the mean values of the internal standard normalized matrix factors of the low and high QCs were 101.67% and 94.74%, with the coefficients of variation of 4.69% and 2.27%, respectively; no significant matrix difference was observed; the relative extraction recoveries of FCN-411 in low and high QCs were both between 85% and 100%, whose coefficients of variation were 5.78% and 1.23%, respectively; plasma samples were stable when preserved under the following conditions: ambient temperature for 2 h, -80°C for 7 days, three freeze-thaw cycles, and extracts in 10°C autosampler for 26 h. Besides, FCN-411 and internal standard in stock solution stored in - 20°C were both stable for 4.8 months. This method was successfully used to detect a clinical plasma sample from a patient administrated 4 mg dose in a phase Ⅰ clinical trial, which demonstrated a good clinical application. The method was ready to be applied to measure FCN-411 in subsequent clinical pharmacokinetic study.

Chapter 2: Development and validation of a UPLC-MS/MS method for detecting toripalimab in human plasma, in comparison with electrochemiluminescence immunoassay (ECLIA). A unique, sensitive and stable peptide was selected as a surrogate peptide of toripalimab to be monitored. Meanwhile, an extended stable isotope-labeled surrogate peptide was synthesized as internal standard. The method was fully validated and successfully applied to determine 77 plasma samples from 29 patients in a phase I clinical trial. Afterwards, method comparison was performed based on 56 samples, which had been previously measured by ECLIA. A good consistent trend observed between the two methods (Spearman correlation r=0.96), but constant and proportional biases were observed by Passing-Bablok regression and Bland-Altman analysis. This discrepancy could be mainly attributed to different forms of toripalimab determined: free and total forms of drug were quantified by ECLIA and UPLC-MS/MS, respectively. As a result, this UPLC-MS/MS method may be complementary to ECLIA to monitor different forms of toripalimab.

Section 2: Study on plasma autoantibodies related to early detection of CRC

Chapter 1: Meta-analysis of anti-p53 autoantibodies for exploring the diagnostic value on colorectal cancer. The PubMed, Cochrane Library, and Embase databases were searched to explore the diagnostic potential of autoantibodies on CRC . A total of 145 autoantibodies published in 80 articles was reviewed. About 70% of the articles reported a sensitivity lower than 30%, and about 80% showed the specificity more than 90%. Of these, anti-p53 antibody was the most commonly studied, thus we performed a meta-analysis on anti-p53 antibody in 27 studies from 24 articles. According to the different cut-off difinitions, we divided the included studies into five groups. Owing to the presence of threshold effect and non-threshold effect in the other four groups, pooled analysis was focused on group 3 using a fixed-effect model. Group 3 was comprised of five articles including 733 patients with CRC and 172 controls (126 healthy individuals and 46 benign diseases). The pooled sensitivity, specificity, positive and negative likelihood ratios, diagnostic odds ratio, and summary area under the receiver operating characteristic curves were 0.21, 0.99, 12.26, 0.80, 15.46, and 0.87, respectively. Serum anti-p53 autoantibody may potentially help distinguish CRC from healthy controls or benign diseases. Additionally, further study was needed to explore optimized combination with other markers due to the low sensitivity.

Chapter 2: Screening autoantibody for early diagnosis of CRC based on high-throughput protein microarray. In the discovery phase, 35 patients with CRC (TNM stage I-II), 25 patients with benign colorectal tumors and 20 healthy controls were included. And 198 differential autoantibodies were screened. In the validation phase, 305 patients with CRC (stage I-II), 61 patients with benign colorectal tumors and 240 healthy controls were included to validate these differential autoantibodies. Firstly, we assessed 4 positive cut-off definitions based on data from early CRC and healthy controls, and found Youden index + at least 90% specificity method could ensure high specificity and sensitivity. Thus, this method was used to assess diagnostic efficacies of autoantibodies. The results were as follows: (1) 11 autoantibodies related to CRC early detection were obtained, and the positive rates of single autoantibody were between 12.46% and 20.00%, of which p53, GATA6, MAP3K5, SOX9-AS1_frag and TIMM8A were selected in a Logistic regression model with the sensitivity of 51.47% and specificity of 75.42%; (2) 13 autoantibodies for distinguishing early CRC from benign colorectal tumors were obtained, and the positive rates were between 14.10% and 28.20%, of which HIST1H1C, OSGIN1, CRY2, PRR7, REG3A and SLC43A2 were included in a Logistic regression model with the AUC of 0.78, sensitivity of 74.10% and specificity of 70.49%; (3) 13 autoantibodies related to benign colorectal tumors were obtained, and the positive rates were between 1.64% and 24.59%, of which p53, RXFP2 and UROS were selected in a Logistic regression model with the sensitivity of 34.43% and specificity of 86.67%；(4) compared with healthy controls and benign colorectal tumors, early CRC mainly expressed ≥2 positive autoantibodies. Therefore, the specificity would be significantly improved when the individuals expressing ≥2 positive autoantibodies were defined as positive individuals, and its diagnostic value was comparable to above Logistic regression model. In summary, several plasma autoantibodies were significantly increased in patients with CRC and benign tumors than normal controls, with greater potential for early diagnosis of CRC and differential diagnosis of benign and malignant tumors. Further study with a larger cohort is needed to explore the optimal combination of joint detection for identifying early CRC patients.

Keywords：FCN-411; Toripalimab; UPLC-MS/MS; Autoantibody; Colorectal cancer