Volume 43 Issue 6
Jan.  2020
Article Contents

Citation:

Preliminary study of 131I-Tyr-Nivolumab for PD-1-related theranostics of colon cancer

  • Objective We synthesized theranostic agent 131I-Tyr-Nivolumab, which targets programmed cell death-1 (PD-1), and studied its preliminary application in the mouse model with orthotopic colon cancer and high PD-1 expression. Methods 131I-Tyr-Nivolumab was prepared through indirect labeling, and the radiochemical purity and stability of products were evaluated. Ten mice with high expression of PD-1 colon neoplasm in situ were randomly categorized into the treatment and untreated groups. In the treatment group, 131I-Tyr-Nivolumab (11.1 MBq/10 μg) was injected into the tail vein, and SPECT/CT was then performed to observe the distribution of the theranostic agent in mice at different time points (2, 4, 24, and 65 h) after injection. After 5 days of treatment, the expression of Bax and Bcl-2 proteins in tumor tissues were quantified by immunohistochemistry. Results 131I-Tyr-Nivolumab had radiochemical purity greater than 99% and in vitro stability greater than 90% for 24 h. The theranostic agent was mainly distributed in the heart, liver, and intestinal tumors, and was eliminated by renal metabolism, and its uptake by the tumor tissue increased gradually at 2 h post-injection. The liver was visualized at 4 h post-injection, and the suspected tumor area of the intestine was clear at 24 h post-injection. Liver non-specific uptake was almost eliminated at 65 h post-injection. The ratios of radioactivity counts in the intestinal suspected tumor area to the whole-body radioactivity counts at 4, 24, and 65 h post-administration were (2.8±0.3)%, (8.4±0.2)%, and (1.8±0.5)%, respectively. The treatment group had significantly higher expression rate of Bax protein [(22.23±1.61)% vs. (13.64±2.43)%, t=−5.476, P=0.006] bur significantly lower expression rate of Bcl-2 protein [(13.81±4.64)% vs. (25.57±2.33)%, t=3.902, P=0.017] compared with the untreated group. Conclusion 131I-Tyr-Nivolumab targeting PD-1 was successfully synthesized and can be used as a theranostic agent for SPECT imaging and internal irradiation therapy. This method could provide a new idea for the theranostics of colon cancer.
  • 加载中
  • [1] Siegel RL, Miller KD, Jemal A. Cancer statistics, 2018[J]. CA Cancer J Clin, 2018, 68(1): 7−30. DOI: 10.3322/caac.214420010.
    [2] Sauer R, Becker H, Hohenberger W, et al. Preoperative versus Postoperative Chemoradiotherapy for Rectal Cancer[J]. N Engl J Med, 2004, 351(17): 1731−1740. DOI: 10.1056/NEJMoa040694.
    [3] Wolmark N, Wieand HS, Hyams DM, et al. Randomized trial of postoperative adjuvant chemotherapy with or without radiotherapy for carcinoma of the rectum: National Surgical Adjuvant Breast and Bowel Project Protocol R-02[J]. J Natl Cancer Inst, 2000, 92(5): 388−396. DOI: 10.1093/jnci/92.5.388.
    [4] Sunshine J, Taube JM. PD-1/PD-L1 inhibitors[J]. Curr Opin Pharmacol, 2015, 23: 32−38. DOI: 10.1016/j.coph.2015.05.011.
    [5] Thara E, Barzi A. Immunotherapeutic Strategies for Colon Cancer: Monoclonal Antibody Therapy[J]. Curr Colorectal Cancer Rep, 2015, 11(2): 84−91. DOI: 10.1007/s11888−015−0260−y.
    [6] Tanaka T, Kohno H, Suzuki R, et al. A novel inflammation-related mouse colon carcinogenesis model induced by azoxymethane and dextran sodium sulfate[J]. Cancer Sci, 2003, 94(11): 965−973. DOI: 10.1111/j.1349−7006.2003.tb01386.x.
    [7] Natarajan A, Mayer AT, Xu LY, et al. Novel Radiotracer for ImmunoPET Imaging of PD-1 Checkpoint Expression on Tumor Infiltrating Lymphocytes[J]. Bioconjug Chem, 2015, 26(10): 2062−2069. DOI: 10.1021/acs.bioconjchem.5b00318.
    [8] England CG, Jiang DW, Ehlerding EB, et al. 89Zr-labeled nivolumab for imaging of T-cell infiltration in a humanized murine model of lung cancer[J]. Eur J Nucl Med Mol Imaging, 2018, 45(1): 110−120. DOI: 10.1007/s00259−017−3803−4.
    [9] Li DN, Wang C, Zhang DK, et al. Preliminary Application of 125I-Nivolumab to Detect PD-1 Expression in Colon Cancer via SPECT[J]. J Radioanal Nucl Chem, 2018, 318(2): 1237−1242. DOI: 10.1007/s10967−018−6124−6.
    [10] 李囡, 林保和, 杨志, 等. CD20阳性B细胞NHL131I-美罗华放射免疫治疗剂量个体化的临床初步研究[J]. 同位素, 2004, 17(4): 204−209. DOI: 10.3969/j.issn.1000−7512.2004.04.003.Li N, Lin BH, Yang Z, et al. The Primary Clinical Study on Individual Radioimmunotherapy Protocol With 131I-Rituximab for CD20 Positive B Cell Non-Hodgkin's Lymphoma[J]. J Isotopes, 2004, 17(4): 204−209. DOI: 10.3969/j.issn.1000−7512.2004.04.003.
    [11] Ychou M, Pelegrin A, Faurous P, et al. Phase-I/Ⅱ radio-immunotherapy study with Iodine-131-labeled anti-CEA monoclonal antibody F6 F(ab')2 in patients with non-resectable liver metastases from colorectal cancer[J]. Int J Cancer, 1998, 75(4): 615−619. DOI: 10.1002/(SICI)1097−0215(19980209)75:4<615::AID−IJC20>3.0.CO;2−6.
    [12] Maier P, Hartmann L, Wenz F, et al. Cellular Pathways in Response to Ionizing Radiation and Their Targetability for Tumor Radiosensitization[J]. Int J Mol Sci, 2016, 17(1) DOI: 10.3390/ijms17010102.
  • 加载中
通讯作者: 陈斌, bchen63@163.com
  • 1. 

    沈阳化工大学材料科学与工程学院 沈阳 110142

  1. 本站搜索
  2. 百度学术搜索
  3. 万方数据库搜索
  4. CNKI搜索

Figures(6)

Article Metrics

Article views(209) PDF downloads(8) Cited by()

Related
Proportional views

Preliminary study of 131I-Tyr-Nivolumab for PD-1-related theranostics of colon cancer

    Corresponding author: Changjing Zuo, cjzuo@smmu.edu.cn
  • 1. School of Medical Imaging, Xuzhou Medical University, Xuzhou 221004, China
  • 2. Department of Nuclear Medicine, Shanghai Changhai Hospital, Shanghai 200433, China

Abstract:  Objective We synthesized theranostic agent 131I-Tyr-Nivolumab, which targets programmed cell death-1 (PD-1), and studied its preliminary application in the mouse model with orthotopic colon cancer and high PD-1 expression. Methods 131I-Tyr-Nivolumab was prepared through indirect labeling, and the radiochemical purity and stability of products were evaluated. Ten mice with high expression of PD-1 colon neoplasm in situ were randomly categorized into the treatment and untreated groups. In the treatment group, 131I-Tyr-Nivolumab (11.1 MBq/10 μg) was injected into the tail vein, and SPECT/CT was then performed to observe the distribution of the theranostic agent in mice at different time points (2, 4, 24, and 65 h) after injection. After 5 days of treatment, the expression of Bax and Bcl-2 proteins in tumor tissues were quantified by immunohistochemistry. Results 131I-Tyr-Nivolumab had radiochemical purity greater than 99% and in vitro stability greater than 90% for 24 h. The theranostic agent was mainly distributed in the heart, liver, and intestinal tumors, and was eliminated by renal metabolism, and its uptake by the tumor tissue increased gradually at 2 h post-injection. The liver was visualized at 4 h post-injection, and the suspected tumor area of the intestine was clear at 24 h post-injection. Liver non-specific uptake was almost eliminated at 65 h post-injection. The ratios of radioactivity counts in the intestinal suspected tumor area to the whole-body radioactivity counts at 4, 24, and 65 h post-administration were (2.8±0.3)%, (8.4±0.2)%, and (1.8±0.5)%, respectively. The treatment group had significantly higher expression rate of Bax protein [(22.23±1.61)% vs. (13.64±2.43)%, t=−5.476, P=0.006] bur significantly lower expression rate of Bcl-2 protein [(13.81±4.64)% vs. (25.57±2.33)%, t=3.902, P=0.017] compared with the untreated group. Conclusion 131I-Tyr-Nivolumab targeting PD-1 was successfully synthesized and can be used as a theranostic agent for SPECT imaging and internal irradiation therapy. This method could provide a new idea for the theranostics of colon cancer.

    HTML

  • 结直肠癌是消化道最常见的恶性肿瘤之一,大多数结直肠癌患者就诊时已属中晚期。结肠癌局部转移相对频繁,肿瘤可穿透肠壁并使区域淋巴结受累[1-3]。中晚期结直肠癌的标准治疗模式以直肠癌根治术为基础,辅以放疗。放疗通过破坏DNA将肿瘤杀灭,可以提高结直肠癌的局部控制率,增加保肛机率,提高患者生存率及生活质量,但仍存在辐射剂量高、对健康组织损伤较大等缺点。具有高度选择性的内照射治疗(selective internal radiotherapy,SIRT)可以降低局部复发率,使肿瘤缩小,减少癌周浸润,同时可以降低放疗所致的生物毒性,减轻放疗引发的不良反应。

    由于阻断T细胞免疫检查点程序性死亡受体1(programmed death receptor, PD-1)/程序性死亡受体-配体1(programmed death receptor-ligand 1, PD-L1)通路的免疫疗法具有高度特异性,因此其在癌症免疫治疗中的重要性被广泛认可[4]。有研究结果证实,结肠癌周间质主要由大量T淋巴细胞浸润[5],所以结直肠癌也成为PD-1/PD-L1治疗的重要应用方向之一。由于PD-1与PD-L1的高度相关性,靶向PD-1的诊疗一体化核素标记的单克隆抗体可用于PD-1表达的评估,同时SIRT作用于T细胞浸润的肿瘤组织有望成为PD-1/PD-L1免疫疗法的有效补充。

    本研究拟合成一种PD-1抗体导向的免疫放疗诊疗剂。在SIRT的同时,131I释放的γ射线可用于SPECT评估PD-1的表达,成为PD-1相关SIRT的诊疗一体化试剂。

1.   材料与方法

    1.1.   动物与试剂

  • 10只SPF级PD-1高表达小鼠(品系全称:C57BL/6J-Pdcd1em1(hPDCD1)/Smoc),雌性,6周龄,体重18~22 g,购自上海南方模式生物科技股份有限公司,实验动物许可证号:SYXK(沪)2018-0002。氧化偶氮甲烷、羟苯基丙酸-N-羟基琥珀酰亚胺酯(Bolton-Hunter,BH试剂)、二甲基亚砜、Iodogen(1,3,4,6-四氯-3α,6α-二苯基甘脲)购自美国 Sigma公司,葡萄糖硫酸钠购自上海翊圣生物科技有限公司,Nivolumab抗体(5 mg/mL)购自美国Selleck公司,核素 131I 购自上海欣科医药有限公司。

  • 1.2.   动物模型的制备

  • 参照文献[6]的方法,先在小鼠腹腔单次注射氧化偶氮甲烷(10 mg/kg),7 d后喂食2%葡萄糖硫酸钠溶液4 d,之后继续喂食纯净水17 d,此为1个周期。以上步骤重复3个周期后,改为普通饮食喂养,模型制备至130 d。将小鼠随机分成治疗组和对照组,每组各5只,两组之间体重的差异无统计学意义(t=−0.472,P=0.649)将部分小鼠处死解剖,肉眼观察腹腔肠道组织有无肿瘤灶,通过苏木精-伊红染色及PD-1免疫组织化学染色验证动物模型制备是否成功。

  • 1.3.   碘-131化酪氨酸标记Nivolumab(131I-Tyr-Nivolumab)的制备

  • 131I-Tyr-Nivolumab的制备采用间接标记法。将1 µg BH试剂溶于1 µL 二甲基亚砜后与111 MBq Na131I(10 µL)分别加入涂有80 µg Iodogen的试管中混合反应15 min。之后将反应体系(无催化剂)与10 µg Nivolumab继续反应1 h,具体标记流程见图1

    Figure 1.  Preparation of 131I-Tyr-Nivolumab

    采用 PD-10(内含物为Sephadex G-25)柱分离标记产物,先用 0.01 mol/L pH = 7.4 的PBS平衡,然后洗脱,每10滴收集一管,共收集40管。使用放射性活度计依次测定每管的放射性活度,第一峰为131I-Tyr-Nivolumab,第二峰为含放射性的杂质(主要成分为游离131I及131I-羟苯基丙酸-N-羟基琥珀酰亚胺酯)。收集第一峰的131I-Tyr-Nivolumab,通过其与全部放射峰之和的比值计算标记率。

    进一步测量产物的放射化学纯度,将Whatman色谱纸制成10 cm×1 cm的色谱纸作为固定相,并使用生理盐水作为流动相。将色谱纸等分成10段,将5 µL第一峰对应样品在距端点1 cm处点样后晾干,置于流动相中。待展开前沿距末端约5 mm处时,取出晾干后沿线剪开,放入试管中编号,用γ计数器分别检测相应的每分钟计数值,近原点处(Rf = 0)为标记蛋白峰。在室温下,将131I-Tyr-Nivolumab分别与0.01 mol/L PBS和1%的胎牛血清共同孵育2、4、8、12、24、48 h,以评估放射性标记Nivolumab的体外稳定性。

  • 1.4.   原位结肠癌小鼠模型的SPECT/CT显像

  • 在治疗组小鼠尾静脉注射含有约11.1 MBq/10 μg的131I-Tyr-Nivolumab。在SPECT/CT成像的每个时间点前,将50 μL 3%利多卡因注射到小鼠的腹腔中进行麻醉。在注射后的不同时间点(2、4、24、65 h),用SPECT/CT(Symbia T16,德国西门子公司)进行扫描,观察不同时间点诊疗剂的分布及清除情况。高能准直器成像采集参数如下,采集矩阵:128×128;变焦:2.67;能峰:364 keV;窗宽:20%;帧数:60 s/帧;总共采集32帧。CT扫描采集参数如下,管电压:130 kV;管电流:35 mA;间距:1.0;重建层厚度:1 mm。对于每个SPECT扫描,在经衰减校正后的图像上绘制肠道肿瘤的ROI,评估全身及肿瘤组织的放射性计数。

  • 1.5.   病理及免疫组织化学检测

  • 给药120 h后,将所有小鼠脱颈处死,根据SPECT/CT探测的PD-1高表达范围,并结合肉眼所见切取肿瘤组织。用4%多聚甲醛溶液固定肿瘤组织,常规脱水,石蜡包埋,切片后苏木精-伊红染色,在光学显微镜下进行组织学观察。将切片交至上海基尔顿生物科技有限公司,采样分析样本相关部位,评估肿瘤组织SIRT凋亡相关的Bax、Bcl-2蛋白表达的改变,与未进行相应治疗的对照组的对应参数进行对比。Bax及Bcl-2染色阳性的肿瘤细胞胞浆内有棕黄色或深棕色颗粒,每张切片随机选取3个高倍镜视野(×200),计数黄染细胞,分别计算Bax及Bcl-2阳性细胞占总细胞数的比例以评估蛋白表达量。

  • 1.6.   统计学分析

  • 采用SPSS 19.0软件对数据进行统计学分析。符合正态分布的实验数据用${\bar{x}}\pm {s}$表示,方差齐的组间数据比较采用配对样本t检验。P<0.05表示差异有统计学意义。

2.   结果

    2.1.   131I-Tyr-Nivolumab的合成与鉴定

  • 131I-Tyr-Nivolumab经PD-10柱分离纯化的洗脱曲线见图2中A。131I-Tyr-Nivolumab的标记率为(30.13±5.36)%。分离后第一峰产品的放射化学纯度>99%(图2中B)。在37 ℃环境下,131I-Tyr-Nivolumab在0.01 mol/L PBS和1%胎牛血清中的24 h稳定性>90%(图2中C)。

    Figure 2.  Purification and radiolabeling stability of 131I-Tyr-Nivolumab

  • 2.2.   131I-Tyr-Nivolumab在原位结肠癌小鼠体内的分布

  • 原位结肠癌小鼠解剖后肉眼可见部分肠道组织充血水肿,部分可见黄豆大小的结节。小鼠肠道肿瘤组织切片,在显微镜下可见核深染色异形细胞,核仁明显(图3)。免疫组织化学法评估PD-1表达量为(19.58±3.23)%(图4)。证明小鼠原位结肠癌模型制备成功。

    Figure 3.  Histopathology and anatomy of mouse intestinal tumor tissue

    Figure 4.  PD-1 positive expression in colon cancer mouse intestinal tumors

    在小鼠尾静脉注射131I-Tyr-Nivolumab后不同时间点(2、4、24、65 h)进行的SPECT扫描显示,131I-Tyr-Nivolumab主要分布在心脏、肝脏及肠道肿瘤区域。随着显像时间的延长,肝脏等非靶器官的放射性摄取逐渐下降,肿瘤组织显影逐渐清晰。给药后2 h,肿瘤组织摄取131I-Tyr-Nivolumab逐渐增加。4 h可见肝脏显影,肠道疑似肿瘤区放射性计数与全身总放射性计数比为(2.8±0.3)%,给药后24 h肠道疑似肿瘤区显影清晰,疑似肿瘤区域与全身放射性计数比为(8.4±0.2)%(图5)。注射药物后65 h,肝脏非特异性摄取几乎被排出,肠道疑似肿瘤区域与全身放射性计数比为(1.8±0.5)%。证明PD-1抗体可较长时间在靶点维持高浓度,即保持较为长效的内照射作用。

    Figure 5.  SPECT/CT image of radioactivity distribution of 131I-Tyr-Nivolumab in colon cancer mice 4 hours and 24 hours post injection

  • 2.3.   内照射疗效

  • Bax及Bcl-2染色阳性的肿瘤细胞表现为胞浆内黄染颗粒(图6)。未治疗组Bax的表达率为(13.64±2.43)%,治疗组为(22.23±1.61)%,两组之间的差异有统计学意义(t=−5.476, P=0.006);未治疗组Bcl-2蛋白的阳性表达率为(25.57±2.33)%,治疗组为(13.81±4.64)%,较未治疗组明显降低,且差异有统计学意义(t=3.902, P=0.017)。

    Figure 6.  Immunohistochemical results of tumor tissues in colon cancer mice before and after treatment

3.   讨论
  • 目前,国外多数研究将PD-1抗体类物质的核素标记应用于核医学诊断及体内分析。例如,Natarajan等[7]基于抗体药物Pembrolizumab建立的抗PD-1免疫PET示踪剂,分别用锆-89(89Zr)和铜-64(64Cu)标记,实现了对人肿瘤浸润淋巴细胞(TILs)上的PD-1表达的特异性成像。England等[8]89Zr-Df-Nivolumab PET有效地显示出表达PD-1的T细胞,以深入了解被各种疗法激活的T细胞转归和肿瘤驻留之间的相互作用,对于深入研究肿瘤免疫治疗过程中微环境的改变提供了可视化参考。类似研究中,我们通过125I标记Nivolumab作为靶向显像剂,经SPECT观察表达PD-1的激活免疫细胞,进而评估Nivolumab在结肠癌模型中的靶向性及代谢特征[9],为本工作中开展基于Nivolumab的核素诊疗一体化研究奠定基础。除PD-1/PD-L1轴特异性显像以外,治疗性或诊疗一体化核素(如131I、177Lu、188Re、225Ac等)标记PD-1抗体用于核医学临床前的研究甚少。迄今为止,临床上尚未批准以PD-1/PD-L1抗体介导的治疗性核素用于肿瘤SIRT,其相对复杂的合成步骤、临床用药安全性等限制了临床转化。

    本研究工作中使用的诊疗一体化核素131I,其物理半衰期约为8 d,因此在体内作用时间较长。由于其组织内有效射程仅为1~2 mm,对周围正常组织影响小,131I作为SIRT核素已用于抗CD20、抗CEA等蛋白的诊疗一体化研究,并取得了较好的临床治疗效果[10-11]。基于以上研究背景,本实验通过131I标记PD-1抗体Nivolumab,探讨放射性碘(131I)标记的 Nivolumab对结肠癌的治疗作用。相应PD-1靶向的高度选择性的内照射治疗通过β-射线直接作用于并破坏肿瘤细胞DNA双链结构,促进肿瘤细胞发生凋亡,发挥抗肿瘤作用[12]。本研究的治疗原理已通过与细胞凋亡相关的Bax、Bcl-2蛋白指标得以验证。当细胞内Bax蛋白较多时,可促进细胞凋亡;细胞内Bcl-2蛋白增多时,细胞凋亡趋势减弱。本实验治疗组与未治疗组免疫组化结果显示,给药120 h后,治疗组比未治疗组的肿瘤组织内Bax蛋白表达率明显增高(22.23±1.61)% vs.(13.64±2.43)%,差异有统计学意义(P<0.05),治疗组比未治疗组肿瘤组织的Bcl-2蛋白表达率明显降低(13.81±4.64)% vs.(25.57±2.33)%,差异有统计学意义(P<0.05)。该结果表明,131I-Tyr-Nivolumab可以诱发结肠癌细胞的凋亡发生,对结肠癌具有一定的治疗作用。在辅助诊断方面,虽然131I发出的高能γ射线可用于脏器的扫描以及功能测定,但相比于123I等发出纯γ射线的放射性碘而言,其单光子扫描图像分辨率较低,图像质量欠佳,不宜单纯作为特异性显像剂使用。

    基于抗体类物质的核素标记应用须建立在尽量不影响其靶向性及免疫原性的前提下,因此本研究采用基于BH试剂的间接标记法来最大化地保留Nivolumab的固有结构。该试剂相对分子量小,以碘标酪氨酸连接到蛋白末端上的形式进行核素标记,既避免了蛋白质和氧化剂直接接触,也避免了与放射性碘原子的直接接触,从而保持Nivolumab的生物活性及对PD-1抗原的高特异性。131I-Tyr-Nivolumab可在PD-1抗体的介导下靶向肿瘤周围激活的T细胞,以实现对肿瘤细胞近距离照射治疗的目的,也可用于体内SPECT非侵入性成像,实现诊疗一体化。

    本研究创新性地将具有免疫阻断治疗效果的抗体与诊疗一体化核素进行结合,实现免疫阻断治疗与免疫内照射治疗的协同治疗作用。虽然本研究只能用于PD-1高表达肿瘤的SIRT,尚无法广泛用于肿瘤治疗,但是依然可以作为PD-1免疫阻断治疗的有力补充,用于精准放疗和对分散性转移病灶的靶向放疗。

    利益冲突 本研究由署名作者按以下贡献声明独立开展,不涉及任何利益冲突。

    作者贡献声明 李丹妮负责研究过程的实施、数据的获取与分析、论文的撰写;任胜男、苏维维、彭烨负责实验的实施和数据、图像的分析;李潇负责研究命题的设计、方法学指导和论文的审阅;左长京负责研究命题的提出、指导、图像的分析及论文的最终审阅。

Reference (12)

Catalog

/

DownLoad:  Full-Size Img  PowerPoint
Return
Return