Chemical Modifications Of Antibodies For Molecular Targeting

分子靶向抗体的化学修饰

• 批准号: 9354067
• 负责人: Chang Hum Paik
• 金额: --
• 依托单位: CLINICAL CENTER
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9354067
• 关键词: 3-Dimensional; 90Y; Address; Affect; Affinity; Algorithms; Anesthesia procedures; Animals; Antibodies; Antigens; Area; Autoradiography; Binding; Binding Sites; Biodistribution; Biological; Biological Sciences; Blood; Blood Circulation; Boston; Breathing; Carbon Dioxide; Cardiac; Cell Survival; Cell surface; Chelating Agents; Chemicals; Computer software; Convection; Deferoxamine; Diagnosis; Dissection; Dose; Drug Kinetics; Enhancing Antibodies; Epithelial; Extracellular Matrix; Extracellular Space; Filtration; Focused Ultrasound Therapy; Freezing; Gamma counter; Half-Life; Healthcare; Hematologic Neoplasms; Human; Image; Image Analysis; Imagery; Imaging Techniques; Immunoglobulin Fragments; Implant; Indium-111; Individual; Injection of therapeutic agent; Intercellular Fluid; Isoflurane; Isothiocyanates; Kinetics; Label; Lead; Liquid substance; Liver; Longitudinal Studies; Malignant Neoplasms; Measures; Methods; Modification; Molecular Target; Monoclonal Antibodies; Motion; Mus; Myocardium; Normal tissue morphology; Nude Mice; Optics; Organ; Oxygen; Paclitaxel; Penetration; Physiologic pulse; Physiological; Play; Positron; Positron-Emission Tomography; Puncture procedure; Radioactivity; Radioimmunoconjugate; Radioimmunotherapy; Radioisotopes; Radiolabeled; Research; Resolution; Role; Sampling; Signal Transduction; Site; Solid Neoplasm; Spleen; Stromal Cells; Surface; Surface Antigens; Thick; Time; Tissues; Treatment Protocols; Tumor Antigens; United States National Institutes of Health; ammonium acetate; attenuation; base; bevacizumab; cancer therapy; expectation; fluorophore; imaging probe; immunoreactivity; improved; in vivo; interest; interstitial; macromolecule; meetings; mesothelin; neoplastic cell; pre-clinical; pressure; prevent; promoter; radiation resistance; radiochemical; radiotracer; research study; response; success; targeted treatment; treatment response; tumor; tumor microenvironment; uptake; whole body imaging

Background: In the past year, we have extended our research to reaffirm the results described above by repeating the experiments with MORAb-009 labeled with Zr-89 (a positron emitter; decay half-life of 3.3 days) which better matches with the biological half-life of mAbs than Cu-64 (decay half-life, 12.7 h) labeled mAb, thus making the Zr-89 labeled mAb more relevant for the diagnosis and the assessment of the therapeutic responses of cancer. As a part of our mechanistic studies on the tumor uptake and microdistribution of MORAb-009 (anti-mesothelin mAb amatuzimab), we compared the results of Zr-89-MORAb-009 studies with those of Zr-89-labeled B3, a monoclonal antibody directed against Lewis-Y antigen which is not shed from tumor surface. Objectives: To develop a method to radiolabel two mAbs (MORAb-009 and anti-Lewis-Y mAb B3) and investigate the effect of the injection dose and the tumor size on the tumor uptake and microdistribution of these two Zr-89-labeled mAbs in nude mice bearing A431/H9 which expresses both mesothelin and Lewis-Y antigen. Methods: The mAbs were radiolabeled with Zr-89 using desferrioxamine with an isothiocyanate linker as a chelating agent. The Zr-89 labeled mAbs were then purified with a PD-10 column eluted with 0.25 M ammonium acetate at pH 5.5. The radiolabeled mAbs with the radiochemical purity >95% and the immunoreactivity >70% were used for in vivo studies. The biodistribution (BD) was performed in groups of nude mice (n=4-5) with A431/H9 tumors (range, 191 265 cubic mm for MORAb-009 and 260 405 cubic mm for B3 study) one day after iv injection of Zr-89-MORAb-009 (3 microCi/2, 10 or 60 microg) or Zr-89-B3 (3 microCi/2, 15 or 60 microg). For the BD studies, the animals were euthanized at 24h post-injection (p.i.) by CO2 inhalation and exsanguinated by cardiac puncture before dissection. Blood and various organs were removed and weighed, and their decay corrected radioactivity counts were measured with a gamma-counter (Wallac, Inc., Perkin-Elmer, Inc., Boston, MA). For PET imaging studies, the longitudinal 15 min static PET scans were performed on athymic mice (n=5) using a Siemens Inveon micro PET scanner (Siemens Preclinical Solutions, Knoxville, TN) at 3, 24, and 48 h post-injection (p.i.). The mice with A431/H9 tumor (range, 195 429 cubic mm for MORAb-009 and 239 700 cubic mm for B3) were injected iv with Zr-89-MORAb-009 (100 microCi/10 or 60 microg) or Zr-89-B3 (80 microCi/15 or 60 microg). All imaging procedures were performed under anesthesia with 1.5% isoflurane in oxygen at 2 L/min. The mice were then euthanized immediately after the 48 h imaging sessions for autoradiography. The PET images were reconstructed with a 3-dimensional ordered-subset expectation maximization/maximum a posteriori (OSEM3D/MAP) algorithm, with no attenuation or scatter correction. The reconstructed pixel size was 0.77 0.77 0.79 mm on a 128 128 159 imaging matrix. The image analysis was performed using ASIPro software (provided by Siemens, v6.8.0.0) on decay-corrected whole-body images. To characterize the accumulation of the probe in tumors, the region-of-interest (ROI) were drawn manually on individual tumor area, liver, spleen, muscle, and heart. The %ID/g was calculated for mice at 3, 24, and 48 h p.i.. For ex vivo autoradiography, the tumors were excised, embedded and frozen in Tissue-Tek CRYO-OCT compound (Sakura Finetek USA Inc., Torrance, CA, USA) at -80C for 3 h. Serial 16 micro-m thick short axis sections were cut in 400 micro-m intervals covering the entire tumor. Two or 3 tumor sections at 16 micro-m thickness were selected in 3 different tumor regions as representative sections throughout of the tumor and exposed in the phosphor screen for 16 h. Signals were obtained by the Typhoon FLA 7000 (GE Healthcare Life Sciences, Pittsburgh, PA, USA) with 25 micro-m pixel resolution and analyzed with Image Quant TL8.1 software. Values were grouped together from the 3 tumor regions to represent a tumor. Each tumor was treated as an independent sample (n=3). To analyze the microdistribution of the radioactivity in the tumor sections, a line was first drawn along a longest axis, and at the center of the longest line a second perpendicular line was drawn along a short axis. The center was selected as the point where two lines meet. Additional lines were drawn evenly and continuously between the two original lines passing through the same center point. Radioactivity profile of each line was analyzed with ImageJ and exported into Excel files to refine values with MATLABs interpolation function interp1. Results: The results of BD studies of Zr-89 MORAb-009 showed that the tumor uptake (2.72+/-0.63 for 2 microg vs. 6.13+/-2.27 %ID/g for 10 microg, p=0.03 and 12.33+/-2.55 %ID/g for 60 microg, p=0.02) and blood retention (1.94+/-0.42 for 2 microg vs. 3.71+/-3.43 %ID/g for 10 microg, p=0.32 and 9.48+/-1.95 %ID/g for 60 microg, p<0.001) increased as the injection dose was increased, whereas the liver uptake (31.68+/-7.47 for 2 microg vs. 25.34+/-4.56 %ID/g for 10 microg, p=0.15 and 19.59+/-3.83 %ID/g for 60 microg, p=0.02) gradually decreased as the injection dose was increased. In contrast, for Zr-89 B3, there was no appreciable dose effects shown on the tumor uptake (14.35+/-4.75 for 2 microg vs. 12.51+/-1.31 %ID/g for 15 microg, p=0.50 and 12.01+/-1.78 %ID/g for 60 microg, p=0.41), blood retention (16.78+/-1.03 for 2 microg vs. 15.80+/-1.49 %ID/g for 15 microg, p=0.27 and 16.29+/-0.96 %ID/g for 60 microg, p=0.46) and liver uptake (13.92+/-0.92 for 2 microg vs. 14.51+/-3.29 %ID/g for 15 microg, p=0.72 and 13.34+/-0.80 %ID/g for 60 microg, p=0.32) among the three different injection doses of Zr-89-B3. The findings from the BD studies were supported by the PET imaging studies as follows: The PET images visualized tumors as early as 3 h p.i. for both 10 and 60 microg MORAb-009 dose. At 24 and 48 h p.i., the radioactivity signal in the tumor remained relatively unchanged compared to that at 3 h p.i. for 10 microg whereas the tumor signal increased over time for 60 microg that is advantageous for the tumor visualization by PET. Compared to Zr-89 MORAb-009, the PET images from Zr-89 B3 did not show any significant dose effects on its uptake and the clearance pharmacokinetics from tumor, blood and liver. The PET images visualized tumors as early as 3 h p.i. for both 15 and 60 microg B3 dose. In addition, unlike Zr-89 MORAb-009, the radioactivity signal steadily increased over time for both B3 doses because Lewis-Y antigen is not shed from the tumor surface. The both BD and PET studies, thus, suggest that the shed antigen in the blood circulation negatively affects the antigen-specific tumor uptake of the mAb. To answer a second question if the shed antigen in the extracellular space of tumor could improve the penetration of mAb, we performed the autoradiography of tumor segments 2 days after the injection of Zr-89 MORAb-009 and Zr-89-B3. Our preliminary results indicate that for Zr-89-B3, higher radioactivity signals were consistently shown in the tumor periphery (>2 times), near the tumor surface than tumor core and diminished rapidly as moving toward the tumor core. In contrast, for Zr-89 MORAb-009, the distribution of the radioactivity was more uniform than that of Zr-64-B3. The high signal intensity in the periphery initially decreased and then increased again near the tumor core with the signal intensity in the tumor core comparable to that in the tumor periphery. Conclusion: The results of our studies provide a message that shed-antigen in the circulation negatively affects the antigen-specific tumor uptake of the antigen-specific antibody, whereas shed-antigen in the extracellular space positively affects the tumor uptake by improving the microdistribution of the antibody, which is important for antibody-based therapy.

背景：在过去的一年中，我们扩展了我们的研究，通过重复 Zr-89（正电子发射体；衰变半衰期为 3.3 天）标记的 MORAb-009 的实验来重申上述结果，Zr-89 标记的 mAb 比 Cu-64（衰变半衰期，12.7 h）标记的 mAb 更符合 mAb 的生物半衰期，从而使 Zr-89 标记的 mAb 与 mAb 更相关。 癌症的诊断和治疗反应的评估。 作为我们对 MORAb-009（抗间皮素单克隆抗体 amatuzimab）肿瘤摄取和微分布机制研究的一部分，我们将 Zr-89-MORAb-009 研究结果与 Zr-89 标记的 B3（一种针对 Lewis-Y 抗原的单克隆抗体，不会从肿瘤表面脱落）的研究结果进行了比较。目的：开发一种放射性标记两种单克隆抗体（MORAb-009和抗Lewis-Y单克隆抗体B3）的方法，并研究注射剂量和肿瘤大小对这两种Zr-89标记单克隆抗体在表达间皮素和Lewis-Y抗原的A431/H9裸鼠中肿瘤摄取和微分布的影响。 方法：使用去铁胺和异硫氰酸酯连接体作为螯合剂，用 Zr-89 对 mAb 进行放射性标记。然后使用 PD-10 柱纯化 Zr-89 标记的 mAb，并用 pH 5.5 的 0.25 M 醋酸铵洗脱。放射化学纯度>95%、免疫反应性>70%的放射性标记单克隆抗体用于体内研究。静脉内注射 Zr-89-MORAb-009（3 microCi/2、10 或 60 microg）或 Zr-89-B3（3微Ci/2, 15 或 60 微克）。对于 BD 研究，在注射后 24 小时（p.i.）通过吸入 CO2 将动物安乐死，并在解剖前通过心脏穿刺放血。取出血液和各种器官并称重，并用伽马计数器（Wallac, Inc., Perkin-Elmer, Inc., Boston, MA）测量它们的衰变校正放射性计数。对于 PET 成像研究，使用西门子 Inveon 微型 PET 扫描仪（田纳西州诺克斯维尔的西门子临床前解决方案）在注射后 (p.i.) 3、24 和 48 小时对无胸腺小鼠 (n=5) 进行纵向 15 分钟静态 PET 扫描。患有A431/H9肿瘤的小鼠（MORAb-009的范围为195 429立方毫米，B3的范围为239 700立方毫米）静脉注射Zr-89-MORAb-009（100 microCi/10或60 microg）或Zr-89-B3（80 microCi/15或60 microg）。所有成像过程均在 1.5% 异氟烷氧气溶液麻醉下以 2 L/min 进行。在 48 小时放射自显影成像后立即对小鼠实施安乐死。 PET 图像使用 3 维有序子集期望最大化/最大后验 (OSEM3D/MAP) 算法重建，没有衰减或散射校正。在 128 128 159 成像矩阵上重建的像素大小为 0.77 0.77 0.79 mm。使用 ASIPro 软件（由西门子提供，v6.8.0.0）对经过衰减校正的全身图像进行图像分析。为了表征探针在肿瘤中的积累，在各个肿瘤区域、肝脏、脾脏、肌肉和心脏上手动绘制感兴趣区域 (ROI)。计算小鼠注射后 3、24 和 48 小时的 %ID/g。对于离体放射自显影，将肿瘤切除、包埋并在 Tissue-Tek CRYO-OCT 化合物（Sakura Finetek USA Inc.，Torrance，CA，USA）中于 -80°C 冷冻 3 小时。以 400 微米间隔连续切割 16 微米厚的短轴切片，覆盖整个肿瘤。在3个不同的肿瘤区域中选择2个或3个16微米厚的肿瘤切片作为整个肿瘤的代表性切片，并在荧光屏中曝光16小时。信号通过 Typhoon FLA 7000（GE Healthcare Life Sciences，匹兹堡，宾夕法尼亚州，美国）以 25 微米像素分辨率获得，并使用 Image Quant TL8.1 软件进行分析。将 3 个肿瘤区域的值分组在一起以代表肿瘤。每个肿瘤被视为独立样本（n=3）。为了分析肿瘤切片中放射性的微观分布，首先沿最长轴画一条线，并在最长线的中心沿短轴画第二条垂直线。选择中心作为两条线相交的点。在穿过同一中心点的两条原始线之间均匀且连续地绘制附加线。使用 ImageJ 分析每条线的放射性分布，并将其导出到 Excel 文件中，以使用 MATLAB 插值函数 interp1 细化值。结果：Zr-89 MORAb-009 的 BD 研究结果显示，肿瘤摄取（2 µg 为 2.72+/-0.63 vs. 10 µg 为 6.13+/-2.27 %ID/g，p=0.03；60 µg 为 12.33+/-2.55 %ID/g，p=0.02）和血液保留（1.94+/-0.42 2微克对比 随着注射剂量的增加，10 微克为 3.71+/-3.43 %ID/g，p=0.32，60 微克为 9.48+/-1.95 %ID/g，p<0.001），而肝脏摄取量（2 微克为 31.68+/-7.47，10 微克为 25.34+/-4.56 %ID/g， p=0.15 且 19.59+/-3.83 %ID/g（60 微克，p=0.02）随着注射剂量的增加而逐渐下降。相比之下，对于 Zr-89 B3，对肿瘤摄取没有明显的剂量效应（2 微克为 14.35+/-4.75，15 微克为 12.51+/-1.31 %ID/g，p=0.50；60 微克为 12.01+/-1.78 %ID/g，p=0.41）、血液滞留（2 微克 vs. 16.78+/-1.03 15 微克为 15.80+/-1.49 %ID/g，p=0.27；60 微克为 16.29+/-0.96 %ID/g，p=0.46）和肝脏摄取（2 微克为 13.92+/-0.92，15 微克为 14.51+/-3.29 %ID/g，p=0.72； 60 微克为 13.34+/-0.80 %ID/g， Zr-89-B3 的三种不同注射剂量之间的差异（p=0.32）。 BD 研究的结果得到了 PET 成像研究的支持，如下：PET 图像早在注射后 3 小时即可显示肿瘤。 10 微克和 60 微克 MORAb-009 剂量。注射后 24 和 48 小时，肿瘤中的放射性信号与注射后 3 小时相比保持相对不变。 10 微克的肿瘤信号随时间的推移而增加，60 微克的肿瘤信号有利于 PET 的肿瘤可视化。与 Zr-89 MORAb-009 相比，Zr-89 B3 的 PET 图像未显示对其从肿瘤、血液和肝脏的摄取和清除药代动力学有任何显着的剂量影响。 PET 图像早在注射后 3 小时即可显示肿瘤。 15 微克和 60 微克 B3 剂量。此外，与 Zr-89 MORAb-009 不同，两种 B3 剂量的放射性信号随着时间的推移稳步增加，因为 Lewis-Y 抗原不会从肿瘤表面脱落。因此，BD 和 PET 研究表明，血液循环中脱落的抗原会对 mAb 的抗原特异性肿瘤摄取产生负面影响。为了回答第二个问题，肿瘤细胞外间隙中脱落的抗原是否可以提高 mAb 的渗透性，我们在注射 Zr-89 MORAb-009 和 Zr-89-B3 后 2 天对肿瘤片段进行放射自显影。我们的初步结果表明，对于Zr-89-B3，在肿瘤外围（>2倍）、靠近肿瘤表面的区域一致显示出比肿瘤核心更高的放射性信号，并且随着向肿瘤核心移动而迅速减弱。 相反，对于Zr-89 MORAb-009，放射性分布比Zr-64-B3更均匀。周边的高信号强度最初降低，然后在肿瘤核心附近再次增加，肿瘤核心的信号强度与肿瘤周边的信号强度相当。结论：我们的研究结果提供了一个信息，即循环中的脱落抗原对抗原特异性抗体的抗原特异性肿瘤摄取产生负面影响，而细胞外空间中的脱落抗原通过改善抗体的微观分布对肿瘤摄取产生积极影响，这对于基于抗体的治疗很重要。