Chemical Modifications Of Antibodies For Molecular Targeting

分子靶向抗体的化学修饰

• 批准号: 8565290
• 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/8565290
• 关键词: 90Y; Aftercare; Antibodies; Antigens; Appearance; Architecture; Area; Avastin; Binding; Binding Sites; Blood Vessels; Breathing; Carbon Dioxide; Cardiac; Cell Death; Cell Division Process; Cell Nucleus; Chemicals; Clinic; Combined Modality Therapy; Computer software; Custom; Daclizumab; Dissection; Dose; Employee Strikes; Enhancing Antibodies; FDA approved; Fluorescence; Fluorescence Microscopy; Focused Ultrasound Therapy; Formalin; Freezing; Gold; Growth Factor; Harvest; Hematologic Neoplasms; I131 isotope; Image; Image Analysis; Immune response; Immunoglobulin Fragments; Indium-111; Individual; Injection of therapeutic agent; Investigation; Kinetics; Label; Lateral; Lectin; Liquid substance; Maps; Masks; Measures; Methods; Microscopic; Microtubule Polymerization; Mitosis; Mitotic; Modification; Molecular Target; Monoclonal Antibodies; Monoclonal Antibody CD20; Mus; Necrosis; Neoplasms in Vascular Tissue; Nitrogen; Normal saline; Nude Mice; Optics; Paclitaxel; Patients; Penetration; Physiologic pulse; Physiological; Plant alkaloid; Play; Puncture procedure; Radioimmunoconjugate; Radioimmunotherapy; Radioisotopes; Radiolabeled; Reagent; Regimen; Research; Rhodamine; Right-On; Role; Sampling; Scanning; Signal Transduction; Site; Skin; Solid Neoplasm; Staging; Staining method; Stains; Surface; Tail; Testing; Therapeutic; Therapeutic Effect; Thick; Time; Tumor Tissue; United States National Institutes of Health; Vascular Endothelial Growth Factors; Veins; Y 90 Ibritumomab Tiuxetan; arm; base; bevacizumab; cancer cell; cancer therapy; cell growth; cryostat; cyanine dye 5; density; design; fluorescence microscope; fluorophore; imaging probe; improved; interstitial; iodine-131-tositumomab; neoplastic cell; prevent; promoter; radiotracer; response; success; tumor; tumor growth

Objectives: To test the effect of Paclitaxel and Bevacizumab on the tumor blood vessels as well as their effects on the microdistribution of Alexa Fluor 647-B3 in tumors by fluorescence microscopy. We previously found that a combination therapy of Y-90-B3 with Paclitaxel produced a synergistic effect in the shrinkage of tumor size whereas the addition of Avastin treatment to the combined therapy of 60 microCi B3 and Taxol provided some additive effect in survival. While the control mice had a median survival time of 5 days, the Taxol and Avastin treatment delayed tumor growth with a median survival time of 17 days and 11 days, respectively. The Y-90 B3 treatment showed a dose-dependent response with a median survival time of 18 days for the 60 microCi B3 group and 29 days for the 100 microCi B3 group. The addition of Avastin before 60 microCi B3 treatment produced an additive effect in survival with a median survival time of 20 days. The combined therapy involving 60 microCi B3 and 100 microCi B3 with Taxol showed a striking synergistic effect in shrinking tumor and prolonging the survival time. On day 120, 3 of 9 mice (33%) treated with a combined therapy involving 60 microCi B3 and Taxol, and 6 of 6 mice treated with a combined therapy of 100 microCi B3 with Taxol were alive with no tumor. The addition of Avastin treatment to the combined therapy of 60 microCi B3 and Taxol provided some additive effect in survival; 3 of 6 (50%) treated with Avastin were alive with no tumor. Based on these previous studies, we investigated the effect of Paclitaxel and Bevacizumab on the tumor blood vessels as well as their effects on the microdistribution of Alexa Fluor 647-B3 in tumors by fluorescence microscopy. Methods: Groups of nude mice (n = 45 mice/group) were inoculated s.c. with A431 tumor cells expressing the Le-Y antigen on the right hind flank. When the tumor size reached 200 cubic mm, the tumor-bearing mice were divided into three groups and were injected with 1) i.v. Alexa Fluor 647-conjugated B3 (150 micro-g in 0.2 ml of PBS) alone on day 0, 2) i.v. Alexa Fluor 647-B3 on day 0 followed by i.p. Paclitaxel (40 mg/kg in 0.2 ml of normal saline) on day 1, and 3) i.v. Bevacizumab (5 mg/kg in 0.2 ml of PBS) on day 0 followed by i.v. Alexa Fluor 647-B3 on day 1 to investigate the effect of Paclitaxel and Bevacizumab on the tumor microdisribution of Alexa Fluor 647-B3. Two days after the injection of Alexa Fluor 647-B3, the mice received a lateral tail vein injection of rhodamine-lectin (RCA, 1 mg in 0.2 ml of PBS) to delineate the blood vessels and 5 min after the lectin injection, the mice were euthanized by CO2 inhalation and exsanguinated by cardiac puncture before dissection. Tumors were harvested with intact skin and flash-frozen using liquid nitrogen for subsequent sectioning and staining. Tumors were sectioned using a Leica CM1850 cryostat at 8 micro-m thickness in 3 different regions to obtain representative sections throughout the tumor. Tumor sections were fixed with formalin for 20 min and mounted with Prolong Gold antifade reagent with DAPI (Invitrogen, Carlsbad, CA). Imaging was performed with a 10X objective (pixel size = 0.64 micro-m, binning 2x2) using an epi-fluorescent microscope (Zeiss, Axio Imager.M1, Thornwood, NY) equipped with a motorized scanning stage and mosaic stitching software (Axiovision, Zeiss). Three independent channels were obtained: DAPI for nuclei (shown in blue), Rhodamine for blood vessels (shown in red), and Cy5 for Alexa Fluor 647-B3 antibody (constant exposure time of 40 ms, shown in green). A tumor that did not contain B3 antibody was imaged with identical parameters to obtain background signal intensity. Image analysis was performed with a custom-designed MATLAB script (MathWorks, Natick, MA). Individual image channels were exported from Axiovision as 16-bit grayscale tiff images to Photoshop where a tumor region was isolated to create a tumor mask. The tumor mask, blood vessel image and B3 antibody image were loaded into MATLAB and a tumor blood vessel mask and distance map were created. Overall B3 antibody intensity and penetration from the tumor edge and blood vessel surface were calculated with a background intensity subtraction. In addition, vascular parameters and architecture such as micro-vasculature density (MVD), blood vessel size, and median distance from a tumor pixel to the nearest vascular surface were measured. Values were grouped together from the 3 tumor regions to represent a tumor. Each tumor was treated as an independent sample (n= 4-5, one tumor was removed due to excessive necrosis limiting analysis). Results: The fluorescence microscopic determination revealed that Paclitaxel (40 mg/kg) treatment did not significantly change the MVD (67.4 vs 70.3 vessels/mm-squared for the control, P > 0.05), median blood vessel area (92.7 vs 100.1 micro-m-squared for the control), and the median distance to the nearest vascular surface, an indicator of vascular architecture (78.8 vs 69.3 micro-m for the control, P > 0.05) compared to the control one day after the treatment. Comparatively, the treatment with Bevacizumab decreased the MVD (53.4 vessels/mm- squared) by 24% and increased the median distance (95.1 micro-m) from a tumor pixel to the nearest vascular surface by 37% compared to the control, although it is not statistically significant (P > 0.05). Bevacizumab did not change the median blood vessel size (92.22 micro-m-squared). Antibody accumulation was determined by analyzing fluorescence intensity in tumor sections. The analysis qualitatively suggests that B3 antibody accumulation and penetration were improved with Paclitaxel treatment and reduced with Bevacizumab treatment. The image analysis demonstrated that when 150 micro-g of fluorescence-labeled B3 was administered, the highest concentration of fluorescence labeled B3 (2 fold higher than that in the tumor surface) accumulated at 50 micro-m distance from the tumor surface. The B3 concentration decreased rapidly as it moved toward the tumor center: The B3 concentration at 1 mm from the tumor surface became 4 fold lower than the maximum concentration at 50 micro-m from the tumor surface. The fluorescence microscopy study also showed that the B3 concentration diminished rapidly as moving away from blood vessels, and a steady state concentration of mAb was shown at a distance between 50 to 100 micro-m from blood vessels. This steady state mAb concentration was approximately 3.4 fold less than the concentration of mAb in tumor blood vessel surface. The Paclitaxel treatment significantly improved B3 antibody delivery to the tumor by 45% (2570 vs 1775 arbitrary fluorescence units for the control, P < 0.05). The B3 penetration was significantly improved following treatment with Paclitaxel both from the tumor surface and from the blood vessel surface (P < 0.05). The Paclitaxel treatment increased the steady state concentration of B3 by 60% (P < 0.05) compared to that of the control. The Paclitaxel treatment also appeared to impact the integrity of the tumor tissue with a muddled cell nuclei appearance. Comparatively, Bevacizumab had a profound effect on B3 antibody delivery, significantly reducing the accumulation by 96% (73 vs 1775 for the control, P < 0.05). In addition, Bevacizumab significantly reduced the penetration of B3 antibody (P < 0.05). Conclusion: The positive effect of Paclitaxel on the accumulation and penetration of B3 warrants further studies on the effect of the dose of B3 and Paclitaxel on the accumulation and penetration of B3 in larger tumors. The influence of tumor microdistribution on combined tumor radioimmunotherapy regimens represents a promising area for further investigation and optimization to improve radioimmuno-therapy for solid tumors in the clinic.

目的：通过荧光显微镜检测紫杉醇和贝伐单抗对肿瘤血管的影响，以及对肿瘤中Alexa Fluor 647-B3微分布的影响。我们之前发现Y-90-B3与紫杉醇联合治疗在肿瘤大小缩小方面产生协同效应，而在60 microCi B3和紫杉醇联合治疗的基础上加入阿瓦斯汀治疗在生存方面提供了一些附加效应。而对照组小鼠的中位生存时间为5天，紫杉醇和阿瓦斯汀治疗延迟肿瘤生长的中位生存时间分别为17天和11天。Y-90 B3治疗显示出剂量依赖性反应，60微ci B3组的中位生存时间为18天，100微ci B3组的中位生存时间为29天。在60微ci B3治疗前添加阿瓦斯汀对生存产生了附加效应，中位生存时间为20天。60微ci B3和100微ci B3与紫杉醇联合治疗在缩小肿瘤和延长生存期方面具有显著的协同作用。第120天，接受60微ci B3和紫杉醇联合治疗的9只小鼠中有3只（33%）存活，接受100微ci B3和紫杉醇联合治疗的6只小鼠中有6只存活，无肿瘤。在60微ci B3与紫杉醇联合治疗的基础上，加用阿瓦斯汀治疗对生存率有一定的附加效应；接受阿瓦斯汀治疗的6例患者中有3例（50%）存活，无肿瘤。在这些前期研究的基础上，我们通过荧光显微镜研究了紫杉醇和贝伐单抗对肿瘤血管的影响，以及它们对肿瘤中Alexa Fluor 647-B3微分布的影响。方法：将右后腹表达Le-Y抗原的A431肿瘤细胞接种于s.c.裸鼠组（45只/组）。当肿瘤大小达到200立方毫米时，将荷瘤小鼠分为三组，第0天单独注射Alexa Fluor 647偶联B3 （0.2 ml PBS中150微克），第0天单独注射Alexa Fluor 647-B3，第1天静脉注射紫杉醇（0.2 ml生理盐水中40 mg/kg）；3)第0天静脉滴注贝伐单抗（5 mg/kg, 0.2 ml PBS），第1天静脉滴注Alexa Fluor 647-B3，研究紫杉醇和贝伐单抗对Alexa Fluor 647-B3肿瘤微分布的影响。注射Alexa Fluor 647-B3 2 d后，小鼠尾侧静脉注射罗丹明-凝集素（RCA, 1 mg， 0.2 ml PBS中）描绘血管，凝集素注射5 min后，CO2吸入安乐死，心脏穿刺放血后解剖。肿瘤取材于完整的皮肤，用液氮快速冷冻，用于随后的切片和染色。使用徕卡CM1850低温恒温器在3个不同区域以8微米厚度对肿瘤进行切片，以获得整个肿瘤的代表性切片。肿瘤切片用福尔马林固定20分钟，然后用含有DAPI （Invitrogen, Carlsbad， CA）的延长金抗褪色试剂固定。使用放大镜（蔡司，Axio Imager），以10倍物镜（像素大小= 0.64微米，像素大小为2 × 2）成像。M1, Thornwood， NY)配备了一个电动扫描台和马赛克拼接软件（Axiovision, Zeiss）。获得三个独立通道：DAPI用于细胞核（蓝色表示），罗丹明用于血管（红色表示），Cy5用于Alexa Fluor 647-B3抗体（恒定暴露时间为40 ms，绿色表示）。不含B3抗体的肿瘤用相同的参数成像以获得背景信号强度。使用定制的MATLAB脚本（MathWorks, Natick， MA）进行图像分析。单个图像通道从Axiovision导出为16位灰度tiff图像到Photoshop中，在Photoshop中分离肿瘤区域以创建肿瘤蒙版。将肿瘤掩膜、血管图像和B3抗体图像加载到MATLAB中，创建肿瘤血管掩膜和距离图。通过背景强度减法计算总体B3抗体强度和肿瘤边缘和血管表面的穿透度。此外，还测量了血管参数和结构，如微血管密度（MVD）、血管大小和从肿瘤像素到最近血管表面的中位数距离。将3个肿瘤区域的值组合在一起代表一个肿瘤。每个肿瘤作为一个独立的样本处理（n= 4-5, 1个肿瘤因过度坏死而被切除）。结果：荧光显微镜检测显示紫杉醇（40 mg/kg）处理后1天的MVD（对照组67.4 vs 70.3血管/mm平方，P > 0.05）、血管面积中位数（对照组92.7 vs 100.1微-m平方）和血管结构指标最近血管表面的中位数距离（对照组78.8 vs 69.3微-m， P > 0.05）与对照组相比没有显著变化。相比之下，与对照组相比，贝伐单抗治疗使MVD（53.4个血管/mm-平方）降低了24%，使肿瘤像素到最近血管表面的中位距离（95.1微米）增加了37%，尽管没有统计学意义（P < 0.05）。贝伐单抗没有改变中位血管大小（92.22 μ m²）。通过分析肿瘤切片的荧光强度来测定抗体积累。定性分析表明，紫杉醇治疗改善了B3抗体的积累和渗透，贝伐单抗治疗降低了B3抗体的积累和渗透。图像分析表明，当150微g荧光标记的B3被给予时，在距离肿瘤表面50微m处，荧光标记的B3浓度最高（比肿瘤表面高2倍）。B3浓度随其向肿瘤中心移动而迅速下降，距离肿瘤表面1mm处的B3浓度比距离肿瘤表面50微米处的最大浓度低4倍。荧光显微镜研究还表明，B3浓度随着远离血管而迅速降低，并且在距离血管50 ~ 100微米的距离上显示出稳定的单抗浓度。该稳态mAb浓度约为肿瘤血管表面mAb浓度的3.4倍。紫杉醇组B3抗体向肿瘤的递送量显著提高45%（对照组为2570个任意荧光单位，P < 0.05）。紫杉醇治疗后肿瘤表面和血管表面的B3穿透性均有显著提高（P < 0.05）。紫杉醇组B3稳态浓度较对照组提高60% （P < 0.05）。紫杉醇治疗似乎也会影响肿瘤组织的完整性，使细胞核出现混乱。相比之下，贝伐单抗对B3抗体递送有深远的影响，显著减少了96%的积累（73 vs 1775，对照组，P < 0.05）。此外，贝伐单抗显著降低了B3抗体的渗透（P < 0.05）。结论：紫杉醇对B3的积累和渗透有积极作用，值得进一步研究B3和紫杉醇剂量对较大肿瘤中B3的积累和渗透的影响。肿瘤微分布对肿瘤放射免疫联合治疗方案的影响是一个有希望进一步研究和优化的领域，以提高临床对实体瘤的放射免疫治疗。