Chemical Modifications Of Antibodies For Molecular Targeting

分子靶向抗体的化学修饰

• 批准号: 9555566
• 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/9555566
• 关键词: 90Y; Address; Affect; Affinity; Amino Acid Sequence; Antibodies; Antigen-Antibody Complex; Antigens; Autoradiography; Binding; Binding Sites; Biodistribution; Biological Sciences; Blood; Blood Circulation; Blood Vessels; Bypass; Carbohydrates; Cell Survival; Cell surface; Chelating Agents; Chemicals; Computer software; Convection; Deferoxamine; Diffuse; Dose; Drug Kinetics; Extracellular Matrix; Extracellular Matrix Proteins; Extracellular Protein; Extracellular Space; Filtration; Fluorescence Microscopy; Focused Ultrasound Therapy; Freezing; Healthcare; Hematologic Neoplasms; Human; IgG1; Image; Immunoglobulin Fragments; Implant; Indium-111; Individual; Injectable; Injection of therapeutic agent; Intercellular Fluid; Isothiocyanates; Kinetics; Label; Lead; Length; Liquid substance; Liver; Measures; Membrane; Membrane Glycoproteins; Methods; Modeling; Modification; Molecular Target; Monoclonal Antibodies; Motion; Mus; Normal tissue morphology; Nude Mice; Optics; Organ; Paclitaxel; Penetration; Physiologic pulse; Physiological; Play; Positron-Emission Tomography; Radioactivity; Radioimmunoconjugate; Radioimmunotherapy; Radioisotopes; Radiolabeled; Research; Resolution; Reticuloendothelial System; Role; Sampling; Signal Transduction; Site; Slice; Solid Neoplasm; Spleen; Stromal Cells; Surface; Surface Antigens; Thick; Time; Tissues; Treatment Protocols; Tumor Antigens; Tumor Volume; United States National Institutes of Health; ammonium acetate; base; bevacizumab; cancer therapy; chimeric antibody; comparative; density; fluorophore; imaging probe; imaging study; immunoreactivity; improved; in vivo; interstitial; macromolecule; mesothelin; mouse model; neoplastic cell; pressure; prevent; promoter; radiation resistance; radiochemical; reconstruction; response; success; targeted treatment; treatment response; tumor; tumor microenvironment; uptake

Background: In the past year, we have focused on studying the effect of shed-antigen on the microdistribution of Zr-89-labeled amatuximab in tumor, apart from other factors such as tumor vascular density, interstitial fluid pressure (IFP) and extracellular matrix proteins that could affect the microdistibution of mAb. For this study, we used a nude mouse model implanted with A431/H9 tumor which over-expresses both mesothelin and Lewis-Y on the tumor cell surface. Mesothelin is a membrane glycoprotein of 40 kDa. It is actively shed from the tumor cell surface. Lewis-Y is a carbohydrate antigen and is not actively shed from the tumor surface. As model antibodies, we used two mAbs, anti-mesothelin mAb amatuximab (mouse/human chimeric antibody with 82.6% amino acid sequence identity to a human IgG1 and nM KD binding affinity) and anti-Lewis-Y mAb B3 (murine IgG1 with 10 nM KD binding affinity). Comparative autoradiography studies using the A431/H9 tumor model targeted with Zr-89 labeled-amatuximab and -B3 enabled us to define the effects of shed-Ag on the tumor microdistribution of the Zr-89-mAb, apart from the effects of other factors such as vascular density, high interstitial fluid pressure (IFP) and extracellular protein contents. Objectives: To investigate the effect of the injection dose and the tumor size on the tumor microdistribution of these two mAbs radiolabeled with Zr-89 in nude mice bearing A431/H9. 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. For autoradiography studies, the mice with A431/H9 tumor were injected iv with Zr-89-amatuximab (100 micro-Ci/10 or 60 micro-g) or Zr-89-B3 (80 micro-Ci/15 or 60 micro-g). The mice (n=3) were euthanized at 48 h post-injection and the tumors were excised. The tumors were embedded and frozen in Tissue-Tek CRYO-OCT compound (Sakura Finetek USA Inc., Torrance, CA, USA) at -20 degree C for 3 h. Serial 20 micro-m thick short axis sections were cut in 400 micro-m intervals covering the entire tumor. Two or three consecutive tumor slices were selected at 3 tumor regions (25%, 50%, and 75% long axis regions from the tumor surface) as representative sections throughout the tumor and exposed on the phosphor screen for 16 h. Signals were obtained by the use of 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. To analyze the microdistribution of the radioactivity in the tumor sections, we introduced a normalized length analysis method as described below. The first line was drawn along a longest axis, and the second line was drawn along a short axis perpendicularly at the center of the first longest line. The center was selected as the point where the two lines meet. Additional lines were drawn evenly and continuously between the two original lines passing through the same center point (total 8 lines). Radioactivity profile of each line was analyzed with ImageJ (NIH, Bethesda, MD) and exported into Excel files to redefine values with MATLABs interpolation function interp1. The maximum length of each line in x-axis was normalized to 1 to correct for the differences in the length of each line for reconstruction of the radioactivity-vs-tumor penetration distance profiles of each tumor section. The maximum signal intensity within each tumor section in y-axis was also normalized to 100 to correct for the differences in the signal intensity between each tumor section. Mean radioactivity-vs-distance profiles with standard deviation were then reconstructed for tumor sections obtained at 25%, 50%, and 75% regions. Results: The radioactivity-vs-distance profiles of Zr-89 B3 showed the peak radioactivity distributed in the tumor periphery which was then rapidly decreased as moved away from the periphery toward the tumor core for both 15 and 60 micro-g doses. These radioactivity-vs-distance profiles were also similar to that of Alexa-labeled B3 analyzed by fluorescence microscopy after the injection of 150 micro-g Alexa-B3. These findings are consistent with a notion that the IFP is often elevated in solid tumors but declines in the tumor periphery in the outer 0.21.1 mm. This transvascular pressure gradient may cause a mAb against non-shed tumor antigen to extravasate and accumulate preferentially in the tumor periphery rather than in the tumor core. In contrast, the profiles of Zr-89 amatuximab depended on the injected dose levels; the radioactivity distribution was more uniform with the radioactivity distributed in the tumor core similar to that in the tumor periphery for 10 micro-g injection whereas the profile became similar to those of Zr-89 B3 for 60 micro-g injection, showing that the peak radioactivity at the tumor periphery rapidly decreased as moved away from the tumor periphery toward the tumor core. These findings support a notion that for 10 micro-g dose, Zr-89 amatuximab in the blood circulation mostly bound to the shed-mesothelin in blood and a small amount of free Zr-89 amatuximab which entered into tumor extracellular space (ECS) after crossing the tumor vasculature would mostly exist as an antibody/antigen complex and thus, would be distributed more uniformly throughout the entire tumor by bypassing the binding sites on the surface of tumor cells nearest to the vasculature. In comparison, Zr-89 amatuximab injected at 60 g/dose has an estimated concentration of 260 nM in a blood immediately after injection and 28.3 nM at 48 h (based on 6.8 %ID/g of blood) and therefore in a much larger molar excess concentration compared to the steady state concentration of shed-mesothelin in the blood ( 6 nM). Consequently, it would remain mostly as an unbound free mAb form, thereby escaping from the sequestration into the reticuloendothelial system in liver during a 48 h period. Therefore, a large portion of the injected dose (95.2 nM for the tumor uptake of 22.90 % ID/g tumor at 48 h and 952 nM in the ECS because the ECS is 10% of the total tumor volume) would have crossed the tumor vasculature and diffused into the ECS of tumor. This concentration (952 nM) is similar to the steady state concentration of the shed-Ag in the ECS (800 nM in 400 cubic mm tumor). This finding suggests that one of two Ag binding sites of amatuximab entered into the ECS would have been free to bind the membrane-bound mesothelin on tumor cells in periphery (i.e., in the lower IFP) rather than in the tumor core, similar to that shown for Zr-89 B3. Conclusion: A mAb injection dose, that could generate the mAb concentration at a several fold molar excess to the shed-Ag concentration in blood but generate the mAb concentration at a several fold lower than the shed-Ag concentration in the tumor ECS, would provide a beneficial effect in maximizing tumor uptake with a minimum liver and spleen uptakes while allowing deeper tumor penetration. This study, thus, provides an important message that it is important to find an optimum treatment regimen to enhance the delivery and penetration of mAb into tumor with shed-Ag.

背景：在过去的一年里，除了肿瘤血管密度、间质液压力（IFP）和细胞外基质蛋白等可能影响mAb微分布的其他因素外，我们在过去的一年里重点研究了脱落抗原对Zr-89标记的amatuximab在肿瘤中微分布的影响。 在本研究中，我们使用植入 A431/H9 肿瘤的裸鼠模型，该肿瘤在肿瘤细胞表面过度表达间皮素和 Lewis-Y。间皮素是一种 40 kDa 的膜糖蛋白。它从肿瘤细胞表面主动脱落。 Lewis-Y 是一种碳水化合物抗原，不会主动从肿瘤表面脱落。作为模型抗体，我们使用了两种 mAb：抗间皮素 mAb amatuximab（与人 IgG1 和 nM KD 结合亲和力具有 82.6% 氨基酸序列同一性的小鼠/人嵌合抗体）和抗 Lewis-Y mAb B3（具有 10 nM KD 结合亲和力的鼠 IgG1）。使用以 Zr-89 标记的 amatuximab 和 -B3 为靶标的 A431/H9 肿瘤模型进行的放射自显影比较研究使我们能够定义脱落 Ag 对 Zr-89-mAb 肿瘤微分布的影响，除了血管密度、高间质液压力 (IFP) 和细胞外蛋白含量等其他因素的影响。目的：探讨Zr-89放射性标记的两种单克隆抗体在A431/H9裸鼠体内注射剂量和肿瘤大小对肿瘤微分布的影响。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。对于放射自显影研究，对患有 A431/H9 肿瘤的小鼠静脉注射 Zr-89-amatuximab（100 micro-Ci/10 或 60 micro-g）或 Zr-89-B3（80 micro-Ci/15 或 60 micro-g）。注射后48小时对小鼠(n=3)实施安乐死并切除肿瘤。将肿瘤包埋在 Tissue-Tek CRYO-OCT 化合物（Sakura Finetek USA Inc.，Torrance，CA，USA）中并在 -20 摄氏度下冷冻 3 小时。以 400 微米间隔切割连续 20 微米厚的短轴切片，覆盖整个肿瘤。在3个肿瘤区域（距离肿瘤表面25%、50%和75%长轴区域）选择两个或三个连续的肿瘤切片作为整个肿瘤的代表性切片，并在荧光屏上曝光16小时。使用具有 25 微米像素分辨率的 Typhoon FLA 7000（GE Healthcare Life Sciences，匹兹堡，宾夕法尼亚州，美国）获得信号，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。每个肿瘤都被视为独立样本。为了分析肿瘤切片中放射性的微观分布，我们引入了如下所述的归一化长度分析方法。第一条线沿最长轴绘制，第二条线沿垂直于第一条最长线中心的短轴绘制。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连续地绘制附加线（共 8 条线）。使用 ImageJ（NIH、Bethesda、MD）分析每条线的放射性分布，并导出到 Excel 文件中，以使用 MATLAB 插值函数 interp1 重新定义值。 x 轴中每条线的最大长度标准化为 1，以校正每条线长度的差异，以重建每个肿瘤切片的放射性与肿瘤穿透距离分布。 每个肿瘤切片内 y 轴的最大信号强度也标准化为 100，以校正每个肿瘤切片之间信号强度的差异。然后针对在 25%、50% 和 75% 区域获得的肿瘤切片重建具有标准差的平均放射性与距离分布。结果：Zr-89 B3 的放射性与距离曲线显示，在 15 微克和 60 微克剂量下，峰值放射性分布在肿瘤外围，然后随着从外围向肿瘤核心移动而迅速下降。这些放射性与距离的关系曲线也与注射 150 微克 Alexa-B3 后通过荧光显微镜分析的 Alexa 标记的 B3 的关系曲线相似。这些发现与 IFP 在实体瘤中通常升高但在肿瘤外围 0.21.1 mm 处下降的观点一致。这种经血管压力梯度可能导致针对非脱落肿瘤抗原的单克隆抗体外渗并优先积聚在肿瘤外围而不是肿瘤核心。相比之下，Zr-89 amatuximab 的分布取决于注射剂量水平；放射性分布更加均匀，注射10微克时，肿瘤核心的放射性分布与肿瘤周边相似，而注射60微克时，分布与Zr-89 B3相似，表明肿瘤周边的峰值放射性随着远离肿瘤周边向肿瘤核心移动而迅速下降。这些发现支持这样一种观点，即对于10微克剂量，血液循环中的Zr-89阿马妥昔单抗主要与血液中脱落的间皮素结合，穿过肿瘤脉管系统后进入肿瘤细胞外间隙（ECS）的少量游离Zr-89阿马妥昔单抗大部分以抗体/抗原复合物形式存在，因此，将更均匀地分布在整个肿瘤中。 绕过最接近脉管系统的肿瘤细胞表面上的结合位点。相比之下，以 60 g/剂注射的 Zr-89 amatuximab 在注射后立即在血液中的估计浓度为 260 nM，在 48 小时时为 28.3 nM（基于 6.8%ID/g 血液），因此与血液中脱落间皮素的稳态浓度 (6 nM) 相比，摩尔过量浓度要大得多。因此，它大部分仍以未结合的游离单克隆抗体形式存在，从而在 48 小时内从隔离状态逃逸到肝脏的网状内皮系统中。 因此，大部分注射剂量（对于48小时时肿瘤摄取22.90％ID/g肿瘤而言为95.2nM，在ECS中为952nM，因为ECS占总肿瘤体积的10％）将穿过肿瘤脉管系统并扩散到肿瘤的ECS中。该浓度 (952 nM) 与 ECS 中脱落的 Ag 的稳态浓度（400 立方毫米肿瘤中为 800 nM）相似。这一发现表明，进入 ECS 的阿马妥昔单抗的两个 Ag 结合位点之一可以自由地结合外周（即下部 IFP）而不是肿瘤核心的肿瘤细胞上的膜结合间皮素，类似于 Zr-89 B3 所示的情况。结论：单克隆抗体注射剂量可以产生比血液中脱落的抗原浓度高几倍摩尔的单克隆抗体浓度，但产生的单克隆抗体浓度比肿瘤ECS中脱落的抗原浓度低几倍，这将提供有益的效果，以最小的肝脏和脾脏摄取最大化肿瘤摄取，同时允许更深的肿瘤渗透。因此，这项研究提供了一个重要的信息，即找到最佳治疗方案以增强mAb通过脱落的Ag递送和渗透到肿瘤中非常重要。

项目成果

(99m)Tc-labeled porphyrin-lipid nanovesicles.

(99m)Tc 标记的卟啉脂质纳米囊泡。

• DOI: 10.3109/08982104.2014.932379
• 发表时间: 2015
• 期刊: Journal of liposome research
• 影响因子: 4.4
• 作者: Lee,Jae-Ho;Shao,Shuai;Cheng,KennethT;Lovell,JonathanF;Paik,ChangH
• 通讯作者: Paik,ChangH

(99m)Tc-labeled therapeutic inhaled amikacin loaded liposomes.

• DOI: 10.3109/08982104.2013.819889
• 发表时间: 2013-12
• 期刊: Journal of liposome research
• 影响因子: 4.4
• 作者: Lee JH;Cheng KT;Malinin V;Li Z;Yao Z;Lee SJ;Gould CM;Olivier KN;Chen C;Perkins WR;Paik CH
• 通讯作者: Paik CH