开发抗体偶联药物的抗上皮细胞黏附分子抗体特性

图 1. Production of anti-epithelial adhesion molecule (EpCAM) monoclonal antibodies (mAbs) intranschromosomic (TC-mAb) mice, (a) Schematics of partially fragmented EpCAM proteins, The EpCL region,illustrated in blue, contains the N-terminal domain (ND) and a part of the thyroglobulin type-1 domain (TY),whereas the EpRE region, illustrated in green, contains parts of'TY and the cysteine-poor region (CD). AllEpCAM protein fragments used in this study lack 'TM and EplC regions. Truncated fragments are shown asdotted gray lines. The numbers in the box indicate the amino acid number to be translated at the start or endof the truncated protein, (b) Quantitative comparison of the mAbs and their binding regions. The numbersof mAbs that were reactive with the EpCAM native form (blue), as well as mAbs that were ELISA-positive bunon-reactive with the EpCAM native form (sky blue), are indicated, Reactivity of the native form ofEpCAMwas determined by immunocytochemistry, (c) Summary of epitope mapping by western blotting. The epitopedistribution of the mAbs against the EpCAM native form is indicated.

用重组蛋白EpEX（氨基酸24-256）和EpRE通过酶联免疫吸附试验，单抗中有83种、22.2%的与EpCL免疫反应单抗，有293种、77.7%的与EpRE免疫反应单抗（如表1、图1b和图2）。这些测量数据与EpCL分子量（6.2 kDa，22.6%）和EpRE分子量（21.2 kDa，77.4%）有关联性，表明鼠内表达的TC-mAb抗体免疫原性与EpCL、EpRE区域结构相似。

图2. ELISA of monoclonal antibodies. Overall, 80 and 297 mAbs obtained from individuals A (upper)and B (lower) were analyzed by ELISA. The signals of EpEX and EpRE are plotted, showing two groups. The firstgroup that reacts proportionally with the EpEX and the EpRE contains mAbs that react with the EpRE domainof EpCAM. Meanwhile, the other group that reacts with the EpEX but not the EpRE contains mAbs that reactwith the EpCL, domain. Therefore, the first group can be defined as the EpRE-reactive mAbs and the secondgroup as the EpCL-reactive mAbs. Black dot in each graph indicates the value for the negative control valueobtained using the medium without mAb.

   通过杂交瘤制备结合上皮细胞黏附分子（EpCAM）的单克隆抗体，使用截短EpEX蛋白和WB完成表位作图（如图1a,c）。大分部抗原决定簇（表位）靶向的是EpCAM 62-80氨基酸。可识别额外表位的单抗也可获得，但没检测到可结合EpCAM 24-61和185-218区域的单抗（如图1c）。WB只能检测到能识别线性表位的单抗信号，结合构象型表位的单抗信号很难被检测到。

图 3. Assessment of anti-epithelial adhesion molecule (EpCAM) fully human monoconal antibodies.Immunocytochemistry (ICC) of selected mAbs against unfixed HO'T116 cells, HO'T116 cells were stained with5 ug/ml mAb. Scale bar= 100 um. Magnifcation x 40. The clone number of the analyzed mAb is indicated in theupper left of each figure, and the area of the EpCAM extracellular domain recognized by the mAb is shown inparentheses.

图4. Flow cytometry of monoclonal antibodies. Flow cytometry of mAbs against HCT116 and SW480cells. (a) Gating strategy used in this study. Data are presented in the scatter plot of forward scatter (FSC-A)and side scatter (S$C-A), as well as the histogram indicating Alexa Fluor 488-induced fluorescence intensity.(b) Flow cytometry ofthe selected m Abs. HCT1 16 and $W480 cells were stained with fully human mAbs at thein dicated concentration. Negative control (gray color) in dicates the signal with secondary antibody but withoutprimary mAb.

图 5. Internalization analysis of anti-EpCAM fully human mAbs. HCT116 cells were stained with theindicated m Abs followed by anti-human lgG-Fc antibody coniugated with Alexa Fluor 594 (The lacksonLaboratory, Bar Harbor, ME, USA) at a dilution of 1:600 and analyzed using IncuCyte S3 (Sartorius, Gottingen,Germany). Fluorescent images taken immediately after the start of incubation at 37 °C for 0 h (left side) andafter 24 h of incubation (right side) are shown. Scale bar=50 um. Magnification x40.

图6. Cytotoxicity of antibody-drug conjugate. (a) Schematic design of mAb-IgG-BP-DM1. The selectedmonoclonal antibody (m Ab) was modified with azide-attached chemical conjugation by afinity peptide reagentand labeled with mertansine (DM1) via mixing with DBCO-PEG4-DM1. (b) Cytotoxicity of anti-EpCAMmAbs directly labeled with DMl using the CCAP method. The growth of HC'T116 cells was monitored as thepercentage confluence. The percentages at 60 h are indicated (n =3). Data are presented as the mean ± SE.

作者为了研究抗体偶联药物细胞毒性，进一步分析了使用亲和肽CCAP标记法抗癌药物DM1直接标记的单抗（如图6a,b），亲和肽CCAP标记法可完成IgGs的具体位点修饰。因为试剂IgG-BP-CCAP能修饰人IgG1、IgG2和IgG4，不能修饰IgG3,作者发现在细胞表面与EpCAM有不同结合亲和性的四种克隆，分别是1C008（IgG4）,3C066（IgG1）和3C060（IgG1）（如图4）。IgG的修饰用叠氮化合物复合的IgG-BP-CCAP通过点化学与DBCO-PEG4-DM1混合标记上DMI药物。DM1标记IgG的流程图如图6所示。根据SDS-PAGE每个单抗中约50%至70%蛋白被一两个DM1分子标记。几乎所有的同型对照IgG1被两分子DM1标记。结果表明在蛋白偶联物浓度0.25-1 μg/mL时所有直接标记的抗EpCAM而非同型对照IgG1表现出细胞毒性（如图6b）。3C101-DM1、1C008-DM1、3C066-DM1和3C060-DM1偶联物对应的半抑制浓度分别为0.553、0.208、0.402和0.254 μg/mL，推测在浓度1 μg/mL时HCT116细胞失活。1C008-DM1偶联物表现出极大细胞毒性，3C060-DM1和3C066-DM1也表现出明显细胞毒性。CCAP-DM1-3C101单抗偶联物细胞毒性（ICC染色法和流式细胞术显示出其较强显色强度，如图3,4）低于CCAP-DM1-其余单抗偶联物细胞毒性。mAb-CCAP-DM1偶联物细胞毒性与结合亲和性没有重要关联。

    该研究发现CCAP直接标记法不仅能检测到高亲和性单抗例如1C008也能检测到很难被被FCM和ICC检测到的低亲和性单抗例如3C060。重要的是3C066单抗偶联活性药物可通过EpRE反应的抗体获得。