圖 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.

用重組蛋白EpEX（氨基酸24-256）和EpRE通過(guò)酶聯(lián)免疫吸附試驗(yàn)，單抗中有83種、22.2%的與EpCL免疫反應(yīng)單抗，有293種、77.7%的與EpRE免疫反應(yīng)單抗（如表1、圖1b和圖2）。這些測(cè)量數(shù)據(jù)與EpCL分子量（6.2 kDa，22.6%）和EpRE分子量（21.2 kDa，77.4%）有關(guān)聯(lián)性，表明鼠內(nèi)表達(dá)的TC-mAb抗體免疫原性與EpCL、EpRE區(qū)域結(jié)構(gòu)相似。

圖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.

   通過(guò)雜交瘤制備結(jié)合上皮細(xì)胞黏附分子（EpCAM）的單克隆抗體，使用截短EpEX蛋白和WB完成表位作圖（如圖1a,c）。大分部抗原決定簇（表位）靶向的是EpCAM 62-80氨基酸。可識(shí)別額外表位的單抗也可獲得，但沒(méi)檢測(cè)到可結(jié)合EpCAM 24-61和185-218區(qū)域的單抗（如圖1c）。WB只能檢測(cè)到能識(shí)別線性表位的單抗信號(hào)，結(jié)合構(gòu)象型表位的單抗信號(hào)很難被檢測(cè)到。

圖 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.

圖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.

圖 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.

圖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.

作者為了研究抗體偶聯(lián)藥物細(xì)胞毒性，進(jìn)一步分析了使用親和肽CCAP標(biāo)記法抗癌藥物DM1直接標(biāo)記的單抗（如圖6a,b），親和肽CCAP標(biāo)記法可完成IgGs的具體位點(diǎn)修飾。因?yàn)樵噭㊣gG-BP-CCAP能修飾人IgG1、IgG2和IgG4，不能修飾IgG3,作者發(fā)現(xiàn)在細(xì)胞表面與EpCAM有不同結(jié)合親和性的四種克隆，分別是1C008（IgG4）,3C066（IgG1）和3C060（IgG1）（如圖4）。IgG的修飾用疊氮化合物復(fù)合的IgG-BP-CCAP通過(guò)點(diǎn)化學(xué)與DBCO-PEG4-DM1混合標(biāo)記上DMI藥物。DM1標(biāo)記IgG的流程圖如圖6所示。根據(jù)SDS-PAGE每個(gè)單抗中約50%至70%蛋白被一兩個(gè)DM1分子標(biāo)記。幾乎所有的同型對(duì)照IgG1被兩分子DM1標(biāo)記。結(jié)果表明在蛋白偶聯(lián)物濃度0.25-1 μg/mL時(shí)所有直接標(biāo)記的抗EpCAM而非同型對(duì)照IgG1表現(xiàn)出細(xì)胞毒性（如圖6b）。3C101-DM1、1C008-DM1、3C066-DM1和3C060-DM1偶聯(lián)物對(duì)應(yīng)的半抑制濃度分別為0.553、0.208、0.402和0.254 μg/mL，推測(cè)在濃度1 μg/mL時(shí)HCT116細(xì)胞失活。1C008-DM1偶聯(lián)物表現(xiàn)出極大細(xì)胞毒性，3C060-DM1和3C066-DM1也表現(xiàn)出明顯細(xì)胞毒性。CCAP-DM1-3C101單抗偶聯(lián)物細(xì)胞毒性（ICC染色法和流式細(xì)胞術(shù)顯示出其較強(qiáng)顯色強(qiáng)度，如圖3,4）低于CCAP-DM1-其余單抗偶聯(lián)物細(xì)胞毒性。mAb-CCAP-DM1偶聯(lián)物細(xì)胞毒性與結(jié)合親和性沒(méi)有重要關(guān)聯(lián)。

    該研究發(fā)現(xiàn)CCAP直接標(biāo)記法不僅能檢測(cè)到高親和性單抗例如1C008也能檢測(cè)到很難被被FCM和ICC檢測(cè)到的低親和性單抗例如3C060。重要的是3C066單抗偶聯(lián)活性藥物可通過(guò)EpRE反應(yīng)的抗體獲得。