生物活性

Cetuximab,an anti-EGFR monoclonal antibody, is used for the treatment of unresectableadvanced or recurrent head and neck or colorectal cancer.

体外研究

体内研究

激酶实验

细胞实验

Cells, antibodies and drugs[1]

NSCLC cell lines were cultured in RPMImedia in the presence of 10% fetal calf serum (FCS) with (SW-1573, H322 andH441) or without 2 mM L-glutamine (A549, H358 and H292) and incubated at 37 °Cin a humidified atmosphere with 5% CO₂. A431 cells were cultured inDMEM high glucose in the presence of 10% FCS.

For monoclonal antibody treatments a totalconcentration of 20μg/mL was used unless otherwise indicated. Erlotinib andbafilomycin A1 were dissolved in dimethyl sulfoxide (DMSO), stored at −20 °Cand diluted in fresh medium for use. The final concentration of DMSO neverexceeded 0.1% v/v.

Flow cytometry

Analysis of EGFR expression in the cancercell lines was performed using flow cytometry. Cells were harvested in phosphate-bufferedsaline (PBS: 9.7mM Na₂HPO₄, 1.6mM KH₂PO₄,150mM NaCl, pH = 7.2) containing 2% FCS (FACS buffer) and left on ice prior toflow cytometry analysis. EGFR membrane expression levels were measured usingthe anti-EGFR human antibody imgatuzumab or cetuximab at final concentrationsof 20μg/mL in FACS medium. Bound antibody was detected using mouse anti-humanFITC diluted 1:50 in FACS medium.

To determine the effects of anti-EGFRmonoclonal antibody treatment on EGFR membrane levels, cells were treated withimgatuzumab (20μg/mL), cetuximab (20μg/mL) or the combination (10μg/mL each or20μg/mL each) for 24 and 72 hours. Cells were harvested in FACS buffer and lefton ice prior to flow cytometry analysis. To determine the internalization ofEGFR monoclonal antibodies and membranous turnover of EGFR in tumor cells,cells were stained on ice with the primary antibodies against EGFR (20μg/mLfinal concentration). After staining with the primary antibody; 1) cells werewashed with ice-cold FACS buffer and incubated with the secondary antibody for1 hour at 4 ˚C to measure surface expression. 2) Cells were washed withice-cold FACS buffer, incubated in culture medium at 37 ˚C for 1, 2, or 4 hoursand subsequently incubated with the secondary antibody for 1 hour at 4˚C tomeasure non-internalized EGFR-antibody complexes since the secondary antibodiesonly bind to surface bound EGFR antibody. The lower the amount ofnon-internalized EGFR-antibody complexes, the more internalization ofEGFR-antibody complexes. 3) Cells were washed with ice-cold FACS buffer,incubated in culture medium at 37 ˚C for 1, 2, or 4 hours and subsequently incubated with theprimary antibody, followed by the secondary antibody to measurenon-internalized, reappeared receptors and possible de novo synthesis ofreceptors. Duplicate samples were measured for each treatment condition, andcorrected for background fluorescence and unspecific binding of the secondaryantibody. Measurement was performed on a BD FACSCalibur or BD Accuri C6. Dataanalysis was performed with FlowJo v10 and surface receptor expression wasexpressed as mean fluorescent intensity (MFI).

Growth inhibition and migration assays

For the proliferation assays, SW-1573 (2000cells/ well), H292 (2000 cells/well), H322 (8000 cells/well), A549 cells (2000cells/well) and H358 (2000 cells/well) were seeded in 12 wells plates. Cellswere allowed to adhere overnight. Subsequently, cells were treated with theindicated anti-EGFR monoclonal antibodies (20μg/mL total) in complete mediumcontaining 10% FCS with or without the addition of 1 or 10ng/mL EGF andincubated for the indicated time period at 37 °C.

For the wound healing assays, H292 and A549cells (both 150,000 cells/well) were seeded in 24 wells plates. After 24 hourscells were serum starved overnight. Next, a scratch was made in the monolayerwith a pipette tip, wells were washed with PBS, and cells incubated in serumfree medium containing 10ng/mL EGF with or without the anti-EGFR monoclonalantibodies at 37 °C. Scratch area was measured when the scratch was made andafter 16 hours for H292 or 24 hours for A549 using Image J software analysis(1.47v).

ADCC assays

ADCC was measured using the LactoseDehydrogenase Cytotoxicity Detection Kit according to standard protocol. Briefly, humanPBMCs were prepared from healthy blood donors by Ficoll gradientcentrifugation. PBMCs were washed in PBS and resuspended in Aim V medium. Serialdilutions of either imgatuzumab or cetuximab were added to each well of a96-well plate. The final concentration of the monoclonal antibody combinationwas the same as in the single monoclonal antibody experiments. ADCC wasconducted using an effector:target (E:T) cell ratio of 25:1 and 4 hoursincubation at 37 °C. All assays were conducted in triplicate.

CDC assays

CDC was measured using the LactoseDehydrogenase Cytotoxicity Detection Kit. Anti-EGFR monoclonal antibodies (10μg/mL total) were added to 25.000 tumors cells in a 96-well plate. CDC wasconducted using a final concentration of 5% freshly drawn human serum and 4hours incubation at 37 °C. Antibody-dependent cytotoxicity without serum wasnot observed. All assays were conducted in triplicate.

动物实验

Animal studies[2]

Four-week-old, 20 g, female athymic nudemice were allowed to acclimatize for 1 week before experiments. For theorthotopic UM-SCC1-luc model, 100,000 cells were injected into the oral tongue,and tumors were imaged biweekly using a luciferase bioluminescence assaystarting at day 4 after injection. Mice received intraperitoneal injections ofD-luciferin substrate (150 mg/kg) 15 minutes before imaging, and luminescencewas measured in photons per second. For the heterotopic UM-SCC47 model, 3 x 10⁶ cells were injected into the right flank, and tumors were measured by caliperbiweekly starting at day 4 after injection. Mice bearing HNSCC cell line xenograftswere subjected to 3 weekly cycles of prexasertib (Mondays and Thursdays),cetuximab (Mondays), and 2 Gy irradiation (Mondays and Thursdays). Prexasertibwas injected subcutaneously at 4 mg/kg twice a day. Cetuximab was given at 0.1mg/ injection intraperitoneally. Twenty percent Captisol was used as a vehiclecontrol.

不同实验动物依据体表面积的等效剂量转换表（数据来源于FDA指南）

例如，已知某工具药用于小鼠的剂量为88 mg/kg , 则用于大鼠的剂量换算方法：将88 mg/kg 乘以小鼠的Km系数（3），再除以大鼠的Km系数（6），得到该药物用于大鼠的等效剂量44 mg/kg。

参考文献

[1] Kol A, T. v. S. A., Pool M, Gerdes C, de Vries E, de Jong S, ADCC responses and blocking of EGFR-mediated signaling and cell growth by combining the anti-EGFR antibodies imgatuzumab and cetuximab in NSCLC cells. Oncotarget 2017.
[2] Zeng, L.; Beggs, R. R.; Cooper, T. S.; Weaver, A. N.; Yang, E. S., Combining Chk1/2 Inhibition with Cetuximab and Radiation Enhances In Vitro and In Vivo Cytotoxicity in Head and Neck Squamous Cell Carcinoma. Mol Cancer Ther 2017, 16 (4), 591-600.

体内溶解度

Clinical Trial Information ( data from http://clinicaltrials.gov )

注：以上所有数据均来自公开文献，并不保证对所有实验均有效，数据仅供参考。

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