A Novel Anti-EGFR mAb Ame55 with Lower Toxicity and Better Efficacy than Cetuximab When Combined with Irinotecan

To improve efficacy and minimize toxicity of EGFR inhibition treatment, we developed Ame55, a novel anti-EGFR IgG1 with lower affinity to EGFR than cetuximab (C225) from a human phage library. Ame55 had lower bioactivity than cetuximab in vitro but similar antitumor efficacy as cetuximab in vivo. Moreover, Ame55 was more efficacious than cetuximab in a Lovo cell xenograft tumor model when combined with irinotecan (CPT-11). Ame55 concentrates in the mouse xenograft tumor and has less toxicity than cetuximab in cynomolgus monkeys in an overdose study.

SDS-PAGE reducing gel was used confirm antibody purity.

Cell ELISA
A431 cells were seeded in 96 well-plates, from 0.5 × 10 4 /well to 4 × 10 4 /well, and were cultivated overnight at 37°C, 5% CO 2 . Antibodies were diluted to 10 µg/mL with PBS containing 2% FBS and serially two-fold diluted for 8 gradients. After 1 h on ice, plates were washed with PBST. HRP-conjugated goat anti-human IgG (Sigma-Aldrich, A0170) was added and incubated for 30 min on ice. After washing, o-phenylenediamine dihydro-chloride (OPD) substrate was added, and optical density (OD) was read at 492 nm with 630 nm as a reference using a microplate reader (Thermo Multiskan MK3, Piedmont, SC).

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For the binding specificity assay, 200 ng His-EGFR, VEGF, IL-6, BSA, CD4, P-selectin, Aβ-T (all proteins were expressed by our laboratory) and PBS were coated in 96-well plates (Costar 9018,Corning,NY) in PBS buffer and incubated 4°C overnight, with 5% fat-free powdered milk in PBS with 0.1% Tween20 were used for blocking for 30min at 37°C, Cetuximab and Ame55 were used as primary antibodies and incubated for 1 h at 37°C, after 3 washings, HRP-goat anti-human IgG (ZSGB-BIO, Beijing, China) were used as a second antibody and incubated for 30 min at 37°C. Plates were developed with OPD and OD was read at 492/630 nm using a 470 microplate reader (Thermo Multiskan MK3, Piedmont, SC).

Immunofluorescence assay
For EGFR-antibody binding assay, 10000 of exponential phased growth CHO, Lovo and A431 cells were seeded on coverslips in 24-well tissue culture plates. After overnight culture, cells were washed with PBS containing 4% paraformaldehyde (Beijing Chemical Works, Beijing, China) and 0.1% triton X-100 (Sigma, St. Louis, MO), and washed with PBS at room temperature. Cells were then blocked with 5% goat serum in PBS for 30 min, washed three times with PBS, and incubated with 50 g/mL Ame55 or cetuximab, for 2 h at room temperature. PBS was a negative control. Cells were then washed three times with PBS and incubated with FITC-labeled goat anti-mouse IgG (ZSGB-BIO, Beijing, China) diluted at 1:200 in PBS for 30 min. Cells were rinsed in PBS three times and visualized under a microscope with 100× zoom in (Nikon Xi-80, Tokyo, Japan).

CCK8 proliferation assay
A CCK-8 Kit (Dojindo Laboratories, Rockville, MD) was used to measure cell growth of A431 and DiFi cells. Cells (10,000 cells/per well) were seeded in 96-well plates. Antibodies were added and incubated at 37°C in 5% CO 2 for 72 h. CCK-8 solution (12 L) was added to 100 µL culture media, and OD was read at 450/630 nm. Three independent experiments were performed.

FACS analysis of cell cycle and apoptosis
After 24 h treatment with 30 μg/mL Ame55 or cetuximab, A431 cells were harvested, washed with PBS, and fixed in 70% ethanol overnight. Cells were washed with PBS twice, and stained with propidium iodide buffer (50 μg/mL PI Basle, Switzerland) with 20 μg/mL RNase) for 25 min at 37°C in the dark. Cell cycle phase distribution was assessed with flow cytometry (Cytomics FC 500, Beckman Coulter). Cells in each phase were analyzed by using was used to quantify apoptosis. Serum-starved cells were treated with 30 μg/mL Ame55 or cetuximab for 24 h, harvested and resuspended with annexin binding buffer, then 5µL Component A and 1 µL 100 μg/mL PI was added to 100 µL cell sample. Samples were incubated in the dark for 15 min at room temperature, and 400 µL annexin binding buffer was added. Cells were analyzed with FACS cytometry (Cytomics FC 500, Beckman Coulter) and Modifit LT.

Anchorage-independent transformation assay
The effect of antibodies on EGF-induced cell transformation was investigated in HaCaT cells. Cells (10,000 cells/well in 6-well plates) were exposed simultaneously to different concentrations (20, 100, 400 nM) of Ame55 or cetuximab with EGF (10 ng/mL) in 0.35% agar 1640 containing 10% FBS over 0.6% agar 1640 containing 10% FBS for 3 weeks. Three independent experiments were performed, and cell colonies were counted in 5 random fields for each culture dish, scored and statistically assessed.

In vitro wound healing assay
Approximately 1.2 × 10 6 cells were seeded on Falcon 6-well tissue culture plates. After 12 h, confluent cells were incubated in serum-free medium for 24 h (~80% plate coverage). Scratch wounds were created by scraping the confluent monolayers with a sterile 200 µL pipette tip to create an ~1.0-mm gap. Four parallel scrapes per well at the same position were made and cells dislodged by scraping were removed by washing cultures with serum-free medium. Cells were treated with cetuximab or Ame55 at 400 nM. Migration of the cells into the scratch was observed at 0, 12, and 48 h after scraping (200× magnification).

Transwell migration assay
Cells (1 × 10 5 ) were suspended in 200 μL 1640 medium without serum and placed in (8 μmpore size; Millipore, Merck, Darmstadt, Germany) of a companion plate (12-well plate, Corning, Corning, NY) with a prewarmed culture medium containing 10% FBS in the well. Then, 400 nM cetuximab or Ame55 antibodies were added the insert chamber and mixed. PBS was a control. Cells were incubated for 18 h at 37°C in 5% CO 2 . The top of the filter was gently wiped with a cottonswab to remove non-migrated cells. Cell migration was measured by staining cells with 1% crystal violet and photographing cells under a light microscope (400× magnification).

Immunohistochemical Analysis
To assess angiogenesis and cell proliferation in tumors, formalin-fixed paraffin-embedded