Development of the antibody-producing stable cell line and antibody characterization.

(a) Construction of the bicistronic vector for the antibody expression. Three separate pcDNA 3.1 (-) vectors were used to clone the encoding gene sequences of scFvDiathis LC and IgG1 HC. The expression cassette bearing the CMV promoter, scFvDIATHIS1 LC encoding gene and the polyA chain, was amplified by PCR in order to add the sequence of BglII and NruI restriction sites, upstream and downstream the LC gene, respectively. The PCR product and the vector bearing the IgG1 HC encoding gene were both digested by BglII and Nru restriction enzymes. The digestion was followed by the ligation reaction to finally develop the bicistronic vectors for the simultaneous expression of the LC and the HC. Absorbance values obtained by ELISA assays on 100 μl of the supernatants collected from the transfected cellular pools (b), clones (c) and subclones (d) for the production of the IgG1 antibody. The statistical significance of the pool differences in the abs values was tested with unpaired t-test. (e) SEC-HPLC chromatogram of IgG1 antibody purified from the subclone #12.3. The molecular weights were calculated based on a standard curve obtained by calibrating the system with molecules of known molecular weight: thyroglobulin 660 kDa, gamma- globulin 150 kDa, ovalbumin 44.3 kDa, ribonuclease A 43.7 kDa, 4-aminobenzoic acid 13.71 kDa. (f) Freshly purified antibody analyzed by SDS-PAGE under reducing (gel on the left) and non-reducing (gel on the right) conditions. (g) SDS- PAGE analysis of the antibody in reducing conditions after eight months of storage at 4°C.</p

Functional characterization of DIA 12.3 and mtN297A DIA antibodies.

(a) Binding activity of serial dilutions of DIA 12.3 antibody ranging from 100 μg/ml to 0,00038 μg/ml tested by ELISA titration assay on 96 multi-well plates coated with 1μg/ml human N+A1 domains of CEACAM1. The concentration values were log-transformed before performing the nonlinear regression on GraphPad Prism. Absorbance values are presented as mean +/- S.D of duplicate measurements. (b) DIA 12.3 binding analysis on tumor cells by flow cytometry: MelC, TCCSUP, 5637 and HT29 cells were treated with or without 20 and 10 μg/ml of DIA 12.3 antibody. The graphs showing the MFI values are reported on the right. (c) Cytotoxicity assay. MelC, TCCSUP and 5637, HT29 and Head&Neck cancer cells were treated with or without 20 μg/ml of DIA 12.3 antibody, and then co-incubated with NK cells at 10:1 E:T cell ratio. Luminescence was recorded after 60 minutes of incubation at RT. The plot is showing the % of NK cell-mediated cytotoxicity calculated upon antibody treatment in comparison with the basal NK cytotoxic activity against melanoma cells. The % of cytotoxicity was calculated as follows: 100 × (Experimental LDH Release–Medium Background)/(Maximum LDH Release Control–Medium Background). (d) and (e) Binding activity of mtN297A DIA in comparison with DIA 12.3 antibody by ELISA titration and flow cytometry assays. Serial dilutions of DIA 12.3 and mtN297A DIA antibodies ranging from 100 μg/ml to 0,00038 μg/ml were tested on 96 multi-well plates coated with 1μg/ml human N-terminal + A1 domain fragment of CEACAM1 antigen. The concentration values were log-transformed before performing the nonlinear regression on GraphPad Prism. Absorbance values are presented as mean +/- S.D of duplicate measurements. In the flow cytometry assay: MelC cells were treated with or without 20 and 10 μg/ml of DIA 12.3 or mtN297A DIA antibodies. (f) Cytotoxicity assay: MelC cells were treated with or without 20 or 10 μg/ml of DIA 12.3 antibody, and then co-incubated with NK cells at 10:1, 5:1 and 1:1 E:T cell ratios. Luminescence was recorded after 60 minutes of incubation at RT. The plot shows the percent of NK cell-mediated cytotoxicity calculated upon antibody treatment in comparison with the basal NK cytotoxic activity against melanoma cells. The percent cytotoxicity was calculated as described in Fig 2.</p

Photo of original gel taken from Fig 1f.

Carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1), a homotypic cell adhesion molecule glycoprotein with apical expression on normal epithelial cells and activated lymphocytes, is overexpressed on many tumors and acts as an inhibitory receptor on NK cells, preventing their killing of CEACAM1 positive tumors. Production of humanized anti-CEACAM1 antibodies to block the inhibitory activity of CEACAM1 for immunotherapy and immunoimaging. Starting from a scFv, a fully human intact anti-CEACAM1 (DIA 12.3) that recognizes the N-terminal domain of CEACAM1 was developed and shown to bind CEACAM1 positive tumor cells and enhanced NK cell killing of CEACAM1 positive targets. DIA 12.3 bound to human neutrophils without activation, indicating they would be safe for human use. DIA 12.3 exhibited some cross-reactivity to CEACAM5, a tumor marker with high sequence homology to the N-terminal domain of CEACAM1. CEACAM1 PET imaging with 64Cu-COTA-DIA 12.3 showed excellent imaging of CEACAM1 positive tumors with reduced binding to CEACAM5 tumors. Based on its immunoinhibitory an immunoimaging activities, DIA 12.3 shows promise for therapeutic studies in man.</div

Neutrophil activation studies.

(a) Analysis of NO levels released by neutrophils in culture supernatants. Neutrophils were co-cultured with CEACAM1 positive MB231 cells at 10:1, 5:1 and 1:1 E:T ratios. For each E:T cell ratio, three different treatment conditions were tested: (i) neutrophils or (ii) tumor cells alone pre-treated with or without 10 μg/ml of DIA 12.3 for 30 minutes at 37°C and then co-incubated 4 hours at 37°C with tumor cells or neutrophils, respectively; neutrophils and tumor cells co-treated with or without 10 μg/ml of DIA 12.3 for 4 hours at 37°C. Then the supernatants were subjected to nitrite assay according to the Griess reagent system. Bars are representative of duplicate values +/- SD. ** = Pb) Evaluation of DIA 12.3 antibody effects on neutrophil activation. Isolated and enriched human neutrophils were incubated for 1, 2 and 18 hours in RPMI medium at 37°C in absence or presence of 10 μg/ml of DIA 12.3 antibody. Following each time point, neutrophils were then stained with anti-CD66b antibody.</p

Characterization of the antibody-producing stable cell line.

(a) Monoclonality of the subclone #12.3 is evaluated by ELISA assay. The graph shows the absorbance values of 50 clones grown 15 days after subjecting the subclone #12.3 to the limiting dilution. (b) Stability studies of antibody production from the subclone #12.3 by ELISA assay. The graphs report the absorbance values of the clones grown on the 20th, 40th and on the 60th day of culture without the pressure of the selective antibiotic.</p

Effect of DIA 12.3 on spontaneous apoptosis ofneutrophils.

(a) Neutrophil identification by CD66b staining after the isolation and (b) the binding profile of 10 μg/ml of DIA 12.3 in comparison with the commercial anti-CEACAM1 antibody. (c) spontaneous apoptosis in human neutrophils after DIA 12.3 treatment was evaluated. Neutrophils were cultured for 0, 1, 2, 16, 18 and 20 hours in RPMI medium with or without 10μg/ml of DIA 12.3 antibody at 37°C. At the end of each time point, neutrophils were double stained for FITC-annexin V and PI. Results show the % of early apoptotic, late apoptotic and living cells of three independent experiments. Bars are representative of the mean of triplicate values +/- SD.</p

PET tumor imaging with ⁶⁴Cu-DOTAylated-DIA 12.3 antibody in NSG mice bearing parental, hCEACAM1 and hCEACAM5-positive human breast tumor xenografts.

(a) Binding profiles of DIA 12.3 and DOTAylated-DIA 12.3 antibodies on MB231 cell lines. hCEACAM1 and hCEA-expressing MB231 cells were incubated with or without 10 μg/ml of DIA 12.3 and DOTAylated-DIA 12.3 antibodies, anti-CEACAM1 or anti-CEA commercial antibodies. (b) PET images of mice bearing parental, hCEACAM1 or hCEA-positive human breast tumor MDA-MB-231 cells at 46 hours after the via tail vein injection of 6,4 μg/mouse antibody radiolabeled with 3.7 Mbq of 64Cu-DOTAylated-DIA 12.3 antibody + 30 μg/mouse of unlabeled DIA 12.3. Arrows indicated location of tumors. (c) Biodistribution analysis of the radiolabeled antibody 46 hours post-injection. Bars represent the mean +/- S.D. of the %ID/g in various organs and tumors from mice injected with each cell line (n = 3 mice per cell line).</p