Characterization of the established anti-Aggrus neutralizing mAb MS-1.

<p>A, cells were treated with control mouse IgG (gray area) or MS-1 mAb (bold lines). B, cells were lysed and immunoblotted with the indicated antibodies. C, the GST-tagged recombinant human ∆N20-Aggrus protein and its point mutants were expressed in <i>E. Coli</i> and immunoblotted with the indicated antibodies. GST, anti-GST antibody. D, interaction between MS-1 mAb and the human Aggrus protein (red line) or the mouse Aggrus protein (blue line) was estimated by SPR analysis. Equilibrium dissociation constants (<i>K_D</i>) of MS-1 mAb on human Aggrus are shown. The <i>K</i>_<i>D</i> of MS-1 mAb on mouse Aggrus could not be calculated (N.C.).</p

Humanized chimeric ChMS-1 antibody suppressed the <i>in vivo</i> growth of Aggrus-expressing lung squamous cell carcinoma PC-10 cells.

<p>A, interaction between the human Aggrus protein prepared from mammalian cells (R&D systems) and ChMS-1 antibody was estimated by SPR analysis. <i>K</i>_<i>D</i> of ChMS-1 antibody on human Aggrus is shown. B, antimetastatic activity of ChMS-1 antibody. BALB/c-<i>nu/nu</i> mice were intravenously injected with the indicated concentrations of antibodies. After 24 h, CHO/Aggrus cells were intravenously inoculated. After 20 days of tumor inoculation, lung surface metastatic foci were counted. The number of metastatic foci are shown. Bars, mean (n=8). **<i>P</i> < 0.01 by the Mann–Whitney <i>U</i> test. C, antitumor activity of ChMS-1 antibody in NOD-SCID mice. PC-10 (left) and A549 (right) cells were subcutaneously inoculated into the backs of eight-week-old female NOD.CB17-<i>Prkdc</i>^<i>scid</i>/J mice. After 10 days of tumor inoculation, antibodies were intravenously injected into the lateral tail vein and repeated 3 more times once a week (100 µg/mouse, arrow heads). Tumor volume was calculated as described in Materials and Methods. All data are shown as means ± SD. *<i>P</i> < 0.05 by the Mann–Whitney <i>U</i> test. D, PC-10 cells were cultured in the presence of control human IgG1 or ChMS-1 (10 µg/ml). Cell viability was measured by adding the CellTiter-Glo assay reagent. Briefly, a total of 1,500 cells were seeded in 96-well plates in triplet. On the following day, cells were treated with or without antibodies and incubated for another 24, 72, or 120 hours. Cell viability was determined by adding the CellTiter-Glo assay reagent (Promega) for 10 minutes and luminescence was measured using a Centro LB 960 luminometer (Berthold Technologies). E, representative image of infiltrated platelets (stained by anti-CD41 antibody, lower panels) and Aggrus expression in tumors (stained by D2-40, upper panels). PC-10 and A549 tumors were excised after 40 days of tumor inoculation. Bar, 0.1 µm. Magnification, x20.</p

MS-1 mAb suppressed spontaneous pulmonary metastasis and tumor growth <i>in vivo</i>.

<p>A to D, CHO/Aggrus cells were xenografted in BALB/c-<i>nu/nu</i> mice. Antibodies (30 µg/mouse) were intravenously injected on the following day of tumor inoculation and repeated two more times every fourth day (arrow heads). Tumor volume was calculated as described in Materials and Methods. All data are shown as means ± SDs (n=5). **<i>P</i> < 0.01 by the Mann–Whitney <i>U</i> test (A). Representative pictures of CHO/Aggrus tumor-bearing mice on day 18 are shown (B). After 29 days of tumor inoculation, spontaneously pulmonary metastatic foci were counted. The numbers of metastatic foci are shown. Bars, mean (n = 5). **<i>P</i> < 0.01 by the Mann–Whitney <i>U</i> test (C). Representative pictures of the lungs and lung surface metastatic foci are shown (D). E, a lung squamous cell line PC-10 (left) and a lung adenocarcinoma cell line A549 (right) cells were treated with control mouse IgG (gray area) or MS-1 mAb (bold lines). Aggrus expression was detected by flow cytometry. F and G, PC-10 cells were xenografted into BALB/c-<i>nu/nu</i> mice. Antibodies were intravenously injected on 1, 4, 7, and 11 days after tumor inoculation (arrow heads). Tumor volume was calculated as described in Materials and Methods. All data are shown as means ± SDs (n=8). **<i>P</i> < 0.01 by the Mann–Whitney <i>U</i> test (F). Representative pictures of the PC-10 tumor-bearing mice on day 18 are shown (G).</p

Suppression of Aggrus-induced platelet aggregation and tumor metastasis by MS-1 mAb.

<p>A, recombinant CLEC-2 protein was immobilized on an ELISA plate and then incubated with the human IgG Fc-tagged recombinant human Aggrus protein in the presence of the indicated concentrations of control mouse IgG (Mouse IgG) or MS-1 mAb. The value of PBS-treated control was normalized to 100%. Data are means ± SDs of triplicate determinations. B, CHO/Aggrus cells were incubated with 10 µg/mL of antibodies, followed by incubation with mouse PRP. Light transmittance of samples was measured as the aggregation rate. C to F, BALB/c-<i>nu/nu</i> mice were intravenously injected with the indicated concentrations of antibodies (C and D) or F(ab’)₂ fragments (E and F). CHO/Aggrus cells were intravenously inoculated on the following day of antibody administration. After 20 days of tumor inoculation, lung surface metastatic foci were counted. Numbers of metastatic foci in each mouse were shown. Bars, mean (n=8). NS, not significant. **<i>P</i> < 0.01 by the Mann–Whitney <i>U</i> test (C and E). Representative pictures of the lungs and lung surface metastatic foci are shown (D and F).</p

Identification of recognition epitope of MS-1 mAb on human Aggrus.

<p>A, cells were lysed and immunoblotted with the indicated antibodies. B, CHO cells that had been transfected with WT-, G45A- or D49A-Aggrus expression plasmids (CHO/Aggrus, CHO/Aggrus-G45A or CHO/Aggrus-D49A, respectively) were treated with control mouse IgG (0.1 µg/ml, gray area) or MS-1 mAb (0.1 µg/ml, bold lines in upper panels). In some experiments, cells were treated with an anti-human Aggrus mAb D2-40 antibody (0.1 µg/ml, bold lines in lower panels). After incubation with the Alexa Fluor 488-conjugated secondly antibody, Aggrus expression was detected by flow cytometry. C, CHO/mock, CHO/Aggrus, CHO/Aggrus-G45A, and CHO/Aggrus-D49A cells were treated with control mouse IgG (0.1 µg/ml, gray area) or mouse IgG Fc-conjugated recombinant human CLEC-2 protein (4 µg/ml, bold lines). After incubation with the Alexa Fluor 488-conjugated secondly antibody, CLEC-2 binding to Aggrus-expressing cells was confirmed by flow cytometry. D and E, BALB/c-<i>nu/nu</i> mice were intravenously injected with control mouse IgG (Mouse IgG) or MS-1 mAb (3 µg/mouse). After 24 h, cells (2.5 × 10⁵ cells/mouse) were intravenously inoculated into mice. After 20 days of tumor inoculation, lung surface metastatic foci were counted. The number of metastatic foci are shown (D). Bars, mean (n = 7 or 8). NS, not significant. **<i>P</i> < 0.01 by the Mann–Whitney <i>U</i> test. Representative pictures of the lungs and lung surface metastatic foci are shown (E).</p

Involvement of Aggrus-platelet interaction in <i>in vivo</i> tumor growth.

<p>A, establishment of Aggrus-knockdowned PC-10 cells. Aggrus expression of PC-10 transfectants was confirmed by immunoblotting with the indicated antibody. B, <i>in vitro</i> growth of PC-10 transfectants that have been stably transfected with ZsGreen-expressing plasmid. Cells were cultured in medium containing 10% FBS for the indicated times. Relative cell viability was calculated as measuring the fluorescence of ZsGreen. C, <i>in vivo</i> growth of PC-10 transfectants. PC-10 cells were subcutaneously inoculated into the backs of eight-week-old female BALB/c-<i>nu/nu</i> mice (n=3). Tumor volume was calculated as described in Materials and Methods. D, the effects of platelets on PC-10 cell growth <i>in vitro</i>. PC-10 transfectants that have been stably transfected with ZsGreen-expressing plasmid were co-cultured with washed platelets in medium containing 0.5% FBS. Relative cell viability was calculated as measuring the fluorescence of ZsGreen.</p