MS17-57 inhibits BGC823 and MKN45 cell growth.

<p>MS17-57 was added to BGC823 cells (8 µg/mL per well) (<b>A</b>) or MKN45 cells (2 µg/mL per well) (<b>B</b>) on 96-well tissue culture plates at day0. MS17-57 inhibited BGC823 cell growth by about 27.5% for up to 5 days and MKN45 cell growth by about 22.5% for up to 7 days. Irrelevant mAb, used for isotype control, was applied at concentrations about four times higher than that used for MS17-57. There are a significant difference of the percentage of cell growth between irrelevant mAb group (<b>A</b>) and MS17-57 group (<b>B</b>), and both statistical testing <i>P</i> values from day 3 to day 7 were 0.0065 and 0.0066.</p

MS17-57 mAb binding to GC cells, GI cells, and human PBMCs.

<p><b>A</b>.MS17-57 bound to all four GC cell lines that were used for live cell immunization. <b>B</b>. MS17-57 exhibited strong binding signals in GES-1 and AGS cells but no binding signal in human PBMCs. <b>C</b>. MS17-57 did not bind to human PBMCs nor any of the five GI tumor cells. Irrelevant mAb was used as the mAb isotype control. </p

ICC staining for MS17-57 binding to MKN45, BGC823, and GES-1 cells on cytospin slides.

<p>Two ICC assays were performed; photomicrographs from one are shown at 40x and images from the other at 100x). MS17-57 bound to all three types of cells. The binding target (marker) was located on the cell surface. Images of blank and negative (isotype) controls were also obtained but are not shown here. </p

MS17-57 inhibits tumor growth in nude mice.

<p>(<b>A</b>) MS17-57 mAb inhibits the growth of MKN45 tumor cell xenografts. Cells (1x10⁶) mixed with 100 µg of MS17-57 mAb (50 µg/mL in PBS) were injected i.p. into the abdominal cavity of male nude mice and 4 mice in each group. About 6 weeks later, mice with a palpable tumor were counted for an average of 1.5 node/each nude mouse and an average tumor diameter 0.3 cm; (<b>B</b>) The same procedures as in (A) using MKN45 cells in nude mice but with irrelevant mAb and a final count of an average of 8.5 node/each nude mouse and an average tumor diameter of about 0.31 cm; (<b>C</b>) The same procedures as (A) using MKN45 cells in nude mice but with PBS (buffer) and the final tumor nodes count an average of 9.0 node/each nude mouse and an average tumor diameter of about 0.28 cm; (<b>D</b>) The same procedures as (A) but using BGC823 cells in nude mice with MS17-57 mAb and a final tumor nodes count of an average of 1.0 node/each nude mouse and an average tumor diameter of about 0.27 cm; (<b>E</b>) The same procedures as (B) but using BGC823 cells in nude mice with irrelevant mAb and a final tumor nodes count of an average of 7.0 node/each nude mouse and an average tumor diameter of about 0.31 cm; (<b>F</b>) The same procedures as (C) but using BGC823 cells in nude mice with PBS and a final tumor nodes count of an average of 6.5 node/each nude mouse and an average tumor diameter of about 0.30 cm.</p

Binding of MS17-57 to lysates of MKN45, BGC823 and GES-1 cell lines in indirect ELISA.

<p>MKN45, BGC823, SGC7901, and MKN28 cells were used in experiments, but MKN45 and BGC823 cell lines were randomly selected as examples in this figure. MS17-57 expressed strong binding signals to MKN45 cells and moderate binding signals to BGC823 cells and GES-1 cells. Cell lysates were coated with 1.0 µg/mL PBS onto Immulon-II HB 96-well ELISA plates (100 µL/well). The protein concentrations of these cell lysates were balanced, but not for the binding targets (Ags) that could be a big variation. Irrelevant mAb was used as an isotype control.</p

Cells from fresh surgical tissues stained with MS17-57 and isotype control mAb.

<p>Immunofluorescence cell staining with MS17-57 revealed significantly stronger staining of GI tumor tissues than that of normal (adjacent noncancerous) control tissues (<i>P</i><0.03 overall). (Data from patient 3 were omitted for analyses because the tissue had not been properly prepared.).</p

IALP and PALP mRNA expression levels in 10 cell lines by qRT-PCR analysis.

<p>mRNA expression of IALP (<b>A</b>) and PALP (<b>B</b>) was the highest in MKN45, CRL5974, AGS, and GES-1 cell lines. In BGC823 cells, mRNA expression was much higher for PALP than IALP.*Superelevated values compared with average values with qRT-PCR.</p

Titration analysis of live GC cells used to immunize mice and bound to mouse serum.

<p>Human PBMCs were isolated from whole blood of a selected healthy donor for normal cell control. MKN45, BGC823, SGC7901, and MKN28 cells were used in experiments, but SGC7901 and BGC823 cell lines were randomly selected as examples in this figure. The MFI were higher in these cells than in PBMCs. All three mice exhibited strong immune responses to the human GC cell lines.</p

cDNA and amino acid sequences of the variable regions of the MS17-57 light chain (A) and heavy chain (B).

<p>FWRs are located between CDRs.</p

Binding of MS17-57 to purified GI cancer markers and lysates of fresh tissues and cells.

<p>ELISA results showed that MS17-57 bound to lysates of fresh GC tissues (strong binding in lysate from one patient and moderate binding in lysate from another patient) and to lysates of GC MKN45 cells, but not to fresh lysates of adjacent noncancerous tissues from the same patients. MS17-57 bound slightly to the purified CA15-3 protein but not to proteins or lysates of PG-1, PG-2, CEA, or <i>H. pylori</i>. ELISA used two dose-dependent dilutions of antibodies. Normal mouse serum protein was used as a negative control.</p