Measurement of the HGF/c-MET complex by Surface Protein-Protein Interaction by Cross-linking ELISA (SPPICE) assay.

<p>A. SPPICE schemetic. c-MET was chemically cross-linked to expgenous HGF using the membrane impermeable sulfo-EGS cross-linker. Membrane proteins were solublized and HGF/c-MET complexes were measured using the SPPICE procedure (Bi) or by immunoprecipitation with HGF antibodies followed by Western blotting with anti-c-MET antibodies (Bii). C. c-MET and endogenous HGF in glioma cells were cross-linked as described above and SPPICE was used to measure HGF/c-MET complexes.</p

VeraTag FFPE quantification of HGF and correlation with Western blot, ELISA, and IHC in human tumor specimens.

<p>A. VeraTag FFPE quantification of HGF in NSCLC tumors. Isotype IgG control signals (inset) were subtracted from HGF signals. B. Immunoprecipitation/Western blot analysis of HGF in corresponding NSCLC tumor lysates. C. ELISA determinations of HGF in corresponding NSCLC specimens. D. IHC detection in NSCLC specimens.</p

VeraTag FFPE c-MET quantification and correlation with Western blot, ELISA and IHC in cell lines.

<p>A. Quantification of c-MET expression in FFPE preparations of H441, H226, H2170, MCF7, Ln18, U138, U118, Ln229, U87, and H661 FFPE cell lines using the VeraTag assay. Background signals generated using an isotype control IgG (shown in inset) were subtracted from the c-MET assay signals. B. Western blot analysis of c-MET expression in H441, H226, H2170, MCF7, Ln18, U138, U118, Ln229, and H661 cell lysates. β-actin levels in the blot serves as a loading control. C. ELISA measurements of c-MET expression in H441, H226, H2170, MCF7, Ln18, U138, U118, Ln229, and H661 cell lysates. ND: Not detectable. D. IHC detection of c-MET expression in FFPE preparations of H441, H226, MCF7, Ln18, U118, and H661 cell lines.</p

Epitope mapping of c-MET antibodies by peptide scanning.

<p>A. Peptide ELISA. A series of 43 overlapping peptides, each 14–15 amino-acid residues in length, spanning the complete intracellular domain of c-MET (K955-S1370), were evaluated to map the binding epitope of c-MET antibodies. Peptides were spotted on microtiter plates and ELISA assays were performed using standard protocols. The binding properties of peptide 28 and 43 localized the binding sites of c-MET antibody 3D4 and c-MET antibodies CVD13 and SP44, respectively. The sequence of peptide #28 and #43 are shown (inset). B. A schematic representing the c-MET receptor and binding sites for c-MET antibodies.</p

c-MET, HGF, and HGF/c-MET complexes in human carcinomas.

<p>A. NSCLC. B. Gastric carcinoma. C. Head and Neck carcinoma. c-MET, HGF, and HGF/c-MET assays were performed for all FFPE samples. Samples were rank ordered from low to high levels of HGF. The levels of c-MET receptor and HGF/c-MET were also tabulated. Red color-High ≥90^th percentile; Pink color-Low ≤10^th percentile. NSCLC and Gastric tumor exceeded 70% tumor content by pathologic analysis. In Head & Neck carcinoma samples, non-tumor tissue was removed by macrodissection. Di. c-MET was immunoprecipitated from NSCLC lysate samples and immunoblotted using c-MET (Tyr1003) or c-MET antibodies. Dii. c-MET was immunoprecipitated from gastric tumor lysates and immunoblotted using c-MET (Tyr1003) or c-MET antibodies. Samples that were c-MET phosphorylation (pY1003) positive (+) or c-MET (pY1003) negative (−) are summarized in Fig. A, B.</p

VeraTag FFPE quantification of HGF and correlation with Western blot, ELISA and IHC in cell lines.

<p>A. VeraTag FFPE quantification of HGF in unstimulated and HGF stimulated A549 cells. Isotype IgG control signals (inset) were subtracted from the HGF signals. Bi. VeraTag quantification of HGF in Ln18, Ln229, U138, U118, and U87MG glioma cell lines. Isotype IgG control signals (inset) were subtracted from HGF assay signals. ND: Not detectable. Bii. ELISA determinations of HGF in A549, Ln18, Ln229, U138, U118, and U87MG cell lysates. ND: Not detectable. Ci. VeraTag FFPE quantification of HGF in HEK293/HGF (clone 1) and U138 glioma cell lines. Cii. IHC detection of HGF in HEK293/HGF stable clone (clone 1) and U138 glioma cells.</p

c-MET quantification using the VeraTag FFPE assay and correlation with Western blot, ELISA and IHC in human tumor specimens.

<p>A. Quantification of c-MET in NSCLC specimens using the VeraTag FFPE proximity assay. Isotype IgG antibody signal (inset) was subtracted from c-MET assay signals. B. Western blot analysis of c-MET in corresponding NSCLC tumor lysates. β-actin levels in the blot serves as a loading control. Ci. ELISA determinations of c-MET levels in corresponding NSCLC tumor lysates. Cii. Correlation of c-MET measurements in NSCLC specimens by proximity assay and ELISA. Di. c-MET detection in NSCLC by IHC. Dii. Correlation of c-MET measurements in NSCLC by FFPE VeraTag proximity assay and IHC. Histologic score (H-score)  =  %IHC3⁺×3 + %IHC2⁺×2 + %IHC1⁺×1 + %IHC0×0 was calculated as described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015932#pone.0015932-Shi1" target="_blank">[10]</a>. Spearmen r and p values were calculated using graphpad prism software.</p

VeraTag FFPE quantification of HGF/c-MET ligand-receptor complexes and correlation with c-MET (Tyr1003) phosphorylation.

<p>Ai. Schematic of the HGF/c-MET VeraTag FFPE proximity assay. Aii. Quantification of HGF/c-MET levels in unstimulated and HGF stimulated A549 cells. Isotype IgG control signals (inset) were subtracted from the HGF/c-MET assay signals. B. VeraTag quantification of HGF/c-MET ligand receptor complexes in H661, MCF7, HGF stimulated MCF7, Ln18, Ln229, U138, U118, U87MG cell lines. C. Immunoprecipitation/Western blot analysis of c-MET (Tyr1003) phosphorylation and c-MET in Ln18, Ln229, U138, U118, and U87 MG glioma cells. The H596 cell line with exon 14 (L964-D1010) deletion was included as a negative control for c-MET (Tyr1003) phosphorylation.</p

Battling Btk Mutants With Noncovalent Inhibitors That Overcome Cys481 and Thr474 Mutations

The Bruton’s tyrosine kinase (Btk) inhibitor ibrutinib has shown impressive clinical efficacy in a range of B-cell malignancies. However, acquired resistance has emerged, and second generation therapies are now being sought. Ibrutinib is a covalent, irreversible inhibitor that modifies Cys481 in the ATP binding site of Btk and renders the enzyme inactive, thereby blocking B-cell receptor signal transduction. Not surprisingly, Cys481 is the most commonly mutated Btk residue in cases of acquired resistance to ibrutinib. Mutations at other sites, including Thr474, a gatekeeper residue, have also been detected. Herein, we describe noncovalent Btk inhibitors that differ from covalent inhibitors like ibrutinib in that they do not interact with Cys481, they potently inhibit the ibrutinib-resistant Btk C481S mutant <i>in vitro</i> and in cells, and they are exquisitely selective for Btk. Noncovalent inhibitors such as GNE-431 also show excellent potency against the C481R, T474I, and T474M mutants. X-ray crystallographic analysis of Btk provides insight into the unique mode of binding of these inhibitors that explains their high selectivity for Btk and their retained activity against mutant forms of Btk. This class of noncovalent Btk inhibitors may provide a treatment option to patients, especially those who have acquired resistance to ibrutinib by mutation of Cys481 or Thr474