Detection of colonic neoplasia in vivo using near-infrared peptide targeted against overexpressed cmet
A technology for intestinal tumors and antibodies, applied in the field of in vivo detection of colon neoplasia using near-infrared peptides targeting overexpressed cMET, which can solve problems such as high molecular weight, limited ability to penetrate tissues, and shortened circulation half-life
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Embodiment 1
[0096] Cell Lines, Media and Chemicals
[0097] The human CRC cell lines HT29, SW480, CCD841 and the mouse embryonic fibroblast cell line NIH 3T3 were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The S114 cell line comprises NIH 3T3 cells transformed with human HGF / SF and Met and expresses cMet. We used McCoy's 5A medium (Gibco) to grow HT29 cells, and Dulbecco's modified Eagle's medium (Gibco) to grow SW480, NIH3T3 and S114 cells. Eagle minimal essential medium (Lonza) was used for CCD841 cells. All cells were stored at 37°C, 5% CO 2 and supplemented with 10% fetal bovine serum (FBS). Cells were passaged using 0.25% trypsin with EDTA (Mediatech, Manassas, VA). Cell numbers were quantified on a hemocytometer. Peptide synthesis reagents were obtained from Anaspec (Anaspec, Fremont, CA) or AAPPTEC (AAPPTEC, Louisville, KY) as the highest grade available (>99% purity) and were used without further purification. Solvents and other chemicals were p...
Embodiment 2
[0142] cMet-specific peptide
[0143] The linear heptad sequence QQTNWSL (SEQ ID NO: 1 ) was identified by biopanning a high diversity phage display library of linearized heptads against the extracellular domain (ECD) of cMet. Using the structural model of cMet, this peptide showed the lowest P value for the binding interaction. The C-terminus of this peptide (black) is covalently linked to the fluorophore Cy5.5 (red) via a GGGSK linker (blue), henceforth referred to as QQT*-Cy5.5, Figure 1A. A linker separates the peptide from the fluorophore to prevent steric hindrance. The scrambled sequence TLQWNQS (SEQ ID NO: 3) was also labeled with Cy5.5 for use as a control, hereafter referred to as TLQ*-Cy5.5, Fig. IB. The 3D models showed differences in biochemical structure, Figure 1C,D. The peak absorption and emission of the Cy5.5-labeled peptide occurs in the near-infrared (NIR) spectrum, Figure 1E,F, where hemoglobin absorption, tissue scattering, and tissue autofluorescence ...
Embodiment 3
[0145] Validation of binding to cells in vitro
[0146] siRNA knockdown experiments were performed using HT29 human colorectal cancer cells to verify the specific binding of QQT*-Cy5.5 to cMet. Under confocal microscopy, QQT*-Cy5.5 (red) and AF488-labeled anti-cMet antibody (green) strongly bound to the surface of control HT29 cells transfected with siC (control) (arrow), figure 2 A, B, while TLQ*-Cy5.5 shows minimal binding, figure 2 c. A decrease in fluorescence intensity was observed using HT29 knockdown cells (sicMet), figure 2 D, E, and TLQ*-Cy5.5 has almost no signal, figure 2 F. Quantitative results show that this decrease is significant, figure 2g. Western blot showing the effect of cMet expression knockdown in cells, figure 2 H. Furthermore, significantly higher fluorescence intensities were observed for the binding of QQT*-Cy5.5 and anti-cMet-AF488 to HT29 cells (cMet+) compared to SW480 human colorectal cancer (cMet-) cells, Figure 10 . Similar results we...
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