This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Targeting tumor blood vessels (TBV) is a promising strategy to treat cancer. Finding TBV specific antibodies is a critical step and is the focus of this work. Phage display technology is a powerful method to generate and identify ligands to cancers. Laser capture microscopy (LCM) is a technique to select pure cells population from histological specimens. Our lab has established a practical method to combine phage display and LCM. Using this method, high specific tumor stroma binding clones were selected with high selectivity binding to this case tumor stroma, but not to a panel of normal human tissue and other cancer specimens. In this report, an antibody orientated capture strategy was established to accurately capture TBV from tumor sections after biopanning. Briefly, tumor frozen tissue sections were incubated with phage display library. After rinsing off the unbounded phage clones, TBV was identified with blood vessel markers and collected with bounded phage clones by LCM under fluoresce microscopy. The panning output was amplified for next round panning. After two rounds of biopanning, 192 phage clones were harvested and screened on tumor frozen tissue from the same patients. 20% of clones showed some level of binding to part of the tumor blood vessel. DNA sequence was used to found full size scFv phage clones for scFv expression and purification. In this report, we identified some tumor blood vessel binding clones with low specificity to TBV. As tumor blood vessel antigen usually expressed in low quantity in tumor tissue, further work will include using more powerful phage display library (the library with more diversity and high affinity candidates) and more accurate TBV selection methods. Successful outcome of this work will result in selection methods to obtain TBV binding ligands as well as potential TBV binding ligands for tumor therapy.
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