Biomedical subjects

David N Krag

Publications and source records attributed to David N Krag.

21 records · Page 2Linked to original sources

PubMed2002

Phage-displayed random peptide libraries in mice: toxicity after serial panning.

PURPOSE: In vivo screening of phage-displayed random peptide libraries (RPLs) has been used to identify peptide ligands to targets found on endothelial cells of blood vessels supplying specific tissues such as brain, kidney, and tumor tissue. Peptides that bind specifically to blood vessels supplying tumor tissue have been conjugated to cytotoxic agents and used to successfully eradicate tumors in a mouse model. With the ultimate goal of developing similar methods for treating human cancer, we describe an in vivo RPL screening process that, unlike previous in vivo experiments, does not harm the animal being screened. METHODS: RPLs were administered to FVB, BalbC, and tumor-bearing MRL/MpJ-fas(LPR) mice in a variety of dosing formats. Tumor nodules were excised 10 min following infusion and phage were amplified from the specimens. Phage were reinjected into the same animal within 48 h. This process was repeated twice for a total of three in vivo screens of mouse tumor tissue within the same animal. Mice were observed for systemic side effects, histopathologic damage, and presence of phage in organs. Peptide sequences were determined from several third-pan phage clones. RESULTS: Overall there was minimal toxicity from administration of single or repeat doses of RPLs. Amino acid consensus sequences were identified and some of the sequences were similar to those of peptide ligands that bind matrix metalloproteinases. CONCLUSIONS: Serial administration of an RPL is well tolerated and serial panning in individual mice leading to consensus sequence motifs is possible. Based on these preclinical data the Food and Drug Administration has approved the implementation of human clinical trials with this technique.

Amino Acid Sequence↗

PubMed2002

Identification of novel non-phosphorylated ligands, which bind selectively to the SH2 domain of Grb7.

Grb7 is an adapter-type signaling protein, which is recruited via its SH2 domain to a variety of receptor tyrosine kinases (RTKs), including ErbB2 and ErbB3. It is overexpressed in breast, esophageal, and gastric cancers, and may contribute to the invasive potential of cancer cells. Molecular interactions involving Grb7 therefore provide attractive targets for therapeutic intervention. We have utilized phage display random peptide libraries as a source of small peptide ligands to the SH2 domain of Grb7. Screening these libraries against purified Grb7 SH2 resulted in the identification of Grb7-binding peptide phage clones that contained a non-phosphorylated Tyr-X-Asn (YXN) motif. The tyrosine-phosphorylated form of this motif is characteristic of Grb7 SH2 domain binding sites identified in RTKs and other signaling proteins such as Shc. Peptides that are non-phosphorylated have greater potential in the development of therapeutics because of the instability of a phosphate group in vivo. Using a biased library approach with this conserved YXN motif, we identified seven different peptide phage clones, which bind specifically to the SH2 domain of Grb7. These peptides did not bind to the SH2 domain of Grb2 (which also selects for Asn at pY(+2)) or Grb14, a closely related family member. The cyclic structure of the peptides was required to bind to the Grb7 SH2 domain. Importantly, the synthetic Grb7-binding peptide G7-18 in cell lysates was able to specifically inhibit the association of Grb7 with the ErbB family of RTKs, in particular ErbB3, in a dose-dependent manner. These peptides will be useful in the development of targeted molecular therapeutics for cancers overexpressing Grb7 and in the development of Grb7-specific inhibitors to gain a complete understanding of the physiological role of Grb7.

Amino Acid Motifs↗

PubMed2002

Pathological and molecular assessment of sentinel lymph nodes in solid tumors.

Sentinel nodes (SNs) are the first set of nodes to receive drainage and cancer cells from a primary tumor. While there may be a single SN, frequently there are one to three or more SNs. The development of sentinel node surgery over the last decade has led to dramatic changes in the surgical approach to regional nodes draining solid tumors. The surgeon can now identify, to a level of accuracy previously impossible, the regional nodes most likely to be involved with cancer in any individual patient. This new capability comes at a cost; the principles guiding the extent of nodal surgery must be completely re-examined. The extent of surgical resection required to achieve each of the goals of regional node surgery-(1) establishing prognosis, (2) obtaining regional control, and (3) improving overall survival-may no longer be simply the default "regional node resection" and may vary depending on the clinical goals. Inseparable from this new surgical technology is the methodology employed for pathologic evaluation of SNs. It is critical that pathologists and clinicians conduct definitive clinical research directed toward defining the role and impact that SN surgery has on each of these surgical goals.

Breast Neoplasms↗