N-telopeptide of type II collagen interacts with annexin V on human chondrocytes.

Type II collagen binds to chondrocytes through integrins and annexin V. While the potential integrin binding sites have been identified, it is unclear which domains bind to annexin V. Proteolytic fragments of collagen are known to modulate cell signaling pathways resulting in degradation of articular cartilage; it is unknown whether annexin V binds to the fragments. The focus of our study was to determine the binding of type II collagen and its fragments to chondrocytes using flow cytometry and fluorescence microscopy. The N-telopeptide binds to annexin V, whereas the C-telopeptide and triple helical peptides do not. These data suggest that the binding of the N-telopeptide of type II collagen is through annexin V, whereas binding of the C-telopeptide and the triple helical peptide to the surface of chondrocytes are potentially facilitated through other collagen receptors, such as integrins or cell-associated matrix proteins.

In vivo expression of a GST-fusion protein mediates the rapid generation of affinity matured monoclonal antibodies using DNA-based immunizations.

Previously we demonstrated the rapid generation of affinity matured monoclonal antibody (MAb) producing cell lines following gene gun delivery of DNA using a mammalian expression vector (pAlpha/hFc), which enables the expression of human Fc-chimera proteins in vivo. Here we compare the pAlpha/hFc vector to modified vectors that replace human IgG(1) with either a Glutathione-S-Transferase (GST) fusion protein or a mouse IgG(2c) (mFc) fusion protein. We report that in vivo expression of a GST-chimera results in the rapid generation of affinity matured MAbs, comparable with antibodies raised using the pAlpha/hFc vector, that were reactive with annexin V. The mFc vector failed to induce early antigen-specific B-cell responses suitable for MAb development.

Antibody targeting of the EphA2 tyrosine kinase inhibits malignant cell behavior.

EphA2 is a transmembrane receptor tyrosine kinase that is up-regulated on many aggressive carcinoma cells. Despite its overexpression, the EphA2 on malignant cells fails to bind its ligand, ephrinA1, which is anchored to the membrane of adjacent cells. Unlike other receptor kinases, EphA2 demonstrates kinase activity that is independent of ligand binding. However, ligand binding causes EphA2 to negatively regulate tumor cell growth and migration. Herein, we translate knowledge of EphA2 into strategies that selectively target malignant cells. Using a novel approach to preserve extracellular epitopes and optimize antibody diversity, we generated monoclonal antibodies that identify epitopes on the extracellular domain of EphA2. EphA2 antibodies were selected for their abilities to inhibit behaviors that are unique to metastatic cells while minimizing damage to nontransformed cells. A subset of EphA2 monoclonal antibodies were found to inhibit the soft agar colonization by MDA-MB-231 breast tumor cells but did not affect monolayer growth by nontransformed MCF-10A breast epithelial cells. These EphA2 antibodies also prevented tumor cells from forming tubular networks on reconstituted basement membranes, which is a sensitive indicator of metastatic character. Biochemical analyses showed that biologically active antibodies induced EphA2 phosphorylation and subsequent degradation. Antisense-based targeting of EphA2 similarly inhibited soft agar colonization, suggesting that the antibodies repress malignant behavior by down-regulating EphA2. These results suggest an opportunity for antibody-based targeting of the many cancers that overexpress EphA2. Our studies also emphasize how tumor-specific cellular behaviors can be exploited to identify and screen potential therapeutic targets.