A humanized, affinity-matured
IgG1 antibody, called D93, and its parental murine
IgM HUI77 have been shown to specifically bind denatured
collagens and thereby inhibit angiogenesis and
tumor growth in various animal models. In this study, we have identified
epitopes for both HUI77 and D93 on human
collagen type IV. Several tryptic D93-binding
peptides were identified by Western blot analysis and
protein sequencing.
Epitopes for D93 were ultimately identified by screening a synthetic 16-mer
peptide array spanning immunoreactive tryptic
peptides. D93 reacted with a
peptide corresponding to alpha1(IV) P(1337)-Y(1352) that could inhibit binding of both D93 and HUI77 to denatured
collagen IV in a concentration-dependent manner. A 9-mer
peptide corresponding to alpha1(IV) G(1344)-Y(1352) showed maximum inhibition of D93 and HUI77 antibody binding to denatured
collagen IV, and was critically dependent on the presence of
hydroxyproline. D93 bound with similar affinity to denatured
collagen IV and synthetic
peptides with a K(D) of 1-10 microM for monovalent and of 30-63 nM for bivalent binding. Potential
epitopes for D93 are highly repeated in multiple
collagen types of diverse vertebrate species explaining reactivity of D93 with denatured
collagens types I-V from chicken to man. Our data suggest that D93 inhibits angiogenesis and
tumor growth by blockade of cryptic bioactive signals on proteolyzed
collagens with importance for growth of
tumors and new blood vessels.