Targeting of humanized antibody D93 to sites of angiogenesis and tumor growth by binding to multiple epitopes on denatured collagens

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 α1(IV) P1337–Y1352 that could inhibit binding of both D93 and HUI77 to denatured collagen IV in a concentration-dependent manner. A 9-mer peptide corresponding to α1(IV) G1344–Y1352 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 KD of 1–10 μM 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.