In recent years monoclonal antibodies (mAbs) recognizing tumor-associated antigens have increasingly been integrated into the clinical management of neoplasia and this trend is expected to continue. Prominent targets for antibody-based therapies are ErbB family members, including the epidermal growth factor receptor (EGFR/ErbB1) itself and ErbB2. EGFR-specific antibodies currently in clinical use typically inhibit EGFR-dependent signal transduction and induce antibody-dependent cell-mediated cytotoxicity. Administration of these agents to tumor patients is, however, associated with dose-limiting adverse side effects due to inhibition of EGFR signaling in normal tissues, specifically skin and the gastrointestinal tract. It is, therefore, highly desirable to develop antibody-based therapeutics that specifically recognize their target antigen in the tumor environment but not on normal tissues. Here we propose a novel approach to achieve this goal. It is based on the construction of antibody derivatives in which antigen recognition sites are reversibly masked by antigen fragments. Association of the antigen fragment with the mAb CDRs is expected to occlude the antigen recognition site of the fusion protein and, thus, reduce normal tissue reactivity. To restore high affinity antigen- antibody interaction at tumor sites, a proteolytic cleavage site is engineered into the linker between the blocking moiety and the antibody fragment. This site is designed to be susceptible to proteases with high activity in tumor tissues (i.e. matrix metalloproteases (MMPs)) but little or no activity in normal tissues including peripheral blood under homeostatic conditions. Upon cleavage at tumor sites, the 'unmasked'antibody is predicted to partition to the tumor-associated antigen due to mass action principles. The proposed work will test biological properties of antibody derivatives based on the EGFR antagonistic mAbs C225 and 425 that recognize distinct epitopes on the extracellular domain of the human EGFR (EGFRdIII). In preliminary work dual (Ab-Ag)2 complexes were made whereby C225 and 425 scFvs were covalently linked to EGFRdIII sequences engineered to encourage dimerization and reciprocal occlusion (crossmasking) of the antigen recognition sites of each other. Upon MMP9 treatment these complexes were shown to dissociate followed by markedly enhanced binding to purified EGFR and to tumor cells expressing EGFR. These promising results form the basis for the reformatting and in vitro testing of crossmasked Abs in an IgG-like format (Specific Aim 1) and the determination of tumor homing and growth inhibition human tumor xenotransplants in mice by such constructs in vivo (Specific Aim 2). In future work, the utility of these reagents as diagnostic agents (imaging) will be further tested in appropriate animal models, i.e. human tumor xenotransplants in mice. PUBLIC HEALTH RELEVANCE: In recent years monoclonal antibodies (mAbs) have been used increasingly in the clinical management of certain forms of cancer and select other diseases. Most mAbs currently used in cancer therapy recognize tumor-associated antigens, which are also present on normal tissues. This circumstance leads to adverse side effects that limit the application and efficacy of these agents in patients. This proposal describes a novel concept to engineer antibody derivatives that overcome these limitations. To this end, we will focus on molecular modification of two EGFR antagonistic monoclonal antibodies currently in clinical use or clinical trials, i.e. C225 and 425, respectively. If successful, the concept to be tested here may be applicable to a wide range of mAbs currently in clinical use or development.