Antibodies are versatile proteins that can be raised to recognize a nearly limitless array of target molecules with exquisite specificity, making them useful for the targeted therapy of cancers that express surface antigens that differentiate them from other cells. The target molecules recognized by antibodies can range from naturally occurring proteins or peptides to synthetic polymers and small molecules. Antibody fragments retain antibody function, and Fab, Fc, and variable regions (scFv) have been incorporated into biopharmaceuticals for the targeted imaging or therapy of diseases. Our lab has been investigating the application of bispecific antibodies that incorporate fragments of the mutant monoclonal antibody 2D12.5 G54C, which has been engineered in the binding pocket to bind irreversibly with moieties of its metal chelate hapten, S-2-(4-aminobenzyl)-1, 4, 7, 10-tetraazacyclododecanetetraacetate (DOTA) (Corneillie, et al. 2004. DOI:10.1021/bc049824m.) In Chapter one, the application of anti-cell surface protein targeting antibodies and antibody fragments for the targeted therapy of cancers is discussed, with a special focus on the potential applications of the covalent probe capturing monoclonal antibody 2D12.5 G54C for pretargeted radioimmunotherapy of the hematologic malignancies Acute Myeloid Leukemia (AML) and Non Hodgkin's Lymphoma (NHL). This work was modified from Day, J.J., Marquez, B.M., Beck, H.E., Aweda, T.A., Gawande, P.D., and Meares, C.F. Current Opinion in Chemical Biology (2010). Chapter two discusses the design, engineering and characterization of a bispecific tetravalent antibody (scFv)2-Fc-(scFv)2, called CTBD, that combines the CD45 targeting scFv of mAb BC8 with the covalent radiometal DOTA probe capturing properties of mAb 2D12.5 G54C, with applications for the pretargeted imaging and therapy of Acute Myeloid Leukemia. The fusion protein was expressed in mammalian cells, and purified to a high degree using recombinant protein A. The CD45 targeting ability of CTBD was confirmed by flow cytometry, by targeting Ramos (Burkitt's Lymphoma) cells. The metal-benzyl-DOTA probe binding properties of CTBD were confirmed using Yttrium-DOTA-ELISA and the irreversible metal-DOTA probe capturing capabilities was investigated using Western blotting techniques. The results of the infinite metal-DOTA probe binding assays suggest that the CTBD fusion is expressed with a free-sulfhydryl in the binding pocket of the scFv 2D12.5 G54C, indicating that electrophilic or disulfide bonded-benzyl DOTA moieties should be used as irreversible capture probes for in vivo experiments. Described in this chapter is also the purification and characterization of an Yttrium-ABD-(PEG)8-maleimide reagent that was synthesized for applications in constructing Yttrium-ABD-labeled clearing agents for in vivo studies, or for labeling fluorescent proteins with Yttrium-ABD as reagents for assaying the bispecific binding of CTBD using flow cytometry. The synthesis of a reversible 5 kDa PEG-DOTA reagent for reversible PEGylation of the CTBD fusion protein for solubility enhancement of the CTBD fusion protein is also described. Chapter three discusses challenges faced when trying to apply antibody based therapies in vivo, and describes the modification of the pharmacokinetic properties of a bispecific, N-terminal Anti-CD20 targeting (scFv1F5)2-chimeric 2D12.5 G54C Fab fusion protein (FP) that was designed for applications in the pretargeted radioimmunotherapy of NHL. The FP performed well when assayed using in vitro methods, binding with both targets, but performed poorly in vivo, clearing from the blood in 1.4 hours and localizing mainly in the liver, kidneys and spleen of nude mice bearing Ramos tumor xenografts (McCoy, M.R., 2009. AAT 3362500). The FP was modified using amine specific PEGylation using a branched 40 kDa PEG-NHS reagent, and purified using cation exchange. The PEGylated FP proved difficult to assay using standard techniques used to assay the properties of unmodified FP. PEGylated-FP was labeled with 111In-MABD and studied in mice where it extended the blood half-life to roughly 12 hours and greatly improved biodistribution properties but failed to improve CD20 targeting capabilities. Strategies to improve future outcomes of PEGylation are discussed. Chapter four discusses the humanization of the 2D12.5 G54C antibody and the incorporation of the humanized 2D12.5 G54C Fab into a humanized 2D12.5 G54C Fab-(scFv BC8)2 fusion protein, designated CLCH-BC8. The CLCH-BC8 protein was expressed in Drosophila (S2) cells, using the pMT/BiP/V5/6His-A, CuSO4 inducible protein expression system from Invitrogen (Carlsbad, CA.) The benefits and detriments of using the S2 cells as an expression host for 2D12.5 G54C fusion proteins are briefly discussed. Chapter five includes the publication Rates and equilibria for probe capture by an antibody with infinite affinity, with supplemental information that confirms covalent probe capture by DAbR1 by Tolulope A. Aweda, Heather Beck, Anna Wu, Liu Wei, Wolfgang Weber,§ and Claude F. Meares*, with a supplementary chapter included that describes the investigation of the probe capturing properties of DAbR1 reporter gene using cultured mutant U87 cells. Using these supplementary methods, it was determined that DAbR1 is expressed with the 2D12.5 G54C as a free sulfhydryl, and binds covalently with AABD(90Y). The results of these studies helped establish valuable methods for assaying 2D12.5 G54C in our lab, provided a proof of concept for the application of 2D12.5 G54C and AABD(Y) for cancer pretargeting, and helped direct the future design of DOTA probes for in vivo experiments using DAbR1 based reporter probes (Aweda, T.A., et al. DOI 10.1021/bc2002049.)