Irreversible chemical programming of monoclonal aldolase antibody (mAb) 38C2 has been accomplished with β-lactam equipped mono- and bifunctional targeting modules, including a cyclic-RGD peptide linked to either the peptide (D-Lys6)-LHRH or another cyclic RGD unit and a small-molecule integrin inhibitor SCS-873 conjugated to (D-Lys6)LHRH. We also prepared monofunctional targeting modules containing either cyclic RGD or (D-Lys6)-LHRH peptides. Binding of the chemically programmed antibodies to integrin receptors α(v)β(3) and α(v)β(5) and to the luteinizing hormone releasing hormone receptor were evaluated. The bifunctional and bivalent c-RGD/LHRH and SCS-783/LHRH, the monofunctional and tetravalent c-RGD/c-RGD, and the monofunctional bivalent c-RGD chemically programmed antibodies bound specifically to the isolated integrin receptor proteins as well as to integrins expressed on human melanoma M21 cells. c-RGD/LHRH, SCS-783/LHRH, and LHRH chemically programmed antibodies bound specifically to the LHRH receptors expressed on human ovarian cancer cells. This approach provides an efficient, versatile, and economically viable route to high-valency therapeutic antibodies that target defined combinations of specific receptors. Additionally, this approach should be applicable to chemically programmed vaccines.

Irreversible chemical programming of monoclonal aldolase antibody (mAb) 38C2 has been accomplished with β-lactam equipped targeting modules. A model study was first performed with β-lactam conjugated to biotin. This conjugate efficiently and selectively modified the catalytic site lysine (LysH93) of mAb 38C2. We then conjugated a β-lactam to a cyclic-RGD peptide to chemically program mAb 38C2 to target integrin receptors α v β 3 and α v β 5 . The chemically programmed antibody bound specifically to the isolated integrin receptor proteins as well as the integrins expressed on human melanoma cells. This approach provides an efficient and versatile solution to irreversible chemical programming of aldolase antibodies.Monoclonal antibody (mAb) 38C2 was generated by reactive immunization with a 1,3-diketone hapten. 1 This antibody possesses a lysine residue at position 93 of the heavy chain, LysH93, that has an unusually low pK a of ~6.0 and is essential for the catalytic mechanism of the antibody. 2 mAb 38C2 can be chemically programmed to selectively bind to cells or other targets by selective reaction of LysH93 with β-diketone (Scheme 1A) or an acetone aldol adduct of a vinylketone (Scheme 1B) linker equipped with a targeting ligand or module. 3-8 Many of our studies have used antagonists of α v β 3 and α v β 5 integrins as targeting modules for chemical programming of aldolase antibody specificity. 3-7 In the first case, a reversible covalent attachment of a diketone compound is achieved through the formation of an imine, which isomerizes to the more stable enaminone derivative. In the latter case, an acetone aldol adduct of the vinylketone is used as a prolinker, because the vinylketone linker is highly reactive. mAb 38C2 catalyzes the retro-aldol reaction of the prolinker to give free vinylketone linker, which then undergoes Michael addition, in situ, with the catalytic Lys residue in the antibody binding site. This approach provides irreversible attachment of the targeting modules; however, preparation of the prolinker equipped targeting modules requires complicated multistep synthesis and is not compatible with a wide range of substrates.In earlier studies we had explored a β-lactam based modification strategy for covalent attachment of thiazolium cofactor into the active site of aldolase antibodies in order to prepare the first thiazolium-dependent catalytic antibodies. 9 Given the success of this study and the chemical stability of the resulting covalent modification, we were encouraged to examine this approach as a route to chemically programmed antibodies. We now report the selective reaction of the catalytic site LysH93 with β-lactam-equipped targeting modules (Scheme 1C); the synthesis of the targeting module was straightforward and this strategy provides for irreversible Correspondence to: Carlos F. Barbas, III. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manu...

CD3 bispecific therapies retargeting T cells to tumors have recently demonstrated striking activity in patients. Several CD3 bispecific antibodies directed against various tumor targets are currently being investigated in the clinic across different tumors. One limitation of these therapies is the risk of target-related toxicity due to low-level expression of tumor antigen in normal tissue. In this study we have engineered a bispecific CD3/HER2 FynomAb, COVA420, which redirects T cells with high potency and selectivity to tumor cells with high HER2 expression in vitro and in vivo. COVA420 activity depends on high HER2 density as no activity was observed on cells with lower HER2 levels as found in human normal tissue. These results suggest that COVA420 may spare normal tissue expressing low levels of HER2 while still having uncompromised efficacy on tumor cells OPEN ACCESSAntibodies 2015, 4 427 with high HER2 expression. This concept may be applied to other cancer antigens that otherwise cannot be targeted by T cell redirecting approaches, and may therefore expand the applicability of CD3 bispecific FynomAbs to a larger number of solid tumors.

Chemically programmed antibodies represent a new class of biologic drugs that acquire their specificity through chemistry rather than through biology. To date, this approach has used small molecules and peptides to direct targeting and to extend the pharmacokinetics and otherwise enhance the biological function of the small molecule or peptide through Fc-based mechanisms of the antibody. However, other classes of therapeutically active molecules, such as aptamers, should benefit from antibody conjugation and the chemically programmed antibody approach. Aptamers are structured nucleic acid ligands often selected using the 'selective evolution of ligands by exponential enrichment' (SELEX) procedure. Although aptamers are a promising class of therapeutics because of their excellent binding and inhibitory properties, only a single VEGFtargeting aptamer is an approved drug. For in vivo applications, aptamers suffer from low chemical stability (these molecules are readily degraded by nucleases in serum) and poor pharmacokinetic properties (circulatory half lives are on the order of several minutes). Nuclease resistance can be enhanced significantly by incorporating 2' ribose modified nucleobases; 2'-O-methyl modified oligonucleotides have acceptable serum stabilities. Other oligonucleotide modifications are also being explored to solve this difficult problem.Here, we demonstrate for the first time site-specific conjugation of an aptamer to the aldolase antibody 38C2 to produce aptamer programmed cpAbs. Conjugation of the VEGF-targeting aptamer ARC245 to the well-characterized chemically programmable antibody 38C2 resulted in a biologically active aptamer-antibody conjugate that had significantly increased functional affinity and circulatory half-life as compared to the free aptamer. The aptamer-cpAb strategy developed here should be general and readily transferable to other aptamers. Aptamer-based cpAbs of the type developed here represent a promising new class of aptamer immunotherapeutics that combine the favourable characteristics of aptamers with those of antibodies. The extent and site of PEGylation must be evaluated for each aptamer since not all aptamers tolerate chemical conjugation to PEG molecules above a certain size. Keywords[13] Antibody programming could provide an attractive alternative to current strategies for extending aptamer half lives. By attaching an aptamer to the chemically programmable antibody the therapeutically valuable binding specificity of the aptamer should be combined with the bivalency, the long in vivo half-life and effector functions of the antibody. In order to explore the potential of aptamer-based programming of antibodies, we synthesized the β-lactam based heterobifunctional linker 3 (Fig. 2) with a reactive maleimide portion for attachment to a thiol modified aptamer.[14] The synthetic scheme for antibody aptamer conjugation is outlined in Figure 3. For our proof of concept experiments, we chose the thiol-modified anti-VEGF aptamer ARC245 since this aptamer is fully 2-O...