Antibody-dependent cellular cytotoxicity plays a pivotal role in antibody-based tumor therapies and is based on the recruitment of natural killer cells to antibody-bound tumor cells via binding of the Fc␥ receptor III (CD16). Here we describe the generation of chimeric DNA aptamers that simultaneously bind to CD16␣ and c-Met, a receptor that is overexpressed in many tumors. By application of the systematic evolution of ligands by exponential enrichment (SELEX) method, CD16␣ specific DNA aptamers were isolated that bound with high specificity and affinity (91 pM-195 nM) to their respective recombinant and cellularly expressed target proteins. Two optimized CD16␣ specific aptamers were coupled to each of two c-Met specific aptamers using different linkers. Bi-specific aptamers retained suitable binding properties and displayed simultaneous binding to both antigens. Moreover, they mediated cellular cytotoxicity dependent on aptamer and effector cell concentration. Displacement of a bi-specific aptamer from CD16␣ by competing antibody 3G8 reduced cytotoxicity and confirmed the proposed mode of action. These results represent the first gain of a tumoreffective function of two distinct oligonucleotides by linkage into a bi-specific aptamer mediating cellular cytotoxicity.Aptamers are structured single-stranded oligonucleotides that can bind to a large variety of targets with high affinity and specificity (1, 2). Aptamers can be isolated by an in vitro selection and an evolution process referred to as systematic evolution of ligands by exponential enrichment (SELEX) 2 (3, 4). Because aptamers have the capacity to inhibit protein-protein interactions with potencies similar to those observed with antibodies, aptamers can also trigger inhibition signals, e.g. by blocking receptor multimerization, and consequently act as therapeutic antagonists. Reversely, bi-and multivalent aptamers can activate co-stimulatory receptors, e.g. to enhance T cell reactivity (5, 6). Finally, aptamers can be applied in ligand-based targeted therapies to specifically deliver cytotoxic payloads (7,8) or siRNA (9) to tumor cells. Monoclonal antibodies serve as established and successful tumor therapeutics. However, naturally bivalent antibody formats comprise the risks of immunogenicity (10) and undesired activation by receptor dimerization (11). Development of monovalent therapeutic antibodies is elaborate and time-intensive (MetMAb (12)). Although antibodies exceed aptamers with proof as therapeutic molecules, high stability, and good pharmacokinetics, the potential advantages of aptamers are a rapid optimization, cost-effective and uniform synthesis, and a high probability of an absence of immunogenicity (5, 13). Approval of Macugen (pegaptanib sodium (14)) as the first therapeutic aptamer in 2007 as well as promising approaches in preclinical development and clinical trials (15, 16) only a few years after inception of the technology indicate aptamers as a promising new class of targeted therapeutics.Antibody-dependent cellular cytotoxicity (A...

Based on the crystal structure of a natural protein substrate for microbial transglutaminase, an enzyme that catalyzes protein crosslinking, a recognition motif for site-specific conjugation was rationally designed. Conformationally locked by an intramolecular disulfide bond, this structural mimic of a native conjugation site ensured efficient conjugation of a reporter cargo to the therapeutic monoclonal antibody cetuximab without erosion of its binding properties.

Spontaneous isopeptide bond formation, a stabilizing posttranslational modification that can be found in gram-positive bacterial cell surface proteins, has previously been used to develop a peptide-peptide ligation technology that enables the polymerization of tagged-proteins catalyzed by SpyLigase. Here we adapted this technology to establish a novel modular antibody labeling approach which is based on isopeptide bond formation between two recognition peptides, SpyTag and KTag. Our labeling strategy allows the attachment of a reporting cargo of interest to an antibody scaffold by fusing it chemically to KTag, available via semi-automated solid-phase peptide synthesis (SPPS), while equipping the antibody with SpyTag. This strategy was successfully used to engineer site-specific antibody-drug conjugates (ADCs) that exhibit cytotoxicities in the subnanomolar range. Our approach may lead to a new class of antibody conjugates based on peptide-tags that have minimal effects on protein structure and function, thus expanding the toolbox of site-specific antibody conjugation.

The human receptor tyrosine kinase c-Met plays an important role in the control of critical cellular processes. Since c-Met is frequently over expressed or deregulated in human malignancies, blocking its activation is of special interest for therapy. In normal conditions, the c-Met receptor is activated by its bivalent ligand hepatocyte growth factor (HGF). Also bivalent antibodies can activate the receptor by cross linking, limiting therapeutic applications. We report the generation of the RNA aptamer CLN64 containing 2’-fluoro pyrimidine modifications by systematic evolution of ligands by exponential enrichment (SELEX). CLN64 and a previously described single-stranded DNA (ssDNA) aptamer CLN3 exhibited high specificities and affinities to recombinant and cellular expressed c-Met. Both aptamers effectively inhibited HGF-dependent c-Met activation, signaling and cell migration. We showed that these aptamers did not induce c-Met activation, revealing an advantage over bivalent therapeutic molecules. Both aptamers were shown to bind overlapping epitopes but only CLN3 competed with HGF binding to cMet. In addition to their therapeutic and diagnostic potential, CLN3 and CLN64 aptamers exhibit valuable tools to further understand the structural and functional basis for c-Met activation or inhibition by synthetic ligands and their interplay with HGF binding.