Bi-specific Aptamers Mediating Tumor Cell Lysis

Abstract

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...

Nucleic acid aptamers: an emerging frontier in cancer therapy

“…However, while the use of antibodies is always associated with complicated and time-consuming genetic engineering, limited stability, and high cost, In contrast, bi-specific aptamer or aptamer-drug conjugate engineering is fairly straightforward as a consequence of facile modification, high stability and programmability. 57, 61–62, 72, 79, 113–114 With further understanding and improvement of aptamers, as well as the advancement of DNA synthesis technology, nucleic acid aptamers are poised to revolutionize the next generation of therapeutics in the context of disease diagnosis and prevention.…”

Methodologies for the isolation of alternative binders with improved clinical potentiality over conventional antibodies

“…Instead, the fraction of singly bound antigens is reduced with the physical vicinity of two independent binders on the same molecule. Biand multi-specific antibodies and aptamers constitute potent reagents since they improve the binder avidity by increasing the binding valency and allow for in vivo inhibition of two independent epitopes and even signaling pathways (Figure 1) (Conrath et al, 2001;Stone et al, 2007;Hmila et al, 2010;Jackman et al, 2010;Boltz et al, 2011). The advantages of targeting two functional domains of the same protein are still to be explored in detail but the following example clearly indicates the potential of this approach.…”

Development of Cell-SELEX Technology and Its Application in Cancer Diagnosis and Therapy

“…The first cell-SELEX process in hybrid-SELEX aimed to select aptamers against target in its native state on the surface of the cells. The following additional rounds of selection with purified target were to enrich the high-affinity aptamers, which were rare in cell aptamer pool [39]. …”