Aptamer Specificity

The specificity of a therapeutic agent is critical as it will drive both the potency of the molecule and its side effect profile. Aptamers are highly discriminating binders; the factors that contribute to their potent affinities bolster highly specific binding as well (Eaton et al., 1995). Generally, aptamers selected against a member of a family of proteins are specific for the target protein versus other family members. For example, an aptamer (14F3'T) to keratinocyte growth factor (KGF), a member of the fibroblast growth factor (FGF) family, discriminates by over 10000-fold between KGF and other FGF family members, all of which, notably, are heparin-binding proteins (Pagratis et al., 1997). In fact, aptamers against heparin-binding members of the growth factor family are without exception highly specific, as illustrated by the examples in Table 17.2. Aptamers to VEGF, PDGF-AB, KGF, and bFGF each bind their targets with high affinity and discriminate against other proteins in that set (Green et al., 1995; Jellinek et al., 1995; Pagratis et al., 1997). Similarly, aptamers to heparin-binding members of the coagulation cascade are remarkably specific for their targets versus other members of the coagulation cascade (Bock et al., 1992; Rusconi et al., 2000, 2002).

This degree of discrimination is not limited to aptamers that target heparin-binding proteins. An anti-L-selectin aptamer exhibits approximately 8000- to 15000-fold and 200- to 500-fold specificity versus P-selectin and E-selectin, respectively (O'Connell et al., 1996) and a P-selectin aptamer is similarly discriminating (Jenison et al., 1998).

As discussed above, specific binding is a general requirement of therapeutic aptamers. While most aptamers bind their targets specifically, discrimination between homologous targets, for example, proteins that share a subdomain, can be programmed into the aptamer through a subtractive SELEX process. Using this procedure, the undesired aptamer target is incubated with the random pool and nucleic acid:protein complexes are then partitioned and discarded. The non-binding pool members are then incubated with the desired target and binders are isolated and amplified. The pool is subjected to iterative rounds of

Table 17.2 Example specificities of aptamers to heparin-binding proteins

Aptamer

VEGF

bFGF

KGF

PDGF-AB

Thrombin

Reference

Kd (nmol/L)

Kd (nmol/L)

Kd (nmol/L)

Kd (nmol/L)

Kd (nmol/L)

VEGF

0.14

286

NA

91

3060

Green

(NX-213)

et al., 1995

bFGF

426

0.35

450

140

>10 mmol/L

Jellinek

(m21a)

et al., 1995

KGF

N.A.

10

0.0008

50

>10 mmol/L

Pagratis

(14F3'T)

et al., 1997

this process, until it is enriched with molecules that discriminate between the desired and undesired targets. White et al. (2003) used this technique to generate an aptamer that discriminates between the Tie2 receptor tyrosine kinase ligands an-giopoietin-2 and angiopoietin-1, which share 60% sequence identity. When aptamers that block specific antibody:antigen binding were sought for applications in autoimmunity, pooled polyclonal antibodies were included in a subtractive steps to eliminate aptamers that target the Fc domain (Lee and Sullenger, 1996; Kim et al., 2003).

Demonstration of efficacy in an animal disease model is a key step in the development of therapeutic agents. Aptamer cross-reactivity between the human target and the homologous protein from the model species will therefore be a key feature of many candidate aptamers. For example, cross-reactivity between the human and canine homologs of thrombin enabled assessment of ARC183 as an anticoagulant in a canine model of coronary artery bypass graft (CABG) surgery (DeAnda et al., 1994). In cases where the target sequence identity is not well conserved between species, the exquisite specificity of aptamers may pose a challenge. ADR58 binds with 7 nmol/L affinity to human OSM but shows no detectable binding to murine OSM (43% identical), limiting its evaluation in murine models of rheumatoid arthritis (Rhodes et al., 2000). In such cases, aptamers to the human and relevant non-human target may be developed in parallel, with the second aptamer acting as a surrogate for the anti-human aptamer in animal models. Alternatively, xenogenic models may be used where applicable.

Finally, in some cases SELEX may be used to build species cross-reactivity into a candidate aptamer. For example, White et al. (2001) used porcine and human thrombin as the target protein in alternating rounds of SELEX and identified cross-reactive aptamers. This was not necessarily a stringent test of the method, since broadly cross-reactive anti-thrombin aptamers have been selected without using this technique (Bock et al., 1992). In an earlier example, Janjic and Gold (2004) demonstrated the utility of sequential SELEX with homologous targets in their efforts to identify aptamers that specifically recognize VEGF receptor 2, encoded by the KDR (human) and flk-1 (mouse) genes. Five rounds of SELEX against the extracellular domain of the KDR protein yielded individual clones which bound with nanomolar affinity to the human but not the mouse protein. Starting with the KDR-enriched pool, five additional rounds of SELEX were carried out using flk-1 as a target. A different set of sequences were isolated as a result and many individual clones in this second effort successfully bound to both human and mouse proteins. This "cross-SELEX" approach is likely to be successful when used with targets which have low overall homology but that contain highly conserved functional regions or subdomains (e.g. Lorger et al., 2003).

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