In this study, we have selected TAs that specifically bind to the CD44’s HABD with the goal of using these TAs for targeted delivery of therapeutic drugs to cancer cells expressing CD44. Targeted delivery of anti-cancer drugs specifically to the cancer cells will greatly enhance the efficacy and reduce adverse effects. Since CD44 is a membrane protein that is over-expressed on the plasma membrane of cancer cells and internalized into the cells upon ligand binding, CD44 targeted delivery presents a powerful strategy for selective cancer cell targeting. Since the HABD is highly conserved among all the splicing variants and all splicing variants contain the HABD in their extracellular domain, TAs specific to the HABD are valuable tools to recognize cancer cells that express CD44 splicing variants.
Previous studies have reported using HA for selective targeting of cancer cells expressing CD44. HA-bound liposomes were used to deliver encapsulated doxorubicin to tumor cells expressing high levels of CD44 (41
). However, there are several limitations in using HA for targeted delivery. HA is naturally present in the human body and can bind to several proteins besides CD44 (42
), leading to non-specific binding to other proteins. Our TAs showed specific binding to CD44, and failed to bind to another HA binding protein, HABP. HA is a large polymer and difficult to synthesize compared to oligonucleotides. Furthermore, several studies have reported that HA interactions with the CD44 splice variants are diminished compared to those with CD44S (20
). The binding constants for the selected TAs reported in this study, are significantly lower compared to the binding constants for HA to CD44. Since TAs show higher affinity and specificity than the HA, TAs would be a better ligand of HABD to achieve higher level of refined targeting.
HA is known to regulate tumor progression through different mechanisms. Several studies have shown that HA binding activates CD44 and stimulates tumor progression (11
). HA, in its very large, high molecular weight form is part of the extra-cellular matrix and is reported to promote cancer initiation and progression by activating signaling pathways and host-tumor interactions (45
). Since the TAs are selected against the HABD of CD44, binding of the TAs would disrupt any further binding of HA to CD44 and could potentially interfere with tumor progression that depends on the HA binding mediated activation of CD44. While the high molecular weight forms of HA have been shown to promote tumor progression, the small, low molecular weight forms of HA have been shown to suppress tumor progression (47
). The TAs are similar to the low molecular weight forms of the HA, and therefore, might not trigger the same signaling pathways as the high-molecular weight HAs, and promote tumor progression. Furthermore, binding of small HA oligomers to the surface of CD44 appears to enhance the HA uptake (48
). The selected TAs, being small oligonuelcotides, might increase the internalization upon binding to CD44. TAs have several advantages over unmodified oligonucleotide aptamers. Thio-modified oligonucleotides exhibit higher level of resistance to digestion by cellular nucleases (28
). TAs also show tighter binding towards target proteins compared to unmodified aptamers (30
). It appears that the thiophosphates do not bind sodium ions as effectively as the unmodified phosphates, and thus the thio-substituted phosphate esters act as nearly bare anions (50
). Since less energy is required to strip the cations from the backbone of thio-substituted oligonucleotides, these agents can in principle bind more tightly to proteins. However, complete thio-substitution of oligonucleotides could lead to non-specific binding to other cellular proteins (31
). Therefore, to achieve selective, high-affinity binding, optimization of the number of thio substitutions is critical. Our combinatorial selection methods allow us to select TAs with hybrid backbones where the number and the position of the sulfur substitutions are optimized. This approach allows us to simultaneously select for both the sequence and the best monothiophosphate substitutions. In contrast, almost all modified aptamers are first selected by SELEX and then post-selection modified. As we have shown previously (49
), monothiothiophosphate substitutions can perturb both the structure and interaction with the target and therefore, it is better to select both sequence and modifications simultaneously. While the TAs selected showed specific binding to CD44, unmodified oligonucleotides containing the same primary sequences failed to bind to the CD44 proteins or to the CD44+ cells (data not shown). This observation shows that sulfur substitution at specific positions of the oligonucleotides led to specificity and higher affinity binding to the CD44.
We have used recombinant CD44-HABD for the iterative selection cycles. For membrane binding assays, we have used both the CD44-Fc chimera and CD44-HABD recombinant protein. It has been shown that the CD44 HABD (20–178) and CD44-Fc are structurally similar and share a common tertiary fold. Based on the similar reactivity of CD44-HABD and CD44-Fc towards the conformation sensitive mAbs BRIC-235 and F10.44.2, Teriete et al (52
) showed these two constructs are structurally similar. The concentration-dependent binding of HA to CD44-Fc is very similar to that of CD44-HABD. Our data demonstrated that TAs selected against CD44-HABD bound to both CD44-HABD and CD44-Fc with similar dissociation constants, supporting the notion of bioequivalence between these two protein constructs.
All TAs showed rapid and selective binding to cultured CD44+ cells, but not to the CD44− cells. Although pre-incubation of the CD44+ cells with HA (1 mM, 10 kD) failed to block the binding of CD44 TA (data not shown), it does not preclude the HABD selective binding of CD44 TA since there are substantial difference in the binding affinities between TA and HA to CD44. We identified TAs that bind to the CD44 HABD at nanomolar affinity, significantly higher binding than its natural ligand HA, and will be of great interest for further development as a targeting or imaging agent to deliver therapeutic payloads for cancer tissues.