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Superior Performance of Aptamer in Tumor Penetration over Antibody: Implication of Aptamer-Based Theranostics in Solid Tumors.

Xiang D, Zheng C, Zhou SF, Qiao S, Tran PH, Pu C, Li Y, Kong L, Kouzani AZ, Lin J, Liu K, Li L, Shigdar S, Duan W - Theranostics (2015)

Bottom Line: Targeted drug delivery to solid tumors followed by complete drug penetration and durable retention will significantly improve clinical outcomes of cancer therapy.To explore whether aptamers are superior to antibodies in terms of tumor penetration, we carried out the first comprehensive study to compare the performance of an EpCAM aptamer with an EpCAM antibody in theranostic applications.We found that the EpCAM aptamer can not only effectively penetrate into the tumorsphere cores but can also be retained by tumor sphere cells for at least 24 h, while limited tumor penetration by EpCAM antibody was observed after 4 h incubation.

View Article: PubMed Central - PubMed

Affiliation: 1. School of Medicine, Deakin University, Pigdons Road, Waurn Ponds, Victoria 3216, Australia.

ABSTRACT
Insufficient penetration of therapeutic agents into tumor tissues results in inadequate drug distribution and lower intracellular concentration of drugs, leading to the increase of drug resistance and resultant failure of cancer treatment. Targeted drug delivery to solid tumors followed by complete drug penetration and durable retention will significantly improve clinical outcomes of cancer therapy. Monoclonal antibodies have been commonly used in clinic for cancer treatment, but their limitation of penetrating into tumor tissues still remains because of their large size. Aptamers, as "chemical antibodies", are 15-20 times smaller than antibodies. To explore whether aptamers are superior to antibodies in terms of tumor penetration, we carried out the first comprehensive study to compare the performance of an EpCAM aptamer with an EpCAM antibody in theranostic applications. Penetration and retention were studied in in vitro three-dimensional tumorspheres, in vivo live animal imaging and mouse colorectal cancer xenograft model. We found that the EpCAM aptamer can not only effectively penetrate into the tumorsphere cores but can also be retained by tumor sphere cells for at least 24 h, while limited tumor penetration by EpCAM antibody was observed after 4 h incubation. As observed from in vivo live animal imaging, EpCAM aptamers displayed a maximum tumor uptake at around 10 min followed by a rapid clearance after 80 min, while the signal of peak uptake and disappearance of antibody appeared at 3 h and 6 h after intravenous injection, respectively. The signal of PEGylated EpCAM aptamers in xenograft tumors was sustained for 26 h, which was 4.3-fold longer than that of the EpCAM antibody. Consistently, there were 1.67-fold and 6.6-fold higher accumulation of PEGylated aptamer in xenograft tumors than that of antibody, at 3 h and 24 h after intravenous administration, respectively. In addition, the aptamer achieved at least a 4-time better tumor penetration in xenograft tumors than that of the antibody at a 200 μm distances from the blood vessels 3 h after intravenous injection. Taken together, these data indicate that aptmers are superior to antibodies in cancer theranostics due to their better tumor penetration, more homogeneous distribution and longer retention in tumor sites. Thus, aptamers are promising agents for targeted tumor therapeutics and molecular imaging.

No MeSH data available.


Related in: MedlinePlus

Cell binding and internalization of EpCAM aptamer and antibody in vitro. (a) Particle size of EpCAM aptamer and EpCAM antibody as determined by dynamic light scattering. (b) Determination of the equilibrium dissociation constants (K'd) of EpCAM aptamer and EpCAM antibody to HT29 cells using flow cytometry by incubating cells at varying concentrations of aptamer and antibody (1-200 nmol/L). (c) Localization of EpCAM aptamer, control EpCAM aptamer or EpCAM antibody in acidic organelles (late endosome and lysosomes). Following incubation with 100 nM EpCAM aptamer or EpCAM antibody at 37 °C for 15 min and three time washes, HT29 cells were incubated with LysoTracker® Green in the first 90 min of a further 2 h incubation followed by confocal microscopy imaging. (d) Quantification of fluorescence signals from localized aptamer or antibody in acidic organelles (late endosome and lysosomes) as in (c). (e) Specificity of EpCAM aptamer binding and internalization. Three EpCAM-positive cell lines (HT29, Huh-7 and PLC/PRF/5) and the control EpCAM-negative HEK293T cells were incubated with 100 nM EpCAM aptamer or control EpCAM aptamer at 37 °C for 15 min, followed by washing and confocal microscopy imaging. (f) Quantification of fluorescence signals from internalized aptamers in various cell lines as in (e). Ab, antibody; Apt, aptamer, RFI, relative fluorescence intensity; MFI, mean fluorescence intensity. Data are means ± SEM, n=3. Scale bar = 5 μm.
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Figure 1: Cell binding and internalization of EpCAM aptamer and antibody in vitro. (a) Particle size of EpCAM aptamer and EpCAM antibody as determined by dynamic light scattering. (b) Determination of the equilibrium dissociation constants (K'd) of EpCAM aptamer and EpCAM antibody to HT29 cells using flow cytometry by incubating cells at varying concentrations of aptamer and antibody (1-200 nmol/L). (c) Localization of EpCAM aptamer, control EpCAM aptamer or EpCAM antibody in acidic organelles (late endosome and lysosomes). Following incubation with 100 nM EpCAM aptamer or EpCAM antibody at 37 °C for 15 min and three time washes, HT29 cells were incubated with LysoTracker® Green in the first 90 min of a further 2 h incubation followed by confocal microscopy imaging. (d) Quantification of fluorescence signals from localized aptamer or antibody in acidic organelles (late endosome and lysosomes) as in (c). (e) Specificity of EpCAM aptamer binding and internalization. Three EpCAM-positive cell lines (HT29, Huh-7 and PLC/PRF/5) and the control EpCAM-negative HEK293T cells were incubated with 100 nM EpCAM aptamer or control EpCAM aptamer at 37 °C for 15 min, followed by washing and confocal microscopy imaging. (f) Quantification of fluorescence signals from internalized aptamers in various cell lines as in (e). Ab, antibody; Apt, aptamer, RFI, relative fluorescence intensity; MFI, mean fluorescence intensity. Data are means ± SEM, n=3. Scale bar = 5 μm.

Mentions: Efficient delivery of a therapeutic ligand to a tumor requires a sufficient amount of the agent to reach tumors, associated with accompanied rapid elimination from healthy tissues. Such effective tumor delivery is dictated by a number of factors including a suitable binding affinity of the ligand to its targets, the expression of the target antigen at the tumor and the size of the therapeutic ligand 14, 16, 35. The EpCAM antibody used in this study has high molecular weight (150 kDa) with a size of around 15 nm, while the EpCAM aptamer has a smaller size of around 2.09 nm (Fig. 1a). The equilibrium dissociation constant of EpCAM aptamer or the EpCAM antibody to HT29 cells was determined to be 39.42 nM and 5.18 nM, respectively (Fig. 1b). Adams et al. reported that for a tumor-targeting ligand, the K'd value lower than 1 nM does not necessarily improve tumor penetration but rather limits their further transport and perfusion in a tumor 16, 35. This phenomena was explained by the “binding site barrier” model which postulated that monoclonal antibodies with very high affinity (K'd < 1 nM) stably bind to the first interacted tumor antigens with a slow rate of dissociation, resulting in a reduction of further diffusion and thus poor penetration into tumors 36, 37. The K'ds for both the EpCAM aptamer and the antibody used in this study are well above the 1 nM threshold for “binding site barrier”. Therefore, it is extremely unlikely that the K'd of either the aptamer or the antibody used will have a profound negative influence on tumor penetration. Here, we sought to investigate the tumor penetration and retention of an aptamer with a 7-fold lower binding affinity than its antibody counterpart.


Superior Performance of Aptamer in Tumor Penetration over Antibody: Implication of Aptamer-Based Theranostics in Solid Tumors.

Xiang D, Zheng C, Zhou SF, Qiao S, Tran PH, Pu C, Li Y, Kong L, Kouzani AZ, Lin J, Liu K, Li L, Shigdar S, Duan W - Theranostics (2015)

Cell binding and internalization of EpCAM aptamer and antibody in vitro. (a) Particle size of EpCAM aptamer and EpCAM antibody as determined by dynamic light scattering. (b) Determination of the equilibrium dissociation constants (K'd) of EpCAM aptamer and EpCAM antibody to HT29 cells using flow cytometry by incubating cells at varying concentrations of aptamer and antibody (1-200 nmol/L). (c) Localization of EpCAM aptamer, control EpCAM aptamer or EpCAM antibody in acidic organelles (late endosome and lysosomes). Following incubation with 100 nM EpCAM aptamer or EpCAM antibody at 37 °C for 15 min and three time washes, HT29 cells were incubated with LysoTracker® Green in the first 90 min of a further 2 h incubation followed by confocal microscopy imaging. (d) Quantification of fluorescence signals from localized aptamer or antibody in acidic organelles (late endosome and lysosomes) as in (c). (e) Specificity of EpCAM aptamer binding and internalization. Three EpCAM-positive cell lines (HT29, Huh-7 and PLC/PRF/5) and the control EpCAM-negative HEK293T cells were incubated with 100 nM EpCAM aptamer or control EpCAM aptamer at 37 °C for 15 min, followed by washing and confocal microscopy imaging. (f) Quantification of fluorescence signals from internalized aptamers in various cell lines as in (e). Ab, antibody; Apt, aptamer, RFI, relative fluorescence intensity; MFI, mean fluorescence intensity. Data are means ± SEM, n=3. Scale bar = 5 μm.
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Figure 1: Cell binding and internalization of EpCAM aptamer and antibody in vitro. (a) Particle size of EpCAM aptamer and EpCAM antibody as determined by dynamic light scattering. (b) Determination of the equilibrium dissociation constants (K'd) of EpCAM aptamer and EpCAM antibody to HT29 cells using flow cytometry by incubating cells at varying concentrations of aptamer and antibody (1-200 nmol/L). (c) Localization of EpCAM aptamer, control EpCAM aptamer or EpCAM antibody in acidic organelles (late endosome and lysosomes). Following incubation with 100 nM EpCAM aptamer or EpCAM antibody at 37 °C for 15 min and three time washes, HT29 cells were incubated with LysoTracker® Green in the first 90 min of a further 2 h incubation followed by confocal microscopy imaging. (d) Quantification of fluorescence signals from localized aptamer or antibody in acidic organelles (late endosome and lysosomes) as in (c). (e) Specificity of EpCAM aptamer binding and internalization. Three EpCAM-positive cell lines (HT29, Huh-7 and PLC/PRF/5) and the control EpCAM-negative HEK293T cells were incubated with 100 nM EpCAM aptamer or control EpCAM aptamer at 37 °C for 15 min, followed by washing and confocal microscopy imaging. (f) Quantification of fluorescence signals from internalized aptamers in various cell lines as in (e). Ab, antibody; Apt, aptamer, RFI, relative fluorescence intensity; MFI, mean fluorescence intensity. Data are means ± SEM, n=3. Scale bar = 5 μm.
Mentions: Efficient delivery of a therapeutic ligand to a tumor requires a sufficient amount of the agent to reach tumors, associated with accompanied rapid elimination from healthy tissues. Such effective tumor delivery is dictated by a number of factors including a suitable binding affinity of the ligand to its targets, the expression of the target antigen at the tumor and the size of the therapeutic ligand 14, 16, 35. The EpCAM antibody used in this study has high molecular weight (150 kDa) with a size of around 15 nm, while the EpCAM aptamer has a smaller size of around 2.09 nm (Fig. 1a). The equilibrium dissociation constant of EpCAM aptamer or the EpCAM antibody to HT29 cells was determined to be 39.42 nM and 5.18 nM, respectively (Fig. 1b). Adams et al. reported that for a tumor-targeting ligand, the K'd value lower than 1 nM does not necessarily improve tumor penetration but rather limits their further transport and perfusion in a tumor 16, 35. This phenomena was explained by the “binding site barrier” model which postulated that monoclonal antibodies with very high affinity (K'd < 1 nM) stably bind to the first interacted tumor antigens with a slow rate of dissociation, resulting in a reduction of further diffusion and thus poor penetration into tumors 36, 37. The K'ds for both the EpCAM aptamer and the antibody used in this study are well above the 1 nM threshold for “binding site barrier”. Therefore, it is extremely unlikely that the K'd of either the aptamer or the antibody used will have a profound negative influence on tumor penetration. Here, we sought to investigate the tumor penetration and retention of an aptamer with a 7-fold lower binding affinity than its antibody counterpart.

Bottom Line: Targeted drug delivery to solid tumors followed by complete drug penetration and durable retention will significantly improve clinical outcomes of cancer therapy.To explore whether aptamers are superior to antibodies in terms of tumor penetration, we carried out the first comprehensive study to compare the performance of an EpCAM aptamer with an EpCAM antibody in theranostic applications.We found that the EpCAM aptamer can not only effectively penetrate into the tumorsphere cores but can also be retained by tumor sphere cells for at least 24 h, while limited tumor penetration by EpCAM antibody was observed after 4 h incubation.

View Article: PubMed Central - PubMed

Affiliation: 1. School of Medicine, Deakin University, Pigdons Road, Waurn Ponds, Victoria 3216, Australia.

ABSTRACT
Insufficient penetration of therapeutic agents into tumor tissues results in inadequate drug distribution and lower intracellular concentration of drugs, leading to the increase of drug resistance and resultant failure of cancer treatment. Targeted drug delivery to solid tumors followed by complete drug penetration and durable retention will significantly improve clinical outcomes of cancer therapy. Monoclonal antibodies have been commonly used in clinic for cancer treatment, but their limitation of penetrating into tumor tissues still remains because of their large size. Aptamers, as "chemical antibodies", are 15-20 times smaller than antibodies. To explore whether aptamers are superior to antibodies in terms of tumor penetration, we carried out the first comprehensive study to compare the performance of an EpCAM aptamer with an EpCAM antibody in theranostic applications. Penetration and retention were studied in in vitro three-dimensional tumorspheres, in vivo live animal imaging and mouse colorectal cancer xenograft model. We found that the EpCAM aptamer can not only effectively penetrate into the tumorsphere cores but can also be retained by tumor sphere cells for at least 24 h, while limited tumor penetration by EpCAM antibody was observed after 4 h incubation. As observed from in vivo live animal imaging, EpCAM aptamers displayed a maximum tumor uptake at around 10 min followed by a rapid clearance after 80 min, while the signal of peak uptake and disappearance of antibody appeared at 3 h and 6 h after intravenous injection, respectively. The signal of PEGylated EpCAM aptamers in xenograft tumors was sustained for 26 h, which was 4.3-fold longer than that of the EpCAM antibody. Consistently, there were 1.67-fold and 6.6-fold higher accumulation of PEGylated aptamer in xenograft tumors than that of antibody, at 3 h and 24 h after intravenous administration, respectively. In addition, the aptamer achieved at least a 4-time better tumor penetration in xenograft tumors than that of the antibody at a 200 μm distances from the blood vessels 3 h after intravenous injection. Taken together, these data indicate that aptmers are superior to antibodies in cancer theranostics due to their better tumor penetration, more homogeneous distribution and longer retention in tumor sites. Thus, aptamers are promising agents for targeted tumor therapeutics and molecular imaging.

No MeSH data available.


Related in: MedlinePlus