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Aptamer nanostructures dual-targeted to the HER receptor family for cancer therapy

Periodic Reporting for period 1 - ADHERE (Aptamer nanostructures dual-targeted to the HER receptor family for cancer therapy)

Reporting period: 2016-03-01 to 2018-02-28

In cancer immunotherapy, antibodies that target specific antigens expressed by cancer cells are used as therapeutic agents. Among these, antibodies directed to HER1, better known as EGFR, and HER2 have shown a favorable outcome in the clinical management of several types of cancer. There is, however, evidence that targeting multiple receptors at the same time is beneficial for improving treatment efficacy and reducing drug resistance. Aptamers are short single-stranded oligonucleotides that can bind a target molecule in a similar fashion as antibodies, and they may represent, due to their physicochemical properties and modular design capacity, a superior alternative strategy for targeted cancer therapy.
In ADHERE, which is funded for period of 2 years, aptamer nanostructures with bispecific (against two different receptors, i.e. EGFR and HER2) and multivalent (up to 4 binding ligands on a single nanostructure) targeting properties will be designed (WP1), and evaluated for their cancer cell targeting activity in vitro (WP2) and in vivo (WP3). The research project will be carried out in the Crielaard research group at the Zernike Institute for Advanced Materials, University of Groningen, The Netherlands.
At the time of termination, this project has achieved 2 out of 3 research objectives, i.e. objective 1 (development of dual HER family-targeted DNA nanostructures) and objective 2 (in vitro characterization of dual HER-family targeted DNA nanostructures in human EGFR/HER2 expressing cancer cell lines). We have selected previously reported DNA aptamer sequences against EGFR (TuTu22)1 and HER2 (2-2(t))2, and extended these sequences to allow the development of multispecific and multivalent DNA nanostructures. The length of the EGFR and HER2 aptamers are around 40 nucleotides. First, we evaluated the toxicity profile of the individual aptamers in several human cancer cell lines with different expression levels of EGFR and HER2. Comparing the measured cell toxicity of these aptamers to clinically used antibodies, which show substantial toxicity in the 50-100 nM range, it was concluded that the cytotoxic activity of aptamers is substantially less then that of antibodies.
Next, to evaluate whether dual bispecific or bivalent aptamers have an improved cytotoxic activity as compared to the single aptamers, we treated two EGFRhi expressing tumor cell lines with linear hybridized aptameric structures possessing EGFR/EGFR (2:0), EGFR/HER2 (1:1), or HER2/HER2 domains. Bivalent targeting of HER2 did in fact decrease the viability of the HER2lo A431 cells, suggesting that bivalent HER2 aptamers might be beneficial for these cell types. However, it is important to note that the scrambled structures also displayed substantial cytotoxic activity, indicating that the effect of the specific structure is rather limited.
Finally, we prepared DNA-lipid micelles to investigate whether a multivalent aptameric nanostructure strategy would be more effective than the single aptamers. The cytotoxic activity of EGFR aptamer-functionalized micelles in MDA-MB-468 and HER2 aptamer-functionalized micelles in NCI-N87 was similar to that of micelles decorated with the scrambled sequences, suggesting that there is no sequence-specific cytotoxic effect of these nanostructures.
The expression of EGFR and HER2 by different human cancer cell lines was analyzed by flow cytometry using fluorescently labeled antibodies. EGFR is expressed on the surface of MB468 cells, while HER2 is expressed on the surface of SKBR3 cancer cells and moderately by A431 cells. The binding of the published DNA sequences to EGFR was evaluated by fluorescently labeled aptamers. As anticipated, we found a sequence-specific binding of the EGFR aptamer to MDA-MB-468 cells (EGFRhi/HER20), and a sequence-specific binding of the HER2 aptamer to SKBR3 cells (EGFR0/HER2hi). However, the binding of the EGFR aptamer to A431 (EGFRhi/HER2lo) was not higher than that of the scrambled control. The difference in fluorescence of MB468 cells labeled with EGFR aptamers, as compared to that of MB468 cells labeled with EGFR antibodies, together with the absence of EGFR aptamer binding to A431 cells, suggests that the EGFR aptamer has lower affinity than the EGFR antibody.
The flow cytometry analysis was then confirmed by confocal microscopy. There was limited association of the EGFR aptamer with the A431 cell line, while the association of the HER2 aptamer with the same cell type is similar or even somewhat stronger. Interestingly, in NCI-N87 cells, the cellular association of the EGFR aptamer appeared to be stronger than that of the HER2 aptamer. Taken together, the cellular binding of EGFR and HER2 aptamers to EGFR- and HER2-expressing cells suggests that further optimization is needed to improve the cellular association and uptake of such aptamers.
The inhibition of the selected aptamers against EGFR and HER2, alone or in combination, on the proliferation of the EGFR/HER2 expressing cancer cell lines, is not as anticipated. In the final stage of the project, we have been working on circumventing this by using different aptamers and setting up a drug resistance cancer model (as described in the contingency plan of the grant proposal). Until now, we have not managed to acquire reliable data from these approaches, and the final goal of the project has not been achieved
Only a fraction of HER2-expressing cancers is sensitive to HER2-targeted immunoglobulins when administered as a monotherapy: less than 1/3 of patients with HER2-positive metastatic breast cancer actually respond to trastuzumab treatment, and of these responsive patients, a large majority eventually develops treatment resistance and relapses into metastatic disease within one year. A number of studies have demonstrated that a subgroup of trastuzumab-resistant HER2-positive cancer cells express higher amounts of EGFR, which is associated with a low disease-free survival in patients with primary breast cancer. And conversely, in a subset of patients with colorectal cancer, activation of HER2 signaling has been implicated in the acquired resistance to the EGFR-targeted antibody cetuximab (Erbitux®). Importantly, dual targeting to EGFR and HER2 has shown positive effects in preclinical and clinical studies, leading to improved clinical outcome and reduced drug resistance, indicating that the dual-targeted aptamers may form a valuable approach for cancer therapy.
In addition, when compared to conventional antibodies, aptamers are produced synthetically, are highly tailorable and allow for relatively straightforward multimerization through complementary hybridization, enabling multivalent and multispecific targeting with limited development and production costs.