Protocol for purification of cells in their native state using reversible aptamer-antidote pairs.

Cell isolation from complex mixtures is a key step in many clinical and research applications, but standard isolation methods may affect the cell's biology and are difficult to reverse. Here, we present a method to isolate and restore cells to their native state using an aptamer that binds epidermal growth factor receptor (EGFR+)cells and a complementary antisense oligonucleotide to reverse binding. For complete details on the use and execution of this protocol, please refer to Gray et al.1.

An Aptamer That Rapidly Internalizes into Cancer Cells Utilizes the Transferrin Receptor Pathway.

Strategies to direct drugs specifically to cancer cells have been increasingly explored, and significant progress has been made toward such targeted therapy. For example, drugs have been conjugated into tumor-targeting antibodies to enable delivery directly to tumor cells. Aptamers are an attractive class of molecules for this type of drug targeting as they are high-affinity/high-specificity ligands, relatively small in size, GMP manufacturable at a large-scale, amenable to chemical conjugation, and not immunogenic. Previous work from our group revealed that an aptamer selected to internalize into human prostate cancer cells, called E3, can also target a broad range of human cancers but not normal control cells. Moreover, this E3 aptamer can deliver highly cytotoxic drugs to cancer cells as Aptamer-highly Toxic Drug Conjugates (ApTDCs) and inhibit tumor growth in vivo. Here, we evaluate its targeting mechanism and report that E3 selectively internalizes into cancer cells utilizing a pathway that involves transferrin receptor 1 (TfR 1). E3 binds to recombinant human TfR 1 with high affinity and competes with transferrin (Tf) for binding to TfR1. In addition, knockdown or knockin of human TfR1 results in a decrease or increase in E3 cell binding. Here, we reported a molecular model of E3 binding to the transferrin receptor that summarizes our findings.

Rapid molecular imaging of active thrombi in vivo using aptamer-antidote probes.

Pathological blood clotting, or thrombosis, limits vital blood flow to organs; such deprivation can lead to catastrophic events including myocardial infarction, pulmonary embolism, and ischemic stroke. Prompt restoration of blood flow greatly improves outcomes. We explored whether aptamers could serve as molecular imaging probes to rapidly detect thrombi. An aptamer targeting thrombin, Tog25t, was found to rapidly localize to and visualize pre-existing clots in the femoral and jugular veins of mice using fluorescence imaging and, when circulating, was able to image clots as they form. Since free aptamer is quickly cleared from circulation, contrast is rapidly developed, allowing clot visualization within minutes. Moreover, administration of an antidote oligonucleotide further enhanced contrast development, causing the unbound aptamer to clear within 5min while impacting the clot-bound aptamer more slowly. These findings suggest that aptamers can serve as imaging agents for rapid detection of thrombi in acute care and perioperative settings.

Aptamers as Reversible Sorting Ligands for Preparation of Cells in Their Native State.

Although antibodies are routinely used to label and isolate a desired cell type from a more complex mixture of cells, via either fluorescence-activated cell sorting (FACS) or magnetic-activated cell sorting (MACS), such antibody labeling is not easily reversible. We describe an FACS and MACS compatible method to reversibly label and purify cells using aptamers. Magnetic beads loaded with the epidermal growth factor receptor (EGFR)-binding antagonistic aptamer E07 specifically isolated EGFR-expressing cells, and pure, label-free cells were recovered via treatment with an "antidote" oligonucleotide complementary to the aptamer. Additionally, while FACS sorting cells with E07 or EGFR antibody yielded EGFR(+) cells with impeded EGFR signaling, stripping off the aptamer via antidote treatment restored receptor function, returning cells to their native state, which was not possible with the antibody. The ability to reversibly label or isolate cells without compromising their function is a valuable, versatile tool with important implications for both the laboratory and clinic.

Identification and characterization of a suite of tumor targeting peptides for non-small cell lung cancer.

Tumor targeting ligands are emerging components in cancer therapies. Widespread use of targeted therapies and molecular imaging is dependent on increasing the number of high affinity, tumor-specific ligands. Towards this goal, we biopanned three phage-displayed peptide libraries on a series of well-defined human non-small cell lung cancer (NSCLC) cell lines, isolating 11 novel peptides. The peptides show distinct binding profiles across 40 NSCLC cell lines and do not bind normal bronchial epithelial cell lines. Binding of specific peptides correlates with onco-genotypes and activation of particular pathways, such as EGFR signaling, suggesting the peptides may serve as surrogate markers. Multimerization of the peptides results in cell binding affinities between 0.0071-40 nM. The peptides home to tumors in vivo and bind to patient tumor samples. This is the first comprehensive biopanning for isolation of high affinity peptidic ligands for a single cancer type and expands the diversity of NSCLC targeting ligands.

A liposomal drug platform overrides peptide ligand targeting to a cancer biomarker, irrespective of ligand affinity or density.

One method for improving cancer treatment is the use of nanoparticle drugs functionalized with targeting ligands that recognize receptors expressed selectively by tumor cells. In theory such targeting ligands should specifically deliver the nanoparticle drug to the tumor, increasing drug concentration in the tumor and delivering the drug to its site of action within the tumor tissue. However, the leaky vasculature of tumors combined with a poor lymphatic system allows the passive accumulation, and subsequent retention, of nanosized materials in tumors. Furthermore, a large nanoparticle size may impede tumor penetration. As such, the role of active targeting in nanoparticle delivery is controversial, and it is difficult to predict how a targeted nanoparticle drug will behave in vivo. Here we report in vivo studies for αvß6-specific H2009.1 peptide targeted liposomal doxorubicin, which increased liposomal delivery and toxicity to lung cancer cells in vitro. We systematically varied ligand affinity, ligand density, ligand stability, liposome dosage, and tumor models to assess the role of active targeting of liposomes to αvß6. In direct contrast to the in vitro results, we demonstrate no difference in in vivo targeting or efficacy for H2009.1 tetrameric peptide liposomal doxorubicin, compared to control peptide and no peptide liposomes. Examining liposome accumulation and distribution within the tumor demonstrates that the liposome, and not the H2009.1 peptide, drives tumor accumulation, and that both targeted H2009.1 and untargeted liposomes remain in perivascular regions, with little tumor penetration. Thus H2009.1 targeted liposomes fail to improve drug efficacy because the liposome drug platform prevents the H2009.1 peptide from both actively targeting the tumor and binding to tumor cells throughout the tumor tissue. Therefore, using a high affinity and high specificity ligand targeting an over-expressed tumor biomarker does not guarantee enhanced efficacy of a liposomal drug. These results highlight the complexity of in vivo targeting.

From phage display to nanoparticle delivery: functionalizing liposomes with multivalent peptides improves targeting to a cancer biomarker.

Phage display is commonly used to isolate peptides that bind to a desired cell type. While chemical synthesis of selected peptides often results in ligands with low affinity, a multivalent tetrameric presentation of the peptides dramatically improves affinity. One of the primary uses of these peptides is conjugation to nanoparticle-based therapeutics for specific delivery to target cell types. We set out to optimize the path from phage display peptide selection to peptide presentation on a nanoparticle surface for targeted delivery. Here, we examine the effects of peptide valency, density, and affinity on nanoparticle delivery and therapeutic efficacy, using the α(v)ß(6)-specific H2009.1 peptide as a model phage-selected peptide and liposomal doxorubicin as a model therapeutic nanoparticle. Liposomes displaying the higher affinity multivalent H2009.1 tetrameric peptide demonstrate 5-10-fold higher drug delivery than liposomes displaying the lower affinity monomeric H2009.1 peptide, even when the same number of peptide subunits are displayed on the liposome. Importantly, a 6-fold greater toxicity is observed toward α(v)ß(6)-expressing cells for liposomes displaying tetrameric verses monomeric H2009.1 peptides. Additionally, liposomal targeting and toxicity increase with increasing concentrations of H2009.1 tetrameric peptide on the liposome surface. Thus, both the multivalent peptide and the multivalent liposome scaffold work together to increase targeting to α(v)ß(6)-expressing cells. This multilayered approach to developing high affinity targeted nanoparticles may improve the utility of moderate affinity peptides. As tetramerization is known to increase affinity for a variety of phage-selected peptides, it is anticipated that the tetrameric scaffold may act as a general method for taking peptides from phage display to nanoparticle display.

Synthesis and biological evaluation of a peptide-paclitaxel conjugate which targets the integrin αvß6.

The integrin α(v)ß(6) is an emergent biomarker for non-small cell lung cancer (NSCLC) as well as other carcinomas. We previously developed a tetrameric peptide, referred to as H2009.1, which binds α(v)ß(6) and displays minimal affinity for other RGD-binding integrins. Here we report the use of this peptide to actively deliver paclitaxel to α(v)ß(6)-positive cells. We synthesized a water soluble paclitaxel-H2009.1 peptide conjugate in which the 2'-position of paclitaxel is attached to the tetrameric peptide via an ester linkage. The conjugate maintains its specificity for α(v)ß(6)-expressing NSCLC cells, resulting in selective cytotoxicity. Treatment of α(v)ß(6)-positive cells with the conjugate results in cell cycle arrest followed by induction of apoptosis in the same manner as free paclitaxel. However, initiation of apoptosis and the resultant cell death is delayed compared to free drug. The conjugate demonstrates anti-tumor activity in a H2009 xenograft model of NSCLC with efficacy comparable to treatment with free paclitaxel.