Combining independent protein and cellular SELEX with bioinformatic analysis may allow high affinity aptamer hit discovery

Screening for hits against a target may be performed with various screening methods, like phage display for peptides and antibodies or SELEX (Systematic Evolution of Ligands by EXponential enrichment) for aptamers (Figure 1). Aptamers are small oligonucleotides (typically 20–100 bases) with various 3D structures that may bind to protein targets. Aptamers may be used as diagnostic or therapeutic tools, for example to perform targeted drug delivery or to block a protein function. From a statistical point of view, hit identification may lead to false positives, particularly in case of large libraries. Aptamer libraries have a large diversity, typically 1015 (versus 1011 for phage display). In this context, identification of aptamers with good properties is rather challenging and usually requires a large number of selection rounds. Screening may be performed against the purified protein target1Tuerk C. Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.Science. 1979; : 1990-2249Google Scholar (Table 1), usually leading to high-affinity aptamers but is at risk of selecting aptamers that bind to an incorrectly folded protein. On the other side, selection may be performed directly on cells,2Morris K.N. Jensen K.B. Julin C.M. Weil M. Gold L. High affinity ligands from in vitro selection: Complex targets.Proc. Natl. Acad. Sci. USA. 1998; 95: 2902-2907Crossref PubMed Scopus (296) Google Scholar coupling a positive selection on cells expressing the protein of interest, and a negative selection on cells that do not express the target protein. This typically leads to selection of lower affinity aptamers but specific to the normally folded protein target.Table 1SELEX methodsBasal techniquesComplex/combined techniquesProtein SELEXCell SELEXLigand guided SELEXToggle SELEXHybrid SELEXIndependent and combined SELEXConceptUse a purified protein target for selectionUse a cell expressing the protein of interest for positive selection, needs to be coupled to a negative selection with similar cells that do not express the protein of interestUse a ligand (e.g., antibody) with high affinity directed against an epitope of the target protein during positive selection to remove aptamers specific of this epitopeAlternating targets (e.g., two protein isoforms) or methods (e.g., cell and protein SELEX) at each roundPerforming cell and protein SELEX one after anotherPerforming cell and protein SELEX independently and select aptamers with secondary structure that are shared and enriched in both casesRef.Tuerk and Gold1Tuerk C. Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase.Science. 1979; : 1990-2249Google ScholarMorris et al.2Morris K.N. Jensen K.B. Julin C.M. Weil M. Gold L. High affinity ligands from in vitro selection: Complex targets.Proc. Natl. Acad. Sci. USA. 1998; 95: 2902-2907Crossref PubMed Scopus (296) Google ScholarZumrut et al.3Zumrut H.E. Ara M.N. Fraile M. Maio G. Mallikaratchy P. Ligand-guided selection of target-specific aptamers: A screening technology for identifying specific aptamers against cell-surface proteins.Nucleic Acid Therapeut. 2016; 26: 190-198Crossref PubMed Scopus (47) Google ScholarWhite et al.4White R. Rusconi C. Scardino E. Wolberg A. Lawson J. Hoffman M. Sullenger B. Generation of species cross-reactive aptamers using “toggle” SELEX.Mol. Ther. 2001; 4: 567-573Abstract Full Text Full Text PDF PubMed Scopus (230) Google ScholarUemachi et al.5Uemachi H. Kasahara Y. Tanaka K. Okuda T. Yoneda Y. Obika S. Hybrid-type selex for the selection of artificial nucleic acid aptamers exhibiting cell internalization activity.Pharmaceutics. 2021; 13888Crossref PubMed Scopus (5) Google ScholarSantana-Viera et al.6Santana-Viera L. Dassie J.P. Rosàs-Lapeña M. Garcia-Monclús S. Chicón-Bosch M. Pérez-Capó M. Pozo L.D. Sanchez-Serra S. Almacellas-Rabaiget O. Maqueda-Marcos S. et al.Combination of protein and cell internalization SELEX identifies a potential RNA therapeutic and delivery platform to treat EphA2-expressing tumors.Mol. Ther. Nucleic Acids. 2023; 32: 758-772https://doi.org/10.1016/j.omtn.2023.05.003Abstract Full Text Full Text PDF PubMed Scopus (1) Google ScholarInterest•Cheap•Fast•High affinity•Target the in vivo tertiary structure of the protein•Select aptamers with epitope specificity•Select cross reactive aptamers•Select aptamers with good affinity and cell internalization property•Select aptamers with good affinity and cell internalization property•May select fewer false positive hitsLimitPotentially nonspecific aptamer (cross reactive to other proteins)•May not target the in vivo tertiary structure of the protein•Complex•Low affinity•Low affinity•Needs to be performed on a pre-enriched aptamer bank•Complex•Lower affinity•Complex•Not cross reactive•Complex•Not cross reactive•Time-consuming Open table in a new tab In the June issue of MTNA, Santana-Viera et al.6Santana-Viera L. Dassie J.P. Rosàs-Lapeña M. Garcia-Monclús S. Chicón-Bosch M. Pérez-Capó M. Pozo L.D. Sanchez-Serra S. Almacellas-Rabaiget O. Maqueda-Marcos S. et al.Combination of protein and cell internalization SELEX identifies a potential RNA therapeutic and delivery platform to treat EphA2-expressing tumors.Mol. Ther. Nucleic Acids. 2023; 32: 758-772https://doi.org/10.1016/j.omtn.2023.05.003Abstract Full Text Full Text PDF PubMed Scopus (1) Google Scholar propose to combine these two selection methods by performing each of them independently and then use a new bioinformatic tool to select conserved and structurally similar sequences. Other authors proposed to combine cellular and protein SELEX, either by changing the method at each round, called “Toggle” SELEX(4), or first performing several rounds of protein SELEX and then several rounds of cellular SELEX, also called “Hybrid” SELEX(5) (Table 1). The authors’ approach led to the identification of a 10−10 M affinity aptamer targeting EphA2, a receptor commonly overexpressed in several types of cancer.7Xiao T. Xiao Y. Wang W. Tang Y.Y. Xiao Z. Su M. Targeting EphA2 in cancer.J. Hematol. Oncol. 2020; 13: 114Crossref PubMed Scopus (55) Google Scholar This aptamer has an in vivo binding capability in a mice model of Ewing sarcoma expressing high levels of EphA2 as well as interesting intrinsic antiproliferative properties, even if not drug loaded. The “independent and combined” SELEX allowed discovery of a good affinity hit, making it a potentially interesting method. It is also expected to be reproducible as it is based on independent experiments. Identification of a candidate EphA2 targeting aptamer may be of interest in Ewing sarcoma but also in many other EphA2 expressing cancers. Compared with antibodies, aptamers are produced by chemical synthesis, a process that is industrially more reliable, less expensive, and less variable than cell-based production. Aptamers are also expected to be less prone to the development of anti-drug antibodies and have better tumor-penetrating capabilities due to their small size (20 kDa versus 150 kDa8Xiang D. Zheng C. Zhou S.F. Qiao S. Tran P.H.L. Pu C. Li Y. Kong L. Kouzani A.Z. Lin J. et al.Superior performance of aptamer in tumor penetration over antibody: Implication of aptamer-based theranostics in solid tumors.Theranostics. 2015; 5: 1083-1097Crossref PubMed Google Scholar). On the other side, aptamers do not benefit from Antibody Dependent Cytotoxicity (ADCC) or antibody’s interesting long half-lives. Aptamers are usually produced using modified nucleotides that resist blood nucleases and typically have a short half-life of 5–15 h that may be increased to 2–4 days using PEGylation. The short half-life of aptamers may potentially be of interest in some indications. EphA2 is a tyrosine kinase receptor whose expression level is linked with tumor aggressiveness. Once bound to its ligand ephrin, it activates a canonical signaling pathway that negatively regulates cell proliferation and induces EphA2 degradation. Ephrin is a membrane protein that activates EphA2 when cells are in close contact. Cancer cells tend to have a looser cell-to-cell contact, leading to EphA2 overexpression. When not bound to ephrin, EphA2 also has an autonomous activation that switches on a non-canonical signaling pathway leading to an increased cell invasiveness and proliferation. EphA2 is therefore an interesting target as it is both a cancer overexpressed target and its targeting induces an anti-proliferative effect even in the absence of combination with a cytotoxic payload. In their article, Santana-Viera et al. identified a 2′-fluoro-modified RNA aptamer that reduced cell proliferation and migration in vitro, as well as in vivo where it reduced primary tumor and metastasis growth in a xenografted Ewing sarcoma murine model. Their aptamer was the top candidate obtained using parallel and independent protein and cellular SELEX whose results were analyzed together using a new bioinformatic pipeline. Briefly, aptamer’s secondary structure is predicted and a clustering algorithm grouped them based on their structural similarity. The protein SELEX aptamer that exhibited the highest number of connections with the cellular SELEX was identified, and its neighbors most common sequences/motifs were used to design the final candidate aptamer. The proposed aptamer has higher affinity than previously published ones targeting EphA2. The proposed method couples the interest of both protein and cellular SELEX, just like the « toggle » and « hybrid » SELEX methods (Table 1). From a statistical point of view, the two experiments are in that case performed independently, probably leading to fewer false positive results. On another side, this study is only a proof of concept on a single case, and real head-to-head comparisons of SELEX methods are required to better understand the relative interest of each method and optimize parameter selection in the bioinformatic pipeline. The authors’ concept of dual independent screening combined with the use of structural or sequence similarity analysis may also be of interest for other screening methods like phage display. The authors chose to demonstrate their aptamer in vivo properties in a xenografted murine model of human Ewing sarcoma, implying that the human EphA2 target is only expressed in the tumor. The relatively high tumor-specific targeting ability observed may be substantially lower in humans where EphA2 is also expressed at a basal level in many epitheliums. Toxicity evaluation, especially if loaded with cytotoxic drugs, will in this context require further evaluation. Other EphA2 targeting drugs or CAR T cells are in preclinical or clinical development (phase I trials) but usually with limited, unpublished, or negative results.7Xiao T. Xiao Y. Wang W. Tang Y.Y. Xiao Z. Su M. Targeting EphA2 in cancer.J. Hematol. Oncol. 2020; 13: 114Crossref PubMed Scopus (55) Google Scholar Only one aptamer (Pegaptanib) has been approved by the Food and Drug Administration since 2004 as the first anti-VEGF agent used in neovascular age-related macular degeneration.9Vinores S.A. Pegaptanib in the treatment of wet, age-related macular degeneration.Int. J. Nanomedicine. 2006; 1: 263-268PubMed Google Scholar It has since been replaced by anti-VEGF antibodies (Lucentis and Avastin) that have shown better clinical efficacy. This may interestingly be explained by the fact that Pegafitinib specifically targets one isoform of VEGF, whereas anti-VEGF antibodies are cross-reactive to all isoforms. Interestingly, this aptamer was PEGylated to achieve longer half-life. PEGylation may also be of interest for EphA2 targeting aptamers in the future, as well conjugation with cytotoxic drugs or antisense oligonucleotides. The authors declare no competing interests.