Table 2.

Aptamers to tumor cell-surface biomarkers selected by protein-based SELEX (updated since 2011 [42,43,44]).

Biomarker	Target Used for Protein-SELEX	Aptamer Library	Number of Variable Nucleotides	Number of Rounds of Selection	Separation Method	Name of Selected Aptamers	KD	Applications	Year	References
Cell-adhesion molecules
Epithelial cell adhesion molecule-EpCAM (CD326)	His-tagged C-terminal domain of EpCAM	2′F-RNA	40	12	affinity chromatography	Truncated EpCAM RNA aptamer	55 nM to human cells	Target stem cell marker. Potential applications: development of targeted cancer nanomedecine and molecular imaging agents	2011	[45]
EpCAM	His-tagged C-terminal domain of EpCAM	DNA	40	12	affinity chromatography	Truncated aptamer SYL3C	38 and 67 nM to cells	Imaging (confocal) Cancer cell capture	2013	[46]
Carcinoembryonic antigen- CEA (CD66e)	His-tagged recombinant protein of full-length CEA	2′F-RNA	40	17	affinity chromatography	Group I, II and III	low nM range	Inhibition of cell migration/invasion in vivo. Promotion of cell anoikis resistance in vitro Inhibition of liver metastasis in vivo	2012	[47]
Integrin αvβ3	purified αvβ3 integrin	2′F-RNA	50	15–17	affinity chromatography	Apt-avb3	nM range	Inhibition of endothelial cell adhesion and proliferation Reduction of endothelial cell tube formation Inhibition of cancer cell proliferation and adhesion (HUVEC) Increases endothelial cell apoptosis	2005	[48,49]
Integrin αvβ3	purified αvβ3 integrin	2′F-RNA	50	6	MAI-SELEX 1	αV-1 and β3-1	8.9–10.5 nM	Recognizes distinct binding sites on a single target ( αV or β3) with minimal cross-reactivity Potential applications: molecular diagnosis and targeted therapies	2012	[50]
E-and P-Selectin	recombinant human E-selectin/IgG-Fc-chimeras	DNA	50	17	affinity chromatography	SDA	100 nM	Inhibition of cancer cell adhesion Potential applications in therapies during metastasis formation	2014	[51]
L-Selectin	L-selectin-Ig chimera	2′-NH2 RNA	40	14	affinity chromatography	14.12	0.2–3 nM range	Preferential blokade of a specific selectin	1996	[52]
L-Selectin	L-selectin–IgG fusion protein	DNA	40	17	affinity chromatography	LD201, LD174 and LD196	1.8, 5.5 and 3.1 nM	Inhibition of lymphocyte rolling on endothelial cells	1996	[53]
Tyrosine kinase receptors
EGFR	purified extracellular domain of human EGFR	RNA	62	12	not documented	J18	7 nM	Drug delivery (internalization of gold nanoparticules) Potential application: delivery of siRNA and cancer detection	2010	[54]
EGFR	human EGFR-Fc protein	2′F-RNA	62	9 + 7–9 rounds with a 30% doped sequence (from aptamer E01)	affinity chromatography	E07 Internalized	2.4 nM	Prevention of proliferation of tumor cells (blocks receptor autophosphorylation) Drug delivery (Gemcitabine) and induces cell death	2011	[55,56]
EGFR	purified extracellular domain of EGFR	DNA	40	11	affinity chromatography	Tutu-22	56 nM	Regognizes EGFR-positive cancer cells with strong affinity and selectivity Potential applications: development of novel targeted cancer detection, imaging and therapy	2014	[57]
EGFRVIII	histidine-tagged EGFRvIII ectodomain (E. coli. system)	2′F-RNA	40	12	affinity chromatography	E21	33 nM	Disruption of post-translational modifications of immature EGFRvIII Induction of apoptosis	2009	[58]
HER-2	recombinant glutathione S-transferase (GST)-tagged ErbB2 protein (22–122 amino acids)	2′F-RNA	50	15	affinity chromatography	SE15-8	low nM range	High specificity to ErbB2 and not to other members of the ErbB family Potential applications: drug delivery and imaging for in vivo diagnosis	2011	[59]
HER-2	peptide from the juxtamembrane region of HER2 extracellular domain	DNA	40	multiple	affinity chromatography	HB5	18.9 nM	Drug delivery (Doxorubicin)	2012	[60]
HER-2	20-amino acid HER2 peptide	Thio-DNA	21	12	affinity chromatography	HY6	172 nM	Potential application: targeted therapy	2015	[61]
HER-2	His-tagged Her2–extra cellular domain (E. coli system)	DNA	40	15	membrane filtration	ECD_Apt1	6.33 nM	Potential applications: theranostic (non invasive cancer diagnosis), therapeutics and monitoring patient compliance	2017	[62]
HER-3	extracellular domains of HER3 produced in S2 insect cells	RNA	49	15	membrane filtration and gel shift assay	A30	0.1 nM range	Inhibition of HER3 activation and growth of tumor cells Potential application: anticancer drug	2003	[63]
c-MET	c-Met-Fc	DNA	40	12	membrane filtration	CLN3 and CLN4	91 pm and 11 nM	Mediates tumor cell lysis Recruits NK cells to tumor and induces ADCC	2011	[64]
c-MET	c-Met-Fc	2′F-RNA	40	16	membrane filtration	CLN64	1 nM	Inhibition of tumor cell migration Potential application: therapeutics and diagnosis	2015	[65]
Cell membrane-associated enzyme
Prostate specific membrane antigen-PSMA	706 extracellular amino acids of PMSA	2′F-RNA	40	6	affinity chromatography	A9, A10, A10-3 after minimization & optimization	low nM range	Promotion of tumor regression Delivery of siRNA Potential application: diagnosis and therapies	2002	[66,67]
Mucins
MUC-1	MUC-1 peptides of 2 lenghts: 9 and 60 amino acids	DNA	25	10	affinity chromatography	S1.3/S2.2	low nM range to 0.1 nM	Potential application: detection by fluorescent microscopy	2006	[68]
MUC-1	His-tagged unglycosylated form of the MUC1 protein containing five tandem repeats of the VTR (E. coli system)	DNA	25	10	affinity chromatography	MUC1-5TR-1, 2, 3, 4	47–85 nM	Potential application: diagnosis assays for early or metastatic diseases	2008	[69]
Tumor necrosis factor receptor (TNF-R) and co-stimulatory receptors
T-cell receptor OX40	extracellular domain of OX40-Fc fusion protein	2′F-RNA	40	9–11	affinity chromatography	9C7, 11F11	2-10 nM for purified OX40 protein and # 50 nM for OX40 on activated T cells	Increasing proliferation of T lymphocytes and production of IFN-γ. Potential application: therapeutics in association with dendritic cell-based vaccines (adoptive cellular therapy)	2013	[70]
T-cell receptor OX40	murine extracellular domain of OX40-Fc fusion protein	2′F-RNA	40	11	affinity chromatography	9.8	8 nM	Induces nuclear localization of NFκB, cytokine production and cell proliferation. Increases dendritic cell based tumor vaccine effects	2008	[71]
T-cell receptor 4-1BB	murine extracellular domain of 4-1BB-Fc fusion protein	2′F-RNA	40	12	affinity chromatography	M12-23 (multimeric aptamer)	40 nM	Inhibition of tumor growth in vivo. Potential application: therapeutic manipulation of the immune system	2008	[72]
Receptor activator of NF-κB-RANK	recombinant human soluble RANK/IgG1Fc chimera	RNA	40	7	affinity chromatography	apt1, apt2 and apt3	0.33, 1.8 and 5.8 μM. 100 nM for the 2′-F version of aptamers	Potential application: therapeutics against osteoclastogenesis	2004	[73]
CD28 2	murine recombinant CD28-Fc fusion protein	2′F-RNA	25	9	affinity chromatography	CD28Apt2 and CD28Apt7	60 nM for CD28Apt7-dimer	Potentialisation of antitumor vaccine efficacy Reduction of tumor progression and increased overall survival (in vivo) Potential application: enhancing vaccine-induced immune responses	2013	[74]
Others
Cytotoxic T cell Antigen-4-CTLA-4	murine CTLA-4/Fc fusion protein	2′F-RNA	40	9	membrane filtration	M9-9	30–60 nM	Increases tumor immunity (in vivo) Potential application: immunotherapy	2003	[75]
B-cell–activating factor (BAFF)-receptor (BAFF-R)	Human recombinant BAFF-R protein	2′F-RNA	50	12	membrane filtration	R-1, R-2 and R-14	47, 95 and 96 nM	Delivery of siRNA. Potential application: combinatorial therapeutics	2013	[76]
CD124 (IL-4Rα)	recombinant ILR4α protein enzymatically cleaved	2′F-RNA	40	5	affinity chromatography	cL42	14 nM for recombinant protein and 788 nM for MCS2 cells	Induction of MDSCS apoptosis Promotes CD8+ T cell infiltration and reduces the number of MDSCs infiltration. Reduction of tumor progression in vivo	2012	[77]
VCAM-1	N-terminal fragment of VCAM-1	2′F-RNA	40	12	affinity chromatography	12.11	10 nM	Potential application: imaging	2007	[78]
Toll-like receptor 3 ectodomain	Toll-like receptor 3 ectodomain with N-terminal FLAG and C-terminal His	RNA	40	7	membrane filtration	Family-I and Family II	# 3 nM	Aptamer without agonist and antagonist effects	2006	[79]
hyaluronic acid (HA) binding domain of CD44	HA-binding domain of human CD44 (cell-free expression system)	Thio-DNA	30	10	affinity chromatography	TA1-TA6	180–295 nM	Potential applications targeted therapy and imaging	2010	[80]
CD44	GST-tagged human recombinant full length CD44 protein	2′F-RNA	45	11	affinity chromatography	Apt1	81.3 nM	Potential applications therapeutic (targeted delivery againt stem cells) and diagnosis	2013	[81]
Angiopoietin-1	recombinant human Ang1	2′F-RNA	40	9	membrane filtration	ANG9-4	2.8 nM	Inhibition of cell endothelial cell survival	2008	[82]
Angiopoietin-2	recombinant human Ang2	2′F-RNA	40	11	membrane filtration	11-1 and truncated 11-1.41	3.1 and 2.2 nM	Inhibition of angiogeneis (in vivo)	2003	[83]

¹ Integrin αvβ3 is a heterodimeric transmembrane protein composed of α and β chains, for which the selection procedure of a 2′-fluoro aptamer has been patented [48]. In order to select for aptamers specific to homodimer αv and β3, Gong et al [50], developed a strategy called MAI-SELEX (MAI for multivalent aptamer isolation). Two distinct selection stages were employed, the first being a classical affinity selection on the purified full-length αvβ3 integrin. The second module, for specificity, leads selection to β3 as integrin αIIbβ3 served as a protein decoy. Two aptamers, specific for αv and β3 were identified with affinities in the low nanomolar range. This selection strategy applied to heterodimeric proteins is limited to the availability of decoy proteins. ² Aptamer, GR1, targets CD28. This G-rich oligonucleotide, which, alike AS1411 [84], has not selected by SELEX, inhibits CD28 T cell responses in vitro and in vivo [85].