Volume 20, Issue 6, Pages 1242-1250 (June 2012) Isolation and Optimization of Murine IL-10 Receptor Blocking Oligonucleotide Aptamers Using High-throughput Sequencing  Alexey Berezhnoy, C. Andrew Stewart, James O. Mcnamara II, William Thiel, Paloma Giangrande, Giorgio Trinchieri, Eli Gilboa  Molecular Therapy  Volume 20, Issue 6, Pages 1242-1250 (June 2012) DOI: 10.1038/mt.2012.18 Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Characterization of aptamers following 5 rounds of selection. (a). Frequency of aptamers represented over 100 times. 8.3 × 106 aptamers analyzed were grouped into four families of related sequences (A–D) as well as orphan aptamers that did not exhibit significant sequence similarities to other aptamers. The scale on the right y-axis corresponds to frequencies (copy number) of family A and the scale on the left y-axis corresponds to the frequencies of families B, C, D, and orphans. (b) Affinity of selected aptamers determined by the double filter-binding assay.20 Specific aptamers shown in panel a that were analyzed in panel b are indicated in red or blue color. Molecular Therapy 2012 20, 1242-1250DOI: (10.1038/mt.2012.18) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 Affinity distribution of aptamers following increasing rounds of selection. (a) Affinity of aptamers identified by high-throughput sequencing (HTS) after 5 rounds of selection and sequencing/cloning after rounds 6, 9, 12, and 16. (b) Frequency distribution of high (black), medium (diagonal lines), and low (vertical lines) affinity aptamers with increasing rounds of selection. Molecular Therapy 2012 20, 1242-1250DOI: (10.1038/mt.2012.18) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Binding of a tetrameric form of the high-affinity R5A1 aptamer to interleukin-10R (IL-10R) expressed on the cell surface. (a) Phycoerythrin (PE)-labeled anti-IL-10R 1B1.3a antibody or tetrameric R5A1 (blue) and isotype control antibody or control aptamer (black) were incubated with IL-10R-expressing MC/9 cells and EL-4 cells which do not express IL-10R, and analyzed by flow cytometry. (b) MC/9 cells were pretreated with polyclonal anti-IL-10R antibody or R5A1 aptamer (blue), or with anti-goat polyclonal antibody or with scrambled aptamer (red). Precoated cells were then incubated with PE-labeled anti-IL-10R or anti-CD16/32 antibody and analyzed by flow cytometry. Molecular Therapy 2012 20, 1242-1250DOI: (10.1038/mt.2012.18) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 R5A1 interleukin-10R (IL-10R)-binding aptamer inhibits IL-10 signaling in vitro. (a) Inhibition of IL-10 dependent proliferation of MC/9 cells. MC/9 cells cultured in the presence of 1 ng/ml IL-10 were incubated with increasing concentrations of either anti-IL-10 antibodies, tetrameric R5A1 aptamers or tetrameric R5B1 aptamers and proliferation was determined 48 hours later by measuring 3H-thymidine incorporation. (b) Rescue of IL-12 secretion by lipopolysaccharide (LPS)-stimulated dendritic cells (DC) cultured in the presence of IL-10. Bone marrow-derived dendritic cells were incubated for 6 days in presence 12 ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and 10 ng/ml IL-4. Cells were plated in triplicates into flat-bottom 96-well plate and cultured in the presence of 100 ng/ml of LPS, 20 ng/ml interferon-γ (IFNγ), and 1 ng/ml of IL-10. As indicated anti-IL-10R antibody, dimeric R5A1 or monomeric R5A1 aptamers were added and 18 hours later supernatants were harvested and IL12p70 concentration was measured. Molecular Therapy 2012 20, 1242-1250DOI: (10.1038/mt.2012.18) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 5 Secondary structure prediction by base-substitution analysis. Three secondary structure configurations of comparable stability were predicted for the R5A0 “founder” sequence using the RNA Structure 5.03 program (The A1 sequence is shown). Effect of substitution on stability (percent change) occurring in R5A1 (T40G), R5A2 (A34G), R5A3 (C37T), R5A4 (C36A), and R5A5 (T13C) shown in insert table. Color-coded changes are indicated in the secondary structure configuration. Molecular Therapy 2012 20, 1242-1250DOI: (10.1038/mt.2012.18) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 6 Secondary structure-guided truncation of the R5A1 aptamer. Truncation of R5A1 aptamer was guided by the secondary structure shown in Figure 5 as configuration gamma to yield truncate aptamers A1.1, A1.2, and A1.3. Binding to interleukin-10R (IL-10R) was determined using the double filter-binding assay. See text for additional details. Molecular Therapy 2012 20, 1242-1250DOI: (10.1038/mt.2012.18) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 7 Interleukin-10R (IL-10R) aptamer-mediated inhibition of tumor growth. (a) The 48-nt long A1.2 aptamer was synthesized chemically with all but four of the 2′fluoro-modified pyrimidines (red) replaced with 2′ O-methyl-modified pyrimidines (green). Balb/c mice were implanted with 1.0 × 105 live CT26 cells. Ten days later mice were injected intratumorally at day 10, 11, and 12 with 10 µg of CpG oligonucleotide and intraperitoneally with 100 µg of anti-IL-10R 1B1.3a antibody, or intravenously with 30 µg of A1.2-4FL aptamer or with scrambled aptamer. (b) Tumor area measured at the days indicated. Arrows indicated day of treatment with antibody or aptamer. Difference between scrambled aptamer and A1.2-4FL aptamer or IBI.3a antibody treated groups.*P < 0.05; **P < 0.01. (c) 72 hours after treatment animals were sacrificed and tumor were examined morphologically and weighted. Molecular Therapy 2012 20, 1242-1250DOI: (10.1038/mt.2012.18) Copyright © 2012 The American Society of Gene & Cell Therapy Terms and Conditions