1. Volume 25, Issue 10, Pages 2280-2288 (October 2017)
A TIM-3 Oligonucleotide Aptamer Enhances T Cell Functions and Potentiates Tumor Immunity in Mice 
Tal Gefen, Iris Castro, Darija Muharemagic, Yvonne Puplampu-Dove, Shradha Patel, Eli Gilboa 
Molecular Therapy 
Volume 25, Issue 10, Pages (October 2017)
DOI: /j.ymthe
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

2. Figure 1 Binding of Aptamers to Murine TIM-3
Aptamers were biotinylated and mixed with StreptAvidin-PE as described in the Materials and Methods. (A) Six aptamers that bind to recombinant TIM-3-Fc fusion protein. (B) b0 aptamer binding to TIM-3-expressing CHO cells. (C) b0 aptamer binding to activated BALB/c lymphocytes. (A–C) Black, b0 aptamer incubated to naked beads, parental CHO cells, or non-activated lymphocytes. Blue, scrambled aptamer binding to TIM-3 targets. Red, TIM-3-specific aptamers. (D–F) TIM-3 antibody binding to TIM-3-Fc fusion-coated beads (D), TIM-3 expressing CHO cells (E) and activated lymphocytes (F). Blue, binding of isotype control antibody. Red, TIM-3-specific antibody.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

3. Figure 2 TIM-3 Aptamer Modulation of T Cell Activation
(A) b0 TIM-3 aptamers with complementary sequences at their 3′ end annealed to form a dimeric aptamer. (B) Three b0 monomers were annealed to three repeats of 2′-O-methyl oligoribonucleotide scaffold, separated with 18 carbon linkers, to form a trimeric aptamer, as described in the Materials and Methods. (C and D) Proliferation (C) and IFN-γ in the supernatant (D) of antigen-activated CD8+ OT-1 cells incubated with TIM-3 antibody or b0 TIM-3 aptamer dimer or trimer. (E and F) Intracellular staining (E) and proliferation (F) of splenocytes isolated from BALB/c mice treated with anti-CD3 antibody (See Materials and Methods for experimental details). (G) Proliferation of isolated CD4+ or CD8+ T cells. (Data are expressed as the mean ± SD of triplicate cultures, *p < 0.05, **p < 0.01, ***p < The data are from one representative experiment out of three with similar results. Ab, antibody).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

4. Figure 3
Competition of TIM-3 Antibody and Trimeric TIM-3 Aptamer with Galectin-9 for Binding to TIM-3
(A) TIM-3-coated beads were incubated with antibody or aptamer for 30 min, followed by the addition of galectin-9. Binding of galectin-9 was monitored by flow cytometry (data are expressed as the mean ± SD of duplicates). (B) Following 6 days of CD8+ activation using CD3 antibody, CD8+ T cells incubated with TIM-3 antibody or trimeric TIM-3 aptamer followed by the addition of galectin-9, cell viability and apoptosis (Annexin-V) were measured by flow cytometry. (The data are from one representative experiment out of two with similar results. See Materials and Methods for details).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

5. Figure 4 TIM-3 Aptamer Inhibition of Tumor Growth
(A) CT-26 tumor-bearing mice were treated with 300 pmol/dose trimeric b0 TIM-3 aptamer three times or with TIM-3 antibody at the same dose (1X, 45 μg/dose) or 4.5 higher dose (4.5X, 200 μg/dose), and tumor growth was monitored (n = 7, tumor volumes are expressed as the mean ± SD, *p < The data are from one representative experiment out of three with similar results. See Materials and Methods for details). (B–D) On day 12 (3 days following the last treatment), tumor-infiltrating lymphocytes were analyzed by flow cytometry for CD8+ to Treg ratio (B), PD-1+TIM-3+ double positive CD8+ cells (C), or PD-1+TIM3+ double positive CD4+ cells (D). Treg were defined as CD4+CD25+Foxp3+ cells. (Each dot represent one mouse; Ab, antibody; ns, not significant; **p < 0.01; ***p < )
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

6. Figure 5 Trimeric TIM-3 Aptamer Synergizes with PD-1 Antibody
CT-26 tumor-bearing mice were treated with 300 pmol/dose trimeric b0 TIM-3 aptamer three times, with 200 μg/dose TIM-3 or PD-1 antibody. (A and B) Mice were monitored for tumor volume (A) and survival (B) (n = 8, tumor volumes are expressed as the mean ± SEM; Ab, antibody; ns, not significant; *p < 0.05; ****p < ).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions