1. Volume 25, Issue 1, Pages 62-70 (January 2017)
Intracellular Cleavable CpG Oligodeoxynucleotide-Antigen Conjugate Enhances Anti- tumor Immunity 
Katrin Kramer, Nicholas J. Shields, Viola Poppe, Sarah L. Young, Greg F. Walker 
Molecular Therapy 
Volume 25, Issue 1, Pages (January 2017)
DOI: /j.ymthe
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

2. Figure 1
High-Resolution Size-Exclusion Chromatography of the Purified OVA-CpG Conjugates
(A–D) The conjugates HYN (B), HYN-SS (C), and SS (D) as well as a mixture of OVA and CpG (A) as control were adjusted to a concentration of 75 μg/mL in PBS. 25 μL of each conjugate or control were injected onto a Yarra-2000 SEC column. The absorbance at 260 nm was recorded for the run time of 10 min at a flow rate of 0.35 mL/min. The chromatographs shown are representative for analysis of synthesized conjugates after purification by preparative size-exclusion chromatography.
Molecular Therapy  , 62-70DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

3. Figure 2 In Vitro Immune Cell Activation by Conjugates
(A–C) BMDCs were pulsed with 3.5 μg/mL of HYN, HYN-SS, or SS conjugate, 3.5 μg/mL OVA, and 0.2 μM CpG, either 2.5 μg/mL LPS as positive control for the activation assay or the peptides SIINFEKL or OVA323–339 as positive controls for co-culture assays or media as negative control. (A) Representative gating strategy for CD11c-positive BMDCs. Expression of BMDC activation markers MHC class II, CD86, and CD40 after 24 hr of activation; results are presented as median fluorescent intensity (MFI). (B) Sorted CFSE-stained CD8+ and CD4+ T cells were co-cultured with activated BMDCs for 48 hr and 72 hr, and percent proliferation was determined by gating on proliferation peaks. (C) IFN-γ measured in supernatant of co-culture determined by ELISA. Bars represent the mean of three independent experiments ± SEM, and statistical significance was determined by (A) two-way Student’s t test and (B and C) one-way ANOVA with Dunnett’s post hoc test, ****p < , ***p < 0.001, **p < 0.01, *p < Statistical significance displayed in (A) is the comparison of all treatment groups to untreated cells and (B) and (C) is the comparison of all treatment groups to CpG/OVA treated cells.
Molecular Therapy  , 62-70DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

4. Figure 3 In Vivo Cytotoxicity Induced by Conjugates
(A) Timeline of cytotoxicity assay. On day 0, n = 6 mice per treatment group were vaccinated with 10 μg of HYN, HYN-SS, SS, a mixture of CpG and OVA, or PBS as a negative control. 24 days later, mice received a boost with the same treatment and, on day 31, mice were injected with 1 × 107 CFSE-labeled target cells, either pulsed with SIINFEKL peptide or left unpulsed. Mice were culled 48 hr after target cell injection, and CFSE-labeled splenocytes were analyzed. (B) Specific lysis of target cells was calculated by comparing unpulsed to target cell ratio. Results show percent specific lysis of vaccinated treatment groups normalized against PBS treatment group ± SEM. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test, ****p < , *p < 0.05.
Molecular Therapy  , 62-70DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions

5. Figure 4
Comparison of Conjugates on Survival in Therapeutic Tumor Trial
C57/BL6 mice with n = 10 mice per treatment group were injected with 5 × 105 B16-OVA tumor cells. On day 5, after tumor challenge, mice were vaccinated with 10 μg of HYN, HYN-SS, SS, a mixture of CpG and OVA, or PBS as a negative control. On day 80, surviving mice received a re-challenge of tumor cells as depicted in the study setup in (A). Survival of mice per treatment group (B) and tumor size up to a maximum of 150 mm2 were recoded (C). Statistical significance was determined by a Log-rank (Mantel-Cox) test ****p < , **p < 0.01.
Molecular Therapy  , 62-70DOI: ( /j.ymthe )
Copyright © 2017 The American Society of Gene and Cell Therapy Terms and Conditions