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Cell-Penetrating Peptide Enhanced Antigen Presentation for Cancer Immunotherapy Hanfei Wu, Qi Zhuang, Jun Xu, Ligeng Xu, Yuhuan Zhao, Chenya Wang, Zongjin Yang, Fengyun Shen, Zhuang Liu, and Rui Peng Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.9b00245 • Publication Date (Web): 24 Jul 2019 Downloaded from pubs.acs.org on July 25, 2019

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Bioconjugate Chemistry

Cell-Penetrating Peptide Enhanced Antigen Presentation for Cancer Immunotherapy

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Hanfei Wu‡, Qi Zhuang‡, Jun Xu, Ligeng Xu, Yuhuan Zhao, Chenya Wang, Zongjin Yang, Fengyun

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Shen, Zhuang Liu* and Rui Peng*

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Institute of Functional Nano & Soft Materials (FUNSOM), Jiangsu Key Laboratory for Carbon-Based

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Functional Materials & Devices, Soochow University, 199 Ren'ai Rd., Suzhou, Jiangsu, 215123, P. R.

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China

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* E-mail: [email protected] (R. Peng), [email protected] (Z. Liu)

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‡

These authors contributed equally to this work.

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ABSTRACT: The development of effective cancer vaccines is an important direction in the area of

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cancer immunotherapy. Although certain types of preventive cancer vaccines have already been used

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in clinic, therapeutic cancer vaccines for treatment of already-established tumors are still highly

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demanded. In this study, we develop a new type of cancer vaccines by mixing cell-penetrating peptide

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(CPP) conjugated antigen as the enhanced antigen, together with CpG as the immune adjuvant. A

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special CPP, cytosol-localizing internalization peptide 6 (CLIP6), which has the ability to enter cells

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exclusively via a non-endosomal mechanism, i.e. direct translocation across the cell membrane, is

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conjugated with model antigen ovalbumin (OVA). Compared to naked OVA, the obtained CLIP6-

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OVA conjugates shows greatly increased uptake by dendritic cells (DCs), and more importantly,

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remarkably enhanced antigen cross-presentation, eliciting stronger cytotoxic T lymphocyte (CTL)

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mediated immune responses with the help of CpG. This CLIP6-OVA/CpG formulation offers effective

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protection for mice against challenged B16-OVA tumors, and is able to further function as a

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therapeutic vaccine, which, in combination with immune checkpoint blockade therapy, can

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significantly suppress the already-established tumors. Such CLIP6-based cancer vaccine developing

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strategy shows promising potential towards clinical practice owing to its features in easy preparation,

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low cost, and remarkable biocompatibility.

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KEYWORDS: Immunotherapy, cancer vaccines, cell-penetrating peptide, immune checkpoint

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blockade

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INTRODUCTION

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Cancer immunotherapy, which could evoke the patients’ own immune system to fight cancer, is

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drawing tremendous interests over the past decade. The present-day cancer immunotherapies including

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cancer vaccines, immune checkpoint blockade and chimeric antigen receptor T-Cell immunotherapy

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(CAR-T) have achieved many exciting clinical results.1-2 In particular, cancer vaccines to elicit tumor-

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specific immune responses have been extensively explored in recent years.3-4An effective cancer

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vaccine requires tumor-specific antigen, adjuvant, and possibly a suitable delivery system.5-6 However,

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as exogenous protein antigen after uptake by antigen-presenting cells (APCs) via endocytosis would

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often be degraded inside lysosomes with limited escape into the cell cytosol,7-8 they may not be able

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to access the major histocompatibility complex (MHC) class I antigen processing pathway.9 Instead,

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most exogenous protein antigens would usually go through the MHC class II antigen processing

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pathway to induced CD4+ T cell mediated immune response,10-11 inducing production of antibodies


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and cytokines rather than the activation of cytotoxic T lymphocytes (CTLs),12 the latter of which,

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however, are crucial for antitumor immune attack.13-14 Therefore, protein-based cancer vaccines that

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are able to induce MHC class I antigen cross-presentation are urgently needed. To realize efficient

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protein antigen cross-presentation, it would be important to deliver protein antigens into the cytosol,

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such as triggering endosomal escape of antigens, or shutting antigens into APCs via non-endocytosis

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pathways.15

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Cell-penetrating peptides (CPPs) are peptides with 8-40 residues that are able to effectively cross

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the cell membrane.16-17 By conjugating CPPs to biomacromolecules or nanoparticles, their cellular

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uptake could often be greatly enhanced.18-19 However, on one hand, most CPPs can enter cells via more

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than one mechanisms, including endocytosis which often leads to endosomal entrapment, on the other

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hand they possess potential membrane-lytic activity, therefore limiting their applications in delivery

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strategy.20 In 2016, Schneider et al. discovered a special cell-penetrating peptide, cytosol-localizing

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internalization peptide 6 (CLIP6; KVRVRVRVDPPTRVRERVK-NH2, DP: D-proline), which has

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remarkable biocompatibility and the ability to enter cells exclusively via a non-endosomal mechanism,

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i.e. direct translocation across the cell membrane.21 Based on the special function of CLIP6, a new

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kind of CPP-based cancer vaccine is developed in this study by conjugating CLIP6 to a model antigen,

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ovalbumin (OVA) (Scheme 1). Owing to the excellent membrane-penetrating ability of CLIP6, the

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obtained CLIP6-OVA shows greatly enhanced antigen uptake by APCs such as dendritic cells (DCs).

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Moreover, with the help of CpG, an agonist of toll-like receptor 9 (TLR9),22 as immune adjuvant, the

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CLIP6-OVA/CpG formulation exhibits much higher efficiency in antigen cross-presentation,

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compared to the mixture of OVA and CpG. At the in vivo level, the CLIP6-OVA/CpG formulation

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could act as a preventive vaccine to delay the growth of challenged B16-OVA tumors in the vaccinated



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mice. Moreover, such CLIP6-OVA/CpG formulation is also able to function as a therapeutic vaccine,

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which, when combined with programmed death-1 (PD-1) immune checkpoint blockade, would lead to

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significant tumor regression for the already-established tumors.23-24

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Scheme1. Schematic illustration to show the CLIP6-conjugation-based protein vaccine developing strategy for cancer immunotherapy.

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RESULTS and DISCUSSION

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Successful conjugation of CLIP6 peptide to OVA

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The original CLIP6 peptide was modified with a cysteine (C, underlined) to generate sulfhydryl-

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containing CLIP6 (KVRVRVRVDPPTRVRERVKC, DP: D-proline), which would provide a thiol

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group for conjugation. The conjugation process between sulfhydryl-containing CLIP6 and OVA is

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illustrated in Figure 1a by using the cross-linker sulfosuccinimidyl 4-(N-maleimidomethyl)-

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cyclohexane-1-carboxylate (Sulfo-SMCC), via a well-established bioconjugation method.25 The

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successful conjugation was verified by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). As

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shown in Figure 1b, compared to free OVA, all the CLIP6-OVA conjugates show slower migration


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induction.26-27 Therefore, the antigen-presentation efficiencies of different CLIP6-OVA conjugates

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were compared by incubating them with mouse bone marrow-derived DCs (BMDCs) and analyzed

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using flow cytometer. As shown in Figure 2, at the same OVA concentration, the efficiency of these

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CLIP6-OVA conjugates to induce antigen cross-presentation increases with increasing number of

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CLIP6 per OVA molecule, which also correlates well with their cell uptake efficiency. Given the

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saturated levels of CLIP6 conjugation, antigen cross-presentation efficiency, and cell uptake efficiency

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of the CLIP6-OVA conjugates obtained at the feeding molar ratio of 1:10 (compare that of 1:10 and

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1:20 in Figure 1 and 2), the CLIP6 conjugation was fixed at this ratio and such CLIP6-OVA conjugates

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were chosen to serve as the cancer vaccine.

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Enhanced non-endosomal internalization of CLIP6-OVA conjugates

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We next investigated the cellular uptake mechanism of CLIP6-OVA conjugates using FITC-

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labeled free OVA (OVA-FITC) and CLIP6-OVA (CLIP6-OVA-FITC) by incubating them with DC2.4

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cells at 4 oC or 37 oC for 15 mins (Figure 3). Notably, at both temperatures, CLIP6-OVA-FITC showed

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rapid and remarkably enhanced cellular uptake when compared with OVA-FITC, and its uptake

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efficiency was not affected by the low temperature (4 oC) as shown in the statistical data (Figure 3a &

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3b). Similar results were observed when we extended the incubation time to 4 h (Supporting

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Information Figure S1a & S1b). Again, CLIP6-OVA-FITC showed similarly high cellular uptake at

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both 4 oC and 37 oC, while OVA-FITC showed 6-fold lower cellular uptake at 37 oC and barely

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detectable cellular uptake at 4 oC. Furthermore, pre-incubation of DC2.4 cells with three endocytosis

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inhibitors, Cytochalasin,28 Nocodazole and Dynasore,29 showed no inhibitory effect on cellular uptake

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of CLIP6-OVA-FITC at both 4 oC and 37 oC, while significant inhibition of cellular uptake of OVA-


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S2), suggesting good biocompatibility of CLIP6-based antigen delivery system.

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Enhanced antigen cross-presentation and cytokine secretion in vitro

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The enhanced non-endosomal cellular uptake of CLIP6-conjugated protein antigen may be

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favorable for promoting antigen cross-presentation through the MHC I presenting pathway.30 We next

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evaluated the DC stimulation and antigen cross-presentation efficiency of CLIP6-OVA. CpG

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oligodeoxynucleotides, the agonist of Toll-Like Receptor 9 (TLR9) which could further induce

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immune responses,31-32 were served as vaccine adjuvant and mixed with CLIP6-OVA to prepare the

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CLIP6-OVA/CpG vaccine formulation. BMDCs were incubated with OVA, CpG, OVA+CpG, CLIP6-

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OVA or CLIP6-OVA/CpG for 24 h and analyzed by flow cytometry. Lipopolysaccharide (LPS) was

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used as the positive control. As shown in Figure 5a & 5b, naked OVA could induce DC maturation as

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expected33 while CLIP6-OVA showed slightly increased DC maturation stimulation effect, and the

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addition of CpG would further trigger DC maturation. The antigen cross-presentation experiment was

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further conducted using anti-SIINFEKL-PE to detect the antigen presentation of OVA257-264

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(SIINFEKL) on MHC I (Figure 5c-5e). Notably, conjugation of CLIP6 on OVA significantly enhanced

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the antigen cross-presentation of OVA by DCs, and the highest level of OVA cross-presentation was

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observed in DCs treated with CLIP6-OVA/CpG formulation. The antigen cross-presentation

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efficiency was lower than that of cellular uptake, suggesting not all CLIP6-OVA in DCs were

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processed via the MHC I pathway, part of them were still presented via the MHC II pathway (Figure

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5f), and it is likely that CLIP6 conjugation on the OVA surface would change its natural structure and

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might partially impair its processing in DCs. Neverthless, the ratio of OVA257–264/MHC I+ cells to

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MHC II+ cells in BMDCs treated with CLIP6-OVA was significantly increased compare to that in


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evaluate antigen cross-presentation after various treatments. (f) The statistical data showing the ratio of OVA257–264/MHC I+ cells to MHC II+ cells in BMDCs treated with OVA or CLIP6-OVA. (g&h) Secretion of ?# -R (g) and IL-12p40 (h) from BMDCs after different treatments as tested by ELISA. Error bars represent the standard deviations (n = 3, ***P < 0.001, **P < 0.01, or *P < 0.05).

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We further evaluated the level of cytokine secretion from treated DCs upon DC stimulation by

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enzyme-linked immunosorbent assay (ELISA). Cell culture medium were collected after DCs were

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incubated with OVA, CpG, OVA+CpG, CLIP6-OVA, CLIP6-OVA/CpG or LPS for 24 h, and the

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secreted levels of tumor necrosis

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response34-35) and interleukin-12 (IL-12p40; highly relevant to antigen-specific CD8+ T cell immune

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response36-37) were quantified. Compared to that observed after free OVA treatment, the secretion

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levels of both cytokines were significantly enhanced after DCs being treated with CLIP6-OVA. In

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combination with CpG, the CLIP6-OVA/CpG formulation could further promote secretion of both

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cytokines from treated DCs to the highest level (Figure 5g & 5h). Taken together, our data demonstrate

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that CLIP6 conjugation could not only greatly enhance the cellular uptake of antigen, but also

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effectively improve the efficiencies of DC stimulation and antigen cross-presentation, which could

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subsequently elicit antigen-specific cell-mediated immune response.

! &-R '?# -RL closely related to tumor-specific immune

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Enhanced lymph node migration

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We next investigated the ability of CLIP6 conjugation to facilitate antigen presentation in vivo.

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PBS, OVA-Cy5.5, or CLIP6-OVA-Cy5.5 were intradermally injected in the back skin of C57 mice.

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Upon antigen uptake, skin APCs will be activated and migrate to lymph nodes for antigen presentation.

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As shown in Figure 6a, the migration was monitored at 0, 6, and 12 h post injection by in vivo

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fluorescence imaging, and increasing Cy5.5 fluorescence signal could be detected in the lymph node

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region. At 12 h post injection, lymph nodes were dissected and imaged ex vivo to confirm the lymph


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7 days after the last vaccination, B16-OVA tumor cells were inoculated (set as Day 0), and the tumor

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volumes were closely monitored from Day 9. Compared to the PBS group, due to the existence of

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tumor-specific antigen OVA, tumor growth was slightly delayed for mice immunized with free OVA

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as antigen (the OVA group), while the anti-tumor efficacy could be further enhanced upon addition of

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CpG in the OVA+CpG group. Notably, conjugation of CLIP6 on OVA was found to be helpful to

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further enhance the tumor growth inhibition effects in both the CLIP6-OVA group and the CLIP6-

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OVA/CpG group (Figure 7b). Two out of six mice became tumor free after immunization with the

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CLIP6-OVA/CpG formulation, whereas mice immunized with OVA or CLIP6-OVA all died within

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31-39 days post tumor inoculation (Figure 7c).

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Next, the immunized mice were sacrificed at Day 7 to further evaluate and understand the

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underlying mechanisms of the CLIP6-based cancer vaccine in inhibiting tumor growth. Given the

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important role of CD8+ T cell-mediated immunity in the immunotherapy against cancer,38 the amount

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of

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activity of CD8+ T cells, was quantified using enzyme-linked immunospot assay (ELISPOT). Notably,

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after mice were immunized with the CLIP6-OVA/CpG formulation, the secretion of

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significantly enhanced (Figure 7d & 7e). Another aspect of CD8+ T cell-mediated immunity is the T

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cell response, e.g. T cell proliferation, after exposure to the same antigen or pathogen again, which is

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important for the efficacy of preventive vaccines.39-40 As shown in Figure 7f & 7g, the proliferation

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abilities of T lymphocytes from spleens of mice immunized with CLIP6-based formulations were

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significantly enhanced (5-fold increase when comparing the CLIP6-OVA group with the free OVA

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group; nearly 3-fold increase when comparing the CLIP6-OVA/CpG group with the OVA+CpG group)

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with the CLIP6-OVA/CpG group being the most effective group. Taken together, our data demonstrate

& &

-U ' #-U* secreting cells from re-stimulated splenocytes, which reflects the cytotoxic


#-U1

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of mice treated with different formulations. (d&e) The ELISPOT picture (d) and statistical data (e) to show the increased #-U spot-forming cells after different treatments (1: PBS; 2: OVA; 3: OVA+CpG; 4: CLIP6-OVA; 5: CLIP6-OVA/CpG). (f&g) Representative flow cytometry plots (f) and the statistical data (g) to show activated T cell proliferation after various treatments as determined by the CFSE method. Error bars represent the standard errors (n=6, ***P < 0.001, **P < 0.01).

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Antitumor efficacy of CLIP6-OVA/CpG formulation as a therapeutic cancer vaccine

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In recent years, immune checkpoint blockade therapy has exhibited great promises in clinical

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cancer treatment, especially when combined with other kinds of cancer therapies.41-42 In particular, the

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immune checkpoint protein PD-1 expressed on the T cell surface could be blocked by anti-PD-1,

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reactivating T cells to fight tumor cells.43 Since cancer vaccines function through inducing tumor-

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specific, T cell-mediated immune responses, when combined with immune checkpoint blockade

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therapy, they may show further improved therapeutic outcomes.44-45 Therefore, we investigated the

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potential of CLIP6-OVA/CpG formulation as a therapeutic cancer vaccine in combination with anti-

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PD-1 immune checkpoint blockade. As depicted in Figure 8a, the B16-OVA tumor bearing mice were

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vaccinated with different formulations intradermally for three times on Day 4, 11, and 18, and treated

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with anti-PD-1 injections ( intravenous, 20 P F;

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Figure 8, when compared with the control group (PBS), mice treated with OVA+CpG, CLIP6-

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OVA/CpG or only anti-PD-1 groups exhibited slight tumor growth suppression at first but the

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treatments were not effective enough, and rapid tumor growth resumed later. In contrast, for mice

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treated with CLIP6-OVA/CpG in combination with anti-PD-1, the most significant tumor growth

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suppression effect was achieved, and two out of six mice in this group survived for more than 60 days

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(Figure 8c & 8d), demonstrating promising therapeutic efficacy. Our results illustrate that such CLIP6-

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based cancer vaccine formulation in combination with immune checkpoint blockade therapy can be

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effective to suppress the growth of already-established tumors by antigen-specific anti-tumor immune

* on Day 5, 8, 12, 15, 19, and 22. As shown in


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responses.

a

Day 0 Inoculation

Day 4 1st

Day 18 3rd

Day 11 2nd

Ab

Ab

Ab

Ab

Ab

Ab: Anti-PD-1

Ab

b

c

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d

Figure 8. Antitumor efficacy of CLIP6-OVA/CpG as a therapeutic vaccine in combination with antiPD1 immune checkpoint blockade therapy. (a) A scheme to illustrate the treatment process. (b) The tumor growth curves of individual groups post various treatments indicated. (c) The average tumor growth curves of mice after treated with different formulations (n=6). CLIP6-OVA/CpG combined



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with anti-PD-1 could effectively suppress the tumor growth. Error bars represent the standard errors (n=6, *P < 0.05). (d) Survival curves of mice post various treatments as indicated.

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CONCLUSION

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In summary, we have shown that conjugation of protein antigens with CLIP6, a special cell-

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penetrating peptide, could be an promising strategy to develop more effective cancer vaccines for

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enhanced cancer immunotherapy. Using OVA as a model antigen, conjugation with CLIP6 can greatly

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promote its uptake by APCs (such as DCs) through a non-endosomal mechanism, and thus triggering

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efficient antigen cross-presentation to induce T cell-mediated immune responses. Together with CpG

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as the immune adjuvant, the CLIP6-OVA/CpG formulation is able to trigger strong antigen-specific

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immune responses in immunized mice. It can serve as a preventive vaccine, offering effective

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protection for mice against challenged B16-OVA tumors, and can be further employed as a therapeutic

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vaccine, which, in combination with anti-PD-1 checkpoint blockade therapy, can effectively suppress

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the already-established B16-OVA tumors. Our study highlights the promises of CLIP6-conjugation-

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based cancer vaccines with non-endocytosis APC internalization behaviors to trigger effective antigen

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cross-presentation for cancer immunotherapy. Such strategy could be further extended to deliver other

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types of protein or peptide antigens used in clinical practice (e.g. neoantigens).46 Because of the easy

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preparation and perfect biocompatibility, such a CLIP6-conjugation-based vaccine developing strategy

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shows great potential in clinical translation.

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EXPERIMENTAL SECTION

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Conjugation of CLIP6 to OVA: Firstly, 10 mg of OVA was activated with 2 mg of sulfo-SMCC in

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1 ml PBS, pH 7.2 for 1 h. The obtained maleimide-activated OVA was purified by applying the

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reaction mixture to a centrifugal spin column (Thermo Fisher) at 4000 rpm for 10 min to remove the


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excess sulfo-SMCC. The purified maleimide-activated OVA was then immediately added into the

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conjugation reaction with 5 mg of sulfhydryl-containing CLIP6 (KVRVRVRVDPPTRVRERVKC-

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NH2, DP: D-proline, KE Biochem) in 1 ml PBS, pH 7.2. After reaction for 4 h at room temperature

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with periodic mixing, CLIP6-OVA was purified by ultrafiltration. The successful conjugation of

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CLIP6 to OVA was confirmed by 12% SDS-PAGE using SDS-PAGE Gel Quick Preparation Kit

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(Beyotime).47

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Animals and cell lines: Animal experiments were exerted with seven-week-old female C57BL/6 mice

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according to the procedures formulated by Soochow University Laboratory Animal Center. B16-OVA

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melanoma cells were cultured in Dulbecco's modified Eagle medium (DMEM, HyClone)

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supplemented with 10% fetal bovine serum (FBS) and 1% penicillin streptomycin at 37 oC in 5% CO2.

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DC2.4 cells were cultured in RPMI 1640 medium (HyClone) containing 10% FBS and 1% penicillin

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streptomycin at 37 oC in 5% CO2. BMDCs were collected from the bone marrow of 8-week-old

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C57BL/6 mice following a well-established protocol.48-49

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Cell viability assay: For the cell viability assay, BMDCs and DC2.4 cells (1×104 cells) were incubated

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with different concentrations of CLIP6-OVA ranging from 0 to 50 P F; for 24 h. Then the relative

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cell viabilities were tested by the standard 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium

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bromide (MTT) assay (Sigma).

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Confocal fluorescence imaging: To verify the non-endosomal penetrating mechanism of CLIP6-

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OVA by CLSM imaging, OVA was firstly labeled with FITC, then conjugated with CLIP6 to prepare



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CLIP6-OVA-FITC. 8×104 DC2.4 cells were stained with 5 P F; Hoechst (Beyotime) for 30 min

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then washed with PBS containing 0.12 mg/mL heparin for three times, the cells were then stained with

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10 P% Lyso-tracker red (Beyotime) for 1 h. The prestained DC2.4 cells were washed again, then

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incubated with OVA-FITC or CLIP6-OVA-FITC (10 P F; * at 4 oC or 37 oC for 15 min, and imaged

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under the confocal microscopy (LSM 800, Zeiss).

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Cellular uptake analysis by flow cytometry assay (FACS): To determine the cellular uptake

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efficiency of OVA, 10 g/ml (OVA concentration) OVA-FITC and CLIP6-OVA-FITC were incubated

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with DC2.4 cells for 15 min or 4 h at different temperatures w/wo endocytosis inhibitors, Cytochalasin

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(2 M), Nocodazole (10 M), or Dynasore (80 M). After incubation, the cells were washed with PBS

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containing 1% FBS (FACS buffer) before FACS assay (BD Calibur). For in vivo cellular uptake

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analysis, 100 P PBS or CLIP6-OVA-FITC (100 P OVA) were injected intradermally in the back

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skin of C57 mice with hair removed. 3 h post injection, the skin injection site was dissected and

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crumbled in 1 mL PBS. After washing step, the crumbled skin tissue was incubated in 1 mL mixture

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of hyaluronidase (0.8 mg/mL), collagenase IV (0.8 mg/mL) and collagenase I (0.8 mg/mL)50 at 37 oC

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for 1 h, followed by filtering the tissue fluid with nylon fabric to discard tissue block and centrifugation

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to gather skin cells. Collected cells were stained with anti-CD11c-APC in dark for 30 min before FACS

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assay.

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In vitro antigen presentation and DC stimulation: BMDCs (5×105 cells) were incubated with OVA,

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OVA+CpG, CLIP6-OVA, or CLIP6-OVA/CpG (10 P F; OVA, 0.4 P F; CpG) for 24 h. After the

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washing step, the cells were stained with anti-CD11c-APC and anti-SIINFEKL-PE for 30 min at room


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temperature in the dark. After washing off the excess antibodies, the FACS assay was conducted to

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determine the efficiency of antigen cross-presentation in vitro. For DC maturation assay, the cells were

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stained with anti-CD11c-FITC, anti-CD86-PE and anti-CD80-APC for 30 min at room temperature in

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the dark before FACS assay. For antigen presentation via MHC II pathway assay, BMDCs (5×105 cells)

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were incubated with OVA or CLIP6-OVA (10 P F; OVA) for 24 h. After the washing step, the cells

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were stained with anti-CD11c-FITC, anti-SIINFEKL-PE and anti-MHC II-PE-Cy5.5 for 30 min at

371

room temperature in the dark. After washing off the excess antibodies, the FACS assay was conducted.

372

To quantify the secretion of ?# -R and IL-12p40, the BMDC supernatants were tested by ELISA

373

according to the vendor’s instructions (eBiosciences).

374 375

In vivo and ex vivo fluorescence imaging: PBS, OVA-Cy5.5, or CLIP6-OVA-Cy5.5 (100 P OVA

376

each mouse) were intradermally injected in the back skin of C57BL/6 mice with hair removed. Live

377

imaging (Lumina III) were conducted at 0 h, 6 h, 12 h post injection. The inguinal lymph nodes were

378

dissected at 12 h and imaged to verify the source of Cy5.5 signal and for quantification.

379 380

Tumor challenge experiments: C57BL/6 mice were divided into five groups (6 per group) and

381

immunized with different samples intradermally including PBS, free OVA, OVA+CpG, CLIP6-OVA

382

and CLIP6-OVA/CpG (100 P OVA, 4 P CpG each mouse) once a week for three times. Seven days

383

post the last vaccination, 4 × 105 B16-OVA cells were inoculated subcutaneously. The tumor sizes

384

were monitored every other day starting from Day 9, with the measuring method referring to the

385

following formula (length × width2 ×0.5). Mice were euthanized when the tumor volume reached 1500

386

mm3.



Page 22 of 29

387 388

Evaluation of immune response in vivo: To evaluate the extent of T cell proliferation, C57BL/6 mice

389

were divided into five groups (6 per group) and immunized with different samples intradermally

390

including PBS, free OVA, OVA+CpG, CLIP6-OVA and CLIP6-OVA/CpG (100 P OVA, 4 P CpG

391

each mouse) once a week for three times. The mice were sacrificed one week post the last vaccination.

392

Thereafter, 1 × 106 splenocytes from each mouse were collected and stained with carboxyfluorescein

393

succinimidyl amino ester (CFSE) using the cell proliferation kit (Invitrogen). After washing cells for

394

three times to remove the excess dye, cells were stimulated with 3 P F; of OVA257-264 (Invivogen)

395

for 3 days. Cells were then stained with anti-CD3e (eBioscience) for 30 min at room temperature in

396

the dark, and then washed with PBS containing 1% FBS (FACS buffer) before FACS assay. The level

397

of

398

bioscience) following the vendor’s protocol.

399

Immunospot Analyzer ELISPOT reader (Cellular Technologies Ltd.).

#-U secretion by splenocytes was quantified by the ELISPOT assay using the ELISPOT set (BD#-U- !&

cell spots were counted by the

400 401

The therapeutic effect of CLIP6-OVA/CpG cancer vaccine in combination with anti-PD-1

402

immune checkpoint blockade: C57BL/6 mice were divided into six groups (6 per group) and

403

inoculated subcutaneously with 4 × 105 B16-OVA cells (set as Day 0). Mice were immunized with

404

different samples intradermally including PBS, free OVA, OVA+CpG, CLIP6-OVA and CLIP6-

405

OVA/CpG (100 P OVA, 4 P CpG each mouse) once a week for three times on Days 4, 11, 18. For

406

specific groups, mice were injected with anti-PD-1 (20 P each mouse every injection) intravenously

407

on Days 5, 8, 12, 15, 19, and 22. The tumor sizes were monitored every other day starting from Day

408

9. Mice were euthanized when the tumor volume reached 1500 mm3.


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409 410 411 412

ASSOCIATED CONTENT

413

Supporting Information

414

The supporting information is available free of charge via the Internet at http://pubs.acs.org.

415

Relative viabilities of BMDCs and DC2.4 cells after incubation with increasing concentrations of

416

CLIP6-OVA for 24 h, enhanced cell uptake and non-endocytic internalization of CLIP6-OVA in

417

DC2.4 cells after 4 h incubation under different temperatures w/wo endocytosis inhibitors, in vivo

418

cellular uptake of CLIP6-OVA in skin cells 3 h post injection.

419

AUTHOR INFORMATION

420

Corresponding Authors

421

*E-mail: [email protected]

422

*E-mail: [email protected]

423

Notes:

424

The authors declare no competing X

! " interest.

425 426

ACKNOWLEDGMENTS

427

This article was partially supported by the National Key Research and Development (R&D) Program

428

of China (2016YFA0201200, 2017YFE0131700), the National Natural Science Foundation of China

429

(51525203), the Collaborative Innovation Center of Suzhou Nano Science and Technology, and a



430

project funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education

431

Institutions.


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