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Enhanced class I tumor antigen presentation via cytosolic delivery of exosomal cargos by tumor cell-derived exosomes displaying a pH-sensitive fusogenic peptide Masaki Morishita, Yuki Takahashi, Makiya Nishikawa, Reiichi Ariizumi, and Yoshinobu Takakura Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00760 • Publication Date (Web): 04 Oct 2017 Downloaded from http://pubs.acs.org on October 5, 2017

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Molecular Pharmaceutics

Title Enhanced class I tumor antigen presentation via cytosolic delivery of exosomal cargos by tumor cell-derived exosomes displaying a pH-sensitive fusogenic peptide Short title Enhanced tumor antigen presentation by GALA-exosomes Authors Masaki Morishita1; Yuki Takahashi1*; Makiya Nishikawa1; Reiichi Ariizumi1 and Yoshinobu Takakura1 1

Department of Biopharmaceutics and Drug Metabolism, Graduate School of

Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan *Corresponding author: Yuki Takahashi, Ph.D., Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan. Tel: +81-75-7534616; Fax: +81-75-7534614. E-mail: [email protected]

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Abstract Tumor cell-derived exosomes contain endogenous tumor antigens and can be used as a potential cancer vaccine without requiring identification of the tumor-specific antigen. To elicit an effective antitumor effect, efficient tumor antigen presentation by MHC class I molecules on dendritic cells (DC) is desirable. Because DC endocytose exosomes, an endosomal escape mechanism is required for efficient MHC class I presentation of exosomal tumor antigens. In the present study, efficient cytosolic delivery of exosomal tumor antigens was performed using genetically engineered tumor cell-derived exosomes and pH-sensitive fusogenic GALA peptide. Murine melanoma B16BL6 cells were transfected with a plasmid vector encoding a streptavidin (SAV; a protein that binds to biotin with high affinity)–lactadherin (LA; an exosome-tropic protein) fusion protein to obtain SAV–LA-modified exosomes (SAV-exo). SAV-exo was mixed with biotinylated GALA to obtain GALA-modified exosomes (GALA-exo). Fluorescent microscopic observation using fluorescent-labeled GALA showed that the exosomes were modified with GALA. GALA-exo exerted a membrane-lytic activity under acidic conditions and efficiently delivered exosomal cargos to the cytosol. Moreover, DC treated with GALA-exo showed enhanced tumor antigen presentation capacity by MHC class I molecules. Thus, genetically engineered GALA-exo are effective in controlling the intracellular traffic of tumor cell-derived exosomes and for enhancing tumor antigen presentation capacity. Keywords: Exosome; GALA; Cytosolic delivery; Tumor antigen presentation; MHC class I molecule

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1. Introduction Tumor antigen-based cancer immunotherapy can elicit an immune response by activating tumor antigen-specific cytotoxic T cells 1. However, a limited number of particular tumor antigens have been identified and used in clinical trials for cancer immunotherapy 2 3. Therefore, novel materials that can be used as a cancer vaccine and do not require identification of a particular tumor antigen need to be utilized. Exosomes are small membrane vesicles secreted from cell and have diameters of 30– 120 nm 4. It has been demonstrated that exosomes deliver cargo such as proteins and nucleic acids to exosome-receiving cells 5 6. Tumor cell-derived exosomes contain endogenous tumor antigens and induce antitumor immunity by transferring tumor antigens to antigen-presenting cells (APC) such as dendritic cells (DC) 7 8 9 10. Therefore, development of cancer immunotherapy using tumor cell-derived exosomes against a variety of cancers is required 11. Clonal expansion of antigen-specific cytotoxic CD8+ T cells as well as helper CD4+ T cells is required for induction of antigen-specific immune response. CD8+ T cells and CD4+ T cells recognize peptide-major histocompatibility complex (MHC) class I and II molecules presented on APC 12. It has been demonstrated that exogenous tumor antigens endocytosed by DC are primarily processed within the endosomal/lysosome compartments, which result in antigen presentation by MHC class II molecules on DC and subsequent priming of helper CD4+ T cells 13 14. However, a part of tumor antigen endocytosed by DC is delivered to the cytosol and presented by MHC class I molecules in the endoplasmic reticulum and activates cytotoxic CD8+ T cells, a process known as cross presentation 15 16. Because the effective generation of cytotoxic CD8+ T cells response is essential for effective cancer immunotherapy, acquisition of cross presentation by cytosolic delivery of tumor antigen is required to elicit potent antitumor immunity 17. It have been reported that exosomes are endocytosed by cells and transferred to the endosomal compartment 6 18. Therefore, it is desirable to facilitate cytosolic delivery of exosomal cargos, including tumor antigens for the enhancement of tumor antigen presentation by MHC class I molecules of DC and subsequent induction of cytotoxic CD8+ T cells. However, a method that control the intracellular trafficking of tumor cell-derived exosomes endocytosed by DC to achieve tumor antigen presentation by MHC class I molecules has not yet been reported. As for the control of intracellular trafficking, it have been demonstrated that GALA, a pH-sensitive fusogenic peptide have been used since GALA forms an amphipathic α-helix when the pH is reduced from 7 to 5 and induces pore formation by association with the lipid membrane 19 20. 3



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Here, we aimed to enhance the tumor antigen presentation capacity of tumor cell-derived exosomes by facilitating cytosolic delivery of tumor antigens. Murine melanoma B16BL6 tumor cells were genetically engineered to obtain exosomes modified with SAV–LA, a fusion protein of streptavidin (SAV; a protein that binds to biotin with strong affinity) and lactadherin (LA; a protein that locates on the exosomes membrane) 21. SAV–LA modified exosomes (SAV-exo) were modified with GALA using the SAV–biotin interaction. The membrane lytic activity of GALA-modified exosomes (GALA-exo) was evaluated by a calcein leakage assay. After the treatment of murine dendritic DC2.4 cells with GALA-exo, cytosolic delivery of exosomal cargos was evaluated using a confocal microscope. Then, tumor antigen presentation capacity by MHC class I molecules of DC2.4 cells after treatment with GALA-exo was evaluated. 2. Materials and Methods 2.1 Chemicals Roswell Park Memorial Institute (RPMI) medium and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Nissui Pharmaceutical, Co., Ltd. (Tokyo, Japan). Opti-modified Eagle’s medium (Opti-MEM), penicillin/streptomycin/L-glutamine (PSG), and sodium pyruvate were purchased from Thermo Fisher Scientific (Waltham, MA, USA). Fetal bovine serum (FBS) was obtained from HyClone Laboratories, Inc. (South Logan, UT, USA). MEM non-essential amino acids (NEAA) were purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Monothioglycerol (MTG) and chloroquine were purchased from Wako Pure Chemical (Osaka, Japan). 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) was obtained from Tokyo Chemical Industry (Tokyo, Japan). Cholesterol was obtained from Nacalai Tesque. The fluorescent dye Exo-Green was purchased from System Biosciences Inc. (Mountain View, CA, USA). The fluorescent membrane dye PKH26 was purchased from Sigma-Aldrich (St. Louis, MO, USA). Bis [N,N-bis (carboxymethyl)aminomethyl] fluorescein (calcein) was purchased from Tokyo Kasei Co., Ltd (Tokyo, Japan). LysoTracker Red DND-99 was purchased from Thermo Fisher Scientific. 2.2 Peptide GALA (NH2-WEAALAEALAEALAEHLAEALAEALEALAA-COOH) modified with biotin–aminohexanoic acid (Ahx) at the N-terminus was purchased from GenScript (Piscataway, NJ, USA). GALA modified with biotin–Ahx at the N-terminus and

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fluorescein isothiocyanate (FITC) at the C-terminus was also purchased from GenScript and used to evaluate the modification efficiency of GALA on exosomes. 2.3 Cell culture Murine melanoma B16BL6 cells were obtained from the Riken BRC (Tsukuba, Japan). Murine dendritic DC2.4 cells were kindly provided by Dr. K. L. Rock (University of Massachusetts Medical School, Worcester, MA, USA). BUSA14 cells (murine T cell hybridoma cells specific for melanoma antigen gp100) were a generous gift from Prof. L. Eisenbach (Weizmann Institute of Science, Rehovot, Israel) 22. B16BL6 cells were cultured in DMEM supplemented with 10% FBS and PSG. DC2.4 cells and BUSA14 cells were cultured in RPMI supplemented with 10% FBS and PSG and NEAA and MTG. 2.4 Collection of SAV-exo from B16BL6 cells pCMV-SAV–LA plasmid vector was constructed as described in a previous report 19. SAV–LA modified exosomes (SAV-exo) were isolated from the culture supernatants of B16BL6 cells at a density of 4 x 106 cells/dish transfected with the plasmid vector. In brief, 1 µg of the plasmid vector was mixed with 2.58 µg of PEI “Max” (Polysciences, Warrington, PA, USA) at a final concentration of 16 µg of plasmid vector/mL. The resulting complex was added to B16BL6 cells seeded on culture plates, followed by a 1-h incubation period. Subsequently, the remaining complex was eluted, and cells were cultured in a medium supplemented with exosome-depleted FBS for the next 24 h. The culture supernatant was eluted by sequential centrifugation at 300 × g for 10 min, 2,000 × g for 20 min, and 10,000 × g for 30 min, followed by filtration using 0.2-µm syringe filters. Subsequently, the cleared sample was spun at 100,000 × g for 1 h to sediment the exosomes. Exosomes were washed twice with PBS. Exosome amounts were estimated by measuring the protein concentrations using the Bradford assay. 2.5 Preparation of GALA-modified exosomes (GALA-exo) SAV-exo (1 µg of protein) was incubated with 10 pmol of biotinylated GALA in PBS for 10 min at room temperature. Subsequently, samples were washed with PBS and ultracentrifuged to remove free GALA. To evaluate the modification efficiency of GALA on SAV-exo, fluorescent-labeled GALA-exo were prepared by mixing FITC-labeled biotinylated GALA with SAV-exo as described above. After washing with PBS and ultracentrifugation, exosome membranes were stained with PKH26 dye for 5 min. Thereafter, the exosomes were washed with PBS and ultracentrifuged to 5



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remove the unbound/free dye and observed under fluorescence microscope (Biozero BZ-X710, Keyence, Osaka, Japan). To evaluate the cytosolic delivery of exosomal cargos by GALA-exo, internal exosome proteins of GALA-exo were fluorescent-labeled with Exo-Green, according to the manufacturer’s instruction. In brief, 50 µg of exosomes suspended in PBS were incubated with Exo-Green for 30 min at 37°C. Then, the sample was washed with PBS followed by ultracentrifugation. 2.6 Preparation of calcein-encapsulating liposomes Calcein-encapsulating liposomes were prepared as follows. In brief, 41 mg of DSPC and 10 mg of cholesterol were dissolved in chloroform and dried under reduced pressure. The lipid membranes were hydrated in an aqueous calcein (60 mM) solution at pH 7.4 in Tris buffer (100 mM). Hydration of lipids was facilitated by shaking for 30 min at 70°C. Thereafter, liposomes were extruded through 0.1-µm pore size polycarbonate membranes using a mini-extruder device (Avanti Polar Lipids, LOCATION). Sephadex G50 gel filtration was performed to separate un-encapsulated calcein from liposomes using citrate (pH 5.0) buffer or PBS (pH 7.4). 2.7 Particle size distribution and zeta potential of exosomes A qNano instrument (Izon Science Ltd., Christchurch, New Zealand) was used to measure the number of exosome particles and particle size distribution using a NP100 nanopore, according to the manufacturer’s instructions. Collected data were processed using the Izon Control Suite software, version 3.2. A Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) was used to determine the zeta potential of the exosomes. 2.8 Transmission electron microscopy Exosomes were mixed with an equal volume of 4% paraformaldehyde in PBS. The mixture was applied to a carbon Formvar-film coated transmission electron microscopy (TEM) grid (Alliance Biosystems, Osaka, Japan) and was incubated for 20 min at room temperature. After washing with PBS, the samples were fixed with 1% glutaraldehyde for 5 min. After washing with distilled water, the grid was stained with 1% uranyl acetate for 2 min, and the samples were observed using TEM (Hitachi H-7650; Hitachi High-Technologies, Tokyo, Japan) 2.9 Western blotting Cell lysates were prepared by a freezing-and-thawing cycle performed four times, followed by centrifugation at 15,000 ×g for 15 min to remove cell debris. Exosomes and 6


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cell lysates (5 µg of protein) were reduced with 0.1-M dithiothreitol and heat treatment at 95°C for 3 min. The samples were then loaded onto a 10% sodium dodecyl sulfate– polyacrylamide gel electrophoresis (SDS–PAGE) and were electrophoretically transferred to a polyvinylidene fluoride transfer membrane. The membrane was incubated with rabbit anti-streptavidin antibody (1:100; Sigma-Aldrich), rabbit anti-HSP70 antibody (1:1,000; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-CD81 antibody (1:200; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-Pmel17 (also known as gp100) antibody (1:200; Santa Cruz Biotechnology), and rabbit anti-Calnexin antibody (1:1,000; Santa Cruz Biotechnology). The membranes were then incubated with horseradish peroxidase (HRP)-conjugated rabbit anti-mouse IgG antibody (1:2000 dilution; Thermo Fisher, Waltham, MA, USA), goat anti-rabbit IgG antibody (1:5000 dilution; Santa Cruz Biotechnology), or donkey anti-goat IgG-HRP (1:5,000; Santa Cruz Biotechnology) for 1 h at room temperature. The membrane was reacted with Immobilon Western Chemiluminescent HRP Substrate (Merck Millipore, Billerica, MA, USA), and chemiluminescence was detected using an LAS-3000 instrument (FUJIFILM, Tokyo, Japan). 2.10 Calcein leakage assay To evaluate the membrane lytic activity, calcein-encapsulating liposomes added to 96-well plates at a total lipid concentration of 100 µg/mL were incubated with the indicated concentration of unmodified exosomes (Exo) or GALA-exo for 1 h at 37°C. The fluorescence intensity (FI) of samples was measured using a multilabel counter (ARVO MX; PerkinElmer, Waltham, MA, USA) with excitation at 490 nm and emission at 520 nm. Complete disruption of liposomes was obtained by adding 1% (v/v) Triton X-100 solution. Calcein leakage was calculated as follows:

% Calcein leakage =

− × 100

−

2.11 Cellular uptake of GALA-exo by DC2.4 cells DC2.4 cells were seeded onto 96-well plates at a density of 5 × 104 cells/well. To evaluate the uptake of exosomes, cells were added with Exo-Green-labeled Exo, SAV-exo, and GALA-exo and incubated for 12 h at 37°C. Cells were then washed twice with PBS and harvested. Subsequently, the FI of the cells was determined by flow cytometry (Gallios Flow Cytometer, Beckman Coulter, CA, USA) using the Kaluza 7



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software (version 1.0, Beckman Coulter), and the mean FI (MFI) was calculated as an indicator of cellular uptake. 2.12 Confocal fluorescence microscopic observation DC2.4 cells were seeded on 13-mm diameter glass coverslips and incubated at 37°C for 4 h. The culture medium was removed and replaced with Opti-MEM containing GALA-exo labeled with Exo-Green. After 12 h of incubation at 37°C, cells were washed twice with PBS and treated with 1 µM LysoTracker Red DND-99 (Thermo Fisher Scientific) and incubated in Opti-MEM for further 2 h. Then, the cells were washed twice with PBS, fixed with 4% paraformaldehyde for 10 min, and the washed twice with PBS. Next, the cells were treated with 300 nM 4′, 6-diamino-2-phenylindole (DAPI) for 10 min. After washing twice with PBS, the coverslips were mounted on glass slides using SlowFade Gold (Thermo Fisher Scientific). Cells were observed under a confocal microscope (A1R MP, Nikon Instech Co., Ltd., Tokyo, Japan). 2.13 Cytokine release from DC2.4 cells DC2.4 cells were seeded onto 96-well plates at a density of 5 × 104 cells/well. After 24 h of incubation at 37°C, Exo, SAV-exo, or GALA-exo were diluted in 0.1 mL of Opti-MEM and added to each well. DC2.4 cells treated with 1ng/mL lipopolysaccharide (LPS, Sigma-Aldrich St. Louis, MO, USA) or Opti-MEM were prepared as a positive and negative control, respectively. The cells were incubated at 37°C for 8 h, and the levels of tumor necrosis factor (TNF)-α in the supernatants were determined by enzyme linked immunosorbent assay (ELISA) using OptEIATM sets (Pharmingen, San Diego, CA, USA). 2.14 Antigen presentation assay DC2.4 cells cultured in Opti-MEM were seeded onto 96-well plates at a density of 5 × 104 cells/well and incubated for 4 h. Then, Exo, SAV-exo, or GALA-exo along with 5 × 104 of BUSA14 cells cultured in Opti-MEM were added to each well. BUSA14 cells co-cultured with DC2.4 cells treated with mouse gp100 25-33 (EGSRNQDWL; Anaspec Inc., Fremont, CA, USA) or Opti-MEM were prepared as a positive and negative control, respectively. After a 24-h incubation period at 37°C, the levels of interleukin (IL)-2 in the supernatant were determined by ELISA using OptEIATM sets (Pharmingen). To examine the effect of endosomal acidification in antigen presentation, DC2.4 cells were pre-incubated with 10 µM chloroquine for 30 min. Then, the culture 8


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medium was eluted and exosomes and BUSA14 cells were added to each well. After 24 h of incubation at 37°C, the levels of IL-2 in the supernatant were measured. 2.15 Statistical analysis Statistical differences were evaluated using a one-way analysis of variance (ANOVA), followed by the Tukey–Kramer multiple comparison test, and p < 0.05 was considered statistically significant.

3. Results 3.1 Development of GALA-modified exosomes Exosomes collected from the B16BL6 cells were positive for the exosome marker proteins HSP70, and CD81 (Fig. 1A). Calnexin, an endoplasmic reticulum marker protein, was not detected in the exosomes, suggesting that the exosome samples contained negligible levels of cell debris 23. SAV was also detected in the exosomes collected from B16BL6 cells transfected with SAV–LA-expressing plasmid vector. Moreover, B16BL6 cell-derived exosomes also contained gp100, a well-known melanoma antigen. Next, to confirm the modification of exosomes with GALA, PKH26-labeled SAV– LA-modified exosomes (SAV-exo) were mixed with fluorescein-labeled biotinylated GALA and observed under a fluorescent microscope (Fig. 1B). When SAV-exo was mixed with biotinylated GALA, green signals derived from FITC-labeled biotinylated GALA were co-localized with red signals of the PKH26-labeled exosomes. In contrast, only red signals were observed when unmodified exosomes (Exo) were mixed with FITC-labeled biotinylated GALA. By measuring the exosome particle number, protein level, and the fluorescence intensity of GALA, it was estimated that 1 µg of exosomes was modified with approximately 1.96 pmol of GALA, corresponding to 571 GALA molecules per exosome (Table 2). 3.2. GALA modification barely altered the physicochemical properties of the exosomes The influence of GALA modification on the physicochemical properties of the exosomes was evaluated. Both the particle size distribution and morphology were comparable among Exo, SAV-exo, and GALA-exo (Fig. 2). Table 1 summarizes the mean particle size and zeta potential of these exosomes. The mean particle size of Exo, SAV-exo, and GALA-exo were 107, 107, and 106 nm, respectively. All exosomes 9



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possessed a negative zeta potential of approximately −32 mV. These results indicated that GALA modification barely alters the physicochemical properties of exosomes. 3.3. GALA-exo exerted membrane lytic activity under acidic conditions To confirm the pH-dependent membrane lytic activity of GALA-exo, calcein-encapsulated liposomes were incubated with GALA-exo at different pH values, and the release of calcein from liposomes was evaluated (Fig. 3). Calcein leakage from liposomes was observed after incubation of the liposomes with GALA-exo at pH 5. Moreover, GALA-exo showed a concentration-dependent membrane lytic activity at pH 5. However, no significant calcein leakage was observed after incubation of the liposomes with Exo at pH 5. Furthermore, neither GALA-exo nor Exo induced calcein leakage from the liposomes at pH 7.4, indicating that GALA-exo exerted membrane lytic activity under acidic conditions. Moreover, the membrane lytic activity of a simple mixture of GALA and unmodified exosomes (GALA+ Exo) did not largely differ from that induced by GALA alone (Supplementary Figure 1). 3.4. Cytosolic delivery of exosomal cargo in DC2.4 cells was facilitated by GALA modification To evaluate the intracellular traffic of exosomes endocytosed by DC2.4 cells, exosomal cargos were fluorescently stained with Exo-Green and added to cells (Fig. 4A). Confocal microscopy showed that green signals from Exo-green were co-localized with the red signals from lyso-tracker, when Exo or SAV-exo were added to DC2.4 cells. In contrast, dispersed green signals in the cytosol were observed after treatment of DC2.4 cells with GALA-exo. This effect was abolished by pretreatment of the cells with chloroquine, an inhibitor of the endosome acidification. Although GALA modification facilitated cytosolic delivery, cellular uptake of exosomes by DC2.4 cells was comparable among all the samples (Fig. 4B), indicating that the modification of exosomes with SAV–LA fusion protein and GALA barely affected the cellular uptake efficiency of exosomes by DC2.4 cells. 3.5. Tumor antigen presentation by MHC class I molecules on DC2.4 cells was enhanced by GALA modification To evaluate the degree of presentation of exosomal tumor antigen by MHC class I molecules of DC2.4 cells, we measured the concentration of IL-2 released from BUSA14 cells, T-cell hybridoma cells specific for the melanoma antigen gp100 presented on MHC class I molecules, after co-culture with DC2.4 cells treated with 10


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exosomes. GALA-exo treatment significantly increased the IL-2 secretion from BUSA14 cells co-cultured with DC2.4 cells (Fig. 5A). Moreover, GALA + Exo hardly enhanced the IL-2 production, suggesting that GALA itself has no stimulating activity on IL-2 secretion (Supplementary Fig. 3). We further examined the effect of endosomal acidification of DC2.4 cells on the MHC class I presentation capacity using chloroquine (Fig. 5B). When GALA-exo was added to DC2.4 cells pretreated with chloroquine, the IL-2 production from BUSA14 cells in co-culture decreased significantly. However, IL-2 release from BUSA14 cells after the addition of Exo or SAV-exo to DC2.4 cells was barely altered by the addition of chloroquine. Although MHC class I presentation capacity of DC2.4 cells was enhanced by treatment of GALA-exo, the production of TNF-α from DC2.4 cells treated with GALA-exo was comparable to those induced by Exo or SAV-exo (Supplementary Fig. 2). 4. Discussion For exosome-based cancer immunotherapy, induction of antigen-specific cytotoxic CD8+ T cells is required to elicit potent antitumor immunity 8 9. So far, adjuvants have been widely used and have successfully enhanced antitumor immunity by activating DC incorporating tumor cell-derived exosomes 24 25 26. Clinical trials for cancer immunotherapy, utilizing tumor cell-derived exosomes from cancer patients, have also demonstrated that the vaccination of tumor cell-derived exosomes in combination with adjuvants is effective in generating tumor-specific antitumor cytotoxic CD8+ T cells response 27. However, a method to control the intracellular traffic of tumor cell-derived exosomes endocytosed by DC to achieve tumor antigen presentation by MHC class I molecules has not yet been reported. In this study, we have presented a valuable method to enhance tumor antigen presentation capacity of DC by facilitating cytosolic delivery of exosomal cargos using GALA, a pH-sensitive fusogenic peptide. Controlling the intracellular traffic of exosomes is important for exosome-based cancer immunotherapy and for the development of drug delivery vehicles using exosomes 28. Given that exosomes act as an endogenous delivery vehicle for nucleic acids, they are promising candidates as delivery vehicles for nucleic acid therapeutics such as small interfering RNA and micro RNA 29 30. As these nucleic acid therapeutics act in the cytosol, cytosolic delivery from the endosomal compartment is recognized as a major challenge 31. Because the modification of exosomes with GALA based on SAV–biotin interaction can be utilized irrespective of the types of exosome-producing cells, controlling the intracellular traffic using this method will be also helpful for the development of exosome-based delivery vehicles. 11



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The results of fluorescent microscopy suggested that exosomes were successfully modified with GALA by the SAV–biotin interaction, and TEM observation as well as analysis of physicochemical properties revealed that the collected exosomes were approximately 100 nm in diameter and modification with GALA had minor effects on the physicochemical properties of exosomes (Fig. 1–2 and Table 1). Although there are no preceding studies investigating the membrane lytic activity of GALA presented on exosomes, the calcein leakage assay demonstrated that the membrane lytic activity of GALA peptide was not impaired after modification with exosomes (Fig. 3 and Supplementary Fig.1). GALA forms an amphipathic α-helix when the pH is reduced from 7 to 5 and associates with lipid membrane, which results in pore formation 19 20 32. Moreover, a previous study has demonstrated that GALA exhibits a concentration-dependent membrane lytic activity under acidic conditions 32, which was also confirmed by the calcein leakage assay using liposomes treated with GALA-exo (Fig. 3). Furthermore, cytosolic delivery of exosomal cargos and antigen presentation by MHC class I molecules after the addition of GALA-exo to DC2.4 cells were suppressed by chloroquine, an inhibitor of endosomal acidification (Fig. 4 and 5), suggesting that GALA-exo exerted membrane lytic activity under acidic conditions of the endosome compartment. We used BUSA14 cells that recognize melanoma gp100 presented on MHC class I molecules for antigen presentation assay 22 33. Measurement of IL-2 released from BUSA14 cells demonstrated that the tumor antigen gp100 was effectively presented by MHC class I molecules of DC2.4 cells after treatment with GALA-exo (Fig. 5A). However, tumor cell-derived exosomes contain tumor antigens and other endogenous autologous antigens. GALA modification may enhance antigen presentation capacity of these endogenous autologous antigens, which could result in autoreactive CD8+ T cells priming 34. Therefore, in exosome-based cancer immunotherapy, elicitation of the autoimmune response after the immunization with GALA-exo should be considered. Tumor antigen presentation capacity was enhanced by GALA modification, whereas the inflammatory cytokine release from DC2.4 cells was barely altered by GALA modification (Supplementary Fig. 2). It is also important to stimulate DC, incorporating tumor cell-derived exosomes, for subsequent antigen presentation. Therefore, vaccination of GALA-exo in combination with adjuvants will be a valuable strategy to enhance antitumor immunity. In conclusion, we demonstrated that GALA-modified exosomes developed by the SAV–biotin interaction are useful for controlling intracellular traffic of tumor cell-derived exosomes for enhanced tumor antigen presentation capacity by MHC class 12


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I molecules. These findings will be helpful for further development of exosome-based cancer immunotherapy. Acknowledgments This work was partly supported by a Research Fellow from the Japan Society for the Promotion of Science (grant number: 16J11152), The Mochida Memorial Foundation for Medical and Pharmaceutical Research and Terumo Foundation for Life Sciences and Arts.

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Table of Contents Graphic

Figures Figure. 1

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Figure legends Figure 1. Development of GALA-modified exosomes (GALA-exo). (A) Western blotting analysis of gp100, HSP70, CD81, calnexin, and SAV in cell lysates and exosomes from non-treated (NT) or SAV–LA transduced B16BL6 cells. (B) Fluorescent microscopic observation of GALA-exo or Exo mixed with biotinylated GALA (Exo + GALA) (green, FITC-labeled biotinylated GALA; red, PKH26-labeled exosomes). Scale bar = 20 µm. Figure 2. Physicochemical properties of exosomes. Histograms of particle size distribution images and transmission electron microscopy (TEM) images of Exo, SAV-exo, and GALA-exo. Scale bar = 100 nm. Figure 3. Membrane-lytic activity of GALA-exo. Calcein-encapsulating liposomes added to 96-well plates were incubated with the indicated concentration of Exo or GALA-exo for 1 h at 37°C. The fluorescence intensity of the samples was measured with excitation at 490 nm and emission at 520 nm. Results are expressed as means ± standard deviations (n = 4). *P < 0.05 compared with the Exo in pH 5 at same concentration. #P < 0.05 compared with the GALA-exo in pH 7.4 at same concentration. Figure 4. Cellular uptake of GALA-exo by DC2.4 cells. (A) Upper, confocal fluorescent microscopic image of DC2.4 cells 12 h after incubation with Exo, SAV-exo, or GALA-exo; Bottom, DC2.4 cells were pre-incubated with 10 µM chloroquine for 60 min to inhibit endosomal acidification. Then, the cells were incubated with Exo, SAV-exo, or GALA-exo for 12 h and observed under confocal fluorescent microscopy. Green (Exo-Green); cargos of exosomes, red (Lysotracker); endosome, and blue (DAPI); nucleus. Scale bar = 20 µm. (B) Flow cytometry analysis of DC2.4 cells after the addition of Exo, SAV-exo, or GALA-exo in the absence or presence of chloroquine. DC2.4 cells were added with Exo-Green-labeled Exo, SAV-exo, and GALA-exo and incubated for 12 h at 37°C. Cells were then washed twice with PBS and the fluorescence intensity of the cells was determined by flow cytometry. The mean fluorescence intensity (MFI) of cells was calculated as an indicator of cellular uptake. Results are expressed as means ± standard deviations (n = 4).

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Figure 5. Tumor antigen presentation capacity by MHC class I molecules on DC2.4 cells. (A) DC2.4 cells treated with Exo, SAV-exo, or GALA-exo were co-cultured with BUSA14 cells and incubated for 24 h. IL-2 secretion from BUSA14 cells induced by a gp100-specific response were measured. Results are expressed as means + standard deviations (n = 4). *P < 0.05 compared with the Exo group at the same concentration. (B) Effect of chloroquine on the MHC class I-restricted presentation of GALA-exo. DC2.4 cells were incubated with 10 µM chloroquine for 30 min prior to the addition of GALA-exo. After 24 h, IL-2 production from BUSA14 cells was measured. Results are expressed as means ± standard deviations (n = 4). *P < 0.05.

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Table Table 1. Particle size and zeta potential of Exo, SAV-exo, and GALA-exo. Results are expressed as means ± standard deviations (n = 3).

Particle size (nm)

Zeta potential (mV)

Exo

107 ± 10

−32.1 ± 0.6

SAV-exo

107 ± 5

−32.3 ± 0.4

GALA-exo

106 ± 4

−32.8 ± 1.9

Table 2. Modification efficiency of GALA on exosomes. Fluorescently labeled GALA-exo were prepared by mixing FITC-labeled biotinylated GALA with SAV-exo. Number of GALA molecules on exosomes were calculated by measuring the exosome protein amount, particle number and the fluorescence intensity of GALA. Results of three independent experiments are shown.

Protein amount GALA

GALA-

Particle number

of exosomes

amount

of exosomes (×

(µg)

(pmol)

1010 particles)

Modification efficiency

pmol/µg

GALA molecules/exosome

16.2

32.1

2.9

1.98

666

25.3

41.6

3.7

1.64

677

16.0

36.2

5.9

2.26

369

exo #1 GALAexo #2 GALAexo #3

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