Cholesterol-modified DP7 enhances the effect of individualized cancer immunotherapy based on neoantigens

Journal Pre-proof Cholesterol-modified DP7 enhances the effect of individualized cancer immunotherapy based on neoantigens Zhang Rui, Tang Lin, Tian Yaomei, Ji Xiao, Hu Qiuyue, Zhou Bailing, Zhenyu Ding, Heng Xu, Yang Li PII:

S0142-9612(20)30098-3

DOI:

https://doi.org/10.1016/j.biomaterials.2020.119852

Reference:

JBMT 119852

To appear in:

Biomaterials

Received Date: 27 September 2019 Revised Date:

1 February 2020

Accepted Date: 7 February 2020

Please cite this article as: Rui Z, Lin T, Yaomei T, Xiao J, Qiuyue H, Bailing Z, Ding Z, Xu H, Li Y, Cholesterol-modified DP7 enhances the effect of individualized cancer immunotherapy based on neoantigens, Biomaterials (2020), doi: https://doi.org/10.1016/j.biomaterials.2020.119852. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.

1

Cholesterol-modified DP7 Enhances the Effect of Individualized Cancer

2

Immunotherapy based on Neoantigens Running title: DP7-C enhances the effect of cancer immunotherapy

3 4 5

Zhang Rui1, Tang Lin1, Tian Yaomei1, Ji Xiao1, Hu Qiuyue1, Zhou Bailing1, Zhenyu

6

Ding2, Heng Xu3,Yang Li1*

7 1

8

State Key Laboratory of Biotherapy and Cancer Center/Collaborative Innovation

9

Center for Biotherapy, West China Hospital, Sichuan University, Chengdu 610041,

10

People’s Republic of China. 2

11 12

Department of Biotherapy, Cancer Center, West China Hospital, Sichuan University,

Chengdu, China. 3

13

Precision Medicine Center, State Key Laboratory of Biotherapy, and Precision

14

Medicine Key Laboratory of Sichuan Province, West China Hospital, Sichuan

15

University and Collaborative Innovation Center, Chengdu, China.

16 17

*Corresponding author: Yang Li; State Key Laboratory of Biotherapy and Cancer

18

Center/Collaborative Innovation Center for Biotherapy, West China Hospital, Sichuan

19

University, Chengdu, China.

20

E-mail address: [email protected]

21 22

Conflicts of interest

23

The authors declare that they have no competition of interests.

24 25

Funding

26

Funding was provided by the National Key Research and Development Program (No.

27

2016YFC1303502) and the National Natural Science Foundation of China (No.

1

28

31570927). This work was also supported by the 1.3.5 project for disciplines of

29

excellence, West China Hospital, Sichuan University.

30 31

Abstract

32

Personalized cancer vaccines based on neoantigens have become an important

33

research direction in cancer immunotherapy. However, their therapeutic effects are

34

limited by the efficiency of antigen uptake and presentation by antigen presenting

35

cells. Here, the low-toxicity cholesterol-modified antimicrobial peptide (AMP) DP7

36

(DP7-C), which has dual functions as a carrier and an immune adjuvant, improved the

37

dendritic cell (DC)-based vaccine efficacy. As a delivery carrier, DP7-C can

38

efficiently delivery various antigen peptides into 75-95% of DCs via caveolin- and

39

clathrin-dependent pathways. As an immune adjuvant, DP7-C can induce DC

40

maturation and proinflammatory cytokine release via the TLR2-MyD88-NF-κB

41

pathway and effectively increase antigen presentation efficiency. In addition, DP7-C

42

enhanced the efficacy of DC-based individualized cancer immunotherapy and

43

achieved excellent antitumor effects on mouse tumor models using the OVA antigen

44

peptides and LL2-neoantigens. Excitingly, after DP7-C stimulation, the antigen

45

uptake efficiency of monocytes-derived DCs (MoDCs) in patients with advanced lung

46

cancer increased from 14-40% to 88-98%, the presentation efficiency increased from

47

approximately 15% to approximately 65%, and the proportion of mature MoDCs

48

increased from approximately 20% to approximately 60%. These findings suggest

49

that our approach may be a potentially alternative strategy to produce cancer vaccines

50

designed for individual patients.

51 52

Key words: DP7-C; Dendritic cell; Neoantigen; Personalized immunotherapy;

53

Antitumor

2

54 55

Figure abstract

56 57

Introduction

58

Tumor immunotherapy refers to the use of the body's own immune system to

59

eliminate tumor cells and has recently received widespread attention. DC vaccination

60

is one of the most important research directions for tumor immunotherapy. Since the

61

first use of DCs containing a melanoma-associated antigen to treat melanoma in vitro

62

in 1995, more than 400 DC-based cancer treatment clinical trials for various

63

malignant tumors have been conducted or completed[1-3]. DC-based tumor

64

immuno-therapies are mainly divided into in vivo-targeted DC vaccines and in

65

vitro-prepared antigen-loaded DC vaccines. In vivo-targeted DC vaccines include

66

antigen-adjuvant vaccines, in situ tumor vaccines, and oncolytic virus-based

67

therapies[4]. However, antigen presenting cell targeted vaccines have an inherent risk

68

of inducing harmful cytokine storms due to their broad targeting[4]. In addition,

69

antigen-adjuvant vaccines may cause immune tolerance due to the sustained release of

70

the antigen at vaccine sites[4, 5]. Studies have shown that antigen-pulsed DC vaccines

71

are more likely to cause CTL (Cytotoxic T lymphocyte) activity than antigen

72

peptide-adjuvant vaccines[6, 7]. Preparation of antigen-loaded DC vaccines in vitro is

73

an effective and selective method for treating human cancers such as B-cell 3

74

lymphoma,

renal

cell

carcinoma

and

metastatic

75

antigen-loaded DC vaccines warrant further study.

melanoma[8-10].

Thus,

76

DC vaccines loaded with individual neoantigens have become a research hotspot in

77

tumor immunotherapy. Neoantigens are considered as an attractive vaccine target

78

because they are predicted to have a strong affinity for major histocompatibility

79

complex (MHC) molecules and are not expressed in healthy tissues[11]. Recent

80

studies have shown that neoantigen vaccines can elicit T cells specifically targeting

81

tumors[12]. However, the low immunogenicity and rapid clearance characteristics of

82

these vaccines limit DC presentation efficiency and subsequent T cell responses[13].

83

Successful cancer immunotherapy requires activation of innate immune receptors to

84

promote DC maturation, cytokine secretion and antigen presentation, leading to

85

activation of tumor antigen-specific CTLs. Effective introduction of antigens into

86

DCs, stimulation of DC maturation, induction of inflammatory cytokine secretion,

87

and efficient migration of DCs to lymph nodes are key features of highly efficient

88

antigen-loaded DC vaccines. In response to the above problems, researchers have

89

developed a variety of synthetic particle-based antigen delivery systems, including

90

polymeric nanoparticles and microparticles, liposomes and inorganic nanoparticles, to

91

enhance the response of CD8+ T cells to protein- and antigen peptides-based vaccines,

92

with varying degrees of efficacy in preclinical animal models[14-16]. However, for

93

patients with advanced diseases who are receiving personalized cancer vaccines, time

94

is crucial, and the time from tissue acquisition to vaccination may be several

95

months[17]. Therefore, nanoparticles-based delivery system for personalized

96

neoantigen vaccines should be able to adapt to fast and scalable preparations,

97

reflecting the disadvantage of most nanoparticles vaccine preparations, which usually

98

require labor-intensive, time-consuming and/or non scalable processing and

99

manufacturing steps[18, 19]. Thus, it is necessary to develop a fast synthetic and

100 101

simple nano delivery system to overcome the above problems. Here, we describe a general "mix" method that can quickly mix a simple and low 4

102

toxic cholesterol modified AMP DP7 (VQWRIRVAVIRK)[20] with antigen peptides

103

(without

104

nanocomposites with dual functions of delivery carrier and immune adjuvant. On the

105

one hand, DP7-C can be used as an antigen delivery carrier, which can efficiently

106

deliver antigen into DCs through specific pathways, and then effectively carry out

107

lysosomal escape. On the other hand, DP7-C can be used as an immune adjuvant to

108

stimulate DC maturation and cytokine secretion so as to promote antigen

109

cross-presentation efficiency. In animal experiments, DC vaccines loaded with

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DP7-C/OVA peptides or DP7-C/LL2-neoantigens showed superior antitumor effects

111

and activated antigen-specific lymphocyte reaction than DC vaccines loaded with

112

OVA peptides or LL2-neoantigens. More importantly, MoDCs isolated from

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peripheral blood of patients with advanced lung cancer also showed the effect of

114

high-efficient uptake of DP7-C/antigen complex. And after DP7-C stimulation, the

115

proportion of mature MoDCs and the efficiency of antigen presentation by MoDCs

116

were significantly improved. Therefore, our approach may be a potentially alternative

117

strategy to produce cancer vaccines designed for individual patients.

chemical

modification

or

physical

emulsification)

to

produce

118 119

Materials and methods

120

Cells and animals

121

EG7-OVA and LL2 tumor cell lines were obtained from the American Tissue Type

122

Collection (ATCC). Six- to eight-week-old female C57BL/6J mice purchased from

123

Beijing HFKBio-technology Co., Ltd. (Beijing, China) were used in this study.

124

Bone marrow-derived DCs (BMDCs) were generated from 4-6-week-old C57BL/6J

125

female mice according to a previous report[19]. Briefly, after treatment with red blood

126

cell lysis buffer, fresh Roswell Park Memorial Institute (RPMI)-1640 medium

127

containing 10% fetal bovine serum (FBS) and 20 ng/ml granulocyte-macrophage

128

colony-stimulating factor (GM-CSF) was added to 3×106 mouse bone marrow cells.

129

At day 8, the BMDCs were collected for further use. MoDCs were generated from 5

130

human peripheral blood mononuclear cells (PBMCs) according to a previous

131

report[21]. Briefly, plasma was separated from whole blood by centrifugation, and the

132

white blood cells were separated by human lymphocyte separation medium. Then, the

133

cells were washed, and CSTTM AIM VTM medium containing 5% autologous serum

134

was added. On day 0, day 3 and day 5, fresh culture medium supplemented with 1,000

135

U/ml GM-CSF (Primegene; Shanghai, China), 500 U/ml recombinant human IL-4

136

(Primegene; Shanghai, China) and 5% autologous serum was added to the cells.

137

RPMI-1640 medium, FBS and PS were all purchased from Thermo Fisher Scientific

138

(Waltham, MA, USA).

139

In this study, all of the OVA peptides were purchased from Sigma Aldrich. The LL2

140

neoantigens Elfn2_P762L (mutant/wild-type sequence: LSPRHYYSGYSSS(L/P))

141

and

142

by GL Biochem (Shanghai, China), and the purity ≥ 98%.

Mastl_D366Y (mutant/wild-type sequence: LSPIH(Y/D)SSA) were synthesized

143 144

Characterization of DP7-C

145

Cholesterol modified peptides DP7-C (VQWRIRVAVIRK) and CPP-C (CPP, which

146

is

147

RKRRQRRRPPQ) were synthesized using standard solid-phase peptide synthesis

148

protocols by GL Biochem (Shanghai, China)[22]. To prepare DP7-C micelles, 1 ml of

149

aqueous solution was added to 10 mg of DP7-C for 30 min. Because of its

150

amphiphilic properties, DP7-C was self-assembled into micelles without using any

151

additives. The prepared DP7-C micelles were lyophilized or stored at 4 ℃. The

152

Malvern ZetaSizer Nano-ZS Zen3600 (Malvern Instruments, UK) was used to

153

characterize the DP7-C micelles (20 µg/ml) and DP7-C (20 µg/ml)/antigen peptide (8

154

µg/ml) complexes, including the particle size distribution and zeta potential. The

155

morphological characteristics of the DP7-C micelles (20 µg/ml) were examined by

156

transmission electron microscopy (TEM) (JSM-7500F; FEI). To detect the stability of

a

commonly

recognized

cell

penetrating

6

peptide

with

the

sequence

157

DP7-C solution, we tested the particle size distribution of DP7-C solution stored at

158

4 ℃ for 1 day, 1 month, 3 months, 6 months and 12 months.

159 160

Cell viability assay

161

To verify the cytotoxicity of DP7-C to DCs, cell viability was measured with a cell

162

counting kit-8 (CCK-8; Sigma, UK) assay. BMDCs (5×104 cells) were incubated with

163

various concentrations of DP7-C and CPP-C for 24 h, and CCK-8 (10 µl/well) was

164

added to each well for an additional 1 h since incubation at 37 ℃. Then, the

165

absorbance of each sample at 450 nm was measured using a microplate reader

166

(Bio-Rad). Additionally, the effects of 10 µg/ml of DP7-C and CPP-C on BMDC

167

viability were analyzed by flow cytometry with propidium iodide (PI)/Annexin-V

168

staining to detect apoptotic cells.

169 170

Flow cytometry assay

171

To verify whether DP7-C and antigens were absorbed by BMDCs in the form of

172

complexes, we designed three groups of experiments: (1) BMDCs were treated with 5

173

µg/ml DP7-C for 1 h, and then DP7-C was washed out and 2 µg/ml FITC-antigen was

174

added; (2) 2 µg/ml FITC-antigen and 5 µg/ml DP7-C were directly added to the

175

BMDC culture medium; and (3) 2 µg/ml FITC-antigen was incubated with 5 µg/ml

176

DP7-C in 1640 complete medium for 5 min, and then they were added to the BMDC

177

culture medium. After 24 h, the uptake efficiency were detected by flow cytometry

178

analysis. To verify the optimal ratio of antigen peptide and DP7-C, we used different

179

concentrations of FITC-antigen peptides for incubation with different concentrations

180

of DP7-C, which were added to BMDCs for 24 h, and then flow cytometry was used

181

to detect the uptake efficiency of BMDCs to select the antigen peptides and DP7-C

182

concentration used in the subsequent test. To detect intracellular antigen signaling,

183

individual FITC-OVA257-264 or DP7-C/FITC-OVA257-264 or CPP-C/FITC-OVA257-264

184

were incubated with BMDCs for different times (0, 2, 4, 12, and 24 h) at a final 7

185

antigen concentration of 2 µg/ml and a DP7-C and CPP-C concentration of 5 µg/ml.

186

The uptake efficiency was measured by flow cytometry analysis. To detect DC

187

maturation after treatment, DCs were treated with 2 µg/ml OVA257-264, 5 µg/ml DP7-C,

188

5 µg/ml CPP-C, 100 ng/ml LPS or DP7-C(5 µg/ml)/OVA257-264(2 µg/ml) for 24 h,

189

followed

190

anti-mouse-CD80 or anti-human-CD11c, anti-human-CD86 and anti-human-CD83

191

antibodies (BD, US) for 40 min, and then detected by flow cytometry. To detect the

192

efficiency of antigen presentation, BMDCs/MoDCs were incubated with 2 µg/ml

193

OVA257-264 or DP7-C(5 µg/ml)/OVA257-264(2 µg/ml) or CPP-C(5 µg/ml)/OVA257-264(2

194

µg/ml) for 24 h, then stained with the monoclonal antibody 25-D1.16 for 40 min, and

195

then detected by flow cytometry. To detect CD103+/CD141+ DCs, the treated DCs

196

were stained with anti-mouse/human CD11c and anti-mouse CD103 or anti-human

197

CD141 antibodies (BD, US) for 40 min and then detected by flow cytometry. To

198

detect the efficiency of treated BMDC migration to the lymph nodes, 2 µg/ml FITC-

199

OVA257-264, DP7-C(5 µg/ml)/FITC-OVA257-264(2 µg/ml), or CPP-C(5 µg/ml)/FITC-

200

OVA257-264(2 µg/ml) pulsed DCs (2×106) were intradermally injected into mice at the

201

base of the tail. After 24 h, the lymph nodes were removed to detect the proportion of

202

fluorescent DCs.

by

staining

with

anti-mouse-CD11c,

anti-mouse-CD86

and

203 204

Uptake pathway inhibitor experiment and confocal microscopy analysis

205

To investigate the pathways through which DCs take up the DP7-C/antigen complex,

206

2×105 DCs were spread on a 24-well plate and cultured for 24 h. Then, the cell was

207

treated with 5 µM chlorpromazine (an inhibitor of the caveolin-mediated endocytosis

208

pathway), 20 µM amiloride (an inhibitor of the macropinocytosis pathway), or 30 µM

209

genistein (an inhibitor of the clathrin-mediated endocytosis pathway) for 2 h. Next,

210

FITC-OVA peptides (2 µg/ml) or DP7-C(5 µg/ml)/FITC-OVA peptides (2 µg/ml) or

211

CPP-C (5 µg/ml)/FITC-OVA peptides (2 µg/ml) were added to the cells for another 4

212

h. Cells were then harvested and stained with CD11c for flow cytometry analysis. 8

213

To further investigate the pathways through which DCs take up the DP7-C/antigen

214

complex, FITC- or Cy3-antigens, DP7-C/FITC- or Cy3-antigens, CPP-C/FITC- or

215

Cy3-antigens were added to DCs for 4 h. Then, the macropinocytosis pathway

216

co-localization probe Dextran Texas Red (20 mg/ml, stained at 37°C for 20 min,

217

Invitrogen), caveolin pathway co-localization probe CT-B (10 µg/ml, stained at 4°C

218

for 15 min, Invitrogen) and clathrin pathway co-localization probe Transferrin (50

219

µg/ml, stained at 37 °C for 2 h, Jackson) were used for uptake pathway staining. For

220

lysosomal staining, DCs were incubated with FITC-antigens or DP7-C/FITC-antigens

221

for 4 h or 22 h, respectively, and then subjected to LysoTracker Red (Beyotime

222

Biotechnology) staining at 37°C for 2 h. The stained cells mentioned above were

223

fixed with 4% paraformaldehyde, sealed with a DAPI-containing anti-fluorescence

224

quencher (Solarbio) and imaged with laser confocal microscopy (Leica).

225 226

Cytokine detection

227

The supernatants of BMDCs (2×106/ml) treated with 5 µg/ml DP7-C, 2 µg/ml

228

OVA257-264 or DP7-C(5 µg/ml)/OVA257-264(2 µg/ml) were diluted in a gradient, and the

229

levels of IL-6, IL-12p70, IL-1β and CXCL2 were detected by ELISA kits (Novus and

230

abcam) according to the vendor's instructions. All samples were measured in

231

triplicate.

232 233

Transcriptomic sequencing

234

BMDCs were treated with PBS, DP7-C (5 µg/ml), OVA257-264 (2 µg/ml) or DP7-C (5

235

µg/ml)/OVA257-264 (2 µg/ml) for 2 h and 4 h (n=3). Residual drug was washed away,

236

and Trizol-lysed cells were sent to Novogene (Beijing, China) for transcriptomic

237

sequencing. Cluster analysis was used to analyze the expression of genes related to

238

DC antigen uptake- and maturation-related signaling pathways. Principal component

239

analysis (PCA) was used to test the degree of dispersion between groups and the

240

repeatability within groups. GO analysis was used for functional enrichment analysis 9

241

of differentially expressed genes.

242 243

Western Blot analysis

244

BMDCs were treated with 5 µg/ml DP7-C for 30 min, 1 h, 2 h, 4 h, and 24 h, and then

245

total protein was extracted for western Blot analysis. The protein samples were

246

probed with anti-β-actin (Abcam), anti-MyD88 (Abcam), and the NF-κB Pathway

247

Sampler Kit (Cell Signaling Technology) for analysis. BMDCs were treated with 2

248

µg/ml OVA257-264, 5 µg/ml DP7-C, 5 µg/ml CPP-C, DP7-C(5 µg/ml)/OVA257-264(2

249

µg/ml) and CPP-C(5 µg/ml)/OVA257-264(2 µg/ml) for 2 h, then total protein was

250

extracted for Western Blot analysis. The protein samples were probed with

251

anti-protease-activated complex subunit 2 (Psme2) (Abcam), anti-calreticulin

252

(Abcam), and anti-antigen peptide transporter 1 (Tap1) (Abcam) antibodies and then

253

incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (Cell

254

Signaling Technology). When pathway inhibitors were used, DCs were pretreated

255

with 50 µM MG-132 (NF-κB inhibitor) (Selleckchem, US) for 1 h, 40 µM ST2825

256

(MyD88 inhibitor) (MCE, US) for 3 h, 150 µM C29 (TLR2 inhibitor) (MCE, US) for

257

1 h, 8 µM CUCPT22 (TLR2 inhibitor) (MCE, US) for 2 h or 1 µM Tak-242 (TLR4

258

inhibitor) (MCE, US) for 30 min. Then, the samples were incubated with 5 µg/ml

259

DP7-C for 2 h and subjected to Western Blot analysis.

260 261

T cell proliferation assay

262

To verify the effect of DCs loaded with DP7-C/OVA peptides on T cell proliferation,

263

splenocytes from OT-I and OT-II mice were separated using a lymphocyte isolation

264

solution (Dakewe Biotech Co., Ltd.) and stained with a CFSE kit (Beyotime

265

Biotechnology) according to the manufacturer’s protocol[23]. The CFSE-stained

266

splenocytes cells were stimulated with OVA257-264 or OVA323-339-pulsed DCs or

267

DP7-C/OVA257-264 or DP7-C/OVA323-339-pulsed DCs for 3 days and then stained with

268

anti-mouse CD3, anti-mouse CD8 or anti-mouse CD4 fluorescence antibody (BD), 10

269

respectively. Then cell proliferation was measured by flow cytometry.

270 271

In vivo immunization and cancer immunotherapy studies

272

To verify the antitumor effect of the prepared DC vaccine. Mice were subcutaneously

273

inoculated with 2×106 EG7-OVA or 2×106 LL2 tumor cells on day 0. Then, DCs were

274

incubated with PBS, 5 µg/ml DP7-C, 2 µg/ml antigen peptides, or DP7-C(5 µg/ml)/

275

antigen peptides (2 µg/ml) for 24 h. In total, 2×106 DCs were intradermally injected

276

into the mice in each group (n=6) on day 4, 11 and 18. Tumor volume was recorded

277

every two days. Mice were sacrificed on day 23, and the tumors were weighed and

278

photographed. To detect CTLs in the spleen, when mice were sacrificed, the collected

279

splenocytes were separated and stained with CD3, CD4, CD8 and IFN-γ fluorescent

280

antibodies for 40 min and then detected by flow cytometry. To detect the

281

antigen-specific lymphocyte response, 3×106 splenic lymphocytes were stimulated

282

with 10 µg/ml wild-type or mutant antigen peptide for 1 h. Then, the cells were mixed

283

with 4 µl Golgiplug (BD, USA) and incubated for another 11 h. Finally, the cells were

284

collected, and the proportion of positive cells was detected using flow cytometry and

285

staining with CD3, CD4, CD8 and IFN-γ fluorescent antibodies (Biosciences Co.,

286

Ltd., USA) for 40 min. To detect CTLs in tumors, tumors were digested with 1 mg/ml

287

type I and 1 mg/ml type IV collagenase at 37 ℃ for 2 h, followed by staining with

288

fixable viability stain 620 and CD45, CD3, CD4, CD25, CD8 and IFN-γ antibodies

289

for 40 min. Then, the stained cells were analyzed by flow cytometry. When mice were

290

sacrificed, serum was collected from each group of mice, and the level of TNF-α was

291

detected by an ELISA kit (Abcam) according to the vendor's instructions.

292 293

ELISPOT assay

294

When mice were sacrificed, splenic lymphocytes were collected for ELISPOT

295

(Dakewe Biotech Co., Ltd.) analysis. ELISPOT analysis was used to detect IFN-γ

296

secreted by splenic lymphocytes according to the instruction manual. Briefly, to 11

297

activate the precoated plate, RPMI-1640 medium was added for 15 min, and then the

298

wells were emptied by flicking. Splenic lymphocytes (5×105) from each mouse in the

299

different immunization groups were added to the plate and cultured with 10 µg/ml

300

mutant immune peptide or wild-type control peptide for 24 h. Then, the cells were

301

removed, and the plates were washed and incubated with a biotinylated anti-IFN-γ

302

antibody for 1 h at 37℃. Next, HRP-conjugated avidin was added for another hour.

303

Finally, a freshly made AEC coloring solution was added for approximately 25 min

304

and then washed out to terminate the coloration reaction. Then, the IFN-γ spots were

305

imaged and counted using an immunospot analyzer/ELISPOT reader (Cellular

306

Technologies Ltd.).

307 308

Histological analysis

309

The main organs were harvested after sacrificing mice, fixed immediately using 4%

310

paraformaldehyde and embedded in paraffin. The embedded tissue sections were

311

dewaxed and rehydrated before staining with Mayer’s hematoxylin and eosin (H&E)

312

and Masson’s trichrome staining according to the vendor’s instructions (Solarbio,

313

China).

314 315

Statistical analysis

316

All data comparisons were analyzed using Prism 6.0 with a two-tailed t-test or

317

one-way ANOVA. p values ≤ 0.05 were considered statistically significant.

318 319

Results

320

Characterization of DP7-C and its cytotoxicity to BMDCs

321

DP7-C consists of hydrophilic DP7 and hydrophobic cholesterol, and therefore has an

322

amphiphilic structure capable of spontaneously forming micelles in water. We

323

incubated DP7-C/antigen with immature DCs (imDCs), which promoted the

324

efficiency of antigen uptake and presentation and increased the proportion of mature 12

325

DCs (mDCs) (Figure 1a). The particle size distribution of DP7-C was approximately

326

36.06 ± 1.52 nm, the polydispersity index was 0.23, and the zeta potential of DP7-C

327

was approximately 47.82 ± 3.13 mV (Figure 1b-1c). In addition, the prepared 10

328

mg/ml DP7-C stored at 4° C for 1 day to 12 months showed almost the same particle

329

size distribution over time, indicating that DP7-C micelles have good stability (Figure

330

S1a). When DP7-C was incubated with antigen peptides, the particle size was larger

331

than that of DP7-C alone, but their zeta potential was lower than that of DP7-C alone

332

(Figure 1b-1c), indicating that DP7-C can form complexes with antigen peptides.

333

Moreover, using TEM, we found that the shape of DP7-C was spherical, and the size

334

was similar to its particle size (Figure 1d). We observed that DP7-C aqueous solution

335

and the lyophilized DP7-C re-dissolved in water both exhibited a transparent liquid

336

state (Figure 1e). To further determine whether DP7-C and antigen peptides enter into

337

BMDCs in the form of complexes, we pretreated BMDCs with DP7-C and then added

338

antigen peptides to the BMDCs, or antigen peptides and DP7-C were added to the

339

BMDCs separately, or added to the BMDCs after incubation. The results showed that

340

the uptake efficiency was highest after incubation of DP7-C and antigen peptide, the

341

efficiency of DP7-C and antigen peptide added separately was slightly lower than that

342

in the incubation group, and the uptake efficiency of BMDCs pretreated with DP7-C

343

was the lowest but still higher than that in the control group (Figure S1b), suggesting

344

that DP7-C loaded with antigen peptides entering into DCs is a complex process. On

345

the one hand, the formation of nanocomposites increases the uptake efficiency of

346

antigens by DCs. On the other hand, DP7-C may activate the signaling pathways

347

through which DCs take up antigens, leading to increased uptake efficiency of antigen.

348

Finally, we evaluated the cytotoxicity of DP7-C on BMDCs and compared it with

349

CPP-C. The results showed no significant change in the cell survival rate after DP7-C

350

treatment. However, the cell survival rate decreased after the same concentration of

351

CPP-C was applied (the survival rate decreased approximately 20% in the 5 µg/ml

352

CPP-C treatment group; the survival rate decreased approximately 40% in the 10 13

353

µg/ml CPP-C treatment group). Moreover, the number of apoptotic BMDCs treated

354

with 10 µg/ml CPP-C was significantly higher than the number of apoptotic BMDCs

355

treated with DP7-C (Figure 1f-1g). The results indicated that DP7-C has low toxicity

356

to BMDCs.

357 358 359 360 361 362 363 364 365

Figure 1. Characterization of DP7-C and its cytotoxicity to BMDCs. (a) Schematic illustration of the preparation process for the DP7-C/antigen peptide-pulsed DC vaccine. (b) Particle size distributions of DP7-C and DP7-C/antigen peptides complexes. (c) Zeta potentials of DP7-C and DP7-C/antigen peptides complexes. (d) Morphological structure of DP7-C assessed by transmission electron microscopy. (e) Pictures of i) water; ii) DP7-C micelles; iii) lyophilized powder of DP7-C micelles; and iv) lyophilized DP7-C re-dissolved in water. (f) CCK-8 assay of DP7-C and CPP-C-treated BMDCs. (g) Apoptosis assay of DP7-C and CPP-C treated BMDCs.

366 367

Incubation of antigen peptides and DP7-C increased the uptake efficiency of the

368

antigen peptide by BMDCs

369

The efficiency and the mechanism of DP7-C/antigen peptides ingested by BMDCs

370

were described. In this experiment, DP7-C was able to greatly increase the uptake

371

efficiency of antigen peptides by DCs, and the uptake efficiency of DCs was highest 14

372

after incubation with 2 µg/ml antigen peptides and 5 µg/ml DP7-C (Figure S2a-S2f).

373

Therefore, 5 µg/ml DP7-C and 2 µg/ml antigen peptides were used in all subsequent

374

experiments. The uptake efficiency of DCs for the DP7-C/antigen peptides at different

375

time points increased within the incubation time range of 0-4 h (Figure 2a-2c). After

376

antigen peptides are internalized, they must escape lysosomes to exert their effect.

377

Therefore, we detected the lysosomal escape of DP7-C/antigen peptide. The results

378

showed that antigen peptide treatment and DP7-C/antigen peptide treatment resulted

379

in strong co-localization of the FITC signal with lysosomes at 6 h. However,

380

DP7-C/antigen peptide treatment resulted in obvious lysosomal escape at 24 h, while

381

antigen peptides alone treatment still yielded strong co-localization of the FITC signal

382

with lysosomes (Figure 2d).

383

The entry of extracellular substances into cells is a complex process. Endocytosis is

384

the process of transporting extracellular substances into cells through plasma

385

membrane deformation. The uptake pathways for extracellular substances used by

386

cells are mainly divided into macropinocytosis, caveolin-mediated endocytosis and

387

clathrin-mediated endocytosis[24]. Here, we mainly verify the internalization pathway

388

through which DP7-C/antigen peptides were absorbed by BMDCs. Our results

389

showed that the antigen peptides alone and the CPP-C/antigen peptide complex could

390

co-localize with the macropinocytosis pathway probe Dextran Texas Red, and the

391

uptake efficiency decreased after treatment with the macropinocytosis pathway

392

inhibitor amiloride (Figure 2e-2f, S3). Moreover, the DP7-C/antigen peptide complex

393

could co-localize with the caveolin and clathrin pathway probes CT-B and transferrin,

394

and the uptake efficiency was significantly decreased after treatment with the

395

corresponding pathway inhibitors genistein and chlorpromazine, respectively (Figure

396

2e-2f, S3). To further support this result, we performed a molecular docking search

397

for the stable complex structure of DP7-C and CPP-C interacting with clathrin

398

followed by all-atom, explicit water molecular dynamics (MD) simulations. Here

399

TLPWDLWTT, which is a small peptide known to bind to clathrin[25], was used as 15

400

the positive control. The final stable complexes including DP7-C/clathrin,

401

CPP-C/clathrin and TLPWDLWTT/clathrin are shown in Figure 2g-2i. The contact

402

list for DP7-C/clathrin, CPP-C/clathrin and TLPWDLWTT/clathrin are shown in

403

Tables S1-S3. The binding energy (∆Gtotal) of the interaction of DP7-C, CPP-C and

404

TLPWDLWTT with clathrin are shown in Tables S4-S6. The binding free energy of

405

the interaction of DP7-C/clathrin, CPP-C/clathrin and TLPWDLWTT/clathrin are

406

computed to be -30.32 ± 1.68, -20.46 ± 1.43 and -33.14 ± 0.70 kcal/mol in aqueous

407

environments, respectively. The lower binding free energy, the stronger binding ability.

408

From the data, the binding free energy of DP7-C/clathrin and TLPWDLWTT/clathrin

409

are close to each other and lower than CPP-C/clathrin, indicating that the binding

410

ability of DP7-C and clathrin is stronger.

411 412 413 414 415 416

Figure 2. Intracellular delivery of DP7-C/OVA peptides into DCs. (a) The efficiencies of OVA peptides and DP7-C/OVA peptides entering into DCs at different time points. (b-c) Graphs plotting uptake efficiency versus time. (d) BMDCs incubated with free FITC-OVA or DP7-C/FITC-OVA for 4 or 22 h and then stained with LysoTracker red and DAPI. (e-f) Co-localization of three uptake pathway markers with OVA peptides, 16

417 418 419 420 421 422 423 424

DP7-C/OVA peptides, and CPP-C/OVA peptides. (g-i) The DP7-C/clathrin, CPP-C/clathrin and TLPWDLWTT/clathrin complexes. The left panel shows the 3D simulated complex structure of the interaction between DP7-C, CPP-C, TLPWDLWTT and clathrin. The right panel shows the 3D binding model of DP7-C, CPP-C, TLPWDLWTT and clathrin. DP7-C, CPP-C and TLPWDLWTT are colored yellow, the residues of chain A are colored cyan, the residues of chain B are colored magenta, the backbone of chain A is depicted as a cyan-colored cartoon, and the backbone of chain B is depicted as a magenta-colored cartoon.

425 426

DP7-C treatment enhanced BMDC functions and antigen cross-presentation

427

DC maturation and cytokine secretion are very important processes influencing the

428

effectiveness of DC vaccines. Many clinical trials have shown that mDCs have

429

obvious advantages over imDCs[26, 27]. The cytokines secreted by DCs, especially

430

IL-12p70, are closely related to the effectiveness of DC vaccines, which can promote

431

CTL differentiation and CD8+ T cell proliferation[28-30]. Therefore, DCs maturation

432

and cytokine secretion must be induced when preparing DC vaccines in vitro. In this

433

study, we found that DP7-C has an adjuvant function and can effectively promote

434

BMDC maturation (Figure 3a-3c). To further explore the mechanism of DP7-C in

435

promoting DC maturation, we sequenced the transcriptome of DP7-C-treated DCs and

436

analyzed the results. PCA analysis showed significant changes in the BMDC

437

transcriptome after treatment with DP7-C and DP7-C/OVA antigen peptide (Figure

438

S4a-S4b). By gene function enrichment analysis, DP7-C-treated BMDCs showed

439

upregulated expression of proinflammatory immune response- and cytokine

440

secretion-related

441

pathway-related genes were activated in DP7-C-stimulated BMDCs (Figure 3d, S4c).

442

Through

443

MyD88-IkK-IκB-NF-κB signaling pathway was activated in BMDCs after DP7-C

444

treatment, indicating that DP7-C promoted BMDC maturation by acting on this

445

signaling pathway (Figure 3e). MyD88 is known to be a key junction molecule of

446

TLR, therefore we speculate that DP7-C may activate MyD88 by acting on TLR.

447

Through analysis of the transcriptome data, we found that in TLR, only TLR2 was

further

genes

(Figure

verification

of

S4d-S4e).

Furthermore,

transcriptome

17

data,

we

NF-κB

found

signaling

that

the

448

activated in BMDCs after DP7-C treatment. Thus, we used a TLR2 inhibitor to

449

pre-treat DCs and found that DP7-C could not effectively activate MyD88 after TLR2

450

inhibitor (C29, CUCPT22) treatment, while DP7-C can still activate MyD88 after

451

TLR4 inhibitor (TAK242) treatment (Figure 3f). In addition, in DP7-C-treated BMDC

452

supernatant, the secretion of IL-6, IL-1β, IL-12p70 and CXCL2 increased

453

significantly (Figure 3g). To further confirm that DP7-C exerts its effects by acting on

454

TLR2, we further tested whether DP7-C can interact with TLR2 by molecular

455

docking. The final stable complex of DP7-C/TLR2 and CPP-C/TLR2 are shown in

456

Figure 3h-3i. The contact list for DP7-C/TLR2 and CPP-C/TLR2 are shown in Tables

457

S7-S8. The ∆Gtotal of the interaction of DP7-C and CPP-C with TLR2 are shown in

458

Tables S9-S10. The binding free energy of the interaction of DP7-C/TLR2 and

459

CPP-C/TLR2 are computed to be -54.95 ± 0.78 kcal/mol and -42.01 ± 0.84 kcal/mol

460

in aqueous environments. From the data, the binding free energy of DP7-C/TLR2 is

461

lower than CPP-C/TLR2, indicating that the binding ability of DP7-C and TLR2 is

462

stronger.

463

DC maturation is accompanied by an enhanced antigen presenting ability.

464

Antigen-presenting ability is also closely related to the effect of DC vaccines.

465

Therefore, we tested whether DP7-C can promote the internalized antigen to

466

cross-present to BMDC surfaces. The results showed that after incubating DP7-C with

467

OVA257-264 significantly increased the OVA257-264-H-2Kb signal on the BMDC surface

468

(Figure 4a). Furthermore, DP7-C/OVA antigen peptides-treated BMDCs significantly

469

stimulated the proliferation of OT-I and OT-II splenic T cells, respectively (Figure

470

4b-4c). To further detect the mechanism of DP7-C in promoting antigen

471

cross-presentation in DCs, we explored the results of transcriptome sequencing. The

472

results showed that DP7-C/antigen-treated BMDCs showed upregulated antigen

473

uptake- and presentation-related pathway gene expression (Figure 4d). In addition, we

474

selected the antigen processing- and presentation-related genes Psme2 (related to

475

MHC-I peptide antigen processing), Tap1 (involved in promoting epitope-related 18

476

peptide transport during antigen presentation) and calreticulin (involved in processing

477

and presentation of MHC class I antigen peptides) for Western Blot verification. The

478

results showed that DP7-C/OVA257-264 could significantly upregulate the expression of

479

the selected genes (Figure 4e), which initially revealed the mechanism of DP7-C in

480

promoting the cross-presentation for antigens. Furthermore, CD103+ DCs have been

481

reported to contribute to antigen cross-presentation and DC migration to lymph nodes

482

[31-33]. Therefore, we further tested the proportion of CD103+ DCs in DP7-C treated

483

BMDCs. The results showed that DP7-C treatment could significantly increase the

484

proportion of CD103+ DCs (Figure 4f-4g), which explained why DP7-C increased

485

antigen presentation from another perspective and indicated that DP7-C-treated DCs

486

may have a greater migration ability. Thus, by subcutaneous injection of DP7-C/

487

FITC-OVA257-264, we detected the proportion of DCs with fluorescence in the lymph

488

nodes. The proportion of DCs with fluorescence in the lymph nodes increased after

489

DP7-C treatment, indicating that DP7-C-treated DCs have a relatively strong ability to

490

migrate to the lymph nodes (Figure 4h).

491 492

Figure 3. DP7-C treatment enhanced BMDC maturation and cytokine release. (a-c) 19

493 494 495 496 497 498 499 500 501 502 503

DP7-C stimulation enhanced the maturation of BMDCs. (d) Differential gene enrichment results revealed that DP7-C stimulation significantly upregulated the expression of genes related to the NF-κB pathway in BMDCs. (e-f) The TLR2-MyD88-IkK-IκB-NF-κB signaling pathway was activated in DP7-C-stimulated BMDCs. (g) ELISA analysis of CXCL2, IL-6, IL-12p70, and IL-1β concentrations in BMDC supernatants after stimulation with DP7-C. (h-i) The DP7-C and CPP-C complex interacted with TLR2. The left panel shows the 3D simulated complex structure of the interaction of DP7-C/TLR2 and CPP-C/TLR2. The right panel shows the 3D binding model of DP7-C/TLR2 and CPP-C/TLR2. DP7-C and CPP-C is colored yellow, the surrounding residues in the binding pockets are colored cyan, and the backbone of the receptor is depicted as a cyan-colored cartoon.

504 505 506 507 508 509 510 511

Figure 4. DP7-C treatment enhanced BMDC antigen cross-presentation. (a) The antigen presentation efficiency of BMDCs stained with the monoclonal antibody 25-D1.16, which recognizes the OVA257-264-H-2Kb complex. (b-c) DCs treated with DP7-C/OVA peptide could stimulate the proliferation of splenic T cells from OT-I and OT-II mice. (d) DP7-C/OVA257-264 stimulation significantly upregulated the expression of genes related to antigen uptake and presentation pathways in BMDCs. (e) DP7-C/OVA257-264-treated DCs could activate antigen processing-related genes 20

512 513 514

such as Tap1, Psme2, and calreticulin. (f-g) DP7-C stimulation increased the proportion of CD103+ BMDCs. (h) The efficiency of the migration of antigen-loaded DCs to lymph nodes.

515 516

Administration of DP7-C/OVA peptide-pulsed DC vaccines enhanced in vivo

517

immune responses and antitumor effects

518

To investigate whether the DP7-C/OVA model antigen peptide-pulsed DC vaccine can

519

trigger a corresponding immune response in vivo and exert antitumor effects, we

520

established an EG.7-OVA subcutaneous tumor model. After DP7-C/OVA model

521

antigen peptide-pulsed DC vaccination, tumor growth was significantly inhibited, and

522

tumor weight was significantly reduced (Figure 5a-5c). Similar to IFN-γ, TNF-α is

523

also a typical marker of cellular immunity, and compared to its expression level in the

524

serum of mice immunized with antigen alone, its expression level in the serum of

525

mice immunized with DP7-C/OVA model antigens-pulsed DC was significantly

526

increased (Figure 5d). Spleen lymphocyte reactions and CTL infiltration into the

527

tumor microenvironment are key indexes used to evaluate the efficacy of a vaccine.

528

Therefore, we examined intratumoral CD8+ T cell responses and splenic lymphocyte

529

responses by flow cytometry and ELISPOT. The results showed that the proportion of

530

IFN-γ-producing CD8+ T cells in the tumors of the DP7-C/OVA model

531

antigens-pulsed DC groups was significantly higher than that in the other groups

532

(Figure 5e). Additionally, the IFN-γ-secreting splenic T cells in the DP7-C/OVA

533

model antigens-pulsed DC groups as measured by ELISPOT assay were significantly

534

higher than those in the other groups (Figure 6a). The flow cytometry results for the

535

spleen also showed significant activation of splenic T lymphocytes in the DP7-C/OVA

536

model antigens-pulsed DC groups (Figure 6b-6i). These results suggested that the

537

DP7-C/OVA model antigens-pulsed DC vaccine successfully induced and enhanced

538

the cellular immune response.

21

539 540 541 542 543 544

Figure 5. Administration of DP7-C/OVA-based DC vaccines enhanced in vivo immune responses and antitumor effects. (a) Tumor growth curves of each mouse and the mean tumor volume in each group. (b) Pictures of the tumors in each group. (c) The average tumor weight of each group. (d) TNF-α production in each group. (e) The proportion of CD8+ T cells secreting IFN-γ.

22

545 546 547 548 549 550 551 552

Figure 6. Administration of DP7-C/OVA model antigens-pulsed DC vaccines enhanced antigen specific lymphocyte reaction. (a) Collected splenic T cells analyzed by ELISPOT assays to detect the antigen-specific T cells secreting IFN-γ. (b, c) The proportion of splenic CD8+ T cells. (d, e) The proportion of splenic CD8+ T cells secreting IFN-γ. (f, g) The proportion of splenic CD8+ T cells. (h, i) The proportion of splenic CD8+ T cells secreting IFN-γ.

553

Administration of the DP7-C/neoantigen-pulsed DC vaccines enhanced in vivo

554

immune responses and antitumor effects

555

Here, we used DP7-C and two neoantigens screened from LL2 to evaluate whether

556

DP7-C can enhance the efficacy of the DC vaccine stimulated by neoantigens[7]. By

557

measuring the particle size and zeta potential of DP7-C incubated with neoantigens,

558

we found that compared with the characteristics of DP7-C alone, the particle size was

559

increased and the zeta potential was decreased (Figure S5a-S5b), indicating that

560

DP7-C can form complexes with neoantigens. Moreover, the DP7-C/neoantigen

561

complex could be efficiently taken up by DCs via caveolin- and clathrin-dependent

562

pathways, and lysosomal escape was observed at 24 h (Figure S5c-S5h). Then, after

563

establishing

an

LL2

subcutaneous

tumor 23

model,

DCs

loaded

with

the

564

DP7-C/neoantigen were injected subcutaneously as an antitumor treatment, and an

565

excellent antitumor effect was observed (Figure 7a). ELISPOT results showed that

566

IFN-γ secretion by splenic T cells in the DP7-C/neoantigen groups was significantly

567

higher than that in the other groups (Figure 7b-7c), and the proportions of CD4+ T

568

cells and CD8+ T cells in the DP7-C/neoantigen groups were significantly increased

569

after mutant antigen stimulation (Figure 7d-7g), indicating that the DP7-C/neoantigen

570

complexes successfully induced a T cell-mediated immune response. Furthermore, the

571

ratio of activated CD8+ T cells secreting IFN-γ in the DP7-C/neoantigen groups was

572

significantly higher than that in the other groups in the tumor microenvironment

573

(Figure 7h). Finally, to evaluate the safety of the vaccine, we used H&E and Masson’s

574

trichrome staining to detect pathological changes and collagen deposition,

575

respectively. The results showed that the DP7-C/neoantigen-pulsed DC vaccine

576

produced no obvious toxic side effects on the main organs of the mice (Figure S6).

577

These results indicate that DP7-C can enhance the antitumor effect of the DC vaccine

578

stimulated by neoantigens.

24

579 580 581 582 583 584 585 586 587

Figure 7. The antitumor effect of DP7-C/neoantigen-pulsed DC vaccine. (a) Tumor growth curves for individual mice and the mean tumor volume of each group. (b-c) ELISPOT test of splenic T cells from mice stimulated with DP7-C/neoantigen-pulsed DCs. (d-e) Activation of splenic antigen-specific CD8+ T cells from mice immunized with DP7-C/neoantigen-pulsed DC vaccines. (f-g) Activation of splenic antigenspecific CD4+ T cells from mice immunized with DP7-C/neoantigen-pulsed DC vaccines. (h) Statistical analysis of the proportion of CD8+ IFN-γ+ T cells in tumor tissue samples.

588 25

589

DP7-C promotes MoDC from tumor patients to take up and present antigen, and

590

promote it maturation

591

To determine whether the clinical implementation of DP7-C combined with

592

neoantigens may be effective, we collected MoDCs from three lung cancer patients to

593

detect whether DP7-C can effectively promote MoDC to take up and present antigen.

594

The results showed that MoDCs can efficiently ingest DP7-C/FITC-OVA257-264, and

595

the uptake efficiency was about 88-98%. The uptake efficiencies of individual

596

FITC-OVA257-264 varied from person to person, but none of the uptake efficiencies

597

exceeded 40% (Figure 8a-8b, S7a-S7b). Subsequently, we found that after incubating

598

DP7-C/FITC-OVA257-264 with MoDCs, significant lysosomal escape occurred at 24 h

599

(Figure 8c). Furthermore, DP7-C/FITC-OVA257-264 could co-localize with the

600

caveolin- and clathrin-dependent pathway markers, while OVA257-264 could co-localize

601

with the macropinocytosis pathway marker (Figure 8d, S7e-S7f). This result is

602

consistent with the uptake pathway of DP7-C/ FITC-OVA257-264 identified in mouse

603

BMDCs. In addition, DP7-C treatment could stimulate MoDC maturation and

604

promote antigen presentation efficiency (Figure 8e-8f). And after DP7-C stimulation,

605

the proportion of CD141+ MoDCs (with a cross-presentation function in humans[34])

606

increased

607

DP7-C/neoantigen-pulsed DCs vaccine may be effective in future clinical practice.

significantly

(Figure

8g,

S7c-S7d).

26

These

results

suggest

that

608 609 610 611 612 613 614 615 616 617 618

Figure 8. Incubation of an antigen peptide with DP7-C enhanced antigen uptake and presentation efficiency and the function of MoDCs derived from tumor patients. (a, b) The efficiencies of OVA257-264 and DP7-C/OVA257-264 uptake by MoDCs. (c) MoDCs incubated with free FITC-OVA257-264 or DP7-C/FITC-OVA257-264 for 4 or 22 h and then stained with LysoTracker and DAPI. (d) Co-localization of three uptake pathway markers with OVA257-264 and DP7-C/OVA257-264. (e) Enhanced maturation of MoDCs by DP7-C stimulation. (f) Antigen presentation efficiency of MoDCs stained with the monoclonal antibody 25-D1.16, which recognizes the OVA257-264-H-2Kb complex. (g) Enhancement of the proportion of CD141+ MoDCs by DP7-C stimulation.

619 620

Discussion

621

As a promising strategy for individualized immunotherapy, cancer vaccines have

622

generated the need to enhance the immunogenicity of neoantigens. Although a variety

623

of nanoparticles-based antigen delivery platforms have been studied, most are not

624

easy to adapt to the rapid and simple loading of neoantigens that must be customized

625

for each patient[18, 19]. Therefore, a rapid and simple loading method for

626

neoantigens customized for each patient must be developed to resolve the above

627

problem. The methodological schematic of this study is shown in Figure S8. In this 27

628

study, we describe a universal "mix" method that can rapidly mix DP7-C and antigens

629

to produce nanocomplexes with both carrier and adjuvant effects. On the one hand,

630

DP7-C can be utilized as a nanocarrier that effectively enhances DC uptake of

631

antigens through caveolin- and clathrin-mediated endocytosis pathways and can

632

effectively promote the lysosomal escape of antigens in DCs. On the other hand,

633

DP7-C has the ability to further induce DC maturation and cytokine secretion, which

634

can increase the efficiency of antigen presentation and improve the immune effect of

635

the DC vaccine. In mouse model, the DC vaccine pulsed with DP7-C/OVA model

636

antigens or DP7-C/LL2-neoantigens can induce an enhanced antigen-specific

637

lymphocyte response and achieve superior antitumor effects than the DC vaccine

638

pulsed with antigens alone. Moreover, after DP7-C stimulation, the efficiency of

639

antigen uptake and presentation of MoDCs isolated from the peripheral blood of

640

patients with advanced lung cancer were significantly improved, and the proportion of

641

mature functional MoDCs was also significantly increased. The most important is that

642

DP7-C has a simple preparation process and low toxicity to DCs, and DP7-C can

643

efficiently deliver antigens into DCs without chemical modification or physical

644

emulsification of the antigens. Thus, our approach may be a potentially alternative

645

strategy to produce cancer vaccines designed for individual patients.

646

In the process of DC vaccine preparation, the efficiency of antigen uptake by DCs

647

and whether the internalized antigens can escape lysosomes are closely related to the

648

effectiveness of the DC vaccine. In this experiment, we verified that DP7-C can

649

promote the efficient uptake of antigens by BMDCs and MoDCs and can effectively

650

enable lysosomal escape. We further explored the mechanism by which DP7-C

651

promotes internalization of antigens by DCs, and the results showed that the

652

internalization of DP7-C/antigen complexes by DCs mainly occurred via caveolin-

653

and clathrin-depended pathways. In addition, DC maturation and cytokine secretion

654

are important indicators of the effectiveness of a DC vaccine. Many clinical trials

655

have shown that mDCs have significant advantages over imDCs[26]. ImDCs 28

656

effectively absorb extracellular substances such as antigens but do not release

657

cytokines[27]. Although imDCs have the ability to present antigens to T cells, they

658

lack suitable co-stimulatory signals and induce antigen-specific suppressor T cells,

659

ultimately leading to immune tolerance[35, 36]. mDCs have a limited ability to take

660

up antigens, but their surface MHC and co-stimulatory molecules are highly

661

expressed and accompanied by the secretion of a large number of cytokines, thereby

662

promoting antigen presentation and eliciting immune response[31, 37]. The cytokines

663

secreted by DCs, especially IL-1β and IL-12p70, are highly related to vaccine

664

effectiveness, which can promote CTL differentiation and CD8+ T cell

665

proliferation[28-30]. Therefore, when preparing DC-based vaccine in vitro, BMDC

666

maturation and cytokine secretion must be induced. In this experiment, we found that

667

DP7-C has a role in stimulating DC maturation. The mechanism of DP7-C in

668

stimulating DC maturation, cytokines secretion, and increasing antigen cross-

669

presentation were described in detail. These two aspects of research have laid a

670

foundation for the application of DP7-C in the study of DC vaccines based on

671

neoantigens.

672

Although the DC vaccine prepared by mix DP7-C with antigens showed significant

673

inhibition of tumor growth, a single administration of DP7-C/antigen failed to

674

eliminate tumors completely, potentially due to the immunosuppressive pathways in

675

the tumor microenvironment[38-40]. Recent research has shown that mouse tumor

676

tissue disappears completely and does not relapse after treatment with a TLR1/2

677

agonist (Diprovocim) combined with OVA and an anti-PD-L1 antibody [41]. DP7-C,

678

as a TLR2 agonist, may have a similar effect. Therefore, to produce better antitumor

679

effects and maximize the function of T cells, subsequent studies will use

680

DP7-C/neoantigen-pulsed DC vaccines in combination with immune checkpoint

681

inhibitor antibodies (such as anti-PD-1 and anti-CTLA-4 antibodies) for antitumor

682

experiments, and this combination may achieve improved antitumor effect. Although

683

this work has laid a foundation for the clinical application of DCs loaded with a 29

684

DP7-C/antigen complex in the future, some restrictions still exist. For example, the

685

screening of neoantigens and a long production time may limit its applications. With

686

the development of next-generation sequencing technology and improvement of

687

mRNA synthesis technology, introducing neoantigens into DCs in the form of mRNA

688

can overcome the shortcomings of prolonged antigen peptide synthesis and high costs,

689

which may render this system easier to apply in clinical practice[42-44]. Based on the

690

characteristics of DP7-C, we plan to use DP7-C to transfer antigen-based mRNA

691

sequences into DCs and combine this approach with immune checkpoint inhibitors or

692

chemotherapeutics for antitumor experiments, which may achieve more significant

693

antitumor effects than DP7-C/antigen peptides loaded DCs vaccine. These will

694

become our future research direction.

695

In a word, we describe a general "mix" method that can quickly mix DP7-C, which

696

possesses the characteristics of simple synthesis and low toxicity, and antigen peptides

697

to produce nanocomposites with double functions as a delivery carrier and an immune

698

adjuvant. The research method in this experiment may provide a reference for

699

follow-up research on the mechanism of delivery vectors and provide a theoretical

700

basis for further preclinical and clinical development of DP7-C/antigen-pulsed DC

701

vaccines. More importantly, our formula may be a potentially alternative strategy for

702

the production of cancer vaccines designed for individual patients.

703 704

Ethics approval and consent to participate

705

All animal procedures were approved and controlled by the Institutional Animal Care

706

and Treatment Committee of Sichuan University and performed in accordance with

707

the Guidelines for Animal Care and Use of Sichuan University. OT-I and OT-II mice

708

were bred in our laboratory.

709 710

Consent for publication

711

Not applicable. 30

712 713

Ethics approval and ethical standards

714

The work described has been carried out in accordance with The Code of Ethics of the

715

World Medical Association for experiments involving humans. This experimental

716

program was approved by the “West China Hospital Review Committee” and

717

obtained written informed consent from voluntary patients.

718 719

Availability of data and materials

720

The majority of the data obtained and the materials used are presented in this

721

publication or in the supplementary material. Additional data or materials will be

722

provided upon reasonable request and the signing of a material transfer agreement. All

723

animal procedures were approved and controlled by the Institutional Animal Care and

724

Treatment Committee of Sichuan University and conducted according to the Animal

725

Care and Use Guidelines of Sichuan University.

726 727

Authors' contributions

728

ZR and YL designed the study; ZR was responsible for all experiments and articles;

729

TL, TYM and JX helped ZR performed the immunological experiments; HQY and

730

ZBL helped ZR conducted the gene expression analysis; YL and DZY contributed to

731

manuscript corrections. All authors read and approved the final manuscript.

732 733

Acknowledgments

734

Not applicable

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Declaration of interests √ The authors declare that they have no known competing financial interestsor personal relationships that could have appeared to influence the work reported in this paper. ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: