Cell-Penetrating Peptide Enhanced Antigen Presentation for Cancer

Subscriber access provided by BUFFALO STATE

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Bioconjugate Chemistry

Cell-Penetrating Peptide Enhanced Antigen Presentation for Cancer Immunotherapy

1 2 3

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

4

Shen, Zhuang Liu* and Rui Peng*

5 6

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

7

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

8

China

9 10

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

11

‡

These authors contributed equally to this work.

12 13

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

14

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

15

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

16

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

17

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

18

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

19

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

20

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

21

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

22

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

ACS Paragon Plus Environment

Bioconjugate Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

23

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

24

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

25

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

26

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

27

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

28

low cost, and remarkable biocompatibility.

29 30

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

31

blockade

32 33

INTRODUCTION

34

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

35

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

36

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

37

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

38

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

39

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

40

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

41

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

42

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

43

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

44

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


Page 2 of 29

Page 3 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


45

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

46

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

47

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

48

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

49

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

50

pathways.15

51

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

52

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

53

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

54

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

55

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

56

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

57

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

58

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

59

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

60

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

61

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

62

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

63

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

64

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

65

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

66

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



67

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

68

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

69

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

70 71 72

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

73 74

RESULTS and DISCUSSION

75

Successful conjugation of CLIP6 peptide to OVA

76

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

77

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

78

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

79

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

80

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

81

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

82

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


Page 4 of 29

Page 5 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60




97

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

98

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

99

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

100

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

101

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

102

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

103

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

104

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

105

were chosen to serve as the cancer vaccine.

106 107

Enhanced non-endosomal internalization of CLIP6-OVA conjugates

108

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

109

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

110

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

111

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

112

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

113

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

114

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

115

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

116

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

117

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

118

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


Page 6 of 29

Page 7 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60





Page 8 of 29

Page 9 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60




164

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

165 166

Enhanced antigen cross-presentation and cytokine secretion in vitro

167

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

168

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

169

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

170

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

171

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

172

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

173

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

174

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

175

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

176

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

177

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

178

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

179

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

180

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

181

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

182

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

183

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

184

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

185

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


Page 10 of 29

Page 11 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60




194 195 196 197

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).

198

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

199

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

200

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

201

secreted levels of tumor necrosis

202

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

203

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

204

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

205

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

206

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

207

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

208

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

209

subsequently elicit antigen-specific cell-mediated immune response.

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

210 211

Enhanced lymph node migration

212

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

213

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

214

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

215

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

216

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

217

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


Page 12 of 29

Page 13 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60




Page 14 of 29

238

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

239

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

240

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

241

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

242

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

243

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

244

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

245

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

246

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

247

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

248

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

249

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

250

of

251

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

252

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

253

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

254

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

255

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

256

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

257

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

258

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

259

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

Page 15 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60




267 268 269 270 271

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).

272 273

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

274

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

275

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

276

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

277

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

278

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

279

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

280

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

281

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

282

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

283

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

284

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

285

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

286

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

287

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

288

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

289

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

290

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

291

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


Page 16 of 29

Page 17 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

292


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

293 294 295 296 297

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



298 299

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.

300

CONCLUSION

301

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

302

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

303

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

304

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

305

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

306

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

307

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

308

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

309

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

310

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

311

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

312

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

313

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

314

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

315

shows great potential in clinical translation.

316 317

EXPERIMENTAL SECTION

318

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

319

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

320

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


Page 18 of 29

Page 19 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


321

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

322

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

323

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

324

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

325

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

326

(Beyotime).47

327 328

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

329

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

330

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

331

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

332

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

333

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

334

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

335 336

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

337

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

338

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

339

bromide (MTT) assay (Sigma).

340 341

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

342

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



343

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

344

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

345

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

346

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

347

under the confocal microscopy (LSM 800, Zeiss).

348 349

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

350

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

351

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

352

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

353

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

354

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

355

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

356

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

357

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

358

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

359

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

360

assay.

361 362

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

363

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

364

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


Page 20 of 29

Page 21 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


365

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

366

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

367

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

368

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

369

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

370

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.


Page 23 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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.


Page 24 of 29

Page 25 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


432

REFERENCES

433

1. Kirkwood, J. M.; Butterfield, L. H.; Tarhini, A. A.; Zarour, H.; Kalinski, P.; Ferrone, S. (2012) Immunotherapy of Cancer in 2012. CA-Cancer J. Clin. 62, 309-335. 2. Giarelli, E. (2007) Cancer Vaccines: A New Frontier in Prevention and Treatment. Oncology (Williston Park, N.Y.) 21 (11 Suppl Nurse Ed):11-7; discussion 18. 3. Nawrocki, S.; Mackiewicz, A. (1999) Genetically Modified Tumour Vaccines - Where We Are Today. Cancer Treat. Rev. 25, 29-46. 4. Jager, E.; Jager, D.; Knuth, A. (2003) Antigen-Specific Immunotherapy and Cancer Vaccines. Int. J. Cancer 106, 817-820. 5. Liu, Y.; Chen, C. (2016) Role of Nanotechnology in HIV/AIDS Vaccine Development. Adv. Drug Delivery Rev. 103, 76-89. 6. Wen, D.; Chen, G.; Chen, Q.; Li, P. Y.; Cheng, H.; Gu, Z. (2019) Engineering Protein Delivery Depots for Cancer Immunotherapy. Bioconjugate Chem. 3, 515-524 7. Schirmbeck, R.; Reimann, J. (2002) Alternative Processing of Endogenous or Exogenous Antigens Extends the Immunogenic, H-2 Class I-Restricted Peptide Repertoire. Mol. Immunol. 39, 249-259. 8. Zhang, Z. H.; Cao, W. G.; Jin, H. L.; Lovell, J. F.; Yang, M.; Ding, L. L.; Chen, J.; Corbin, I.; Luo, Q. M.; Zheng, G. (2009) Biomimetic Nanocarrier for Direct Cytosolic Drug Delivery. Angew. Chem., Int. Edit. 48, 9171-9175. 9. Kim, D. T.; Mitchell, D. J.; Brockstedt, D. G.; Fong, L.; Nolan, G. P.; Fathman, C. G.; Engleman, E. G.; Rothbard, J. B. (1997) Introduction of Soluble Proteins into the MHC Class I Pathway by Conjugation to an HIV tat Peptide. J. Immunol. 159, 1666-1668. 10. Joffre, O. P.; Segura, E.; Savina, A.; Amigorena, S. (2012) Cross-Presentation by Dendritic Cells. Nat. Rev. Immunol. 12, 557-569. 11. Rowell, J. F.; Ruff, A. L.; Guarnieri, F. G.; Staveleyocarroll, K.; Lin, X. L.; Tang, J.; August, J. T.; Siliciano, R. F. (1995) Lysosome-Associated Membrane Protein-1-Mediated Targeting of the HIV1 Envelope Protein to an Endosomal/Lysosomal Compartment Enhances Its Presentation to MHC Class II-Restricted T-Cells. J. Immunol. 155, 1818-1828. 12. Nierkens, S.; Tel, J.; Janssen, E.; Adema, G. J. (2013) Antigen Cross-Presentation by Dendritic Cell Subsets: One General or All Sergeants? Trends Immunol. 34, 361-370. 13. Zhang, L.; Conejo-Garcia, J. R.; Katsaros, D.; Gimotty, P. A.; Massobrio, M.; Regnani, G.; Makrigiannakis, A.; Gray, H.; Schlienger, K.; Liebman, M. N. et al. (2003) Intratumoral T cells, Recurrence, and Survival in Epithelial Ovarian Cancer. N. Engl. J. Med. 348, 203-213. 14. Hoeffel, G.; Ripoche, A. C.; Matheoud, D.; Nascimbeni, M.; Escriou, N.; Lebon, P.; Heshmati, F.; Guillet, J. G.; Gannage, M.; Caillat-Zucman, S. et al. (2007) Antigen Crosspresentation by Human Plasmacytoid Dendritic Cells. Immunity 27, 481-492. 15. Chen, J. A.; Li, Z. R.; Huang, H.; Yang, Y. Z.; Ding, Q. A.; Mai, J. H.; Guo, W.; Xu, Y. H. (2011) Improved Antigen Cross-Presentation by Polyethyleneimine-Based Nanoparticles. Int. J. Nanomed. 6,

434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469



470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 504 505 506 507 508

77-84. 16. Frankel, A. D.; Pabo, C. O. (1988) Cellular Uptake of the TAT Protein from Human Immunodeficiency Virus. Cell 55, 1189-1193. 17. Pooga, M.; Hallbrink, M.; Zorko, M.; Langel, U. (1988) Cell Penetration by Transportan. Faseb J. 12, 67-77. 18. Asai, T.; Tsuzuku, T.; Takahashi, S.; Okamoto, A.; Dewa, T.; Nango, M.; Hyodo, K.; Ishihara, H.; Kikuchi, H.; Oku, N. (2014) Cell-Penetrating Peptide-Conjugated Lipid Nanoparticles for siRNA Delivery. Biochem. Biophys. Res. Commun. 444, 599-604. 19. Wang, Z.; Chen, Y. Z.; Liu, E. G.; Gong, J. B.; Shin, M. C.; Huang, Y. Z. (2014) CPP-mediated Protein Delivery in a Noncovalent Form: Proof-of-Concept for Percutaneous and Intranasal Delivery. Protein Pept. Lett. 21, 1129-1136. 20. Feni, L.; Neundorf, I. (2017) The Current Role of Cell-Penetrating Peptides in Cancer Therapy. Adv. Exp. Med. Biol. 1030, 279-295. 21. Medina, S. H.; Miller, S. E.; Keim, A. I.; Gorka, A. P.; Schnermann, M. J.; Schneider, J. P. (2016) An Intrinsically Disordered Peptide Facilitates Non-Endosomal Cell Entry. Angew. Chem., Int. Edit. 55, 3369-3372. 22. Chockalingam, A.; Brooks, J. C.; Cameron, J. L.; Blum, L. K.; Leifer, C. A. (2009) TLR9 Traffics through the Golgi Complex to Localize to Endolysosomes and Respond to CpG DNA. Immunol. Cell Biol. 87, 209-217. 23. Taube, J. M.; Klein, A.; Brahmer, J. R.; Xu, H. Y.; Pan, X. Y.; Kim, J. H.; Chen, L. P.; Pardoll, D. M.; Topalian, S. L.; Anders, R. A. (2014) Association of PD-1, PD-1 Ligands, and Other Features of the Tumor Immune Microenvironment with Response to Anti-PD-1 Therapy. Clin. Cancer Res. 20, 5064-5074. 24. Chen, L. P.; Han, X. (2015) Anti-PD-1/PD-L1 Therapy of Human Cancer: Past, Present, and Future. J. Clin. Invest. 125, 3384-3391. 25. Kalia, J.; Raines, R. T. (2010) Advances in Bioconjugation. Curr. Org. Chem. 14, 138-147. 26. Schwartz, R. H.; Yano, A.; Paul, W. E. (1978) Interaction between Antigen-Presenting Cells and Primed T-Lymphocytes-Assessment of IR Gene-Expression in Antigen-Presenting Cell. Immunol. Rev. 40, 153-180. 27. Kapsenberg, M. L.; Stiekema, F. E. M.; Teunissen, M. B. M. (1985) Dendritic Cells and Macrophages as Antigen Presenting Cells in Ovalbumin-Induced Lymphocyte-T Proliferation Invitro. Adv. Exp. Med. Biol. 186, 389-394. 28. Ivanov, A. I. (2014) Pharmacological Inhibitors of Exocytosis and Endocytosis: Novel Bullets for Old Targets. Methods in molecular biology (Clifton, N.J.) 1174, 3-18. 29. Fujimoto, L. M.; Roth, R.; Heuser, J. E.; Schmid, S. L. (2000) Actin Assembly Plays a Variable, but Not Obligatory Role in Receptor-Mediated Endocytosis in Mammalian Cells. Traffic 1 (2), 161171. 30. Albert, M. L.; Sauter, B.; Bhardwaj, N. (1998) Dendritic Cells Acquire Antigen from Apoptotic Cells and Induce Class I Restricted CTLs. Nature 392, 86-89. ACS Paragon Plus Environment

Page 26 of 29

Page 27 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

509 510 511 512 513 514 515 516 517 518 519 520 521 522 523 524 525 526 527 528 529 530 531 532 533 534 535 536 537 538 539 540 541 542 543 544 545 546 547 548


31. Krieg, A. M. (2002) CpG Motifs in Bacterial DNA and Their Immune Effects. Annu. Rev. Immunol. 20, 709-760. 32. Bauer, S.; Kirschning, C. J.; Hacker, H.; Redecke, V.; Hausmann, S.; Akira, S.; Wagner, H.; Lipford, G. B. (2001) Human TLR9 Confers Responsiveness to Bacterial DNA via Species-Specific CpG Motif Recognition. Proc. Natl. Acad. Sci. U. S. A. 98, 9237-9242. 33. Pan, J. B.; Wang, Y. Q.; Zhang, C.; Wang, X. Y.; Wang, H. Y.; Wang, J. J.; Yuan, Y. Z.; Wang, X.; Zhang, X. J.; Yu, C. S. et al. (2018) Antigen-Directed Fabrication of a Multifunctional Nanovaccine with Ultrahigh Antigen Loading Efficiency for Tumor Photothermal-Immunotherapy. Adv. Mater. 30 (8), 8. 34. Beg, A. A.; Baltimore, D. (1996) An Essential Role for NF-Kappa B in Preventing TNF-AlphaInduced Cell Death. Science 274, 782-784. 35. Liu, S. X.; Yu, Y. Z.; Zhang, M. H.; Wang, W. Y.; Cao, X. T. (2001) The Involvement of TNFAlpha-Related Apoptosis-Inducing Ligand in the Enhanced Cytotoxicity of IFN-Beta-Stimulated Human Dendritic Cells to Tumor Cells. J. Immunol. 166, 5407-5415. 36. Brunda, M. J.; Luistro, L.; Warrier, R. R.; Wright, R. B.; Hubbard, B. R.; Murphy, M.; Wolf, S. F.; Gately, M. K. (1993) Antitumor and Antimetastatic Activity of Interleukin-12 against Murine Tumors. J. Exp. Med. 178, 1223-1230. 37. Zaki, M. H.; Wysocka, M.; Everetts, S. E.; Wang, K. S.; French, L. E.; Ritz, J.; Rook, A. H. (2002) Synergistic Enhancement of Cell-Mediated Immunity by Interleukin-12 plus Interleukin-2: Basis for Therapy of Cutaneous T Cell Lymphoma. J. Invest. Dermatol. 118, 366-371. 38. Seder, R. A.; Darrah, P. A.; Roederer, M. (2008) T-cell Quality in Memory and Protection: Implications for Vaccine Design. Nat. Rev. Immunol. 8, 247-258. 39. Zhang, Q. B.; Wei, W.; Wang, P. L.; Zuo, L. P.; Li, F.; Xu, J.; Xi, X. B.; Gao, X. Y.; Ma, G. H.; Xie, H. Y. (2017) Biomimetic Magnetosomes as Versatile Artificial Antigen-Presenting Cells to Potentiate T-Cell-Based Anticancer Therapy. ACS Nano 11, 10724-10732. 40. Parsons, T.; Spendlove, I.; Nirula, R.; Writer, M.; Carter, G.; Carr, F.; Durrant, L. G. (2004) A Novel CEA Vaccine Stimulates T Cell Proliferation, Gamma IFN Secretion and CEA Specific CTL Responses. Vaccine 22, 3487-3494. 41. Mahoney, K. M.; Rennert, P. D.; Freeman, G. J. (2015) Combination Cancer Immunotherapy and New Immunomodulatory Targets. Nat. Rev. Drug Discovery 14, 561-584. 42. Xu, J.; Xu, L. G.; Wang, C. Y.; Yang, R.; Zhuang, Q.; Han, X.; Dong, Z. L.; Zhu, W. W.; Peng, R.; Liu, Z. (2017) Near-Infrared-Triggered Photodynamic Therapy with Multitasking Upconversion Nanoparticles in Combination with Checkpoint Blockade for Immunotherapy of Colorectal Cancer. ACS Nano 11, 4463-4474. 43. Topalian, S. L.; Hodi, F. S.; Brahmer, J. R.; Gettinger, S. N.; Smith, D. C.; McDermott, D. F.; Powderly, J. D.; Carvajal, R. D.; Sosman, J. A.; Atkins, M. B. et al. (2012) Safety, Activity, and Immune Correlates of Anti-PD-1 Antibody in Cancer. N. Engl. J. Med. 366, 2443-2454. 44. Van Hooren, L.; Sandin, L. C.; Moskalev, I.; Ellmark, P.; Dimberg, A.; Black, P.; Totterman, T. H.; Mangsbo, S. M. (2017) Local Checkpoint Inhibition of CTLA-4 as a Monotherapy or in Combination with Anti-PD1 Prevents the Growth of Murine Bladder Cancer. Eur. J. Immunol. 47,



549 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567

385-393. 45. Yang, R.; Xu, J.; Xu, L. G.; Sun, X. Q.; Chen, Q.; Zhao, Y. H.; Peng, R.; Liu, Z. (2018) Cancer Cell Membrane-Coated Adjuvant Nanoparticles with Mannose Modification for Effective Anticancer Vaccination. ACS Nano 12, 5121-5129. 46. Guo, Y. G.; Lei, K. W.; Tang, L. (2018) Neoantigen Vaccine Delivery for Personalized Anticancer Immunotherapy. Front. Immunol. 9, 8. 47. Laemmli, U. K. (1970) Cleavage of Structural Proteins During Assembly of Head of Bacteriophage-T4. Nature 227, 680-+. 48. Chenwoan, M.; Delaney, C. P.; Fournier, V.; Wakizaka, Y.; Murase, N.; Fung, J.; Starzl, T. E.; Demetris, A. J. (1995) A New Protocol for the Propagation of Dendritic Cells from Rat Bone-Marrow Using Recombinant GM-CSF, and Their Quantification Using the Mab OX-62. J. Immunol. Methods 178, 157-171. 49. Inaba, K.; Inaba, M.; Romani, N.; Aya, H.; Deguchi, M.; Ikehara, S.; Muramatsu, S.; Steinman, R. M. (1992) Generation of Large Numbers of Dendritic Cells from Mouse Bone-Marrow Cultures Supplemented with Granulocyte Macrophage Colony-Stimulating Factor. J. Exp. Med. 176, 16931702. 50. Le Belle, J. E.; Harris, N. G.; Williams, S. R.; Bhakoo, K. K. (2002) A Comparison of Cell and Tissue Extraction Techniques Using High-Resolution H-1-NMR Spectroscopy. NMR Biomed. 15 (1), 37-44.

568


Page 28 of 29

Page 29 of 29 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


569 570 571

TOC

572


Cell-Penetrating Peptide Enhanced Antigen Presentation for Cancer

Recommend Documents