PLGA-nanoparticle mediated delivery of anti-OX40 monoclonal antibody enhances anti-tumor cytotoxic T cell responses

Cellular Immunology 287 (2014) 91–99

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PLGA-nanoparticle mediated delivery of anti-OX40 monoclonal antibody enhances anti-tumor cytotoxic T cell responses Mingshui Chen a,b,⇑, Haichao Ouyang a,b, Shangyong Zhou c, Jieyu Li a,b, Yunbin Ye a,b,⇑ a

Laboratory of Immuno-Oncology, Department of Medical Oncology, Fujian Provincial Tumor Hospital, Fuzhou 350014, China Fujian Provincial Key Laboratory of Translational Cancer Medicine, Fuzhou 350014, China c State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou 350000, China b

a r t i c l e

i n f o

Article history: Received 16 September 2013 Accepted 6 January 2014 Available online 13 January 2014 Keywords: OX40 Agonist anti-OX40 monoclonal antibody Cytotoxic T lymphocytes Poly(DL-lactide-co-glycolide) Nanoparticle

a b s t r a c t OX40 (CD134) is a tumor necrosis factor (TNF) receptor expressed mainly on activated T cells and transmits a potent costimulatory signal once engaged. Agonistic anti-OX40 monoclonal antibody (mAb) enhances tumor immune response leading to therapeutic effects in mouse tumor models. However, when tested in phase I clinical trials it did not show objective clinical activity in cancer patients. In this study, we examined the feasibility of nanoparticle (NP)-mediated delivery of anti-OX40 mAb to efficiently induce cytotoxic T lymphocyte (CTL) responses. The biodegradable poly(DL-lactide-co-glycolide) nanoparticle (PLGA-NP) carrying anti-OX40 mAb, anti-OX40-PLGA-NP, was prepared by double emulsion method and showed an average diameter of 86 nm with a loading efficiency of 25%. We found that anti-OX40PLGA-NP induced CTL proliferation and tumor antigen-specific cytotoxicity as well as cytokine production more strongly than free anti-OX40 mAb. These results suggest that PLGA-based nanoparticle formulation may provide efficient delivery system of anti-OX40 mAb for cancer immunotherapy. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction TNFR/TNF superfamily members control a wide array of immune function. Over the past decade, extensive research has uncovered that one of the most important and prominent interactions in this family occurs between OX40 and its ligand, OX40L [1]. These molecules effectively regulate conventional CD4 and CD8 T cells and subsequent studies revealed their ability to modulate the function of other immune cells such as NK, NKT, APC and neutrophils, etc [1,2]. Signaling through OX40 increases T-cell expansion and survival, and augments pro-inflammatory cytokine production [3,4]. Ligation of OX40 could recruit TRAF2 and TRAF3, causing activation of the canonical and noncanonical NF-jB pathways [5,6] that up-regulate the expression of anti-apoptotic Bcl-2 family members (Bcl-2 and Bcl-XL) thus providing the basis

Abbreviations: APC, antigen presenting cells; Bcl, B-cell lymphoma; CFSE, carboxyfluorescein diacetate succimidyl ester; CTL, cytotoxic T lymphocyte; DCM, dichloromethane; EDC, (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; GMCSF, granulocyte macrophage colony-stimulating factor; IFN-c, interferon-c; IL-2, interleukin-2; NF-jB, nuclear factor-kappa B; NK, natural killer cells; NKT, natural killer T cells; NP, nanoparticle; PBMC, peripheral blood mononuclear cells; PLGA, poly(DL-lactide-co-glycolide); PVA, polyvinyl alcohol; RECIST, response evaluation criteria in solid tumors; TNF, tumor necrosis factor. ⇑ Corresponding authors at: Laboratory of Immuno-Oncology, Department of Medical Oncology, Fujian Provincial Tumor Hospital, Fuzhou 350014, China. E-mail addresses: [email protected] (M. Chen), [email protected] (Y. Ye). 0008-8749/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.cellimm.2014.01.003

for clonal expansion and expanded memory pool of activated T cells [7]. Given that OX40 engagement can expand T-cell populations and support T-cell memory, agonists including monoclonal antibodies (mAbs) and soluble forms of OX40L have been used in the treatment of a variety of transplantable tumors in mice. An anti-OX40 agonist antibody appeared to be effective in various tumor models including MC303 sarcomas, CT26 colon carcinomas, SM1 breast cancer, and small B16 melanoma [8]. Mirroring this seminal work, anti-tumor activity of anti-OX40 antibody was also confirmed in additional preclinical tumor models [3,9–12]. Similarly, overexpression of OX40L in tumor cell lines and dendritic cells also induced strong antitumor immunity [13,14]. However, as a monotherapy, triggering of OX40 alone has only marginal effects on established and more clinically relevant poorly immunogenic tumors [15]. Furthermore, in a recent phase I clinical trial, a mouse anti-human OX40 antibody was given to 30 patients on days 1, 3, and 5 at the dose of 0.1, 0.4, and 2.0 mg/kg, respectively. The antibody was well tolerated with minimal toxicity and certain tumor size reduction was observed. However, none of the patients showed an objective response by Response Evaluation Criteria in Solid Tumors (RECIST) criteria [2]. This may not be surprising since OX40 expression on tumor-specific T cells would require sufficient priming and rigorous stimulation by an agonist that are probably not provided by poorly immunogenic tumors and the agonist antibody itself as well. Therefore, approaches that increase priming of activated T cells into the tumor and enhance accessibility of the

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immune cells to the antibody are attractive for further improving anti-OX40 antibody therapy. Nanoparticles (NPs) are defined as particulate dispersions or solid particles with a size in the range of 10–1000 nm and have shown great potential in various biomedical applications. A wide variety of biomolecules can be delivered using NP such as DNA, RNA, proteins and other biological macromolecules either adsorbed or covalently attached to the surface or encapsulated within the particles. NP also allows a targeted delivery of encapsulated payload to specific organs or cells, or sustained release of carried biomolecules [16,17]. Among the nanoparticulate carriers, poly(lactic-co-glycolic acid) (PLGA) is one of the most successfully developed biodegradable polymers whose hydrolysis releases two metabolite monomers, lactic acid and glycolic acid, which are endogenous and readily metabolized by the body via the Krebs cycle. Thus, a minimal systemic toxicity is associated with the use of PLGA for drug delivery or biomaterial applications [18]. PLGAbased NP presents many advantages for delivery of drugs, proteins, peptides or nucleic acids by protecting them from degradation and enhancing their stability [17]. These drug-loaded PLGA-NPs, particularly when polyethylene glycol (PEG) is incorporated to the NP surface (PEGylation), not only prolong the in vivo circulation time of the therapeutics from several minutes to several hours but also reduce cellular uptake along the endocytic route thereby increasing the efficacy of treatments [19,20]. Another major advantage of PLGA over other polymers is that PLGA is approved by the US FDA and European Medicine Agency (EMA) in various drug delivery systems in humans [17,21], leading PLGA-based NP in a good position for clinical trials. Our recent work has demonstrated that PLGA nanoparticlemediated delivery of tumor antigenic peptides induced effective immune response [22]. In this study, we hypothesized that an anti-OX40 agonist antibody conjugated in PLGA-NP would elicit more robust immune response. We report here on the preparation and characterization of anti-OX40 antibody-loaded PLGA-NP and its in vitro efficacy on CTL proliferation, activation and cytotoxicity.

conjugated anti-human CD8, FITC-conjugated anti-human CD3, PE-Cy5 conjugated anti-CD25 and anti-CD69 antibodies were obtained from BD Bioscience. Dynabeads CD3/CD28 T cell expander was purchased from Dynal Biotech (Oslo, Norway). 2.2. Preparation and characterization of anti-OX40 antibody-loaded PLGA-NP PLGA nanoparticles were prepared using an oil-in-water emulsion solvent extraction/evaporation technique as previously described with minor modifications [23]. Briefly, 50 mg of the PLGA was dissolved in 500 ll of acetone and 750 ll of DCM. The polymer solution was added to 10 ml of an aqueous solution containing 3% (w/v) PVA as a stabilizer. The mixture was emulsified for 20 s with a sonicator operated at 70 W. The formed o/w emulsion was poured into 50 ml of a PVA aqueous solution (0.25%, w/v) and magnetically stirred for 24 h at room temperature to completely extract/evaporate the organic solvent and harden the particles. The produced nanoparticles were collected by centrifugation at 11,000 rpm (Optima™ L-100 XP ultracentrifuge, Beckman coulter), washed three times with deionized water and freeze-dried. For the covalent attachment of anti-OX40 antibody onto the nanoparticle surface, 4.5 lg of EDC was added to a 360 ll mixture of 400 lg nanoparticles and 400 lg mAb. The reaction mixture was stirred gently for 2 h at room temperature. Excess linking reagent and soluble byproducts were separated by centrifugation at 13,200 rpm for 10 min, and the sediment was washed three times with 1 ml PBS (pH 7.4). Finally, the antibody-loaded nanoparticles were redispersed in 100 ll of PBS and protein content determined by Bradford assay. For measuring the size of the PLGA-NP, samples were sputter coated with gold/palladium and imaged with a scanning electron microscope (Phillips XL30, FEI, OR). Size distribution was analyzed using MetaMorph (Version 7.1.0.0, Molecular Devices Inc., Sunnyvale, CA). Zeta potential of the PLGA-NP was measured using the ZetasizerÒ Nano ZS90 (Malvern Instruments, Malvern, UK). 2.3. Determination of antibody loading on PLGA-NP

2. Materials and methods 2.1. Materials EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride, sulfo-NHS (sulfosuccinimidyl ester), DCM (dichloromethane) were purchased from Sigma–Aldrich (St. Louis, MO). Cy5.5 NHS ester was obtained from Amersham. COOH-terminated PLGA (inherent viscosity range 0.15–0.25 dL/g, lactide/Glycolide ratio 50:50), polyvinyl alcohol (PVA, average molecular weight 30,000–70,000) were purchased from Sigma–Aldrich (St. Louis, MO). RPMI-1640, L-glutamine, and gentamycin were purchased from Gibco-BRL (Gaithersburg, MD). Tumor necrosis factor-a (TNF-a) was purchased from Promega (Madison, WI). Recombinant human granulocyte macrophage colony-stimulating factor (GMCSF) was purchased from Peprotech Inc (Rocky Hill, NJ). IL-4 were purchased from R&D Systems (Minneapolis, MN). Lipopolysaccharide (LPS) were from Sigma. IL-2, IL-4, IL-10, IL-17 ELISA set and IFN-c ELISA Set were purchased from R&D Systems. Carboxyfluorescein diacetate succimidyl ester (CFSE) was purchased from Invitrogen (Carlsbad, CA). Standard lactate dehydrogenase release assay kit was purchased from Promega. AFP158–166 (FMNKEIYEI) and gp100154–162 peptides were synthesized by GenScript Corp (Piscataway, NJ). Human hepatocellular carcinoma cell line HepG2 was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Anti-human OX40 antibody was purchased from R&D Systems, and the FITC-conjugated anti-human OX40, PE-Cy7 conjugated anti-human CD4, APC-Cy7 or FITC

Anti-OX40-PLGA-NP was identified using Bradford method with Coomassie dye. Briefly, 300 ll of Coomassie Plus reagent was added to 10 ll of dispersion of anti-OX40-loaded PLGA-NP. After 10-min incubation, the absorbance was measured at 595 nm using a microplate reader (Thermo scientific, USA). Based on the optical density, the amount of the antibody conjugated in PLGA-NP was calculated from a standard curve of BSA solution in the concentration range from 10 lg/ml to 750 lg/ml. 2.4. Generation of CTL from human peripheral blood mononuclear cells (PBMC) PBMC were collected from healthy donors. CD14+ cells were purified by positive selection with CD14 MicroBeads (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions. Dendritic cells were generated from CD14+ cells cultured for 7 days in the presence of 500 IU/ml GM-CSF and 500 IU/ml IL-4 in complete medium (RPMI 1640 supplemented with 10% heat inactivated FBS, 2 lM L-glutamine, and antibiotics). On day 7 the maturation agent LPS at 100 ng/ml was added in the culture for 24 h. To generate antigen-specific DC, the mature DC were pulsed with 5 lg/ml AFP158–166 or gp100154–162 peptide in the presence of 5 lg/ml b-2-microglobulin for 2 h at 37 °C and then inactivated by Mitomycin C. CD8+ cells were purified by positive selection with CD8 MicroBeads (Miltenyi Biotec) according to the manufacturer’s instructions. To generate CTL cells, CD8+ cells were stimulated with DC at a ratio of 1:10 in complete culture medium supplemented

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with 1000 IU/ml IL-2 (R&D Systems) in a final volume of 200 ll/ well in 96-well round-bottom plates.

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conjugated anti-CD25 plus FITC-conjugated anti-human CD8 antibodies or PE-Cy5 conjugated anti-human CD69 plus FITC conjugated anti-human CD8 antibodies.

2.5. Nanoparticle degradation and anti-OX40-mAb release 2.7. CFSE proliferation assay To study the degradation profile of the PLGA nanoparticles under physiological conditions, the anti-OX40-PLGA nanoparticle suspension was diluted 1:5 with phosphate buffered saline (PBS), pH 7.4 in a low volume Eppendorf UVette. Hydrodynamic diameter was measured at different time points at 25 °C by photon correlation spectroscopy (PCS) using the Zetasizer, Nano ZS, Malvern Instruments. Each time point was measured in triplicate with total measurements up to 6 h. For the anti-OX40-mAb release studies at pH 7.4, a stock of anti-OX40-PLGA-NP suspension in PBS was incubated at 37 °C and aliquots of 100 ll suspension was transferred at particular time intervals and centrifuged immediately at 10,000 rpm for 10 min. The supernatants were collected and quantified for the amount of anti-OX40-mAb using BCA method. Both degradation and release experiments were repeated for 3 times.

The DC-stimulated CD8+ T cells were stained with 5 lM carboxyfluorescein diacetate succinimidyl ester (CFSE) for 10 min at 37 °C. The labeled T cells were seeded on 96-well plate and incubated with free anti-OX40 mAb, NP or anti-OX40-PLGA-NP for 3 days. Untreated CD8+ T cells and the cells cultured in anti-OX40 antibody-coated plates served as control. The CFSE fluorescence intensity was measured by FACScanII flow cytometer. 2.8. Measurement of cytokine production by ELISA Cytokine IFN-c, IL-2, IL-4, IL-10 and IL-17 production were induced under the same culture condition used for proliferation assay. After 3 days of incubation, supernatants were collected and assayed for the cytokine expression using the respective ELISA kits (BD Biosciences).

2.6. Flow cytometry Cell-surface OX40 expression on CD8+ T cells isolated from PBMCs using CD8 MicorBeads and then stimulated with mature DC was determined by using single-parameter fluorescence-activated cell sorter (FACS) analysis with FITC-conjugated anti-OX40 antibody by a FACSCanII flow cytometer (BD-PharMingen). The changes in T cell phenotype before and after anti-OX40-PLGA-NP treatment were assessed with dual staining of PE-Cy7 conjugated anti-human CD4 plus FITC conjugated anti-human CD3, APC-Cy7 anti-human CD8 plus FITC conjugated anti-human CD3, PE-Cy5

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2.9. CTL cytotoxic assay For cellular cytotoxicity assays, a standard lactate dehydrogenase release assay was performed according to the manufacturer’s instructions. In brief, 2  106 AFP-expressing HepG2 target cells were added to the AFP or gp100 peptide-specific CTLs at the ratios of 1:10,1:20 1:40 in a final volume of 200 ll in the presence of anti-OX40 mAb or anti-OX40-PLGA-NP at 10 lg/ml. The tumor

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Fig. 1. Characterization of anti-OX40-PLGA-NPs. (A) Scanning electron microscopy was used to view the morphology of nano-scale particles. Scale bars = 100 nm. (B) Particle size distributions based on the images taken. (C) Zeta potential of the particles. (D) Schematic representation of the anti-OX40-PLGA-NP formulation. PLGA nanoparticles were produced by double emulsion solvent diffusion method. Anti-OX40 mAb was covalently conjugated to the surface of the nanoparticles using EDC activation method.

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antigen-specific CTLs were generated from the CD8+ T cells that had been stimulated with the AFP158–166 or gp100154–162 pulsed DC in the presence of IL-2. The plates were incubated at 5% CO2, 37 °C for 3 days. 10 ll of 10  lysis buffer was added into the wells for maximum release, and incubation continued for 45 min at 37 °C. 50 ll of supernatant was transferred to a fresh ELISA plate and 50 ll substrate was added to each well and mixed. The mixture was incubated for 30 min at room temperature in the dark and 50 ll stop solution was added to each well. The plate was read at 490 nm and the specific lysis was calculated by the following formula: % Cytotoxicity = 100  (Experiment Effector Spontaneous Target Spontaneous)/(Target Maximum Target Spontaneous) [22]. 2.10. Statistical analysis

A Hydrodynamic diameter (nm)

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Data were expressed as mean ± standard deviation (SD). Student’s t test was used to compare quantitative data and values of p less than 0.05 were considered statistically significant.

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3.1. Characterization of PLGA nanoparticles Nanoparticles were prepared from a PLGA polymer containing free carboxylic end groups using a modified double emulsion solvent diffusion method. Scanning electron microscopy revealed that the formulated nanoparticles had an average diameter of 86.0 ± 14.1 nm (Fig. 1A and B) with a mean zeta potential of 12.8 ± 1.5 mV (Fig. 1C). To generate anti-OX40-PLGA-NP, the EDC and NHS activation method was used to covalently link amine groups of antibody to the carboxylic group of PLGA-NP (Fig. 1D). The encapsulation efficiency was 65.8 ± 5.6% determined by Bradford protein assay where the unconjugated anti-OX40 mAb remaining in the reaction medium after nanoparticle separation was divided by the total amount of anti-OX40 antibody used for the conjugation reaction. A high loading efficiency (25%) was also achieved as there was 248 ± 16.3 lg anti-OX40 mAb in per mg polymers of nanoparticles. The NP stability is always of concern since a possible therapeutic formulation would better have the same integrity when formulated as when administered several hours later. To monitor the integrity of the anti-OX40-mAb conjugated PLGA nanoparticles, the nanoparticle size was measured at various time points up to 6 h on a freshly prepared sample of PLGA nanoparticle suspension. Fig. 2A showed that the hydrodynamic diameter remained essentially unchanged as the particle size of 80 ± 6.2 nm at the end of the experiment was close to the starting particle size of 87 ± 3.6 nm, indicating that the nanoparticle suspensions did not aggregate or undergo gross degradation throughout the 6 h incubation period at 25 °C. In the in vitro release study, the NPs demonstrated a sustained release of the conjugated anti-OX40 antibody, with approximately 55% cumulative antibody in 20 days (Fig. 2B). The NPs gave a 12% of the antibody release from the polymeric matrix in 1 day, indicating no initial burst effect for the release. Further studies to explore longer-term stability or degradation of the nanoparticles and correlation with the release rate are clearly warranted. 3.2. Expression of OX40 on CTL cells OX40 is primarily expressed on activated T cells and transduces a potent costimulatory signal when engaged [1]. In this regard, we first examined the expression of OX40 on CD8+ T cells stimulated with allogeneic dendritic cells (DCs). As expected, naive CD8+ T

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Time (days) Fig. 2. Nanoparticle degradation and anti-OX40 antibody release. (A) Hydrodynamic diameters of the anti-OX40-PLGA-NP as measured by photon correlation spectroscopy at the time points of 0, 10, 20, 40, 60, 120, 240, 360 min. (B) In vitro release kinetics of anti-OX40 antibody from the NPs as determined using BCA method. The line graph shows the percentage release of anti-OX40 antibody from the anti-OX40-PLGA-NP suspensions over a period of time. Values are mean ± SD from 3 independent experiments.

cells did not express OX40 whereas stimulation with allogeneic DCs increased OX40 expression on the CTL cells in a time-dependent manner with almost half population of the cells expressing OX40 (Fig. 3) on day 7. These results demonstrated that OX40 could be up-regulated on antigen-activated CTL cells and thus OX40 agonist would expect to augment anti-tumor immunity mediated by CTLs. 3.3. Anti-OX40-PLGA-NP treatment affects the T cell phenotype Given the ability of anti-OX40-PLGA-NP to increase OX40 expression on the CD8+ T cells stimulated with allogeneic DCs, we next sought to determine whether the NPs affects the early activation phenotype of the T cells following anti-OX40-PLGANP treatment. Generation of activated CD8+ T cells was assessed by analysis of expression of CD25 and CD69, both of which are typical cell surface markers upon the activation. As shown in

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CD8 Fig. 3. Flow cytometric analysis of OX40 expression on the surface of CD8+ T cells stimulated with allogeneic DCs. The quadrants of the flow cytometric contour plots shown are representative of 3 independent experiments.

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C Fig. 4, although treatment of anti-OX40-PLGA-NP had no effect on the expression of CD4 on CD8+ T cells as compared with the untreated controls (Fig. 4A), it did increase the percentage of CD8+ T cells expressing CD8 from 38.4% to 62.9%, CD25 from 11.7% to 23.6%, and CD69 from 1.6% to 5.9%, representing a 1.6-, 2.0-, and 3.7-fold (p < 0.05 for all) increase, respectively. These results indicate that while CD8+ T cells tend to express OX40 following activation further OX40 stimulation may be preferred in order to sustain the activation.

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3.4. Anti-OX40-PLGA-NP induces CTL cell proliferation One of the most recognized and accepted activity of OX40OX40L interactions is to promote the division and survival of conventional T cells [24–26]. To determine whether this effect also applies to agonist stimulation by anti-OX40-PLGA-NP, we incubated CFSE-labeled CTLs with equal concentration of antiOX40 antibody, immobilized anti-OX40 antibody, PLGA-NP, or anti-OX40-PLGA-NP then CTL proliferation was assessed by a CFSE dilution assay followed by flow cytometric analysis. Fig. 5 shows that the percentage of proliferating cells was markedly higher in anti-OX40-PLGA-NP-treated cells (48.9%) compared to cells treated with anti-OX40 mAb or PLGA-NP alone (27.2% and 24.7%, respectively). However, there was no significant difference in the percentage of proliferating cells between the immobilized anti-OX40 antibody-treated group and the anti-OX40-PLGA-NP-treated group (43.2% versus 48.9%). These results indicate that treatment with anti-OX40-PLGA-NP significantly increased the proliferative capacity of the CTLs. 3.5. Effect of anti-OX40-PLGA-NP on activation of CTL cells One marker for CTL stimulation and activation is the production of cytokines. To examine a functional endpoint of CTL activation, expression of a panel of cytokines including Th1-type (IFN-c and IL-2), Th2-type (IL-4 and IL-10) and Th17-type (IL17) cytokines was measured by ELISA assay following exposure to anti-OX40 mAb, PLGA-NP or anti-OX40-PLGA-NP. As shown in Fig. 6, treatment of the CTL cells with anti-OX40-PLGA-NP

5.9%

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CD8 Fig. 4. Activation phenotype of CD8+ T cells following anti-OX40-PLGA-NP treatment. PBMCs were grown in RPMI medium supplemented with 1000 U/ml IL-2 and anti-CD3/CD28 immunobeads to induce activation of CD8 T cells, and cultured either in the presence or absence of 10 lg/ml anti-OX40-PLGA-NP for 3 days at 37 °C in a 5% CO2-humidified incubator. The cultured cells were stained with PECy7 conjugated anti-human CD4 plus FITC conjugated anti-human CD3, APC-Cy7 conjugated anti-human CD8 plus FITC conjugated anti-human CD3, or PE-Cy5 conjugated anti-CD25 antibodies plus FITC conjugated anti-human CD8, and subjected to flow cytometric analysis. Dot plots show untreated or treated CD4+CD3+ (A), CD8+CD3+ (B), CD25+CD8+ (C) and CD8+CD69+ (D) T cells. Numbers indicate the percentage of positive cells in the quadrant.

did not significantly affect the levels of IL-4 (Fig. 6C) and IL10 (Fig. 6D) as compared with those treated with anti-OX40 mAb or PLGA-NP alone. In contrast, CTL incubated with antiOX40-PLGA-NP secreted significantly higher amounts of IFN-c (Fig. 6A), IL-2 (Fig. 6B) and IL-17 (Fig. 6E) than those treated with anti-OX40 mAb or PLGA-NP alone, implicating that conjugation of anti-OX40 mAb in PLGA-NP enhanced the activation of CTLs and subsequently increased secretion of those inflammatory cytokines.

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Fig. 5. The effect of anti-OX40-PLGA-NP on CTL cell proliferation. The ability of cells to proliferate following the indicated treatments was evaluated using the CFSE dilution assay followed by flow cytometric analysis. (A) An analysis of CFSE staining in the CTLs was performed after gating on viable cells, and the percentage of proliferating cells is indicated for each panel. (B) The data from three independent experiments are expressed as the mean percentages of proliferating cells ± SD. ⁄⁄p < 0.01, anti-OX40-PLGA-NPtreated cells versus anti-OX40 mAb-treated cells.

3.6. Anti-OX40-PLGA-NP increases cytotoxic activity of tumor antigenspecific CTLs Given the evidence that anti-OX40-PLGA-NP could efficiently activate CTL through OX40 agonist, we hypothesized that it may also be able to augment antigen-specific T cell response. To test this hypothesis, tumor antigen-specific CTLs were generated by using human mature DCs pulsed with AFP158–166, a peptide derived from hepatocarcinoma antigen a-fetoprotein (AFP). The cytotoxic activity of those CTL/AFP158–166 cells was assessed on AFP-expressing human hepatocellular carcinoma HepG2 cells in the presence of anti-OX40 mAb, PLGA-NP, or anti-OX40-PLGA-NP. As shown in Fig. 7A, the CTL/AFP158–166 cells in the presence of anti-OX40-PLGA-NP exhibited significantly higher cytotoxic activity against the target HepG2 cells than those treated with anti-OX40 mAb or PLGA-NP alone, regardless of the ratio of effector/target at 40/1, 20/1 or 10/1. To further confirm that the CTL

cytotoxic effect on AFP-expressing HepG2 cells was AFP antigenspecific, the CTL/gp100154–162 cells that had been stimulated with melanoma antigen-pulsed DC were used as effector. As shown in Fig. 7B, CTL/gp100154–162 cells had no any killing effect on HepG2 cells irrespective of anti-OX40-PLGA-NP treatment. This observation clearly suggests that incorporation of anti-OX40 antibody into PLGA-NP increases its potency in induction of tumor antigen-specific T cell responses.

4. Discussion Advances in the field of cancer immunotherapy relies on our increased understanding of the mechanisms of the immune system. The ability to elicit immune responses against tumor antigens holds great promise that harnessing the immune system to combat tumors is feasible. However, it is often difficult to efficiently prime

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Fig. 6. Anti-OX40-PLGA-NP affects cytokine secretion from CTL cells. CTL cells were stimulated with equal concentration of PLGA-NP, anti-OX40 mAb, anti-OX40-PLGANP. Three days after the treatment, expression of IFN-c (A), IL-2 (B), IL-4 (C), IL-10 (D) and IL-17 (E) in the culture medium were measured by ELISA. Values are mean ± SD, n = 3. ⁄⁄p < 0.01, anti-OX40-PLGA-NP-treated cells versus anti-OX40 mAb-treated cells.

and/or activate cytotoxic T lymphocytes (CTLs) that recognize and kill tumor cells because tumor-associated self-antigens are often poorly immunogenic [27–29]. A critical step in the development of an immune response is the activation of T cells which requires recognition of cognate antigen by the T-cell receptor (TCR) in conjunction with costimulation signal delivered by antigen-presenting cells. While this initial interaction is important for the successful priming of T-cells, the tumor microenvironment where these primed T cells reside will ultimately dictate their ability to generate clinically relevant anti-tumor immune response and establish immunological memory [30]. Under most circumstances, after T-cell priming, negative regulatory molecules such as CTLA-4 are induced intracellularly resulting in down-regulation of the T-cell response [31]. Therefore, a successful immunotherapy

Fig. 7. Anti-OX40-PLGA-NP increases tumor antigen-specific CTL cytotoxicity. AFP158–166 (A) or gp100154–162 (B) antigen-specific CTL activity against the AFPexpressing HepG2 at various effector/target ratios was assessed by a standard lactate dehydrogenase release assay after 3-day incubation with 10 lg/ml of PLGANP, anti-OX40 mAb or anti-OX40-PLGA-NP. Values are mean ± SD, n = 3. ⁄p < 0.05, anti-OX40-PLGA-NP-treated cells versus anti-OX40 mAb-treated cells.

requires both adequate numbers of tumor-specific T cells and extended period of survival time. Members of TNF superfamily, exemplified by OX40, have been shown to provide such a post-activation signaling for primed T cells to differentiate into effector cells, proliferate, expand and induce productive immune responses [4]. The strong activity of OX40-OX40L interaction in driving CD4 and CD8 T cells as well NK and NKT cells [32,33] suggests that OX40 is a potential adjuvant that could be used as a target in vaccination strategies or therapeutic applications. Indeed, agonistic antibodies to OX40 (aOX40) are an effective adjuvant for both CD4 and CD8 activation. Provision of aOX40 immediately after antigen priming enhances T-cell expansion and effector function, and the number of long-term memory CD4 and CD8 T cells [8,32,34]. Given such substantial evidence from preclinical mouse models demonstrating that OX40 agonists can potentiate an antitumor immune response in multiple settings, a mouse anti-human OX40 mAb has been tested in phase I clinical trial. The clinical results and assessment of immunological effects have showed that the drug was tolerated, increased T cell activation/proliferation, and induced tumor shrinkage in

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some patients [35]. However, overall none of the patients met the criteria of an objective response by Response Evaluation Criteria in Solid Tumors (RECIST) [2]. Therapeutic use of monoclonal antibody is one of emerging areas of biopharmaceutical applications which require more advanced technologies for adequate formulation and delivering of mAbs, requisite for preserving the unstable structure and controlled release of mAbs [36]. Although the injectable particle systems for protein delivery have been developed with microparticles/nanoparticles, mAb delivery based on biodegradable polymers such as PLGA and poly(lactic acid) (PLA) is still in infancy [37]. In this study, the formulation and conjugating conditions to incorporate a potential mAb (anti-OX40 mAb) into biodegradable PLGA-NPs were investigated. We showed that the formulated anti-OX40-PLGA-NP exhibited the smooth surface and homogeneous size distribution with an average diameter of 86 ± 14.1 nm. It is generally believed that control of the size, size distribution and surface morphology is essential for quality, therapeutic effects and biodistribution of PLGA-NP [37]. Small NPs are preferable as they allow a variety of routes of administration but decreasing the NP size should also be carefully considered because the extended sonication time could reduce protein stability. On the other hand, caution should be exercised that nanoparticles smaller than 100 nm are often associated with increased cellular and genomic toxicity due to facile uptake of the small NPs by most organs of the body leading to multi-organ toxicity. Conversely, such increased uptake and toxicity may be true for any tumors as well and therefore antiOX40-PLGA-NP may promote T cell expansion at the site of oncogenesis. High loading efficiency is another fundamentally important parameter for the therapeutic effect using NPs. One of major pitfalls of PLGA-based NPs often relates to the poor loading while high encapsulation efficiency could be readily achieved [17]. In our case, approximately 250 lg anti-OX40 mAb was coupled to per mg polymers of nanoparticles reaching as high as 25% drug loading efficiency. Although OX40 stimulation was initially thought to provide costimulatory signals to a conventional T cell for promoting its division and survival irrespective of antigen specificity, accumulating evidence has demonstrated that addition of OX40 agonists to an immune-based therapy further boosts the tumor antigenspecific T-cell response after the antigen re-encounter [38]. Antigenic stimulation of T cells proceeds through four phases: activation, expansion, contraction and development of long-lived memory [39]. Naive antigen-specific T cells proliferate in response to antigen stimulation and constitute a pool of effector T cells. As the antigenic challenge diminishes, the antigen-specific T cells contracts leaving a smaller pool of memory T cells. The temporal expression of OX40 post the priming events indicates its important role in late proliferation and survival of the effector T cells. In the absence of OX40, antigen-primed T cells proliferate only for a shorter period of time and there is a significant reduction in their survival time as well [40]. In contrast, engagement of OX40 results in recruitment of TNFR-associated (TRAFs) molecules leading to activation of NF-jB and up-regulation of anti-apoptotic molecules, which are responsible for the increased clonal expansion and a larger pool of memory T cells [1,41,42]. Our observation that anti-OX40-PLGA-NP was more effective than anti-OX40 mAb alone at stimulating CTL proliferation and enhancing its tumor antigen-specific cytotoxic activity provides further confirmation of concept that multivalent engagement of OX40 agonist antibody by PLGA-NP on CTLs could warrant the maintenance of survival signals resulting in sustained and augmented antigen-specific immune response. Triggering costimulatory molecules on T cells also modulates the profile of cytokine secretion and thereof influences the

outcome of a T cell-mediated immune response [43,44]. Although early reports point to the role of OX40 signaling in Th2 differentiation, it has become clear that OX40 signaling can promote the development of both Th1 and Th2 [40]. However, T-cell differentiation is more directly related to the strength of TCR stimulation, antigen dose, and local cytokine milieu rather than OX40 signaling [45,46]. We observed that CTL incubated with anti-OX40-PLGANPs secreted significantly higher amounts of IFN-c (9-fold) and IL-2 (13-fold) with no change in IL-4 and IL-10 levels, favoring Th1 differentiation. It should be recognized that since the role of OX40 in CD8+ T-cell differentiation is more complex and less clear, further studies to determine whether other types of cytokines are similarly regulated are necessary. 5. Conclusions A PLGA-based nanoparticle with high loading efficiency for anti-OX40 mAb was successfully prepared and characterized. Those anti-OX40-PLGA-NPs were more effective than free antiOX40 mAb in inducing CTL proliferation and tumor antigen-specific cytotoxicity as well as cytokine production. The results obtained from this study suggest that PLGA-based nanoparticle formulation can provide efficient delivery system of anti-OX40 mAb and possibly other potential mAbs or proteins for cancer immunotherapy, in particular when sustained release and multivalency are needed for T cell stimulation. Acknowledgment This work was supported by the Natural Science Foundation of Fujian Province (No. 2011J01140). References [1] M. Croft, Control of immunity by the TNFR-related molecule OX40 (CD134), Annu. Rev. Immunol. 28 (2010) 57–78. [2] I. Melero, D. Hirschhorn-Cymerman, A. Morales-Kastresana, M.F. Sanmamed, J.D. Wolchok, Agonist antibodies to TNFR molecules that costimulate T and NK cells, Clin. Cancer Res. 19 (2013) 1044–1053. [3] I. Gramaglia, A. Jember, S.D. Pippig, A.D. Weinberg, N. Killeen, M. Croft, The OX40 costimulatory receptor determines the development of CD4 memory by regulating primary clonal expansion, J. Immunol. 165 (2000) 3043–3050. [4] M. Croft, Costimulation of T cells by OX40, 4-1BB, and CD27, Cytokine Growth Factor Rev. 14 (2003) 265–273. [5] R.H. Arch, C.B. Thompson, 4-1BB and Ox40 are members of a tumor necrosis factor (TNF)-nerve growth factor receptor subfamily that bind TNF receptorassociated factors and activate nuclear factor kappaB, Mol. Cell. Biol. 18 (1998) 558–565. [6] S. Kawamata, T. Hori, A. Imura, A. Takaori-Kondo, T. Uchiyama, Activation of OX40 signal transduction pathways leads to tumor necrosis factor receptorassociated factor (TRAF) 2- and TRAF5-mediated NF-kappaB activation, J. Biol. Chem. 273 (1998) 5808–5814. [7] J. Song, T. So, M. Croft, Activation of NF-kappaB1 by OX40 contributes to antigen-driven T cell expansion and survival, J. Immunol. 180 (2008) 7240– 7248. [8] A.D. Weinberg, M.M. Rivera, R. Prell, A. Morris, T. Ramstad, J.T. Vetto, W.J. Urba, G. Alvord, C. Bunce, J. Shields, Engagement of the OX-40 receptor in vivo enhances antitumor immunity, J. Immunol. 164 (2000) 2160–2169. [9] J. Kjaergaard, J. Tanaka, J.A. Kim, K. Rothchild, A. Weinberg, S. Shu, Therapeutic efficacy of OX-40 receptor antibody depends on tumor immunogenicity and anatomic site of tumor growth, Cancer Res. 60 (2000) 5514–5521. [10] L.C. Ndhlovu, N. Ishii, K. Murata, T. Sato, K. Sugamura, Critical involvement of OX40 ligand signals in the T cell priming events during experimental autoimmune encephalomyelitis, J. Immunol. 167 (2001) 2991–2999. [11] W.L. Redmond, A.D. Weinberg, Targeting OX40 and OX40L for the treatment of autoimmunity and cancer, Crit. Rev. Immunol. 27 (2007) 415–436. [12] S. Piconese, B. Valzasina, M.P. Colombo, OX40 triggering blocks suppression by regulatory T cells and facilitates tumor rejection, J. Exp. Med. 205 (2008) 825– 839. [13] S. Andarini, T. Kikuchi, M. Nukiwa, P. Pradono, T. Suzuki, S. Ohkouchi, A. Inoue, M. Maemondo, N. Ishii, Y. Saijo, K. Sugamura, T. Nukiwa, Adenovirus vectormediated in vivo gene transfer of OX40 ligand to tumor cells enhances antitumor immunity of tumor-bearing hosts, Cancer Res. 64 (2004) 3281– 3287.

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