Engineering nanoparticles to locally activate T cells in ... · and immunosuppressive microenvironment of tumor. Tumor resistance to immune checkpoint inhibitors due to immunological

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1State Key Laboratory of Drug Research & Center of Pharmaceutics, Shanghai In-stitute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China.2Yantai Key Laboratory of Nanomedicine & Advanced Preparations, Yantai Insti-tute of Materia Medica, Shandong 264000, China. 3University of Chinese Academyof Sciences, Beijing 100049, China. 4Institute of Biomedical Science and Children'sHospital, Fudan University, Shanghai 200032, China. 5School of Pharmacy, YantaiUniversity, Shandong 264005, China.*Corresponding author. Email: [email protected] (H.Y.); [email protected] (Y.L.)

Wang et al., Sci. Immunol. 4, eaau6584 (2019) 12 July 2019

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Engineering nanoparticles to locally activate T cellsin the tumor microenvironmentDangge Wang1,2,3, Tingting Wang1,3, Haijun Yu1,2*, Bing Feng1,3, Lei Zhou1, Fangyuan Zhou1,Bo Hou1, Hanwu Zhang1, Min Luo4, Yaping Li1,5*

Immunological tolerance of tumors is characterized by insufficient infiltration of cytotoxic T lymphocytes (CTLs)and immunosuppressive microenvironment of tumor. Tumor resistance to immune checkpoint inhibitors due toimmunological tolerance is an ongoing challenge for current immune checkpoint blockade (ICB) therapy. Here,we report the development of tumor microenvironment–activatable anti-PDL1 antibody (aPDL1) nanoparticlesfor combination immunotherapy designed to overcome immunological tolerance of tumors. Combination ofaPDL1 nanoparticle treatment with near-infrared (NIR) laser irradiation–triggered activation of photosensitizerindocyanine green induces the generation of reactive oxygen species, which promotes the intratumoral in-filtration of CTLs and sensitizes the tumors to PDL1 blockade therapy. We showed that the combination ofantibody nanoparticles and NIR laser irradiation effectively suppressed tumor growth and metastasis to thelung and lymph nodes in mouse models. The nanoplatform that uses the antibody nanoparticle alone bothfor immune stimulation and PDL1 inhibition could be readily adapted to other immune checkpoint inhibitorsfor improved ICB therapy.

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INTRODUCTIONImmune checkpoint blockade (ICB) therapy is emerging as a promisingapproach for cancer immunotherapy by modulating the immuno-suppressive tumor microenvironment (1, 2). In particular, ICB therapyby monoclonal antibodies (e.g., pembrolizumab, nivolumab, atezolizu-mab, and durvalumab) targeting the immune checkpoint programmedcell death protein 1 (PD1) or its predominant ligand, programmeddeath ligand 1 (PDL1; B7-H1), has induced durable tumor regressionin a broad variety of cancers (3–6). However, only a subset of treatedpatients respond to current ICB therapies (7–11), likely due to im-munological tolerance of tumors (12, 13). Immunological tolerance oftumors could be attributed to insufficient tumor infiltration of cyto-toxic T lymphocytes (CTLs) (14, 15) and immunosuppressive tumormicroenvironment (6, 16, 17). The development of robust strategies toimprove the response rates of immune-tolerant tumors to ICB therapyhas become a priority.

Recent clinical studies have revealed a positive correlation betweenintratumoral infiltration of CTLs and the response rate to ICB therapy(18–25). Extensive efforts have been devoted to combine systemic ICBtherapy with chemotherapy (26, 27), radiotherapy (28–30), and photo-therapy, which uses photosensitizers to realize photothermal conver-sion or generate reactive oxygen species (ROS) [photodynamictherapy (PDT)] (21, 26, 31, 32), or to co-delivery two antagonistic anti-bodies using one single nanoparticle to elicit antitumor immunity andpromote intratumoral infiltration of CTLs (33). Despite promisingresults, the clinical translation of ICB suffers from several formidablechallenges. For instance, ICB therapy using anti-PDL1 antibodies(aPDL1) displays “on-target but off-tumor” binding with the normaltissues due to PDL1 expression on vascular endothelium, pancreatic

islet cells, hepatocytes, muscle, epithelium, and mesenchymal stemcells (34), which attenuated the therapeutic efficacy and resulted insevere immune-related adverse events when aPDL1 was systemicallyinfused (11, 35). To avoid the nonspecific binding of the checkpointinhibitors with the normal tissues, several systemic or local-regionaldelivery strategies had been developed by integrating the checkpointinhibitors into nanoparticles (33), microparticles (36, 37), microneedles(38, 39), hydrogels (40, 41), or biomimetic vesicles (42–44). Althoughthese delivery strategies display notable benefits for primary tumortherapies, it remains a major challenge for treatment of deep-seatedor metastatic tumors (45).

To address the challenges of current ICB therapy, we hereinpresented a matrix metalloproteinase protein 2 (MMP-2)–sensitiveaPDL1/ICG-based nanoparticle (S-aPDL1/ICG@NP) for combatingthe immunological tolerance of tumors by specifically blocking thePD1/PDL1 cascade at the tumor sites (Fig. 1). S-aPDL1/ICG@NP re-mains stable during the blood circulation and protects aPDL1 frombinding with the normal tissues. S-aPDL1/ICG@NP can passively ac-cumulate at the tumor site through the enhanced permeability and re-tention (EPR) effect and become activated in MMP-2 highly expressedtumor to release aPDL1 for PDL1 blockade at the tumor site (18, 46).In combination with PDT, the aPDL1 nanoparticles triggered tumorantigen release and promoted intratumoral CTLs infiltration by gen-erating ROS (47, 48). The antibody nanoparticle both for immunestimulation and checkpoint inhibition could be readily adapted to otherimmune checkpoint inhibitors for improved ICB therapy, which mightrepresent a robust nanoplatform to overcome immunological toleranceas a barrier to cancer immunotherapy.

RESULTSEngineering and characterization of MMP-2–activatableS-aPDL1/ICG@NPTo construct S-aPDL1/ICG@NP, we first synthesized a dimer of(−)-epigallocatechin-3-O-gallate (dEGCG) by the Baeyer acid–catalyzedcondensation reaction (49). PEGylated dEGCG (PEG-dEGCG) wasthen synthesized by conjugating the dimer with polyethylene glycol

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(PEG) via a MMP-2–liable proline-leucine-glycine-leucine-alanine-glycine (PLGLAG) peptide spacer, which was termed as PEG-PLGLAG-dEGCG (figs. S1 to S3). High-performance liquid chromatographic(HPLC) measurement verified the MMP-2 proteolytic activity upon thePEG-PLGLAG-dEGCG substrate (fig. S4). PEG-dEGCG without thepeptide spacer was also synthesized as a MMP-2–insensitive analogof PEG-PLGLAG-dEGCG (fig. S5).

It has been reported that photosensitizer indocyanine green (ICG)can complexwith various proteins throughnonspecific hydrophobic andelectrostatic interactions (50, 51).MMP-2–liable S-aPDL1/ICG@NPwasthus fabricated by integrating aPDL1, ICG, dEGCG, and PEG-PLGLAG-dEGCG into one nanocomposite. aPDL1/ICG@NP, a MMP-2–insensitive analog of aPDL1/ICG@NP was also prepared by replacingPEG-PLGLAG-dEGCG with PEG-dEGCG. The optimized formula-tions of S-aPDL1/ICG@NP and aPDL1/ICG@NP were fabricated atan ICG to aPDL1 feeding ratio of 0.3 by screening both the ICG en-capsulation efficiency and particle sizes (fig. S6, A to E).

The fabrication of S-aPDL1/ICG@NP was monitored using trans-mission electron microscopic (TEM) and dynamic light scattering(DLS) examinations (Fig. 2, A and B, and fig. S7). The hydrodynamicdiameter of aPDL1 increased from 8.0 ± 1.3 nm to 14.5 ± 1.6 nm afterICG incubation and further increased to 38.7 ± 3.9 nmwith the additionof dEGCG. In the presence of PEG-PLGLAG-dEGCG, the dEGCG-aPDL1/ICG complexes further transformed to S-aPDL1/ICG@NPwith a uniform particle size of 163.4 ± 6.6 nm and a narrow poly-dispersity index (PDI) of 0.061. Upon MMP-2–mediated cleavage of


the PEG corona, the hydrodynamic diameter of S-aPDL1/ICG@NPexpanded to 298.1 ± 146.8 nm (PDI, 0.426) due to the formation ofaPDL1 aggregates. The hydrodynamic diameter of S-aPDL1/ICG@NPcould be readily tuned in the size range between 150 and 750 nm withnarrow particle size distribution by simply adjusting the feeding ratiosbetween dEGCG and PEG-PLGLAG-dEGCG (Fig. 2C).

To clarify the nature of the interactions between aPDL1, dEGCG,and PEG-PLGLAG-dEGCG, we incubated S-aPDL1/ICG@NP withNaCl, urea, Tween 20, or Triton X-100 solution for 2 min. S-aPDL1/ICG@NP was quickly dissociated after the addition of Tween 20 andTriton X-100 but was minimally affected by NaCl and urea incubation,suggesting that dEGCG complexes with aPDL1 through hydrophobicinteraction (Fig. 2D).

To verify the serum stability of S-aPDL1/ICG@NP in serum-containing solutions,weusedultracentrifugation andSDS–polyacrylamidegel electrophoresis analysis todistinguish the freeaPDL1andnanoparticle-encapsulated aPDL1 in 10% bovine serum albumin (BSA)–containingphosphate-buffered saline (PBS) solution. Upon 24-hour incubation,S-aPDL1/ICG@NP shows good stability in 20% of serum-containingPBS solution (fig. S8, A and B). Around 12% of aPDL1 was releasedfrom the S-aPDL1/ICG@NP, slightly higher than that of the BSA-freecontrol (~10%), verifying the good colloidal stability of the S-aPDL1/ICG@NP in serum-equivalent concentration of BSA (fig. S8, C to F).

To investigate whether the reversible compression procedure im-paired the PDL1 binding activity of aPDL1, we tested the binding affin-ity of aPDL1 in S-aPDL1/ICG@NP by enzyme-linked immunosorbent

Fig. 1. S-aPDL1/ICG@NP for improved ICB therapy by combating the immunologic tolerance of tumors. (A) Fabrication of MMP-2–liable S-aPDL1/ICG@NP. The aPDL1 wasfirst complexed with photosensitizer ICG and subsequently stabilized with dEGCG to form the dEGCG-aPDL1/ICG aggregates and further compressed by PEG-PLGLAG-dEGCG.The nanoparticles could be activated in the presence of MMP-2. (B) Schematic illustration of S-aPDL1/ICG@NP–mediated combination ICB and PDT therapy. S-aPDL1/ICG@NPcould be activatedwithin the tumormicroenvironment for sustained release of aPDL1. UponNIR laser irradiation, ICG-mediated PDT induced antitumor immunity and promotedthe intratumoral infiltration of CTLs. PDT and aPDL1-mediated PDL1 blockade cumulatively suppress tumor growth and inhibit metastasis. i.v., intravenous.

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assay (ELISA). aPDL1 antibodies retained only 17.7 ± 7.1% of its PDL1binding capability when theywere compressed into nanoparticles. How-ever, once dissociated by MMP-2, its PDL1 binding affinity was signif-icantly restored to 82.3 ± 2.5%, suggesting that the compression processnegligibly affects the bioactivity of aPDL1 (Fig. 2E).

To elucidate the influence of laser irradiation on the bioactivity ofaPDL1, we examined the PDL1 binding affinity of the antibody in S-aPDL1/ICG@NP before and after laser irradiation (fig. S9). Theresults showed that aPDL1 retained ~85% of their activity upon808-nm laser irradiation at 1.2 W/cm2 in contrast to groups withoutlaser treatment, indicating that PDT affected negligibly the bioactivityof aPDL1.

To examine the influence of the assembly process on protein activity,we further prepared three types of S-aPDL1/ICG@NP–mimickingnanoparticles by condensing three kinds of enzymes including protein-ase K, ribonuclease (RNase), or horseradish peroxidase–conjugated


antibody (HRP antibody) with dEGCG and PEG-PLGLAG-dEGCG.All the enzymes retained ~20% of their catalytic activities when encap-sulated inside the nanoparticles due to the shielding effect of the PEGcorona. In contrast, the enzymes were sufficiently reactivated once thenanoparticles were deconstructed via MMP-2 incubation, implyingthat the bioactivity of the proteins could be reversibly shielded by com-plexation with dEGCG (Fig. 2E). Furthermore, S-aPDL1/ICG@NPdisplayed satisfactory stability against enzyme degradation of aPDL1with proteinase K. For instance, more than 70% of free aPDL1 wasdegraded by proteinase K in 30 min, but less than 10% of aPDL1 inS-aPDL1/ICG@NP was degraded after 24-hour incubation (Fig. 2F).

To measure tumor enzymatic microenvironment–triggered aPDL1release, S-aPDL1/ICG@NP was incubated with 50 nM MMP-2 andmonitored for aPDL1 release by ELISA. It was found that more than45% of aPDL1 was released from S-aPDL1/ICG@NP after 6-hour in-cubation with 50 nM MMP-2, which was 4.6-fold more efficient than

Fig. 2. Chemo-physical characterization of S-aPDL1/ICG@NP. (A) TEM images of aPDL1 and S-aPDL1/ICG@NP. Scale bars, 200 nm. (B) Particle size change of S-aPDL1/ICG@NP during the assembly and disassembly process. (C) Particle size optimization of S-aPDL1/ICG@NP as a function of feeding ratio between dEGCG and PEG-PLGLAG-dEGCG. (D) Stability assay of S-aPDL1/ICG@NP with the addition of NaCl, urea, Tween 20, or Triton X-100 (n = 3). (E) Activity assay of aPDL1, proteinase K,RNase, and HRP antibody (Ab) released from nanoparticles (n = 3). (F) Proteinase K degradation assay for S-aPDL1/ICG@NP, S-aPDL1/ICG@NP + MMP-2, and free aPDL1.Protein suspensions were incubated with proteinase K (20 ng/ml) for the indicated time duration (n = 3). (G) aPDL1 release profiles of S-aPDL1/ICG@NP in the presenceor absence of MMP-2 (n = 3). (H) Fluorescence spectroscopic examination of laser-induced ROS generation of S-aPDL1/ICG@NP and S-ICG@NP as a function of ir-radiation time, singlet oxygen sensor green (SOSG) was used as the fluorescent indicator for ROS (n = 6; photo density, 1.2 W/cm2). Data are means ± SD. Statisticalsignificance was calculated by two-sided unpaired Student’s t test (***P < 0.001). a.u., arbitrary units.

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that of the MMP-2–free group. About 30% of aPDL1 was releasedfrom S-aPDL1/ICG@NP upon 12-hour incubation with 5 nMMMP-2, verifying the superior MMP-2 sensitivity of S-aPDL1/ICG@NP. The release rates dropped slightly along with the decreasedconcentration of MMP-2, but the cumulative release amount reached41.5 ± 1.4% in 24 hours even at an enzyme concentration of 5 nM (Fig.2G). The MMP-2–triggered aPDL1 release profiles of S-aPDL1/ICG@NP suggest that the reversible complexation strategy couldavoid antibody leakage during the blood circulation and become acti-vated in the tumor microenvironment.

The photoactivity of S-aPDL1/ICG@NP was further evaluatedby measuring ROS generation upon 808-nm near-infrared (NIR)laser irradiation. Bulk S-aPDL1/ICG@NP and MMP-2–activatedS-aPDL1/ICG@NP both induced significant ROS generation atphotodensity of 1.2 W/cm2 (Fig. 2H). To eliminate the impact of aPDL1of the photoactivity of ICG, we fabricated analog nanoparticles loadingICG by using mouse immunoglobulin G (IgG) protein to replace theantibodies (namely, S-ICG@NP). Comparable photoactivity was ob-served in S-ICG@NP or MMP-2–pretreated S-ICG@NP group dueto the identical ICG loading efficiency between aPDL1 and mouseIgG protein (fig. S6A). The ROS generation capability of S-aPDL1/ICG@NP was also confirmed by electron spin resonance (ESR) spec-trum. Upon laser irradiation, characteristic ROS signals (g = 2.0058,A = 16.4 G) were detected in both the S-ICG@NP and S-aPDL1/ICG@NP groups (Fig. 2H and fig. S10).

The phototoxicity of S-aPDL1/ICG@NP in vitro was investigated in4T1 murine breast cancer cells. Laser illumination at a photodensity of1.2 W/cm2 and ICG concentration of 20 mg ml−1 significantly reduced~70% of 4T1 cell viability (fig. S11, A and B), due to ROS-inducedcellular apoptosis as confirmed by annexin V–fluorescein isothio-cyanate (FITC)/propidium iodide (PI) staining assay (fig. S11C).

It has been well established that aPDL1 inhibits immune evasion byblocking the cell surface–expressed PDL1 of the tumor cells (52).Therefore, it is crucial to control specific binding of S-aPDL1/ICG@NPwith cell surface PDL1 while avoiding S-aPDL1/ICG@NP internaliza-tion. The interaction between S-aPDL1/ICG@NP and tumor cells wasinvestigated in vitro using 4T1 murine breast tumor cells (fig. S12).Confocal laser scanning microscopic (CLSM) examination revealedthat S-aPDL1/ICG@NP was efficiently internalized by 4T1 tumorcells (fig. S13A). In contrast, MMP-2 preactivation of S-aPDL1/ICG@NP efficiently suppressed cellular uptake of S-aPDL1/ICG@NP(fig. S13B), indicated by membrane colocalization of releasedaPDL1. These results show successful binding of aPDL1 releasedfrom MMP-2–activated S-aPDL1/ICG@NP with the cell surface–expressed PDL1.

Biodistribution of S-aPDL1/ICG@NP in vivoThe pharmacokinetic profile of S-aPDL1/ICG@NP was investigated inSpragueDawley rats by using rhodamineB (Rb)–labeled IgG-basednano-complexes, which were prepared by following the same procedure forfabrication of S-aPDL1/ICG@NP. The blood clearance half-time (t1/2b)of free IgG was 38.24 ± 4.82 hours. In contrast, IgG-Rb–based S-aPDL1/ICG@NP showed much shorter t1/2b of 8.45 ± 1.66 and lowerbioavailability of 544.00 ± 100.85 mg/liter hour than those of free IgG,which could be most likely explained by quick blood clearance of thenanoparticles (fig. S14 and table S2).

The biodistribution of S-aPDL1/ICG@NP was evaluated in 4T1tumor–bearing BALB/cmice. The results showed that freeaPDL1non-specifically distributed in the tumors and other normal organs 2 hours


after injection (Fig. 3A and fig. S15), probably due to the on-target butoff-tumor effect of aPDL1 (34). In contrast, S-aPDL1/ICG@NP andaPDL1/ICG@NP showed more specific accumulation at the tumorsites, which may be attributed to the EPR effect of tumors. TheS-aPDL1/ICG@NP group showed 4.3- and 10.7-fold higher intra-tumoral aPDL1 accumulation than aPDL1/ICG@NP and freeaPDL1, respectively, as determined by quantification of the fluo-rescence intensity 24 hours after injection (Fig. 3B). The increasedintratumor accumulation and retention of S-aPDL1/ICG@NP could bemost likely explained by MMP-2–mediated activation of S-aPDL1/ICG@NP in the tumor microenvironment since MMP-2 is known tobe overexpressed in 4T1 tumor cells (fig. S16).

Accurate distribution of S-aPDL1/ICG@NP in the major organswas also determined in 4T1 tumor–bearing mice. In the lungs of freeaPDL1–treated group, the accumulation of antibodies was 1.5-foldhigher than that of the S-aPDL1/ICG@NP group 4 hours after theinjection and even increased to 3.4-fold 24 hours later (Fig. 3C). Thiscould attribute to the specific binding of aPDL1 in lung tissues whichreduces the clearance rates. For other organs, obvious accumulation ofaPDL1 was detected in the livers of all groups while barely found inhearts, spleens, and kidneys. For instance, aPDL1/ICG@NP and S-aPDL1/ICG@NP both displayed ~2.0-fold higher liver distributionthan that of the free aPDL1 group 4 hours after intravenous injection,which could be attributed to reticuloendothelial system-medicatedclearance of the aPDL1 nanoparticle (fig. S17). The accumulation offree aPDL1 in the liver could result in immune-related adverse effectsdue to the unexpected binding with PDL1. However, compressingantibodies into S-aPDL1/ICG@NP would reduce this risk by usingthe PEG shells to weaken the interactions between the antibody andPDL1 in livers. Together, these findings suggest that S-aPDL1/ICG@NP was highly efficient in suppressing aPDL1 distribution innormal tissues while targeting specific activation in tumor tissues.The tumor-specific distribution and activation of S-aPDL1/ICG@NPmay suppress the immune-related adverse effects of aPDL1 due tobinding of aPDL1 with the normal tissues.

It has been reported that the efficacy of ICB therapy is impairedby the limited tumor penetration of checkpoint inhibitors due to theexistence of tumor burden (53, 54). To assess the penetration andaccessibility of S-aPDL1/ICG@NPwithin tumors, we examined the in-tratumoral distribution of S-aPDL1/ICG@NP by immunofluorescenceimaging. Visible fluorescent signals of aPDL1-Cy5.5 were detected inperivascular areas of tumors 2 hours after intravenous injection in allgroups, and increased signals were found in aPDL1/ICG@NP andS-aPDL1/ICG@NP groups at 24 hours. Unlike the limited diffusionand quick clearance of aPDL1/ICG@NP, released aPDL1 fromS-aPDL1/ICG@NP diffused throughout the whole areas of tumorand retained more than 48 hours, suggesting that S-aPDL1/ICG@NPfacilitated deep tumor penetration of aPDL1 for improved PDL1 block-ade (Fig. 3D).

Dissemination of the tumor cells from the primary tumor to thelymph nodes (LNs) is one of the dominant pathways for tumor metas-tasis, which is a lethal event for patients with cancer (55, 56). To inves-tigate the potential of S-aPDL1/ICG@NP for LN targeting, lymphaticmetastasis tumor model was established by inoculating 4T1 tumor cellsat the right footpad of mice. Footpad injection has been widely used fornanoparticle-mediated drug delivery to the lymphatic metastasis tu-mors (57–59). The metastatic tumor-bearing mice were then injectedwith Cy5.5-labeled aPDL1, S-aPDL1/ICG@NP, or aPDL1/ICG@NPat the right footpad at an identical aPDL1 dose of 0.5 mg/kg when

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the tumors disseminated from the right flank to the LNs 12 days aftertumor inoculation. S-aPDL1/ICG@NP efficiently accumulated atthe lymphatic system as indicated by the strong fluorescence signalsin the popliteal, inguinal, and axillary LNs (Fig. 3E). In contrast, neg-ligible aPDL1 distribution was observed in the free aPDL1 group(Fig. 3F).

The draining LNs were harvested 24 hours after injection for fluo-rescence imaging and CLSM examination ex vivo. Consistent with thefluorescence imaging data in vivo, aPDL1 exhibited marginal accumu-lation in the popliteal, inguinal, and axillary LNs (Fig. 3, G and H). Incontrast, S-aPDL1/ICG@NP was highly efficient in accumulating at allthe draining LNs and distributed throughout the sections of tumoral


metastatic popliteal LNs (Fig. 3I), indicating the potential use ofS-aPDL1/ICG@NP for treatment of lymphatic metastasis tumors.

Elicitation of a durable immune response in vivoThe photoactivity of S-aPDL1/ICG@NP in vivowas examined by usingdichlorofluorescin diacetate (DCF) as a ROS indicator. CLSMexamina-tion demonstrated the appearance of highly diffuse green fluorescenceof DCF upon 808-nm laser illumination of the tumor sections, suggest-ing PDT-triggered ROS generation in the tumor tissue (Fig. 4A).

Dendritic cells (DCs) play crucial roles in initiating and regulat-ing the antitumor immune response by presenting tumor antigens toT lymphocytes. To investigate whether S-aPDL1/ICG@NP–mediated

Fig. 3. Biodistribution of S-aPDL1/ICG@NP in 4T1 tumor–bearing mice. (A) Fluorescence imaging of S-aPDL1/ICG@NP distribution in 4T1 tumor–bearing mice invivo. The fluorescence images were collected at 2, 4, 8, 12, 24, 48, and 72 hours after intravenous injection of Cy5.5-labeled aPDL1, aPDL1/ICG@NP, or S-aPDL1/ICG@NP,respectively. The dosage of aPDL1 is 2.0 mg/kg. White dotted circles indicate the location of tumors. Quantification of aPDL1 distribution in (B) tumors and (C) lungs 4,12, or 24 hours after injection [aPDL1 (5.0 mg/kg), n = 3]. (D) Immunofluorescence of aPDL1 distribution in tumors with various treatments, CD31 was stained with anti–CD31-FITC. Scale bars, 75 mm. DAPI, 4′,6-diamidino-2-phenylindole. (E) Fluorescence examination of S-aPDL1/ICG@NP distribution in the lymphatic metastasis tumorsafter footpad injection [aPDL1 (0.5 mg/kg)]. (F) Time course quantification of S-aPDL1/ICG@NP fluorescence intensity in the lymphatic regions after footpad injection(n = 3). (G) Ex vivo fluorescence imaging and (H) quantification of excised axillary, inguinal, and popliteal LNs 4 hours after subcutaneous injection of S-aPDL1/ICG@NP(n = 4). (I) CLSM examination of S-aPDL1/ICG@NP distribution in the popliteal LNs 4 hours after injection. Scale bars, 100 mm. Data are means ± SD. Statistical signif-icance was calculated by one-way ANOVA with Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001).

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PDT acceleratedDCmaturation in vivo, 4T1 tumors were illuminatedwith an 808-nm laser and were then harvested 3 days after treatmenttomeasure the frequency ofmaturedDCs (CD11c+CD80+CD86+). Toelucidate the influence ofaPDL1 on the immune response in vitro andin vivo, MMP-2–liable nanoparticles S-ICG@NP were prepared ascontrol. S-ICG@NP-based PDTmarkedly accelerated DCmaturationin the tumor tissues to 25.2 ± 1.3%, whereas only 9.2 ± 1.7% of maturedDCs was found in the PBS group. S-aPDL1/ICG@NP in combinationwith PDT treatment inducedmorematuration ofDCs, 2.6- and 1.2-foldmore efficient than free aPDL1 or aPDL1/ICG@NP + 1.2 W/cm2

group (Fig. 4B).The DC maturation–induced systemic immune response was eval-

uated by examining PDT-induced intratumor secretion of proinflam-matory cytokines. S-aPDL1/ICG@NP treatment and 808-nm laserirradiation at 1.2 W/cm2 elevated the intratumoral secretion of tumornecrosis factor–a (TNF-a), interferon-g (IFN-g), and interleukin-


1b (IL-1b). PDT at photodensity of 1.2 W/cm2 increased the secretionof all three cytokines (Fig. 4C). S-aPDL1/ICG@NP–induced immuneresponse was further investigated by examining the frequency, pro-liferation, and function of intratumoral infiltration of CTLs (60).CD3+ T cells in S-aPDL1/ICG@NP + 1.2 W/cm2 group performed a1.3 to 5.0-fold increase in contrast to the other groups (fig. S18A).More-over, the PBS control group showed negligible tumor infiltration of CD8+

T cells (Fig. 4, D to F), while S-aPDL1/ICG@NP–based PDT increasedthe frequency of intratumor infiltrating CD8+ T cells to 34.4 ± 2.3%(Fig. 4E). The infiltration number of CD8+ T cells in S-aPDL1/ICG@NP + 1.2 W/cm2 group was found to be 2.2 to 6.1-fold morethan that of other aPDL1-treated groups, suggesting that a robustCD8+ T cell infiltration was triggered through the co-delivery ofPDT and PDL1 blockade by S-aPDL1/ICG@NP (Fig. 4F).

High infiltration of regulatory T cells (Tregs; CD11b+CD4+CD25+FoxP3+)

has been found to correlate with the poor prognosis of patients with

Fig. 4. S-aPDL1/ICG@NP induced cellular response to antitumor immunotherapy. (A) CLSM examination of PDT-induced ROS generation in vivo. (B) Maturation ofDCs in tumors after treatments [1#, PBS; 2#, aPDL1; 3#, S-ICG@NP; 4#, S-ICG@NP+ laser (S-ICG@NP+L); 5#, aPDL1/ICG@NP; 6#, aPDL1/ICG@NP+ laser; 7#, S-aPDL1/ICG@NP; 8#,S-aPDL1/ICG@NP+ laser; laser power density, 1.2W/cm2;n=3)]. (C) Intratumoral secretion of TNF-a, IFN-g, and IL-1b in BALB/cmice (n=3). (D) Immunofluorescence stainingofintratumor infiltrating CD8+ T cells in 4T1 tumors 5 days after treatments. (E) Flow cytometric quantification of intratumoral infiltration of CD4+ and CD8+ T cells. Micewere treatedat an equal antibody dosage of 5.0 mg/kg, selectively irradiated in PDT groups, and examined 5 days later (n = 3 ~ 5). (F) Normalized intratumoral infiltration of CD8+ T cell as afunction of tumormass. (G) CD8+ T cell–to–Treg ratio in TILs. (H) Proliferation assay of CD8

+ T cells. (I) Frequency of TNF-a and IFN-g dual-positive CD8+ T cells (1, PBS; 2, aPDL1;3, S-ICG@NP; 4, S-ICG@NP + laser; 5, aPDL1/ICG@NP; 6, aPDL1/ICG@NP + laser; 7, S-aPDL1/ICG@NP; 8, S-aPDL1/ICG@NP + laser; laser power density, 1.2 W/cm2; n = 3 ~ 5).Data are means ± SD. Statistical significance was calculated by one-way ANOVA with Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001).

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cancer in a clinic (61). The number of infiltrated CD4+ T cells in tumortissues was enhanced by PDT (fig. S18B), leading to a potential increaseof Tregs. Fortunately, the CD8+ T cell–to–Treg ratio in S-aPDL1/ICG@NP + laser group was determined to be 2.5- and 2.0-fold higherthan those of the S-ICG@NP + laser and aPDL1/ICG@NP + lasergroup, respectively, implying that S-aPDL1/ICG@NP–based PDL1blockade and PDT significantly elicit antitumor immunity (Fig. 4G).

The proliferation of CD8+ T cells was evaluated by Ki-67 immuno-fluorescence staining. S-aPDL1/ICG@NP/PDT–based combinationtherapy significantly promoted the proliferation of tumor-infiltratingCTLs (Fig. 4H). Furthermore, the combination therapy also increasedthe frequency of TNF-a and IFN-g dual-positive effector CD8+ T cellsto 22.3 ± 5.6% (Fig. 4I), a likelymechanism accounting for intratumoralsecretion of proinflammatory cytokines.

Antitumor study and immune memory effects in vivoNext, we evaluated the antitumor efficacy of S-aPDL1/ICG@NP in the4T1 tumor–bearing BALB/c mouse model. The tumor growth inhibi-tion rate was regressed by 39.8% with free aPDL1 injection and furtherincreased to 56.8% by S-aPDL1/ICG@NP (Fig. 5, A to D). Taking theeffects of PDT into consideration, aPDL1/ICG@NP with a 1.2 W/cm2

laser inhibited 66.3% of tumor growth because the activity of aPDL1was attenuated by the PEG corona, which was comparable with theS-ICG@NP + laser group. Systemic-treated free aPDL1 + S-ICG@NPwith a 1.2 W/cm2 laser could further delay the tumor growth andshowed moderate inhibition rate of 68.6% (fig. S19). In contrast, S-aPDL1/ICG@NP treatment in combination with PDT at 0.9 W/cm2

regressed 73.2%of the tumor growth, and the inhibition ratewas furtherimproved along with the increase of photodensity, leading to completetumor regression in ~60% of mice.

FreeaPDL1 treatment showed negligible survival benefits comparedwith the PBS group, and all animals died within 45 days, whereas theS-aPDL1/ICG@NP + 1.2 W/cm2 group achieved durable tumorrejection, with 80% of mice surviving over 70 days (Fig. 5E). No ob-vious body weight loss and histological damage of normal tissueswere found in all groups (figs. S20 and S21). All these results impliedthat optimized PDT, together with PDL1 blockade, sufficiently regu-lated the immunological tolerance and immunosuppressive micro-environment of 4T1 tumors, resulting in significant improvement ofthe therapeutic efficacy of treating primary tumors. Terminal deoxy-nucleotidyl transferase–mediated deoxyuridine triphosphate nick endlabeling (TUNEL) analysis of the 4T1 tumor sections revealed that S-aPDL1/ICG@NP–mediated combination therapy induced significantapoptosis andnecrosis of the tumor cells, amechanism thatmay explainthe disruption of the established tumors (fig. S22). Furthermore, metas-tasis of the primary tumor in the S-aPDL1/ICG@NP + 1.2 W/cm2

group was completely inhibited (Fig. 5F and fig. S23), verifying thepotential of S-aPDL1/ICG@NP/PDT combination for treatment of ad-vanced metastatic tumors.

We further evaluated the antitumor performance of S-aPDL1/ICG@NP + PDT in the B16-F10 melanoma tumor–bearing C57BL/6mouse model. The results showed that S-aPDL1/ICG@NP + PDT sig-nificantly suppressed the tumor growth (fig. S24, A and B). Moreover,S-aPDL1/ICG@NP + PDT treatment significantly elongated the sur-vival of B16-F10 tumor–bearing C57BL/6 mice, indicating a promisingpotential of S-aPDL1/ICG@NP–mediated combination immuno-therapy for treatment of a broad types of solid tumors (fig. S25).

The immune memory effect induced by ICB therapy was crucialfor durable therapeutic benefits, recurrence prevention, and metas-


tasis inhibition. At the end of antitumor study, we examined thefrequency of central memory T cells (TCM; CD3

+CD8+CD44+CD127+)isolated from the draining LNs. The frequency of CD8+ TCM in theS-aPDL1/ICG@NP + 1.2 W/cm2 group was significantly increasedas compared with the control groups (Fig. 5G). Similarly, the nor-malized number of CD8+ TCM in S-aPDL1/ICG@NP + 1.2 W/cm2

group was 2.5- and 1.9-times higher than the S-ICG@N + laser andaPDL1/ICG@NP + laser groups, respectively (Fig. 5H). In addition,this same S-aPDL1/ICG@NP/PDT combination treatment alsoincreased the TNF-a concentration in serum (Fig. 5I). These resultsindicate that the combination of ICB therapy and PDT could induce along-term immune memory effect for immunotherapy.

To demonstrate the immune memory effect induced by S-aPDL1/ICG@NP and PDT, we rechallenged the secondary 4T1 tumors 25 daysafter the resection of the primary 4T1 tumors by combination therapyor surgical resection. The progression of the secondary tumorwas great-ly suppressed by S-aPDL1/ICG@NP + PDT at 1.2 W/cm2 as demon-strated by the highly efficient response of TCM in the draining LNs. Incontrast, PBS or aPDL1 treatment of the first established tumors failedto inhibit the growth of the secondary tumors due to the weak immunememory effects in mice (Fig. 5J). These results suggest that S-aPDL1/ICG@NP–based combination therapy induced long-term immunememory effects for preventing tumor recurrence.

The combination of ICB therapy and PDT-inhibitedtumor metastasisOur results suggest that S-aPDL1/ICG@NP efficiently accumulates intumor-draining LNs and could potentially boost antitumor immunity.To test whether S-aPDL1/ICG@NP–mediated ICB therapy and PDTcould inhibit metastasis to LNs, we performed an antimetastasis studyusing a BALB/cmousemodel (Fig. 6A). Popliteal LNs and lungs in eachgroup were harvested, photographed, weighed, and measured at thestudy endpoint. Tumormetastasis to theLNs and lungswas significantlyinhibited when mice were administrated with S-aPDL1/ICG@NP andirradiated by a laser at the popliteal LNs (Fig. 6, B and C). The averageweight of popliteal LNs in the PBS control group was ~35.5mg, whereasit was only ~1.4 mg in the combination therapy group, supporting theability of S-aPDL1/ICG@NP and PDT combination therapy to activatean immune response to kill metastatic cancer cells (Fig. 6D).

Furthermore, similar results were obtained for S-aPDL1/ICG@NPand PDT combination therapy to inhibit lung metastasis. The averagenumber of metastatic lesions in free aPDL1 or S-aPDL1/ICG@NP–treated mice is 18.3 ± 7.6 or 10.8 ± 4.5, respectively. However, the num-ber of metastatic lesions in S-aPDL1/ICG@NP + PDT group decreasedto 3.2 ± 3.4 per lung, suggesting that the combination therapy could notonly inhibit LN metastasis but also suppress lung metastasis of 4T1cancer cells (Fig. 6E). In addition to effectively inhibiting lung metasta-sis, the overall survival time of S-aPDL1/ICG@NP+1.2W/cm2–treatedmice was significantly prolonged compared with the control groups(Fig. 6F).

Last, we examined the lung sections using hematoxylin and eosin(H&E) staining and several of the metastatic lesions present in the sur-vival group (Fig. 6G). Such outstanding therapeutic benefits were likelyattributed to the remarkable priming of adaptive immune responses byPDT and effective regulation of immunosuppressive tumor micro-environment by PDL1 blockade.

Given the high LN specificity of S-aPDL1/ICG@NP, as demon-strated in the lymphatic metastasis mouse model, we further examinedthe immune responses in tumor metastatic LNs after S-aPDL1/ICG@NP

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treatment. PDL1 blockade along with PDT significantly promotedDCmaturation in the tumor-draining LNs. For instance, S-aPDL1/ICG@NP increased the frequency of matured DCs (CD80+CD86+)to 14.0 ± 1.6%, compared with 4.3 ± 2.5% in the PBS control group.The DCmaturation ratio of the S-aPDL1/ICG@NP group was mark-edly increased to 30.1 ± 2.9% upon laser irradiation at 1.2 W/cm2

(Fig. 6H). DC maturation facilitated the presentation of tumor anti-gens to naive T cells and subsequently boosted the adaptive anti-tumor immunity for inhibiting lymphatic tumor metastasis. Thecombination of S-aPDL1/ICG@NP treatment and laser irradiationat 1.2W/cm2 elicited 2.1 × 104 CD8+ T cells/mg of the draining LNs(Fig. 6I), 3.0-fold higher than that of the aPDL1 control, which could


explain the superior antimetastasis of S-aPDL1/ICG@NP–based com-bination therapy.

DISCUSSIONIn the current study, we have developed a aPDL1-based nanoplatformfor combination immunotherapy of the metastatic tumors. The nano-platform was rationally fabricated by noncovalently compressingaPDL1 with PEG-dEGCG without disturbing the antibody bindingaffinity. We have demonstrated that the nanoplatform integratingaPDL1 and photosensitizer could simultaneously target the primaryand metastatic tumors through tumor-specific accumulation of the

Fig. 5. Antitumor effects of S-aPDL1/ICG@NP–mediated combination immunotherapy. (A andB) Average tumor growth curves of subcutaneously inoculated 4T1 tumor–bearing BALB/cmicewith different treatments (n= 5). (C) Individual tumor growth curves in control and treated groups. (D) H&E staining of tumor sections at the end of theantitumor study. Scale bars, 250 mm. (E) Survival curves of the 4T1 primary tumor–bearing mice with various treatments (1#, PBS; 2#, aPDL1; 3#, S-ICG@NP; 4#, S-ICG@NP +1.2 W/cm2; 5#, aPDL1/ICG@NP + 1.2 W/cm2; 6#, S-aPDL1/ICG@NP; 7#, S-aPDL1/ICG@NP + 0.9 W/cm2; 8#, S-aPDL1/ICG@NP + 1.2 W/cm2; n = 5). (F) Quantification ofpulmonary metastasis nodules at the end of the antitumor study (n = 6). (G) Flow cytometry analysis of CD8+ TCM cells (gated on CD3+CD8+) in tumor-drainingLNs 25 days after the initial treatments (n = 3). (H) Normalized number of CD8+ TCM cells in draining LNs on day 25 after initial treatments. (I) Secretion of TNF-a in seraof mice on day 25. (J) Growth curves of inoculated secondary 4T1 tumor after elimination of the primary tumor (n = 6). Data are means ± SD. Statistical significancewas calculated by one-way ANOVA with Tukey’s post hoc test and log-rank (Mantel-Cox) test (*P < 0.05, **P < 0.01, ***P < 0.001).

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nanoparticles. In combination with NIR laser illumination, the anti-body nanoparticles regulate the immunological tolerance and immu-nosuppressive conditions of tumors by blocking tumor cell surfacePDL1 and recruiting intratumoral infiltration of CTLs.


In comparison with the conventional ICB immunotherapy usingcheckpoint inhibitors alone or the combination with other therapeuticstrategies (e.g., radiotherapy, chemotherapy, or phototherapy), theaPDL1 nanoparticles can passively accumulate inside the tumors and

Fig. 6. Antimetastasis effects of S-aPDL1/ICG@NP–mediated combination immunotherapy. (A) Combination immunotherapy of the lymphatic metastatic tumors inBALB/c mice. (B) Photographs of the popliteal LNs (1#, PBS; 2#, aPDL1; 3#, S-ICG@NP; 4#, S-ICG@NP + 1.2 W/cm2; 5#, aPDL1/ICG@NP + 1.2 W/cm2; 6#, S-aPDL1/ICG@NP; 7#,S-aPDL1/ICG@NP + 0.9 W/cm2; 8#, S-aPDL1/ICG@NP + 1.2 W/cm2) and (C) lungs collected at the end of antitumor studies. (D) Mass of the popliteal LNs (n = 6). (E) Numberof the pulmonary metastasis nodules examined at the end of antimetastasis study (n = 6). (F) Survival curves of mice given different treatments (1#, PBS; 2#, aPDL1; 3#,S-ICG@NP; 4#, S-ICG@NP + 1.2 W/cm2; 5#, aPDL1/ICG@NP + 1.2 W/cm2; 6#, S-aPDL1/ICG@NP; 7#, S-aPDL1/ICG@NP + 0.9 W/cm2; 8#, S-aPDL1/ICG@NP + 1.2 W/cm2;n = 6). (G) H&E staining of the lung sections at the end of the antitumor study. Scale bars, 300 mm. (H) DC maturation in the LNs of lymphatic metastasis BALB/c mice incontrol and treated groups. (I) Normalized number of CD8+ T cells in the LNs (n = 3). Data aremeans ± SD. Statistical significancewas calculated by one-way ANOVAwithTukey’s post hoc test and log-rank (Mantel-Cox) test (*P < 0.05, **P < 0.01, ***P < 0.001).

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be specifically activated at the tumor site to avoid the on-target butoff-tumor effect of free antibody and enhance tumoral accumulationand penetration of aPDL1. The aPDL1 nanoparticles can spatio-temporally co-deliver aPDL1 and photosensitizer ICG to the tumormass for combination immunotherapy. The aPDL1 nanoparticles notonly highly efficiently boost the antitumor immune responses but alsoelicit long-term immune memory effects in BALB/c mice, thus leadingto remarkable therapeutic efficacy for inhibition of the primary andmetastatic tumors. The simplicity of our system, which uses the anti-body nanoparticle alone both for immune stimulation and checkpointinhibition, could be readily adapted to other immune checkpoint inhib-itors for improved ICB therapy.

The checkpoint inhibitor–based nanoplatform provides valuableinsights for revealing the mechanisms of combination immuno-therapy in eliciting antitumor immunity and suggests promising strat-egies to combat advanced metastatic cancer, which has the realpotential of being translated into the future generations of cancer im-munotherapy. Nevertheless, the nanoparticle design still needs to beimproved for the treatment of a broad diversity of solid tumors. Spe-cially designed endoscopes are urgently needed for the deep deliveryof NIR radiation for combination ICB and PDT. Moreover, radioiso-topes or chemotherapeutics could be incorporated into the nanoplatformto overcome the limitation of NIR laser delivery. The tumor-specificdistribution and activation of the aPDL1 nanoparticles is highly depen-dent on MMP-2 expression in the tumor mass. Further work is stillneeded to fully understand how the aPDL1 nanoparticles interact withthe tumor microenvironment. For the potential translation of theaPDL1 nanoparticles, systemic biosafety assay to investigate whetherthe aPDL1 nanoparticles suppress the immune-related adverse eventsof ICB therapy would be of priority.


ag.org/
MATERIALS AND METHODSPreparation and characterization of S-aPDL1/ICG@NPThe MMP-2–activatable S-aPDL1/ICG@NP was formulated bynanoprecipitation method. First, 100 ml of aPDL1 aqueous solution(10 mg/ml) was incubated with 30 ml of ICG solution (10 mg/ml) for2 hours under gentle stirring. The mixture was centrifuged at 8000 rpmfor 10min. The resultant aPDL1/ICGnanocomplexes were redispersedin 1.0 ml of deionized (DI) water at a concentration of 1.0 mg/ml, andthen 10 ml of dEGCG [10 mg/ml in H2O/dimethyl sulfoxide (DMSO)(4:1, v/v)] and 2.5 ml of PEG-PLGLAG-dEGCG [10 mg/ml in H2O/DMSO (4:1, v/v)] were added under ultrasonication. The resultant S-aPDL1/ICG@NP was purified by dialyzing against DI water. The par-ticle size and morphology of S-aPDL1/ICG@NP were examined usingDLS (Malvern, UK) and TEM (JEOL, Japan), respectively. The absorp-tion and fluorescence spectra of S-aPDL1/ICG@NP were recordedusing ultraviolet-visible (Shimadzu UV-2450, Japan) and fluorescence(Hitachi F-4600, Japan) spectrophotometers.
To clarify the interactions between aPDL1, ICG, dEGCG, and PEG-PLGLAG-dEGCG, we incubated S-aPDL1/ICG@NP with NaCl,Tween-20, Triton X-100, or urea solution at desired concentrationsfor 5 min. The particle size change was monitored by DLS.

MMP-2 triggered aPDL1 release from S-aPDL1/ICG@NPTo investigate MMP-2–triggered aPDL1 release from S-aPDL1/ICG@NP, we incubated the nanoparticles with 50 nM MMP-2 and0.72 mM CaCl2 in phosphate buffer (pH 7.4) at 37°C. aPDL1 releasedfrom S-aPDL1/ICG@NPwas collected using ultracentrifugation [Ami-


con centrifugation unit; molecular weight cut-off (MWCO), 300 kDa]and examined by an anti-rabbit total IgG ELISA kit.

Activity analysis of aPDL1To test the ability of S-aPDL1/ICG@NP to retain the activity ofcomplexed proteins, the nanoparticles were pretreated, and thebinding affinity of aPDL1 was determined by ELISA with recom-binant PDL1 protein (Sino Biological, China). To clarify the effectof PDT on aPDL1 activity, S-aPDL1/ICG@NP was first treated by0.9 W/cm2 and 1.2 W/cm2 lasers for 5 min and then examined usingELISA. Three kinds of S-aPDL1/ICG@NP–mimicking nanocom-plexes were prepared from proteinase K, RNase, or HRP antibodyby compacting with dEGCG and PEG-PLGLAG-dEGCG. Then, theprotein nanoparticles were dissociated with Triton X-100 or activatedby MMP-2 for 30 min, thereafter the activities of proteinase K, RNase,and HRP antibody were tested using proteolysis, RNA degradation,and 3,3′,5,5′-tetramethylbenzidine assay, respectively. Untreated pro-tein solutions were set as the positive control.

To examine the stability of S-aPDL1/ICG@NP against proteol-ysis, aqueous solution of free aPDL1 or S-aPDL1/ICG@NP wasmixed with proteinase K solution (20 mg/ml) for desired time inter-vals at 37°C. The solution was then added with phenylmethanesulfonylfluoride to inhibit the bioactivity of proteinase K and subsequently withMMP-2 to dissociate the nanoparticles. The amounts ofaPDL1 residualwere examined using an anti-rabbit total IgG ELISA kit.

Immune response analysis in vivoThe immune response of S-aPDL1/ICG@NP–mediated photoimmu-notherapy was investigated on 4T1 tumor–bearing BALB/c mice. Thetumor-bearing mice were randomly grouped when the tumor volumereached 300 mm3. The mice groups were then systemically injectedwith PBS, free aPDL1, S-ICG@NP, aPDL1/ICG@NP, or the S-aPDL1/ICG@NP suspension with aPDL1 dosage (5.0 mg/kg). TheS-aPDL1/ICG@NP groups were subjected to 808-nm laser irradiationat photodensity of 0.9 or 1.2 W/cm2 for 5 min. S-ICG@NP andaPDL1/ICG@NPgroupswith a 1.2W/cm2 laser at the same timewereset as control. The tumors were harvested and weighted 5 days afterthe final treatment and digested by collagenase IV (175 U/ml), hyal-uronidase (100 U/ml), and deoxyribonuclease (30 U/ml) at 37°C for30 min. The monodispersed tumor-infiltrating lymphocytes (TILs)were prepared by filtering the tumor suspensions through 75-mmfilters and enriched using lymphocyte separation medium. Theseparated TILs were stained with anti–CD3-PerCP Cy5.5, anti–CD4-FITC, and anti–CD8-PE antibodies to investigate the frequency ofCD4+ and CD8+ T cells.

To determine the proliferation and functions of TILs, the TILswere fixed and permeabilized using a FoxP3/Transcription Factorstaining buffer set (eBioscience, San Diego, CA). Next, the TILswere stained with anti–Ki-67-FITC, anti–IFN-g (IFN-g–Alexa Fluor488), and anti–TNF-a (TNF-a–Alexa Fluor 647) antibodies beforeexamined by flow cytometry. The proinflammatory cytokines secretedwithin the tumors, including IFN-g, TNF-a, and IL-1b, weredetermined using ELISA kits. The immune memory effect of CD8+

T cells was examined 25 days after the treatments. CD8+ centralmemory T cells were extracted from draining LNs of BALB/c mice,stained with anti–CD44-PerCP Cy5.5 and anti–CD127-PE antibodies,and examined by flow cytometry analysis.

The immune priming properties of S-aPDL1/ICG@NP were alsoevaluated using an LN metastatic mouse model. Enlarged LNs were

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intratumorally injected with 50 ml of PBS, free aPDL1, S-ICG@NP,aPDL1/ICG@NP, and S-aPDL1/ICG@NP suspensions (2.5 mg/kgof aPDL1) into the right footpad of BALB/c mice. Two hours aftertreatment, the popliteal and inguinal LNs were subjected to lasertreatment at 0.9 or 1.2 W/cm2 for 5 min, respectively. The mice wereeuthanized to collect the drain LNs 5 days after treatments. Next,lymphocytes were gathered by grinding the LNs and enriched forthe following analyses. For DC maturation studies, the lymphocyteswere incubated with anti–CD11c-FITC, anti–CD80-PE, and anti–CD86–Alexa Fluor 647 antibodies for flow cytometry examination.To determine the frequency of CD4+ or CD8+ T cells, the lympho-cytes were stained with fluorescent probe–labeled anti-CD3, anti-CD4, and anti-CD8 antibodies and examined using flow cytometry.

Primary tumor growth inhibition and lymphatic metastasissuppression in vivoBALB/c and C57BL/6 mice (female, 5 to 6 weeks old) were purchasedfrom Shanghai Experimental Animal Center (Shanghai, China) andmaintained under pathogen-free conditions. To investigate the anti-tumor effects of S-aPDL1/ICG@NP,BALB/cmicewere subcutaneouslytransplanted with 5 × 105 4T1 cells at the right flank. The mice wererandomly grouped (n = 5) when the tumor volume reached 200 cm3

(i.e., group 1, PBS; group 2, aPDL1; group 3, S-ICG@NP; group 4,S-ICG@NP + 1.2 W/cm2; group 5, aPDL1/ICG@NP + 1.2 W/cm2;group6, S-aPDL1/ICG@NP; group 7, S-aPDL1/ICG@NP+0.9W/cm2;group 8, S-aPDL1/ICG@NP + 1.2 W/cm2). The mice in differentgroups were intravenously injected with an equal aPDL1 (5.0 mg/kg)and ICG (1.0 mg/kg) dose. Two hours after injection, the tumors ingroups 4, 5, 7, and 8 were irradiated with an 808-nm laser for 5 minat the optimized photodensity. The tumor volume and body weightswere then monitored every 2 days after injection. All the major organs(i.e., heart, liver, spleen, lung, and kidney) and the tumorswere collectedand examined by H&E or TUNEL staining at the end of the antitumorstudy. In a separated experiment, the tumor-bearing mice were treatedas above, and overall survival was also recorded in time.

To test the broad application of S-aPDL1/ICG@NP on differenttypes of tumor, we established a B16-F10 tumor–bearing C57BL/6micemodel by subcutaneously injection of 5 × 105 B16-F10 cells on the backof mice.When the tumor volume reached 200mm3, themice were ran-domly grouped (n = 5) and treated with different suspensions at thesame dose applied for 4T1 tumor therapy. The B16-F10 tumor–bearingmice were then irradiated by an 808-nm laser for 5 min at the desiredphotodensity. The tumor growth rates and body weight change wererecorded during the antitumor study. The overall survival rates wereevaluated at the end of the antitumor studies.

To investigate the immune memory effect, we grouped and treatedBALB/c mice by following the same procedure applied for the anti-tumor study. The primary tumors were surgically removed after thethird administration, and 1 × 105 4T1 tumor cells were rechallengedinto the left flank of each mouse 25 days after the final treatment toestablish the distant metastasis tumor models. The volumes of the dis-tant tumors were monitored for 15 days. The tumor volume wasdetermined following the formula

V ¼ ðL�W �WÞ=2ðL; the longest dimension;W; the shortest dimensionÞ

For lymphatic metastasis inhibition study, 5 × 105 4T1 cells wereinoculated into the right rear footpad of the mice on day 0. The mice


were randomly divided into eight groups when lymphatic metastasiswas confirmed and intratumorally treatedwith 50 ml of different formu-lations [aPDL1 (2.5 mg/kg) and ICG (0.5 mg/kg)]. The popliteal andinguinal LNs of mice in selected groups (4, 5, 7, and 8) were stimulatedby an 808-nm laser for 5min and 2 hours after the injection. All animalswere euthanized on day 22, and the popliteal LNs and lungs werecollected, weighted, and photographed for metastasis inhibition inves-tigation. The lungs were fixed for H&E staining analysis of micrometa-static foci. For the mouse survival study, a separate study wasconducted, and an event was recorded when the mice died or exhibitedany signs of impaired health.

StatisticsData were given as means ± SD. The statistical significance wasperformed by one-way analysis of variance (ANOVA) with Tukey’spost hoc test and two-sided unpaired Student’s t test. Comparisonsof survival rates were calculated by the log-rank (Mantel-Cox) test.Statistical significance was set as follows: *P < 0.05, **P < 0.01, and***P < 0.001.

SUPPLEMENTARY MATERIALSimmunology.sciencemag.org/cgi/content/full/4/37/eaau6584/DC1Materials and MethodsTable S1. List of antibodies used for flow cytometry and immunohistochemistry examination.Table S2. Pharmacokinetic profiles of the antibody nanoparticles.Fig. S1. Synthetic route for MMP-2–liable PEG-PLGLAG-dEGCG.Fig. S2. Electrospray ionization (ESI) mass spectrum of the dimer of EGCG (dEGCG).Fig. S3. Matrix-assisted laser desorption/ionization–time-of-flight (MALDI-TOF) mass spectra ofEGCG conjugates.Fig. S4. HPLC examination of MMP-2–mediated degradation of the PLGLAG spacer.Fig. S5. MALDI-TOF mass spectra of MMP-2–insensitive PEGylated EGCG.Fig. S6. Characterization of S-aPDL1/ICG@NP assembly process.Fig. S7. Representative TEM image of the S-aPDL1/[email protected]. S8. Serum stability of S-aPDL1/[email protected]. S9. PDL1 binding affinity of aPDL1 in S-aPDL1/ICG@NP post PDT.Fig. S10. ESR spectroscopic examination of S-aPDL1/ICG@NP–induced ROS.Fig. S11. S-aPDL1/ICG@NP induced ROS generation and phototoxicity in vitro.Fig. S12. Flow cytometric examination of PDL1 expression in different cell lines.Fig. S13. Intracellular uptake property of S-aPDL1/[email protected]. S14. Pharmacokinetic profiles of nanoparticles in vivo.Fig. S15. Fluorescence imaging of 4T1 tumors at desired time after different treatments.Fig. S16. Immunohistochemical examination of MMP-2 expression.Fig. S17. Quantification of aPDL1 distribution in major organs.Fig. S18. Normalized tumoral infiltration of CD3+ and CD4+ T cells in control and treated groups.Fig. S19. The tumor growth plots of 4T1 tumor–bearing mice in control and treated groups.Fig. S20. Body weight change recorded during the experimental period.Fig. S21. H&E staining of the normal organs examined at the end of the antitumor study.Fig. S22. TUNEL analysis of S-aPDL1/ICG@NP–mediated antitumor effects.Fig. S23. H&E staining of the lung sections at the end of the antitumor study.Fig. S24. Relative growth curves of B16-F10 tumors after various treatments.Fig. S25. Survival curves of B16-F10 tumors in control and treated groups.Data S1. Raw data for Figs. 2 to 6 (Excel).Data S2. Raw data for figs. S6 to S24 (Excel).

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Acknowledgments: We thank X. Yan from Institute of Biophysics, Chinese Academy ofSciences for fruitful discussion. The MS-FACILITY from National Center for Protein ScienceShanghai, SIBCB, CAS, is gratefully acknowledged. Funding: Financial supports from theNational Natural Science Foundation of China (31671024, 31622025, 51873228, and 81521005)and the Youth Innovation Promotion Association of Chinese Academy of Sciences (2019283)are gratefully acknowledged. Author contributions: D.W., T.W., H.Y., and Y.L. conceivedand designed the project. D.W., T.W., B.F., L.Z., F.Z., and H.Z. performed the experiments. D.W.and H.Y. analyzed the data and wrote the initial manuscript. All the authors approved thesubmission. Competing interests: The authors declare that they have no competing financialinterests. Data and materials availability: All data needed to evaluate the conclusions in thepaper are present in the paper or the Supplementary Materials.

Submitted 3 July 2018Accepted 3 June 2019Published 12 July 201910.1126/sciimmunol.aau6584

Citation: D. Wang, T. Wang, H. Yu, B. Feng, L. Zhou, F. Zhou, B. Hou, H. Zhang, M. Luo, Y. Li,Engineering nanoparticles to locally activate T cells in the tumor microenvironment. Sci.Immunol. 4, eaau6584 (2019).

t on October 19

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Engineering nanoparticles to locally activate T cells in the tumor microenvironment

LiDangge Wang, Tingting Wang, Haijun Yu, Bing Feng, Lei Zhou, Fangyuan Zhou, Bo Hou, Hanwu Zhang, Min Luo and Yaping

DOI: 10.1126/sciimmunol.aau6584, eaau6584.4Sci. Immunol.

growth and metastasis of murine tumors. show that these nanoparticles outperform systemic anti-PDL1 in limitinget al.produce reactive oxygen species, Wang

nanoparticle. When used in conjunction with localized near-infrared radiation that activates the photosensitizer tois highly expressed in tumors, and delivery of the nanoparticle to tumors elicits release of the antibody from the

sensitive nanoparticles that carry a photosensitizer. MMP-2−1 (PDL1) with matrix metalloproteinase protein 2 (MMP-2)nanoparticles that can be specifically activated within tumors by conjugating antibodies against programmed death ligand

have attempted to do this by engineering et al.solved, from improving efficacy to limiting side effects. Here, Wang Although immunotherapy has transformed the cancer therapeutics landscape, a number of problems remain to be

Targeted tumor immunotherapy

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MATERIALSSUPPLEMENTARY http://immunology.sciencemag.org/content/suppl/2019/07/08/4.37.eaau6584.DC1

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