Research ArticleIMMUNOTHERAPY

Engineering nanoparticles to locally activate T cells in the tumor microenvironment

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Science Immunology  12 Jul 2019:
Vol. 4, Issue 37, eaau6584
DOI: 10.1126/sciimmunol.aau6584

Targeted tumor immunotherapy

Although immunotherapy has transformed the cancer therapeutics landscape, a number of problems remain to be solved, from improving efficacy to limiting side effects. Here, Wang et al. have attempted to do this by engineering nanoparticles that can be specifically activated within tumors by conjugating antibodies against programmed death ligand 1 (PDL1) with matrix metalloproteinase protein 2 (MMP-2)–sensitive nanoparticles that carry a photosensitizer. MMP-2 is highly expressed in tumors, and delivery of the nanoparticle to tumors elicits release of the antibody from the nanoparticle. When used in conjunction with localized near-infrared radiation that activates the photosensitizer to produce reactive oxygen species, Wang et al. show that these nanoparticles outperform systemic anti-PDL1 in limiting growth and metastasis of murine tumors.

Abstract

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 to immunological tolerance is an ongoing challenge for current immune checkpoint blockade (ICB) therapy. Here, we report the development of tumor microenvironment–activatable anti-PDL1 antibody (αPDL1) nanoparticles for combination immunotherapy designed to overcome immunological tolerance of tumors. Combination of αPDL1 nanoparticle treatment with near-infrared (NIR) laser irradiation–triggered activation of photosensitizer indocyanine green induces the generation of reactive oxygen species, which promotes the intratumoral infiltration of CTLs and sensitizes the tumors to PDL1 blockade therapy. We showed that the combination of antibody nanoparticles and NIR laser irradiation effectively suppressed tumor growth and metastasis to the lung and lymph nodes in mouse models. The nanoplatform that uses the antibody nanoparticle alone both for immune stimulation and PDL1 inhibition could be readily adapted to other immune checkpoint inhibitors for improved ICB therapy.

INTRODUCTION

Immune checkpoint blockade (ICB) therapy is emerging as a promising approach for cancer immunotherapy by modulating the immunosuppressive tumor microenvironment (1, 2). In particular, ICB therapy by monoclonal antibodies (e.g., pembrolizumab, nivolumab, atezolizumab, and durvalumab) targeting the immune checkpoint programmed cell death protein 1 (PD1) or its predominant ligand, programmed death ligand 1 (PDL1; B7-H1), has induced durable tumor regression in a broad variety of cancers (36). However, only a subset of treated patients respond to current ICB therapies (711), likely due to immunological tolerance of tumors (12, 13). Immunological tolerance of tumors could be attributed to insufficient tumor infiltration of cytotoxic T lymphocytes (CTLs) (14, 15) and immunosuppressive tumor microenvironment (6, 16, 17). The development of robust strategies to improve the response rates of immune-tolerant tumors to ICB therapy has become a priority.

Recent clinical studies have revealed a positive correlation between intratumoral infiltration of CTLs and the response rate to ICB therapy (1825). Extensive efforts have been devoted to combine systemic ICB therapy with chemotherapy (26, 27), radiotherapy (2830), and phototherapy, which uses photosensitizers to realize photothermal conversion or generate reactive oxygen species (ROS) [photodynamic therapy (PDT)] (21, 26, 31, 32), or to co-delivery two antagonistic antibodies using one single nanoparticle to elicit antitumor immunity and promote intratumoral infiltration of CTLs (33). Despite promising results, the clinical translation of ICB suffers from several formidable challenges. For instance, ICB therapy using anti-PDL1 antibodies (αPDL1) displays “on-target but off-tumor” binding with the normal tissues due to PDL1 expression on vascular endothelium, pancreatic islet cells, hepatocytes, muscle, epithelium, and mesenchymal stem cells (34), which attenuated the therapeutic efficacy and resulted in severe immune-related adverse events when αPDL1 was systemically infused (11, 35). To avoid the nonspecific binding of the checkpoint inhibitors with the normal tissues, several systemic or local-regional delivery strategies had been developed by integrating the checkpoint inhibitors into nanoparticles (33), microparticles (36, 37), microneedles (38, 39), hydrogels (40, 41), or biomimetic vesicles (4244). Although these delivery strategies display notable benefits for primary tumor therapies, it remains a major challenge for treatment of deep-seated or metastatic tumors (45).

To address the challenges of current ICB therapy, we herein presented a matrix metalloproteinase protein 2 (MMP-2)–sensitive αPDL1/ICG-based nanoparticle (S-αPDL1/ICG@NP) for combating the immunological tolerance of tumors by specifically blocking the PD1/PDL1 cascade at the tumor sites (Fig. 1). S-αPDL1/ICG@NP remains stable during the blood circulation and protects αPDL1 from binding with the normal tissues. S-αPDL1/ICG@NP can passively accumulate at the tumor site through the enhanced permeability and retention (EPR) effect and become activated in MMP-2 highly expressed tumor to release αPDL1 for PDL1 blockade at the tumor site (18, 46). In combination with PDT, the αPDL1 nanoparticles triggered tumor antigen release and promoted intratumoral CTLs infiltration by generating ROS (47, 48). The antibody nanoparticle both for immune stimulation and checkpoint inhibition could be readily adapted to other immune checkpoint inhibitors for improved ICB therapy, which might represent a robust nanoplatform to overcome immunological tolerance as a barrier to cancer immunotherapy.

Fig. 1 S-αPDL1/ICG@NP for improved ICB therapy by combating the immunologic tolerance of tumors.

(A) Fabrication of MMP-2–liable S-αPDL1/ICG@NP. The αPDL1 was first complexed with photosensitizer ICG and subsequently stabilized with dEGCG to form the dEGCG-αPDL1/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-αPDL1/ICG@NP–mediated combination ICB and PDT therapy. S-αPDL1/ICG@NP could be activated within the tumor microenvironment for sustained release of αPDL1. Upon NIR laser irradiation, ICG-mediated PDT induced antitumor immunity and promoted the intratumoral infiltration of CTLs. PDT and αPDL1-mediated PDL1 blockade cumulatively suppress tumor growth and inhibit metastasis. i.v., intravenous.

RESULTS

Engineering and characterization of MMP-2–activatable S-αPDL1/ICG@NP

To construct S-αPDL1/ICG@NP, we first synthesized a dimer of (−)-epigallocatechin-3-O-gallate (dEGCG) by the Baeyer acid–catalyzed condensation reaction (49). PEGylated dEGCG (PEG-dEGCG) was then synthesized by conjugating the dimer with polyethylene glycol (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 the PEG-PLGLAG-dEGCG substrate (fig. S4). PEG-dEGCG without the peptide spacer was also synthesized as a MMP-2–insensitive analog of PEG-PLGLAG-dEGCG (fig. S5).

It has been reported that photosensitizer indocyanine green (ICG) can complex with various proteins through nonspecific hydrophobic and electrostatic interactions (50, 51). MMP-2–liable S-αPDL1/ICG@NP was thus fabricated by integrating αPDL1, ICG, dEGCG, and PEG-PLGLAG-dEGCG into one nanocomposite. αPDL1/ICG@NP, a MMP-2–insensitive analog of αPDL1/ICG@NP was also prepared by replacing PEG-PLGLAG-dEGCG with PEG-dEGCG. The optimized formulations of S-αPDL1/ICG@NP and αPDL1/ICG@NP were fabricated at an ICG to αPDL1 feeding ratio of 0.3 by screening both the ICG encapsulation efficiency and particle sizes (fig. S6, A to E).

The fabrication of S-αPDL1/ICG@NP was monitored using transmission electron microscopic (TEM) and dynamic light scattering (DLS) examinations (Fig. 2, A and B, and fig. S7). The hydrodynamic diameter of αPDL1 increased from 8.0 ± 1.3 nm to 14.5 ± 1.6 nm after ICG incubation and further increased to 38.7 ± 3.9 nm with the addition of dEGCG. In the presence of PEG-PLGLAG-dEGCG, the dEGCG-αPDL1/ICG complexes further transformed to S-αPDL1/ICG@NP with a uniform particle size of 163.4 ± 6.6 nm and a narrow polydispersity index (PDI) of 0.061. Upon MMP-2–mediated cleavage of the PEG corona, the hydrodynamic diameter of S-αPDL1/ICG@NP expanded to 298.1 ± 146.8 nm (PDI, 0.426) due to the formation of αPDL1 aggregates. The hydrodynamic diameter of S-αPDL1/ICG@NP could be readily tuned in the size range between 150 and 750 nm with narrow particle size distribution by simply adjusting the feeding ratios between dEGCG and PEG-PLGLAG-dEGCG (Fig. 2C).

Fig. 2 Chemo-physical characterization of S-αPDL1/ICG@NP.

(A) TEM images of αPDL1 and S-αPDL1/ICG@NP. Scale bars, 200 nm. (B) Particle size change of S-αPDL1/ICG@NP during the assembly and disassembly process. (C) Particle size optimization of S-αPDL1/ICG@NP as a function of feeding ratio between dEGCG and PEG-PLGLAG-dEGCG. (D) Stability assay of S-αPDL1/ICG@NP with the addition of NaCl, urea, Tween 20, or Triton X-100 (n = 3). (E) Activity assay of αPDL1, proteinase K, RNase, and HRP antibody (Ab) released from nanoparticles (n = 3). (F) Proteinase K degradation assay for S-αPDL1/ICG@NP, S-αPDL1/ICG@NP + MMP-2, and free αPDL1. Protein suspensions were incubated with proteinase K (20 ng/μl) for the indicated time duration (n = 3). (G) αPDL1 release profiles of S-αPDL1/ICG@NP in the presence or absence of MMP-2 (n = 3). (H) Fluorescence spectroscopic examination of laser-induced ROS generation of S-αPDL1/ICG@NP and S-ICG@NP as a function of irradiation 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. Statistical significance was calculated by two-sided unpaired Student’s t test (***P < 0.001). a.u., arbitrary units.

To clarify the nature of the interactions between αPDL1, dEGCG, and PEG-PLGLAG-dEGCG, we incubated S-αPDL1/ICG@NP with NaCl, urea, Tween 20, or Triton X-100 solution for 2 min. S-αPDL1/ICG@NP was quickly dissociated after the addition of Tween 20 and Triton X-100 but was minimally affected by NaCl and urea incubation, suggesting that dEGCG complexes with αPDL1 through hydrophobic interaction (Fig. 2D).

To verify the serum stability of S-αPDL1/ICG@NP in serum-containing solutions, we used ultracentrifugation and SDS–polyacrylamide gel electrophoresis analysis to distinguish the free αPDL1 and nanoparticle-encapsulated αPDL1 in 10% bovine serum albumin (BSA)–containing phosphate-buffered saline (PBS) solution. Upon 24-hour incubation, S-αPDL1/ICG@NP shows good stability in 20% of serum-containing PBS solution (fig. S8, A and B). Around 12% of αPDL1 was released from the S-αPDL1/ICG@NP, slightly higher than that of the BSA-free control (~10%), verifying the good colloidal stability of the S-αPDL1/ICG@NP in serum-equivalent concentration of BSA (fig. S8, C to F).

To investigate whether the reversible compression procedure impaired the PDL1 binding activity of αPDL1, we tested the binding affinity of αPDL1 in S-αPDL1/ICG@NP by enzyme-linked immunosorbent assay (ELISA). αPDL1 antibodies retained only 17.7 ± 7.1% of its PDL1 binding capability when they were compressed into nanoparticles. However, once dissociated by MMP-2, its PDL1 binding affinity was significantly restored to 82.3 ± 2.5%, suggesting that the compression process negligibly affects the bioactivity of αPDL1 (Fig. 2E).

To elucidate the influence of laser irradiation on the bioactivity of αPDL1, we examined the PDL1 binding affinity of the antibody in S-αPDL1/ICG@NP before and after laser irradiation (fig. S9). The results showed that αPDL1 retained ~85% of their activity upon 808-nm laser irradiation at 1.2 W/cm2 in contrast to groups without laser treatment, indicating that PDT affected negligibly the bioactivity of αPDL1.

To examine the influence of the assembly process on protein activity, we further prepared three types of S-αPDL1/ICG@NP–mimicking nanoparticles by condensing three kinds of enzymes including proteinase 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 encapsulated inside the nanoparticles due to the shielding effect of the PEG corona. In contrast, the enzymes were sufficiently reactivated once the nanoparticles were deconstructed via MMP-2 incubation, implying that the bioactivity of the proteins could be reversibly shielded by complexation with dEGCG (Fig. 2E). Furthermore, S-αPDL1/ICG@NP displayed satisfactory stability against enzyme degradation of αPDL1 with proteinase K. For instance, more than 70% of free αPDL1 was degraded by proteinase K in 30 min, but less than 10% of αPDL1 in S-αPDL1/ICG@NP was degraded after 24-hour incubation (Fig. 2F).

To measure tumor enzymatic microenvironment–triggered αPDL1 release, S-αPDL1/ICG@NP was incubated with 50 nM MMP-2 and monitored for αPDL1 release by ELISA. It was found that more than 45% of αPDL1 was released from S-αPDL1/ICG@NP after 6-hour incubation with 50 nM MMP-2, which was 4.6-fold more efficient than that of the MMP-2–free group. About 30% of αPDL1 was released from S-αPDL1/ICG@NP upon 12-hour incubation with 5 nM MMP-2, verifying the superior MMP-2 sensitivity of S-αPDL1/ICG@NP. The release rates dropped slightly along with the decreased concentration of MMP-2, but the cumulative release amount reached 41.5 ± 1.4% in 24 hours even at an enzyme concentration of 5 nM (Fig. 2G). The MMP-2–triggered αPDL1 release profiles of S-αPDL1/ICG@NP suggest that the reversible complexation strategy could avoid antibody leakage during the blood circulation and become activated in the tumor microenvironment.

The photoactivity of S-αPDL1/ICG@NP was further evaluated by measuring ROS generation upon 808-nm near-infrared (NIR) laser irradiation. Bulk S-αPDL1/ICG@NP and MMP-2–activated S-αPDL1/ICG@NP both induced significant ROS generation at photodensity of 1.2 W/cm2 (Fig. 2H). To eliminate the impact of αPDL1 of the photoactivity of ICG, we fabricated analog nanoparticles loading ICG by using mouse immunoglobulin G (IgG) protein to replace the antibodies (namely, S-ICG@NP). Comparable photoactivity was observed in S-ICG@NP or MMP-2–pretreated S-ICG@NP group due to the identical ICG loading efficiency between αPDL1 and mouse IgG protein (fig. S6A). The ROS generation capability of S-αPDL1/ICG@NP was also confirmed by electron spin resonance (ESR) spectrum. Upon laser irradiation, characteristic ROS signals (g = 2.0058, A = 16.4 G) were detected in both the S-ICG@NP and S-αPDL1/ICG@NP groups (Fig. 2H and fig. S10).

The phototoxicity of S-αPDL1/ICG@NP in vitro was investigated in 4T1 murine breast cancer cells. Laser illumination at a photodensity of 1.2 W/cm2 and ICG concentration of 20 μg ml−1 significantly reduced ~70% of 4T1 cell viability (fig. S11, A and B), due to ROS-induced cellular apoptosis as confirmed by annexin V–fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining assay (fig. S11C).

It has been well established that αPDL1 inhibits immune evasion by blocking the cell surface–expressed PDL1 of the tumor cells (52). Therefore, it is crucial to control specific binding of S-αPDL1/ICG@NP with cell surface PDL1 while avoiding S-αPDL1/ICG@NP internalization. The interaction between S-αPDL1/ICG@NP and tumor cells was investigated in vitro using 4T1 murine breast tumor cells (fig. S12). Confocal laser scanning microscopic (CLSM) examination revealed that S-αPDL1/ICG@NP was efficiently internalized by 4T1 tumor cells (fig. S13A). In contrast, MMP-2 preactivation of S-αPDL1/ICG@NP efficiently suppressed cellular uptake of S-αPDL1/ICG@NP (fig. S13B), indicated by membrane colocalization of released αPDL1. These results show successful binding of αPDL1 released from MMP-2–activated S-αPDL1/ICG@NP with the cell surface–expressed PDL1.

Biodistribution of S-αPDL1/ICG@NP in vivo

The pharmacokinetic profile of S-αPDL1/ICG@NP was investigated in Sprague Dawley rats by using rhodamine B (Rb)–labeled IgG-based nanocomplexes, which were prepared by following the same procedure for fabrication of S-αPDL1/ICG@NP. The blood clearance half-time (t1/2β) of free IgG was 38.24 ± 4.82 hours. In contrast, IgG-Rb–based S-αPDL1/ICG@NP showed much shorter t1/2β of 8.45 ± 1.66 and lower bioavailability of 544.00 ± 100.85 mg/liter hour than those of free IgG, which could be most likely explained by quick blood clearance of the nanoparticles (fig. S14 and table S2).

The biodistribution of S-αPDL1/ICG@NP was evaluated in 4T1 tumor–bearing BALB/c mice. The results showed that free αPDL1 nonspecifically distributed in the tumors and other normal organs 2 hours after injection (Fig. 3A and fig. S15), probably due to the on-target but off-tumor effect of αPDL1 (34). In contrast, S-αPDL1/ICG@NP and αPDL1/ICG@NP showed more specific accumulation at the tumor sites, which may be attributed to the EPR effect of tumors. The S-αPDL1/ICG@NP group showed 4.3- and 10.7-fold higher intratumoral αPDL1 accumulation than αPDL1/ICG@NP and free αPDL1, respectively, as determined by quantification of the fluorescence intensity 24 hours after injection (Fig. 3B). The increased intratumor accumulation and retention of S-αPDL1/ICG@NP could be most likely explained by MMP-2–mediated activation of S-αPDL1/ICG@NP in the tumor microenvironment since MMP-2 is known to be overexpressed in 4T1 tumor cells (fig. S16).

Fig. 3 Biodistribution of S-αPDL1/ICG@NP in 4T1 tumor–bearing mice.

(A) Fluorescence imaging of S-αPDL1/ICG@NP distribution in 4T1 tumor–bearing mice in vivo. The fluorescence images were collected at 2, 4, 8, 12, 24, 48, and 72 hours after intravenous injection of Cy5.5-labeled αPDL1, αPDL1/ICG@NP, or S-αPDL1/ICG@NP, respectively. The dosage of αPDL1 is 2.0 mg/kg. White dotted circles indicate the location of tumors. Quantification of αPDL1 distribution in (B) tumors and (C) lungs 4, 12, or 24 hours after injection [αPDL1 (5.0 mg/kg), n = 3]. (D) Immunofluorescence of αPDL1 distribution in tumors with various treatments, CD31 was stained with anti–CD31-FITC. Scale bars, 75 μm. DAPI, 4′,6-diamidino-2-phenylindole. (E) Fluorescence examination of S-αPDL1/ICG@NP distribution in the lymphatic metastasis tumors after footpad injection [αPDL1 (0.5 mg/kg)]. (F) Time course quantification of S-αPDL1/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-αPDL1/ICG@NP (n = 4). (I) CLSM examination of S-αPDL1/ICG@NP distribution in the popliteal LNs 4 hours after injection. Scale bars, 100 μm. 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).

Accurate distribution of S-αPDL1/ICG@NP in the major organs was also determined in 4T1 tumor–bearing mice. In the lungs of free αPDL1–treated group, the accumulation of antibodies was 1.5-fold higher than that of the S-αPDL1/ICG@NP group 4 hours after the injection and even increased to 3.4-fold 24 hours later (Fig. 3C). This could attribute to the specific binding of αPDL1 in lung tissues which reduces the clearance rates. For other organs, obvious accumulation of αPDL1 was detected in the livers of all groups while barely found in hearts, spleens, and kidneys. For instance, αPDL1/ICG@NP and S-αPDL1/ICG@NP both displayed ~2.0-fold higher liver distribution than that of the free αPDL1 group 4 hours after intravenous injection, which could be attributed to reticuloendothelial system-medicated clearance of the αPDL1 nanoparticle (fig. S17). The accumulation of free αPDL1 in the liver could result in immune-related adverse effects due to the unexpected binding with PDL1. However, compressing antibodies into S-αPDL1/ICG@NP would reduce this risk by using the PEG shells to weaken the interactions between the antibody and PDL1 in livers. Together, these findings suggest that S-αPDL1/ICG@NP was highly efficient in suppressing αPDL1 distribution in normal tissues while targeting specific activation in tumor tissues. The tumor-specific distribution and activation of S-αPDL1/ICG@NP may suppress the immune-related adverse effects of αPDL1 due to binding of αPDL1 with the normal tissues.

It has been reported that the efficacy of ICB therapy is impaired by the limited tumor penetration of checkpoint inhibitors due to the existence of tumor burden (53, 54). To assess the penetration and accessibility of S-αPDL1/ICG@NP within tumors, we examined the intratumoral distribution of S-αPDL1/ICG@NP by immunofluorescence imaging. Visible fluorescent signals of αPDL1-Cy5.5 were detected in perivascular areas of tumors 2 hours after intravenous injection in all groups, and increased signals were found in αPDL1/ICG@NP and S-αPDL1/ICG@NP groups at 24 hours. Unlike the limited diffusion and quick clearance of αPDL1/ICG@NP, released αPDL1 from S-αPDL1/ICG@NP diffused throughout the whole areas of tumor and retained more than 48 hours, suggesting that S-αPDL1/ICG@NP facilitated deep tumor penetration of αPDL1 for improved PDL1 blockade (Fig. 3D).

Dissemination of the tumor cells from the primary tumor to the lymph nodes (LNs) is one of the dominant pathways for tumor metastasis, which is a lethal event for patients with cancer (55, 56). To investigate the potential of S-αPDL1/ICG@NP for LN targeting, lymphatic metastasis tumor model was established by inoculating 4T1 tumor cells at the right footpad of mice. Footpad injection has been widely used for nanoparticle-mediated drug delivery to the lymphatic metastasis tumors (5759). The metastatic tumor-bearing mice were then injected with Cy5.5-labeled αPDL1, S-αPDL1/ICG@NP, or αPDL1/ICG@NP at the right footpad at an identical αPDL1 dose of 0.5 mg/kg when the tumors disseminated from the right flank to the LNs 12 days after tumor inoculation. S-αPDL1/ICG@NP efficiently accumulated at the lymphatic system as indicated by the strong fluorescence signals in the popliteal, inguinal, and axillary LNs (Fig. 3E). In contrast, negligible αPDL1 distribution was observed in the free αPDL1 group (Fig. 3F).

The draining LNs were harvested 24 hours after injection for fluorescence imaging and CLSM examination ex vivo. Consistent with the fluorescence imaging data in vivo, αPDL1 exhibited marginal accumulation in the popliteal, inguinal, and axillary LNs (Fig. 3, G and H). In contrast, S-αPDL1/ICG@NP was highly efficient in accumulating at all the draining LNs and distributed throughout the sections of tumoral metastatic popliteal LNs (Fig. 3I), indicating the potential use of S-αPDL1/ICG@NP for treatment of lymphatic metastasis tumors.

Elicitation of a durable immune response in vivo

The photoactivity of S-αPDL1/ICG@NP in vivo was examined by using dichlorofluorescin diacetate (DCF) as a ROS indicator. CLSM examination demonstrated the appearance of highly diffuse green fluorescence of DCF upon 808-nm laser illumination of the tumor sections, suggesting PDT-triggered ROS generation in the tumor tissue (Fig. 4A).

Fig. 4 S-αPDL1/ICG@NP induced cellular response to antitumor immunotherapy.

(A) CLSM examination of PDT-induced ROS generation in vivo. (B) Maturation of DCs in tumors after treatments [1#, PBS; 2#, αPDL1; 3#, S-ICG@NP; 4#, S-ICG@NP + laser (S-ICG@NP+L); 5#, αPDL1/ICG@NP; 6#, αPDL1/ICG@NP + laser; 7#, S-αPDL1/ICG@NP; 8#, S-αPDL1/ICG@NP + laser; laser power density, 1.2 W/cm2; n = 3)]. (C) Intratumoral secretion of TNF-α, IFN-γ, and IL-1β in BALB/c mice (n = 3). (D) Immunofluorescence staining of intratumor infiltrating CD8+ T cells in 4T1 tumors 5 days after treatments. (E) Flow cytometric quantification of intratumoral infiltration of CD4+ and CD8+ T cells. Mice were treated at 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 a function of tumor mass. (G) CD8+ T cell–to–Treg ratio in TILs. (H) Proliferation assay of CD8+ T cells. (I) Frequency of TNF-α and IFN-γ dual-positive CD8+ T cells (1, PBS; 2, αPDL1; 3, S-ICG@NP; 4, S-ICG@NP + laser; 5, αPDL1/ICG@NP; 6, αPDL1/ICG@NP + laser; 7, S-αPDL1/ICG@NP; 8, S-αPDL1/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).

Dendritic cells (DCs) play crucial roles in initiating and regulating the antitumor immune response by presenting tumor antigens to T lymphocytes. To investigate whether S-αPDL1/ICG@NP–mediated PDT accelerated DC maturation in vivo, 4T1 tumors were illuminated with an 808-nm laser and were then harvested 3 days after treatment to measure the frequency of matured DCs (CD11c+CD80+CD86+). To elucidate the influence of αPDL1 on the immune response in vitro and in vivo, MMP-2–liable nanoparticles S-ICG@NP were prepared as control. S-ICG@NP-based PDT markedly accelerated DC maturation in the tumor tissues to 25.2 ± 1.3%, whereas only 9.2 ± 1.7% of matured DCs was found in the PBS group. S-αPDL1/ICG@NP in combination with PDT treatment induced more maturation of DCs, 2.6- and 1.2-fold more efficient than free αPDL1 or αPDL1/ICG@NP + 1.2 W/cm2 group (Fig. 4B).

The DC maturation–induced systemic immune response was evaluated by examining PDT-induced intratumor secretion of proinflammatory cytokines. S-αPDL1/ICG@NP treatment and 808-nm laser irradiation at 1.2 W/cm2 elevated the intratumoral secretion of tumor necrosis factor–α (TNF-α), interferon-γ (IFN-γ), and interleukin-1β (IL-1β). PDT at photodensity of 1.2 W/cm2 increased the secretion of all three cytokines (Fig. 4C). S-αPDL1/ICG@NP–induced immune response was further investigated by examining the frequency, proliferation, and function of intratumoral infiltration of CTLs (60). CD3+ T cells in S-αPDL1/ICG@NP + 1.2 W/cm2 group performed a 1.3 to 5.0-fold increase in contrast to the other groups (fig. S18A). Moreover, the PBS control group showed negligible tumor infiltration of CD8+ T cells (Fig. 4, D to F), while S-αPDL1/ICG@NP–based PDT increased the frequency of intratumor infiltrating CD8+ T cells to 34.4 ± 2.3% (Fig. 4E). The infiltration number of CD8+ T cells in S-αPDL1/ICG@NP + 1.2 W/cm2 group was found to be 2.2 to 6.1-fold more than that of other αPDL1-treated groups, suggesting that a robust CD8+ T cell infiltration was triggered through the co-delivery of PDT and PDL1 blockade by S-αPDL1/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 cancer in a clinic (61). The number of infiltrated CD4+ T cells in tumor tissues was enhanced by PDT (fig. S18B), leading to a potential increase of Tregs. Fortunately, the CD8+ T cell–to–Treg ratio in S-αPDL1/ICG@NP + laser group was determined to be 2.5- and 2.0-fold higher than those of the S-ICG@NP + laser and αPDL1/ICG@NP + laser group, respectively, implying that S-αPDL1/ICG@NP–based PDL1 blockade and PDT significantly elicit antitumor immunity (Fig. 4G).

The proliferation of CD8+ T cells was evaluated by Ki-67 immunofluorescence staining. S-αPDL1/ICG@NP/PDT–based combination therapy significantly promoted the proliferation of tumor-infiltrating CTLs (Fig. 4H). Furthermore, the combination therapy also increased the frequency of TNF-α and IFN-γ dual-positive effector CD8+ T cells to 22.3 ± 5.6% (Fig. 4I), a likely mechanism accounting for intratumoral secretion of proinflammatory cytokines.

Antitumor study and immune memory effects in vivo

Next, we evaluated the antitumor efficacy of S-αPDL1/ICG@NP in the 4T1 tumor–bearing BALB/c mouse model. The tumor growth inhibition rate was regressed by 39.8% with free αPDL1 injection and further increased to 56.8% by S-αPDL1/ICG@NP (Fig. 5, A to D). Taking the effects of PDT into consideration, αPDL1/ICG@NP with a 1.2 W/cm2 laser inhibited 66.3% of tumor growth because the activity of αPDL1 was attenuated by the PEG corona, which was comparable with the S-ICG@NP + laser group. Systemic-treated free αPDL1 + S-ICG@NP with a 1.2 W/cm2 laser could further delay the tumor growth and showed moderate inhibition rate of 68.6% (fig. S19). In contrast, S-αPDL1/ICG@NP treatment in combination with PDT at 0.9 W/cm2 regressed 73.2% of the tumor growth, and the inhibition rate was further improved along with the increase of photodensity, leading to complete tumor regression in ~60% of mice.

Fig. 5 Antitumor effects of S-αPDL1/ICG@NP–mediated combination immunotherapy.

(A and B) Average tumor growth curves of subcutaneously inoculated 4T1 tumor–bearing BALB/c mice with 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 the antitumor study. Scale bars, 250 μm. (E) Survival curves of the 4T1 primary tumor–bearing mice with various treatments (1#, PBS; 2#, αPDL1; 3#, S-ICG@NP; 4#, S-ICG@NP + 1.2 W/cm2; 5#, αPDL1/ICG@NP + 1.2 W/cm2; 6#, S-αPDL1/ICG@NP; 7#, S-αPDL1/ICG@NP + 0.9 W/cm2; 8#, S-αPDL1/ICG@NP + 1.2 W/cm2; n = 5). (F) Quantification of pulmonary 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-draining LNs 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-α in sera of 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 significance was 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).

Free αPDL1 treatment showed negligible survival benefits compared with the PBS group, and all animals died within 45 days, whereas the S-αPDL1/ICG@NP + 1.2 W/cm2 group achieved durable tumor rejection, with 80% of mice surviving over 70 days (Fig. 5E). No obvious body weight loss and histological damage of normal tissues were found in all groups (figs. S20 and S21). All these results implied that optimized PDT, together with PDL1 blockade, sufficiently regulated the immunological tolerance and immunosuppressive microenvironment of 4T1 tumors, resulting in significant improvement of the therapeutic efficacy of treating primary tumors. Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) analysis of the 4T1 tumor sections revealed that S-αPDL1/ICG@NP–mediated combination therapy induced significant apoptosis and necrosis of the tumor cells, a mechanism that may explain the disruption of the established tumors (fig. S22). Furthermore, metastasis of the primary tumor in the S-αPDL1/ICG@NP + 1.2 W/cm2 group was completely inhibited (Fig. 5F and fig. S23), verifying the potential of S-αPDL1/ICG@NP/PDT combination for treatment of advanced metastatic tumors.

We further evaluated the antitumor performance of S-αPDL1/ICG@NP + PDT in the B16-F10 melanoma tumor–bearing C57BL/6 mouse model. The results showed that S-αPDL1/ICG@NP + PDT significantly suppressed the tumor growth (fig. S24, A and B). Moreover, S-αPDL1/ICG@NP + PDT treatment significantly elongated the survival of B16-F10 tumor–bearing C57BL/6 mice, indicating a promising potential of S-αPDL1/ICG@NP–mediated combination immunotherapy for treatment of a broad types of solid tumors (fig. S25).

The immune memory effect induced by ICB therapy was crucial for durable therapeutic benefits, recurrence prevention, and metastasis inhibition. At the end of antitumor study, we examined the frequency of central memory T cells (TCM; CD3+CD8+CD44+CD127+) isolated from the draining LNs. The frequency of CD8+ TCM in the S-αPDL1/ICG@NP + 1.2 W/cm2 group was significantly increased as compared with the control groups (Fig. 5G). Similarly, the normalized number of CD8+ TCM in S-αPDL1/ICG@NP + 1.2 W/cm2 group was 2.5- and 1.9-times higher than the S-ICG@N + laser and αPDL1/ICG@NP + laser groups, respectively (Fig. 5H). In addition, this same S-αPDL1/ICG@NP/PDT combination treatment also increased the TNF-α concentration in serum (Fig. 5I). These results indicate that the combination of ICB therapy and PDT could induce a long-term immune memory effect for immunotherapy.

To demonstrate the immune memory effect induced by S-αPDL1/ICG@NP and PDT, we rechallenged the secondary 4T1 tumors 25 days after the resection of the primary 4T1 tumors by combination therapy or surgical resection. The progression of the secondary tumor was greatly suppressed by S-αPDL1/ICG@NP + PDT at 1.2 W/cm2 as demonstrated by the highly efficient response of TCM in the draining LNs. In contrast, PBS or αPDL1 treatment of the first established tumors failed to inhibit the growth of the secondary tumors due to the weak immune memory effects in mice (Fig. 5J). These results suggest that S-αPDL1/ICG@NP–based combination therapy induced long-term immune memory effects for preventing tumor recurrence.

The combination of ICB therapy and PDT-inhibited tumor metastasis

Our results suggest that S-αPDL1/ICG@NP efficiently accumulates in tumor-draining LNs and could potentially boost antitumor immunity. To test whether S-αPDL1/ICG@NP–mediated ICB therapy and PDT could inhibit metastasis to LNs, we performed an antimetastasis study using a BALB/c mouse model (Fig. 6A). Popliteal LNs and lungs in each group were harvested, photographed, weighed, and measured at the study end point. Tumor metastasis to the LNs and lungs was significantly inhibited when mice were administrated with S-αPDL1/ICG@NP and irradiated by a laser at the popliteal LNs (Fig. 6, B and C). The average weight of popliteal LNs in the PBS control group was ~35.5 mg, whereas it was only ~1.4 mg in the combination therapy group, supporting the ability of S-αPDL1/ICG@NP and PDT combination therapy to activate an immune response to kill metastatic cancer cells (Fig. 6D).

Fig. 6 Antimetastasis effects of S-αPDL1/ICG@NP–mediated combination immunotherapy.

(A) Combination immunotherapy of the lymphatic metastatic tumors in BALB/c mice. (B) Photographs of the popliteal LNs (1#, PBS; 2#, αPDL1; 3#, S-ICG@NP; 4#, S-ICG@NP + 1.2 W/cm2; 5#, αPDL1/ICG@NP + 1.2 W/cm2; 6#, S-αPDL1/ICG@NP; 7#, S-αPDL1/ICG@NP + 0.9 W/cm2; 8#, S-αPDL1/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) Number of the pulmonary metastasis nodules examined at the end of antimetastasis study (n = 6). (F) Survival curves of mice given different treatments (1#, PBS; 2#, αPDL1; 3#, S-ICG@NP; 4#, S-ICG@NP + 1.2 W/cm2; 5#, αPDL1/ICG@NP + 1.2 W/cm2; 6#, S-αPDL1/ICG@NP; 7#, S-αPDL1/ICG@NP + 0.9 W/cm2; 8#, S-αPDL1/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 μm. (H) DC maturation in the LNs of lymphatic metastasis BALB/c mice in control and treated groups. (I) Normalized number of CD8+ T cells in the LNs (n = 3). Data are means ± SD. Statistical significance was 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).

Furthermore, similar results were obtained for S-αPDL1/ICG@NP and PDT combination therapy to inhibit lung metastasis. The average number of metastatic lesions in free αPDL1 or S-αPDL1/ICG@NP–treated mice is 18.3 ± 7.6 or 10.8 ± 4.5, respectively. However, the number of metastatic lesions in S-αPDL1/ICG@NP + PDT group decreased to 3.2 ± 3.4 per lung, suggesting that the combination therapy could not only inhibit LN metastasis but also suppress lung metastasis of 4T1 cancer cells (Fig. 6E). In addition to effectively inhibiting lung metastasis, the overall survival time of S-αPDL1/ICG@NP + 1.2 W/cm2–treated mice 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 survival group (Fig. 6G). Such outstanding therapeutic benefits were likely attributed to the remarkable priming of adaptive immune responses by PDT and effective regulation of immunosuppressive tumor microenvironment by PDL1 blockade.

Given the high LN specificity of S-αPDL1/ICG@NP, as demonstrated in the lymphatic metastasis mouse model, we further examined the immune responses in tumor metastatic LNs after S-αPDL1/ICG@NP treatment. PDL1 blockade along with PDT significantly promoted DC maturation in the tumor-draining LNs. For instance, S-αPDL1/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 DC maturation ratio of the S-αPDL1/ICG@NP group was markedly increased to 30.1 ± 2.9% upon laser irradiation at 1.2 W/cm2 (Fig. 6H). DC maturation facilitated the presentation of tumor antigens to naive T cells and subsequently boosted the adaptive antitumor immunity for inhibiting lymphatic tumor metastasis. The combination of S-αPDL1/ICG@NP treatment and laser irradiation at 1.2 W/cm2 elicited 2.1 × 104 CD8+ T cells/mg of the draining LNs (Fig. 6I), 3.0-fold higher than that of the αPDL1 control, which could explain the superior antimetastasis of S-αPDL1/ICG@NP–based combination therapy.

DISCUSSION

In the current study, we have developed a αPDL1-based nanoplatform for combination immunotherapy of the metastatic tumors. The nanoplatform was rationally fabricated by noncovalently compressing αPDL1 with PEG-dEGCG without disturbing the antibody binding affinity. We have demonstrated that the nanoplatform integrating αPDL1 and photosensitizer could simultaneously target the primary and metastatic tumors through tumor-specific accumulation of the nanoparticles. In combination with NIR laser illumination, the antibody nanoparticles regulate the immunological tolerance and immunosuppressive conditions of tumors by blocking tumor cell surface PDL1 and recruiting intratumoral infiltration of CTLs.

In comparison with the conventional ICB immunotherapy using checkpoint inhibitors alone or the combination with other therapeutic strategies (e.g., radiotherapy, chemotherapy, or phototherapy), the αPDL1 nanoparticles can passively accumulate inside the tumors and be specifically activated at the tumor site to avoid the on-target but off-tumor effect of free antibody and enhance tumoral accumulation and penetration of αPDL1. The αPDL1 nanoparticles can spatiotemporally co-deliver αPDL1 and photosensitizer ICG to the tumor mass for combination immunotherapy. The αPDL1 nanoparticles not only highly efficiently boost the antitumor immune responses but also elicit long-term immune memory effects in BALB/c mice, thus leading to remarkable therapeutic efficacy for inhibition of the primary and metastatic tumors. The simplicity of our system, which uses the antibody nanoparticle alone both for immune stimulation and checkpoint inhibition, could be readily adapted to other immune checkpoint inhibitors for improved ICB therapy.

The checkpoint inhibitor–based nanoplatform provides valuable insights for revealing the mechanisms of combination immunotherapy in eliciting antitumor immunity and suggests promising strategies to combat advanced metastatic cancer, which has the real potential of being translated into the future generations of cancer immunotherapy. Nevertheless, the nanoparticle design still needs to be improved for the treatment of a broad diversity of solid tumors. Specially designed endoscopes are urgently needed for the deep delivery of NIR radiation for combination ICB and PDT. Moreover, radioisotopes or chemotherapeutics could be incorporated into the nanoplatform to overcome the limitation of NIR laser delivery. The tumor-specific distribution and activation of the αPDL1 nanoparticles is highly dependent on MMP-2 expression in the tumor mass. Further work is still needed to fully understand how the αPDL1 nanoparticles interact with the tumor microenvironment. For the potential translation of the αPDL1 nanoparticles, systemic biosafety assay to investigate whether the αPDL1 nanoparticles suppress the immune-related adverse events of ICB therapy would be of priority.

MATERIALS AND METHODS

Preparation and characterization of S-αPDL1/ICG@NP

The MMP-2–activatable S-αPDL1/ICG@NP was formulated by nanoprecipitation method. First, 100 μl of αPDL1 aqueous solution (10 mg/ml) was incubated with 30 μl of ICG solution (10 mg/ml) for 2 hours under gentle stirring. The mixture was centrifuged at 8000 rpm for 10 min. The resultant αPDL1/ICG nanocomplexes were redispersed in 1.0 ml of deionized (DI) water at a concentration of 1.0 mg/ml, and then 10 μl of dEGCG [10 mg/ml in H2O/dimethyl sulfoxide (DMSO) (4:1, v/v)] and 2.5 μl of PEG-PLGLAG-dEGCG [10 mg/ml in H2O/DMSO (4:1, v/v)] were added under ultrasonication. The resultant S-αPDL1/ICG@NP was purified by dialyzing against DI water. The particle size and morphology of S-αPDL1/ICG@NP were examined using DLS (Malvern, UK) and TEM (JEOL, Japan), respectively. The absorption and fluorescence spectra of S-αPDL1/ICG@NP were recorded using ultraviolet-visible (Shimadzu UV-2450, Japan) and fluorescence (Hitachi F-4600, Japan) spectrophotometers.

To clarify the interactions between αPDL1, ICG, dEGCG, and PEG-PLGLAG-dEGCG, we incubated S-αPDL1/ICG@NP with NaCl, Tween-20, Triton X-100, or urea solution at desired concentrations for 5 min. The particle size change was monitored by DLS.

MMP-2 triggered αPDL1 release from S-αPDL1/ICG@NP

To investigate MMP-2–triggered αPDL1 release from S-αPDL1/ICG@NP, we incubated the nanoparticles with 50 nM MMP-2 and 0.72 mM CaCl2 in phosphate buffer (pH 7.4) at 37°C. αPDL1 released from S-αPDL1/ICG@NP was collected using ultracentrifugation [Amicon centrifugation unit; molecular weight cut-off (MWCO), 300 kDa] and examined by an anti-rabbit total IgG ELISA kit.

Activity analysis of αPDL1

To test the ability of S-αPDL1/ICG@NP to retain the activity of complexed proteins, the nanoparticles were pretreated, and the binding affinity of αPDL1 was determined by ELISA with recombinant PDL1 protein (Sino Biological, China). To clarify the effect of PDT on αPDL1 activity, S-αPDL1/ICG@NP was first treated by 0.9 W/cm2 and 1.2 W/cm2 lasers for 5 min and then examined using ELISA. Three kinds of S-αPDL1/ICG@NP–mimicking nanocomplexes were prepared from proteinase K, RNase, or HRP antibody by compacting with dEGCG and PEG-PLGLAG-dEGCG. Then, the protein nanoparticles were dissociated with Triton X-100 or activated by 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 protein solutions were set as the positive control.

To examine the stability of S-αPDL1/ICG@NP against proteolysis, aqueous solution of free αPDL1 or S-αPDL1/ICG@NP was mixed with proteinase K solution (20 μg/ml) for desired time intervals at 37°C. The solution was then added with phenylmethanesulfonyl fluoride to inhibit the bioactivity of proteinase K and subsequently with MMP-2 to dissociate the nanoparticles. The amounts of αPDL1 residual were examined using an anti-rabbit total IgG ELISA kit.

Immune response analysis in vivo

The immune response of S-αPDL1/ICG@NP–mediated photoimmunotherapy was investigated on 4T1 tumor–bearing BALB/c mice. The tumor-bearing mice were randomly grouped when the tumor volume reached 300 mm3. The mice groups were then systemically injected with PBS, free αPDL1, S-ICG@NP, αPDL1/ICG@NP, or the S-αPDL1/ICG@NP suspension with αPDL1 dosage (5.0 mg/kg). The S-αPDL1/ICG@NP groups were subjected to 808-nm laser irradiation at photodensity of 0.9 or 1.2 W/cm2 for 5 min. S-ICG@NP and αPDL1/ICG@NP groups with a 1.2 W/cm2 laser at the same time were set as control. The tumors were harvested and weighted 5 days after the final treatment and digested by collagenase IV (175 U/ml), hyaluronidase (100 U/ml), and deoxyribonuclease (30 U/ml) at 37°C for 30 min. The monodispersed tumor-infiltrating lymphocytes (TILs) were prepared by filtering the tumor suspensions through 75-μm filters and enriched using lymphocyte separation medium. The separated TILs were stained with anti–CD3-PerCP Cy5.5, anti–CD4-FITC, and anti–CD8-PE antibodies to investigate the frequency of CD4+ and CD8+ T cells.

To determine the proliferation and functions of TILs, the TILs were fixed and permeabilized using a FoxP3/Transcription Factor staining buffer set (eBioscience, San Diego, CA). Next, the TILs were stained with anti–Ki-67-FITC, anti–IFN-γ (IFN-γ–Alexa Fluor 488), and anti–TNF-α (TNF-α–Alexa Fluor 647) antibodies before examined by flow cytometry. The proinflammatory cytokines secreted within the tumors, including IFN-γ, TNF-α, and IL-1β, were determined using ELISA kits. The immune memory effect of CD8+ T cells was examined 25 days after the treatments. CD8+ central memory 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-αPDL1/ICG@NP were also evaluated using an LN metastatic mouse model. Enlarged LNs were intratumorally injected with 50 μl of PBS, free αPDL1, S-ICG@NP, αPDL1/ICG@NP, and S-αPDL1/ICG@NP suspensions (2.5 mg/kg of αPDL1) into the right footpad of BALB/c mice. Two hours after treatment, the popliteal and inguinal LNs were subjected to laser treatment at 0.9 or 1.2 W/cm2 for 5 min, respectively. The mice were euthanized to collect the drain LNs 5 days after treatments. Next, lymphocytes were gathered by grinding the LNs and enriched for the following analyses. For DC maturation studies, the lymphocytes were 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 lymphocytes 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 metastasis suppression in vivo

BALB/c and C57BL/6 mice (female, 5 to 6 weeks old) were purchased from Shanghai Experimental Animal Center (Shanghai, China) and maintained under pathogen-free conditions. To investigate the antitumor effects of S-αPDL1/ICG@NP, BALB/c mice were subcutaneously transplanted with 5 × 105 4T1 cells at the right flank. The mice were randomly grouped (n = 5) when the tumor volume reached 200 cm3 (i.e., group 1, PBS; group 2, αPDL1; group 3, S-ICG@NP; group 4, S-ICG@NP + 1.2 W/cm2; group 5, αPDL1/ICG@NP + 1.2 W/cm2; group 6, S-αPDL1/ICG@NP; group 7, S-αPDL1/ICG@NP + 0.9 W/cm2; group 8, S-αPDL1/ICG@NP + 1.2 W/cm2). The mice in different groups were intravenously injected with an equal αPDL1 (5.0 mg/kg) and ICG (1.0 mg/kg) dose. Two hours after injection, the tumors in groups 4, 5, 7, and 8 were irradiated with an 808-nm laser for 5 min at the optimized photodensity. The tumor volume and body weights were then monitored every 2 days after injection. All the major organs (i.e., heart, liver, spleen, lung, and kidney) and the tumors were collected and examined by H&E or TUNEL staining at the end of the antitumor study. In a separated experiment, the tumor-bearing mice were treated as above, and overall survival was also recorded in time.

To test the broad application of S-αPDL1/ICG@NP on different types of tumor, we established a B16-F10 tumor–bearing C57BL/6 mice model by subcutaneously injection of 5 × 105 B16-F10 cells on the back of mice. When the tumor volume reached 200 mm3, the mice were randomly grouped (n = 5) and treated with different suspensions at the same dose applied for 4T1 tumor therapy. The B16-F10 tumor–bearing mice were then irradiated by an 808-nm laser for 5 min at the desired photodensity. The tumor growth rates and body weight change were recorded during the antitumor study. The overall survival rates were evaluated at the end of the antitumor studies.

To investigate the immune memory effect, we grouped and treated BALB/c mice by following the same procedure applied for the antitumor study. The primary tumors were surgically removed after the third administration, and 1 × 105 4T1 tumor cells were rechallenged into the left flank of each mouse 25 days after the final treatment to establish the distant metastasis tumor models. The volumes of the distant tumors were monitored for 15 days. The tumor volume was determined following the formulaV = (L × W × W)/2 (L,the longest dimension; W, the shortest dimension)

For lymphatic metastasis inhibition study, 5 × 105 4T1 cells were inoculated into the right rear footpad of the mice on day 0. The mice were randomly divided into eight groups when lymphatic metastasis was confirmed and intratumorally treated with 50 μl of different formulations [αPDL1 (2.5 mg/kg) and ICG (0.5 mg/kg)]. The popliteal and inguinal LNs of mice in selected groups (4, 5, 7, and 8) were stimulated by an 808-nm laser for 5 min and 2 hours after the injection. All animals were euthanized on day 22, and the popliteal LNs and lungs were collected, weighted, and photographed for metastasis inhibition investigation. The lungs were fixed for H&E staining analysis of micrometastatic foci. For the mouse survival study, a separate study was conducted, and an event was recorded when the mice died or exhibited any signs of impaired health.

Statistics

Data were given as means ± SD. The statistical significance was performed by one-way analysis of variance (ANOVA) with Tukey’s post hoc test and two-sided unpaired Student’s t test. Comparisons of 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 MATERIALS

immunology.sciencemag.org/cgi/content/full/4/37/eaau6584/DC1

Materials and Methods

Table 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 of EGCG 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-αPDL1/ICG@NP assembly process.

Fig. S7. Representative TEM image of the S-αPDL1/ICG@NP.

Fig. S8. Serum stability of S-αPDL1/ICG@NP.

Fig. S9. PDL1 binding affinity of αPDL1 in S-αPDL1/ICG@NP post PDT.

Fig. S10. ESR spectroscopic examination of S-αPDL1/ICG@NP–induced ROS.

Fig. S11. S-αPDL1/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-αPDL1/ICG@NP.

Fig. 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 αPDL1 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-αPDL1/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).

REFERENCES AND NOTES

Acknowledgments: We thank X. Yan from Institute of Biophysics, Chinese Academy of Sciences for fruitful discussion. The MS-FACILITY from National Center for Protein Science Shanghai, SIBCB, CAS, is gratefully acknowledged. Funding: Financial supports from the National 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. conceived and 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 the submission. Competing interests: The authors declare that they have no competing financial interests. Data and materials availability: All data needed to evaluate the conclusions in the paper are present in the paper or the Supplementary Materials.
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