High Affinity of Chlorin e6 to Immunoglobulin G for Intraoperative

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High Affinity of Chlorin e6 to Immunoglobulin G for Intraoperative Fluorescence Image-Guided Cancer Photodynamic and Checkpoint Blockade Therapy Jiaojiao Xu, Sheng Yu, Xiaodong Wang, Yuyi Qian, Weishu Wu, Sihang Zhang, Binbin Zheng, Guoguang Wei, Shuai Gao, Zhonglian Cao, Wei Fu, Zeyu Xiao, and Wei Lu ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.9b03466 • Publication Date (Web): 09 Aug 2019 Downloaded from pubs.acs.org on August 10, 2019

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High Affinity of Chlorin e6 to Immunoglobulin G for Intraoperative Fluorescence Image-Guided Cancer Photodynamic and Checkpoint Blockade Therapy

Jiaojiao Xu,†,# Sheng Yu,†,# Xiaodong Wang,‖ Yuyi Qian,† Weishu Wu,† Sihang Zhang,† Binbin Zheng,† Guoguang Wei,† Shuai Gao,† Zhonglian Cao,† Wei Fu,† Zeyu Xiao,*,‡ Wei Lu*,† †Minhang

Hospital & School of Pharmacy, Key Laboratory of Smart Drug Delivery Ministry of

Education, State Key Laboratory of Molecular Engineering of Polymers, Fudan University, Shanghai 201199, China. ‡Department

of Pharmacology and Chemical Biology, & Clinical and Fundamental Research

Center, Institute of Molecular Medicine, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China. ‖ Department

of Biomedical and Pharmaceutical Sciences, College of Pharmacy, The University

of Rhode Island, Kingston, Rhode Island 02881, United States. *Corresponding author: E-mail: [email protected], [email protected] #J.X.

and S.Y. contributed equally to this work.

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ABSTRACT: Cancer photodynamic therapy (PDT) represents an attractive local treatment in combination with immunotherapy. Successful cancer PDT relies on the image-guidance to ensure the treatment accuracy. However, existing nanotechnology for co-delivery of photosensitizers and image contrast agents slow the clearance of PDT agents from the body, and cause a disparity between the release profiles of the imaging and PDT agents. We have found that the photosensitizer Chlorin e6 (Ce6) is inherently bound to immunoglobulin G (IgG) in a nanomolarity range of affinity. Ce6 and IgG self-assembles to form the nanocomplexes termed Chloringlobulin (Chlorin e6 + immunoglobulin G). Chloringlobulin enhances the Ce6 concentration in tumor without changing its elimination half-life in blood. Utilizing the immune checkpoint inhibitor antiprogrammed death-ligand 1 (PD-L1) (αPD-L1) to prepare αPD-L1 Chloringlobulin, we have demonstrated a combination of Ce6-based red-light fluorescence image-guided surgery, stereotactic PDT and PD-L1 blockade therapy of mice bearing orthotopic glioma. In mice bearing orthotopic colon cancer model, we have prepared another Chloringlobulin that allows intraoperative fluorescence image-guided PDT in combination with PD-L1 and cytotoxic T lymphocyte antigen 4 (CTLA-4) dual checkpoint blockade therapy. The Chloringlobulin technology shows great potential for clinical translation of combinatorial intraoperative fluorescence image-guided PDT and checkpoint blockade therapy.

KEYWORDS: immunoglobulin G (IgG), chlorin e6 (Ce6), fluorescence image-guided surgery (FIGS), photodynamic therapy (PDT), programmed death-ligand 1 (PD-L1), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4)


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Immune checkpoint blockade therapy utilizes monoclonal antibodies (mAbs) to release the brakes on the effector T cells.1, 2 Antibodies inhibiting cytotoxic T lymphocyte antigen 4 (CTLA4) or the programmed cell death 1 (PD-1) and its ligand (PD-L1) signaling pathway have shown significant clinical effectiveness.3-5 However, checkpoint blockade monotherapy such as anti-PD1 mAbs receives the objective response rate to ~20-30% in the treatment of several types of cancers.5, 6 This limitation calls for combination therapies to augment the antitumor immunity and broaden the therapeutic benefit.7, 8 Photodynamic therapy (PDT) ablates tumor and provokes immune responses through enhancing tumor antigen presentation,9 thus synergizing the efficacy of the checkpoint blockade such as antiPD-L1 antibody therapy.10-12 Successful cancer PDT relies on the image-guidance for accurate laser irradiation as well as optimal drug-to-light interval.13, 14 Accordingly, delicate nanostructures have been prepared to co-load the photosensitizers and the imaging contrast agents.12,

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However, the long-circulating nanoparticles incurred slow clearance of the photosensitizers from the body with prolonged photosensitivity. The disparity between the in vivo release profiles of the imaging contrast agents and the photosensitizers may lead to inaccuracy of the image-guidance. These pitfalls plus the toxicity of certain nanomaterials remain in the way of the clinical translation of the nanostructured theranostic agents based on the current design.13, 15, 17 Antibodies are immunoglobulin G (IgG) molecules. Here, we find that the photosensitizer Chlorin e6 (Ce6) inherently interacts with the IgG molecule in a nanomolarity range of affinity. The binding is much stronger than the affinity of Ce6 to human serum albumin (HSA) -- the endogenous carrier of Ce6. Ce6 and IgG spontaneously assembles and forms a ~30-nm-sized nanostructure in presence of the pharmaceutical excipient, polyvinylpyrrolidone (PVP). We term this nanostructure Chloringlobulin (Chlorin e6 + immunoglobulin G). Chloringlobulin enhances


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the Ce6 concentration in tumor within the initial hours following the intravenous (i.v.) injection, but does not change its elimination half-life in blood. We hypothesize that the distinctive characteristics of Chloringlobulin could allow to co-deliver Ce6 and single or multiple checkpoint inhibitor mAbs in a single setting for a combination of Ce6-based intraoperative red-light fluorescence imaging, PDT and immunotherapy for cancer. Utilizing anti-PD-L1 antibody (αPDL1) to prepare αPD-L1 Chloringlobulin, we present a combinatorial fluorescence image-guided surgery (FIGS), PDT and PD-L1 blockade of mice bearing orthotopic GL261 glioma, significantly prolonging the survival of the mice. In mice bearing orthotopic CT26 colon cancer model, we use αPD-L1 and anti-CTLA-4 antibody (αCTLA-4), to formulate αPD-L1-αCTLA-4 Chloringlobulin and demonstrate the intraoperative fluorescence image-guided PDT (FIGPDT) of both primary and metastatic lesions in combination with PD-L1 and CTLA-4 dual checkpoint blockade, eliciting potent systemic antitumor immunity and a long-lasting immune memory against tumor rechallenge. RESULTS AND DISCUSSION High Affinity of Ce6 to IgG. With immobilized αPD-L1 (rat IgG2b), surface plasmon resonance (SPR) analysis by Biacore demonstrated a potent binding capability to Ce6 trisodium (Figure 1A). The calculated equilibrium dissociation constants (KD) of Ce6-αPD-L1 was 61 nM. Ce6 was bound to the Control IgG (without PD-L1 binding effect) in a similar high affinity to that to αPD-L1, indicating that the interaction between Ce6 and the IgG molecule was not affected by the variation of the antigen binding site of IgG. By contrast, Ce6 and serum albumin had a relatively lower affinity in a micromolarity range. αPD-L1 (or IgG) exhibited 34.7 (or 28.6)-fold and 26.5 (or 21.9)-fold higher in terms of the Ce6 binding affinity than human serum albumin (HSA) and mouse serum albumin (MSA), respectively. Moreover, Ce6 in presence or absence of


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PVP at a mass ratio of 1:1 displayed similar binding to αPD-L1, suggesting that the presence of PVP did not affect the interaction between Ce6 and αPD-L1. To test the binding stability in serum, 1% HSA or 1% mouse serum (MS) in HBS-EP+ buffer (pH 7.4) was used for the binding analysis. The KD of Ce6 to Control IgG did not significantly change (< 3-fold) with the running buffer in presence or absence of 1% HSA, suggesting that HSA did not significantly affect the binding between Ce6 and IgG (Figure 1B). Comparatively, the KD values using the running buffer with or without 1% MS changed 7-fold, demonstrating that MS affected the binding stability of Ce6 to Control IgG. These results indicated that serumal IgG and/or other components rather than albumin competed the binding of Ce6 to IgG. The results of docking study showed that rat IgG had five binding pockets to Ce6, named P1, P2, P3, P4 and P5 with the total scores of 1.08, 2.75, 1.87, 5.41 and 4.46, respectively (Figure 2A). The representative binding conformation of IgG-Ce6 of P4 and P5 was shown in Figure 2B and 2C, respectively. The results of van’t Hoff thermodynamic analysis demonstrated that the increase of the equilibrium affinity was correlated with the elevated temperature (Figure 2D-F). The KD values were used to calculate the thermodynamic parameters by fitting van’t Hoff equation.18 The binding interaction between Ce6 and IgG was confirmed by the negative value of the free energy (ΔG). Moreover, according to the thermodynamic law,18 the calculated positive values of enthalpy (ΔH) and entropy (ΔS) together with the results of the docking study (Figure 2B and 2C), supported that the binding interaction of IgG-Ce6 involved hydrophobic force and hydrogen bonds. Characterization of Chloringlobulin. Transmission electron microscopic (TEM) imaging depicted spherical nanostructures formed following a simple mixture of αPD-L1, Ce6 trisodium and PVP at a mass ratio of 2:1:1 in phosphate buffered saline (PBS) (Figure 3A and B). αPD-L1 Chloringlobulin had an average particle size of ~30-nm in diameter. Scanning transmission


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electron microscopy (STEM)/energy-dispersive X-ray (EDX) spectroscopy demonstrated a peak corresponding to sulfur element of the nanostructures, confirming the presence of αPD-L1 in the Chloringlobulin (Figure 3C, arrow). We further developed a lyophilized formulation by addition of mannitol as the freeze-drying protective agent. The result of dynamic light scattering (DLS) showed no significant difference in the size distribution of the nanoformulation before and after lyophilization (Figure 3D). Thus, the lyophilized formulation was used for further study. The binding kinetics of αPD-L1 to PD-L1 did not change after αPD-L1 was complexed with Ce6 in the presence of PVP (Figure 3E and Table S1). For the preparation of Chloringlobulin, the Ce6 encapsulation efficiency was 93.55 ± 1.37%. Within the initial 2 h, Chloringlobulin released ~20% of Ce6 in 10% fetal bovine serum (FBS) at the same release rate as that in PBS (Figure 3F), possibly attributed to the Ce6 diffusion. Whereas, after 2 h Ce6 was released in 10% FBS much faster and more than that in PBS, resulting in 91% of cumulative release in 10% FBS within 24 h. This result indicated that, in addition to the diffusion, a competitive binding of the serumal components of FBS to Ce6 was contributed to the Ce6 release after 2 h. DLS analysis illustrated the size distribution of FBS without addition of the nanocomplexes peaked at ~7 nm and ~44 nm, respectively (Figure 3G, blue curve). Addition of PVP-Ce6 or Chloringlobulin did not change the size distribution of FBS within the first hour (Figure S1). However, PVP-Ce6 in 10% FBS started to form aggregation at 2 h (Figure 3G, black curve, arrow, and Figure S1). By contrast, Chloringlobulin did not aggregate in 10% FBS until 12 h (Figure S1, arrow). Previous literature reported that the binding constant (KB) of PVP-Ce6 (PVP K17, MW 10,000 D) was ~3.66 × 104 M-1.19 Accordingly, the KD of PVP-Ce6 was calculated to be 2.73 × 10-5 M. As shown in Figure 1A, the KD of IgG-Ce6 was 7.394 × 10-8 M, which was two orders of magnitude lower than that of PVP-Ce6. The KD of HSA-Ce6 (2.118 × 10-6 M, Figure 1A) was 12.9-fold lower


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than that of PVP-Ce6 but 28.6-fold higher than that of IgG-Ce6, indicating that the order of the stability among the three complexes was IgG-Ce6 > HSA-Ce6 > PVP-Ce6. Due to the existence of much more stable IgG-Ce6 complex, Chloringlobulin showed slower aggregation than PVPCe6 in 10% FBS (Figure 3G). Pharmacokinetics and tumor distribution. C57BL/6 mice i.v. injected with either αPD-L1 Chloringlobulin or Control Chloringlobulin (prepared with Control IgG to replace αPD-L1) exhibited significantly higher blood Ce6 levels in comparison with PVP-Ce6 group within 4 h following the injection (Figure 4A and Figure S2). At 1 h post-injection, the % ID/mL of Ce6 in blood by αPD-L1 Chloringlobulin was 1.64 times as high as that by PVP-Ce6. However, the blood Ce6 concentration among all the three groups did not have difference after 4 h. αPD-L1 Chloringlobulin significantly increased the area under the concentration-time curve (AUC) of Ce6 compared with PVP-Ce6, but did not change the blood elimination half-life (t1/2) or the mean residence time (MRT) (Table 1). Control Chloringlobulin exhibited a similar pharmacokinetic profile as αPD-L1 Chloringlobulin. Live fluorescence imaging of C57BL/6 mice bearing GL261 orthotopic glioma showed that either αPD-L1 Chloringlobulin or Control Chloringlobulin group displayed higher Ce6 accumulation in tumor within 4 h post-injection compared with the PVP-Ce6 group (Figure 4B and Figure S3). Specifically, αPD-L1 Chloringlobulin increased the fluorescence intensity of Ce6 in tumor in comparison with the PVP-Ce6 by 2.18-fold at 1 h and 2.30-fold at 2 h, respectively. At 8 h or after, either Chloringlobulin did not significantly enhance the tumor accumulation of Ce6 in comparison with PVP-Ce6. The tumor distribution of Ce6 did not show statistically significant difference between the αPD-L1 Chloringlobulin group and the Control Chloringlobulin group. This finding was consistent with the ex vivo distribution results at 1 h (Figure S4). The confocal


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laser scanning microscopy confirmed the higher tumor distribution of Ce6 by Chloringlobulin than that by PVP-Ce6 at 1 h post injection (Figure 4C). However, after 24 h the Ce6 signal was indiscernible in tumor among all three groups. Similarly, in BALB/c mice bearing orthotopic CT26-Luc colon cancer the Ce6 concentration in tumor of the αPD-L1-αCTLA-4 Chloringlobulin-treated group was 1.58-fold at 1 h, 1.72-fold at 2 h, and 2.30-fold at 4 h of that of the PVP-Ce6 group, respectively (Figure 4D and Figure S5). αPDL1-αCTLA-4 Chloringlobulin exhibited a similar biodistribution profile as Control Chloringlobulin. Confocal images of tumor further verified this distribution behavior (Figure 4E). A clinically successful photosensitizer is able to target specific tissues or their vasculature. Following PDT, it has to be quickly eliminated from the body to reduce the sunlight photosensitivity.20-22 To achieve the tumor accumulation, the photosensitizer is traditionally designed to be either chemically conjugated or physically entrapped to the nanostructures through the prolonged blood circulation and the enhanced permeability and retention (EPR) effect.11, 12 As a consequence, these nanostructures slow the clearance of the photosensitizer from the body. The design of Chloringlobulin, on the other hand, is based on the high affinity of Ce6 to IgG, enhancing the tumor accumulation of the photosensitizer likely through the “IgG hitchhiking” approach. Our results of the Ce6 release profile reveal that the IgG-Ce6 complex is stable for 2 h in serum. After 2 h, significant amount of Ce6 is released and redistributed to the serumal components, indicating that Ce6 “hitchhikes” on IgG for a short term (Figure 3F). In contrast to conventional nanostructures, this approach offered the ideal pharmacokinetic parameters of Ce6, which elevated its tumor accumulation within the initial a few hours followed by quick elimination without changing its blood elimination half-life. More importantly, Chloringlobulin is formulated by a simple mixture of the clinically approved drug (Ce6),23 the checkpoint inhibitor mAbs as well as


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the pharmaceutical excipients (PVP and the freeze-drying protective agent), showing the possibility for clinical use. Tumor cell in response to PDT in vitro. We firstly examined the binding of αPD-L1 from αPDL1 Chloringlobulin to GL261, a PD-L1 positive cell line,24 following PDT. The results demonstrated that the binding of αPD-L1 to the cells was not significantly changed following the treatment with the tested laser power densities (Figure S6). Besides, we performed the Ce6 uptake study of αPD-L1 Chloringlobulin in GL261 cells (Figure S7). The results showed the significant uptake of Ce6 following the incubation of αPD-L1 Chloringlobulin in the cell culture medium in presence or absence of 10% FBS. Pre-blocking with free αPD-L1 reduced ~7% of Ce6 uptake of αPD-L1 Chloringlobulin in the serum-free culture medium, but did not change the Ce6 uptake in the culture medium plus 10% FBS. These results indicated that αPD-L1 from αPD-L1 Chloringlobulin may not significantly affect the internalization of Ce6 in vivo. We then evaluated the PDT-induced cell-killing effect on GL261 cells. Phase contrast micrographic images showed that a population of cells appeared in spherical shape at 24 h following PDT (Figure 5A, left). The percentage of this population was dependent on the laser dose applied. Flow cytometry plots in forward scatter and side scatter identified two populations of the cells following PDT (Figure 5A, right). Cells without receiving PDT mostly stayed in P1 showing negligible Annexin V+ apoptotic cells (< 0.1%). Following PDT, the cell population shifted to P2 with increased apoptotic cells. The number of Annexin V+ apoptotic cells was proportional to the laser dose (Figure 5B). Noticeably, the apoptotic cells were mostly derived from P2 with decreased signals of forward scatter. The P2 gated in scatter plots corresponded to the cells in spherical shape (PDT-responsive cells) observed in the microscopic images. On the


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other hand, most cells in P1 gate were Annexin V- cells, representing the residual tumor cells following PDT. We further measured the level of PD-L1 expression in GL261 cells following PDT. The mean fluorescence intensity (MFI) of PD-L1 in P2 Annexin V- cells reduced to 6.51%-8.07% of that of the untreated cells, suggesting that PDT drastically inhibited the PD-L1 expression in PDTresponsive cell population (Figure 5C). Although the PD-L1 expression in P1 Annexin V- cells significantly decreased following various doses of PDT, the PD-L1 in this population of cells remained at high levels (78.31%-83.20% of that of the untreated cells). The relatively high expression of PD-L1 in the residual GL261 population following PDT warranted further attempts to combine PDT with PD-L1 blockade therapy. αPD-L1 Chloringlobulin-mediated PDT in combination with PD-L1 blockade. To mimic the procedure of clinical treatment, in mice bearing orthotopic GL261 glioma, we used a quartz fiber optics in 0.9-mm diameter under the guidance of the stereotactic instrument to deliver a 660 nm-laser light at a total dose of 458 mJ to the tumor site for PDT at 1 h post i.v. injection of αPD-L1 Chloringlobulin (Figure 6A). In the PDT-treated group, the tumor-bearing mice received the i.v. injection of Control Chloringlobulin plus PDT after 1 h. In the αPD-L1-treated group, the mice received αPD-L1 Chloringlobulin without PDT. We firstly examined the immune cell populations in the tumor as well as the lymphocyte subsets in spleen and draining cervical lymph nodes (CLNs) at Day 2 following various treatment. As depicted in Figure 6B, the percentage of T regulatory cells (Treg, CD4+CD25+Foxp3+) in the tumor was significantly decreased in all treatment groups compared with the none treatment control. PDT plus PD-L1 blockade had the most significant inhibition effect, which decreased tumor Treg to 26.67% of that in the untreated control groups. Without treatment, the ratio of classic M1 macrophage (CD11b+F4/80+CD206-) to alternative M2


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macrophage (CD11b+F4/80+CD206+) in the tumor was about 0.51, suggesting that the M2 phenotype dominated the tumor and promoted tumor progression. Following PDT alone, although the M1/M2 ratio was increased to nearly 0.9, the M2 population still overweighed in tumor. On the other hand, treatment with αPD-L1 alone reversed the M1/M2 ratio. Specifically, PDT plus αPD-L1 increased the M1/M2 ratio to 1.88, which was 3.64 folds of that in the non-treatment control group. The polarization of tumor associated macrophage toward M1 phenotype suggested an antitumor microenvironment and may suppress tumor growth. In spleen, both CD4+ and CD8+ T cells significantly increased following PDT plus αPD-L1 treatment. Whereas, either PDT or αPD-L1 treatment alone slightly induced CD8+ T cells but not CD4+ T cells. Treatment with PDT plus αPD-L1 induced the highest amount of the activated myeloid dendritic cells (mDCs) (CD11C+CD86+) in CLNs among all the treatment groups, resulting in 3.15 folds as high as that of the non-treatment control. Moreover, a combined effect on the up-regulation of both CD4+ and CD8+ T cells in CLNs was observed following treatment with PDT plus αPD-L1. Next, we measured the effector T cells in the tumor and CLNs at Day 6 after the treatment. PDT or αPD-L1 treatment alone significantly increased CD4+ and CD8+ T cells in CLNs (Figure 6C, left). Noticeably, the percentages of CD4+ and CD8+ T cells in CLNs following PDT in combination with αPD-L1 were the highest among all the treated groups, which were 7.65-fold and 6.97-fold compared with the none treatment group, respectively. Consistently, the percentage of cytotoxic T lymphocytes (CTL, CD8+IFN-γ+) following PDT plus αPD-L1 in CLNs was increased by 6.57-fold in comparison with the none treatment group. Besides, the frequency of tumor-infiltrating CTLs following PDT alone or αPD-L1 alone was elevated (Figure 6C, right). Notably, PDT plus αPD-L1 treatment resulted in a 24.6-fold increase of the tumor-infiltrating CTLs compared with the non-treatment control. Immunofluorescence staining further confirmed


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the enhanced intra-tumor CD8+ T cells (Figure 6D, left) and activated NK cells (NK1.1+CD49b+, Figure 6D, middle) infiltrates, which were the most significant following the combined treatment. Active caspase-3 staining depicted the most apoptotic cells in tumor at Day 6 following PDT plus αPD-L1 treatment compared with other groups (Figure 6D, right). The combined antitumor effect of the PDT plus αPD-L1 therapy was also reflected in the median survival time of mice bearing orthotopic GL261 glioma (Figure 6E). The median survival time of mice treated with PDT alone (23 d) or αPD-L1 alone (27 d) was significantly longer than that of the Control (19 d). Considerably, the treatment with PDT plus αPD-L1 prolonged the median survival time to 32 d (P < 0.05 compared with αPD-L1 alone). Taken together, these results substantiated that PDT in combination with PD-L1 blockade reversed the immunosuppressive microenvironment of tumor as well as elicited potent local and systemic antitumor immune response, thereby enhancing the therapeutic efficacy. αPD-L1 Chloringlobulin-mediated combinatorial FIGS, PDT and αPD-L1 treatment. Since the i.v. injected Chloringlobulin enhanced tumor accumulation of Ce6, we proposed a study on intraoperative imaging based on the fluorescence emitted from Ce6. A home-made intraoperative fluorescence imaging system was set up according to the previous report with modification.25 The system included a 660-nm diode laser illuminator and a scope with a coherent fiber bundle connected to a lens and a 670-nm long-pass emission filter in order to match the excitation and emission wavelength of Ce6 (Figure S8, See Methods). At 1 h after the administration of Chloringlobulin, the glioma bearing mice were anesthetized. An incision was made on the scalp to expose the skull. The fluorescence of Ce6 in mouse brain was clearly imaged without opening the skull (Figure 7A, left, yellow arrows). The position of the fluorescence signal was correlated with the area of the tumor cell inoculation, indicating imaging of the tumor site.


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Following craniotomy, the resected specimen displayed high fluorescence intensity in the imaging (Figure 7A, middle, red arrowheads). The tissue of mice with high fluorescence intensity was further resected under the intraoperative imaging until no discernable fluorescence signal was seen in the imaging (Figure 7A, right). H&E staining verified that the resected tissue was tumor without noticeable amount of normal brain tissue in the tumor margin (Figure 7B, upper row, left). Specifically, the Ce6 fluorescence in the whole tissue section (Figure 7B, upper row, second left) overlapped with the image of H&E staining. Fluorescence micrograph further evidenced the accumulation of Ce6 fluorescence in the tumor cells (Figure 7B, upper row, right). Both H&E staining and fluorescence scanning of the brain section after the intraoperative FIGS revealed that no distinguishable tumor cells remained in the subsurface of the surgical bed area (Figure 7B, middle and lower rows, yellow and green boxes). Only a few number of tumor cells remained deep from the surgical bed (Figure 7B, lower row, black and red boxes, blue arrow). These results supported that the fluorescence signal of the intraoperative imaging corresponded to the tumor tissue, applicable to accurate tumor detection. In a parallel experiment, the tumor was resected through conventional surgery assessed by visual inspection. Besides the tumor cells, H&E staining illustrated a considerable number of normal brain tissue in the resected specimen (Figure 7C, upper row, blue box). Moreover, extensive tumor residues remained adjacent to the surgical bed area (Figure 7C, lower row, yellow box), suggesting the inaccurate brain tumor resection following the conventional surgery. Our data demonstrated that the intraoperative FIGS by Chloringlobulin offered more accurate tumor resection with minimum tumor residue and maximum saving of normal brain tissue. We next evaluated whether the Chloringlobulin-mediated FIGS could favor the prognosis of glioma-bearing mice and whether FIGS combined with PDT and PD-L1 blockade therapy would


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further improve the survival of the mice. In the combination treatment protocol, the tumor bearing mice received an i.v. injection of αPD-L1 Chloringlobulin. At 1 h following the administration, FIGS was performed to resect brain tumor. Following the surgery, a lower dose of laser light (totally 153 mJ) was delivered to the surgical bed area for adjuvant PDT. As shown in Figure 7D, conventional surgery (25 d) did not significantly prolong the median survival time of mice in comparison with Control (24 d) (P = 0.137), while the Chloringlobulin-mediated FIGS considerably extended the survival time (29 d) (P = 0.003 compared with Control). In combination with FIGS, PDT plus αPD-L1 further improved median survival time to 38 d (P = 0.027 compared with FIGS alone). Current clinical treatment protocol of malignant glioblastoma involving maximum safe resection followed by adjuvant radiotherapy and temozolomide chemotherapy only offers 13.4 19 months of the median survival time and about 5% of the 5-year survival rate.26-28 Because the glioma cells grow invasively and infiltrate into neurological structures, the interface of the tumor and normal brain is hardly determined by human eye under an operative microscope.29 The completeness of the tumor removal is the key predictor that maximizes the overall survival and progression-free survival of the patients.30 On the other hand, the existence of anti-tumor immunity in glioma indicates immunotherapy to be an optimistic alternative for glioma management.31-33 Besides the primary absorption at ~407 nm, Ce6 has the secondary absorption peaked at ~658 nm (Figure S9) allowing deeper tissue penetration and less phototoxicity. Based on this optical property, we have built an intraoperative fluorescence imaging system using a 660-nm red light for Ce6 excitation and developed a protocol to treat glioma. αPD-L1 Chloringlobulin enables FIGS to facilitate more complete tumor resection than conventional surgery, followed by stereotactic PDT of the residual tumor cells. Concomitant blockade of PD-L1 by αPD-L1 Chloringlobulin


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remodels the tumor microenvironment, thus potentiating immune responses to further kill the residual tumor cells and to prevent recurrence. Since the specific operating microscope adapted for clinically red-light fluorescence image-guided glioma resection,34 and since the clinically developed intraoperative PDT for intracranial neoplasms and the illumination device,35,

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the

combinatorial FIGS, stereotactic PDT and PD-L1 blockade therapy may offer a great promise to improve the prognosis of glioma. The administration of Control Chloringlobulin and free αPD-L1 separately may exhibit similar antitumor efficacy in comparison with the simultaneous delivery of αPD-L1 and Ce6 using αPDL1 Chloringlobulin. However, in order to prepare the Control Chloringlobulin additional Control IgG are needed to be administered. It is known that intravenous immunoglobulin therapy (IVIG) used the serumal IgG for the treatment of a variety of immune system disorders.37,

38

IVIG-

mediated immunomodulation mainly through two functional domains, F(ab’)2 and Fc fragment, resulted in suppression or activation of the autoimmune diseases or immune deficiency diseases.38, 39

Therefore, infusion of additional serumal IgG may increase the risk of disrupting the balance

between the activated and inhibitory signals in the immune surveillance.39-41 In sight of the αPDL1 Chloringlobulin designed in the present study, since no significant changes of the affinity of αPD-L1 to the PD-L1 in presence of Ce6 were observed (Figure 3E and Table S1), the co-delivery Ce6 and αPD-L1 in one Chloringlobulin system can avoid the administration of additional serumal IgG, reducing the potential risk from serumal IgG. The possible immunoregulative effect of serumal IgG will be investigated in the future study. Chloringlobulin-mediated combinatorial FIGPDT, αPD-L1 and αCTLA-4 dual blockade therapy. Recent clinical trial focus on the development of multiple checkpoint blockade for the treatment of cancer in digestive system in order to improve the response rate.42-44 Accordingly, we


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prepared a Chloringlobulin composed of both αPD-L1 and αCTLA-4. We established BALB/c mice bearing orthotopic CT26-Luc colon cancer model with primary and spontaneous metastatic lesions in cecum. At 1 h post i.v. injection of αPD-L1-αCTLA-4 Chloringlobulin, the tumor bearing mice under anesthetic condition received the fluorescence imaging with the cecum exposed under the home-made imaging scope. Fluorescence imaging clearly delineated both primary and metastatic tumor lesions with dimensions of 1 mm × 2 mm and 0.6 mm × 1 mm, respectively (Figure 8A). Following the FIGPDT, the cecum was repositioned and the incision was closed with suture. Different treatment regimen was scheduled for the survival study (Figure 8B). Bioluminescence imaging (BLI) was used for monitoring the tumor growth following the treatment (Figure S10). Without treatment, the tumor-bearing mice demonstrated a median survival time of 26 d (Figure 8C). FIGPDT alone significantly extended the life-span of mice resulting in 3 out 10 survivals at Day 55, among which two mice did not have significant BLI signals (Figure 8C and Figure S10). Half of mice treated with αPD-L1-αCTLA-4 Chloringlobulin alone survived at Day 55 without significant BLI signals. Significantly, 9 out of 10 mice survived at Day 55 without significant BLI signals following the combined treatment with FIGPDT, αPDL1 and αCTLA-4. All the survivals without significant BLI signals rejected the tumor rechallenge at Day 55 (Figure S11). Collectedly, these results proved the combined antitumor effect of FIGPDT, αPD-L1 and αCTLA-4 mediated by Chloringlobulin, indicating potent systemic antitumor immunity and long-term immune memory effect. Flow cytometry analysis revealed that all three treatments increased the percentage of CD4+ or CD8+ T cells in draining lymph nodes (DLNs) in comparison with the Control at Day 21 (Figure 9A, upper). Particularly, FIGPDT in combination with dual checkpoint inhibition induced higher CD8+ T cells than FIGPDT alone (P = 0.023) or dual checkpoint blockade alone (P < 0.0001).


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This result was in line with the analysis of granzyme B-positive CD8+ T cells (CD8+GranB+) or CTLs. All the three treatments increased the activated mDCs compared with the Control. In spleen, similar trend of CD4+ or CD8+ T cells was observed following different treatment (Figure 9A, lower). Significantly, the triple combination therapy elevated the CD8+GranB+ or CTLs numbers by 3.66-fold and 8.97-fold in comparison with the Control, respectively. This result correlated with the decreased Treg number following the triple treatment, eliciting potent antitumor immunity. Literature has shown that the immunosuppressive myeloid-derived suppressor cells (MDSCs) accumulate in tumor and spleen during malignant progression.45-47 These cells direct tumor metastasis and disturb long-term antitumor immune response.48-50 We then checked the population of these cells in either the monocytic (M-MDSC) or granulocytic (G-MDSC) subpopulations in the spleens of the tumor-bearing mice following different treatment (Figure 9B). All the treated mice showed a significantly reduced populations of M-MDSC or G-MDSC in spleen compared with the Control (P < 0.001). Specifically, αPD-L1-αCTLA-4 Chloringlobulin alone or in combination with FIGPDT was more effective than FIGPDT alone. The percentages of M-MDSC and G-MDSC following the triple therapy were decreased by 82.10% and 85.45% in comparison with the Control, respectively. We next evaluated the immune memory phenotype in spleen of CT26-Luc bearing mice following the different treatment regimen illustrated in Figure 8B. The FIGPDT plus dual checkpoint blockade significantly increased the splenic CD8+ T cells including naïve cells (CD44CD62L+) by 2.94-fold, central memory cells (CD44+CD62L+) by 3.22-fold and the effector memory phenotype (CD44+CD62L-) by 3.60-fold compared with the Control at Day 21 (Figure 9C, left). Specifically, the triple combinatorial treatment induced the largest amount of the central memory cells and the effector memory cells among all three treated groups, respectively. However,


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the proportion of each phenotype did not change significantly (Figure 9C, right). At 45 d after the tumor rechallenge, the numbers of total splenic CD8+ T cells in all three treated groups were drastically increased in comparison with those at Day 21 (Figure 9D, left). The proportion of naïve cell in the splenic T cells was significantly reduced in the three treated groups in comparison with Control (Figure 9D, right), indicating a shift from naïve to memory phenotypes. Specifically, PDL1-αCTLA-4 Chloringlobulin with or without FIGPDT significantly increased the numbers of both central memory and effector memory CD8+ T cells, while FIGPDT only elevated the effector memory cells (Figure 9D, left). The triple combination therapy induced the highest among of effector memory T cells among all three treated groups. These results supported that αPD-L1αCTLA-4 Chloringlobulin in combination with FIGPDT induced a long-term memory response through enhancement of both central and effector memory T cells. Despite positive and promising clinical trial results from I to II/III phase for the use of PDT in colorectal cancer (CRC) treatment, PDT is currently not used as a method for radical treatment of early CRC forms.51 The Chloringlobulin developed herein allows for FIGPDT of both primary and metastatic lesions with sizes as small as 0.6 mm × 1 mm (Figure 8A). Because of the widely used endoscopic treatment, success of FIGPDT in combination with PD-L1 and CTLA-4 dual checkpoint blockade in eradicating mouse tumor shows promise for the clinical application of this protocol to radical treatment of early CRC. CONCLUSIONS Our current findings have unveiled the inherent interaction between Ce6 and IgG molecules in high affinity. By taking this advantage, we have prepared the immune checkpoint inhibitor antibody-Ce6 nanocomplexes termed Chloringlobulin. Chloringlobulin enhances the Ce6 concentration in tumor without changing its elimination half-life in blood. We have developed two


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practical strategies using Chloringlobulin for Ce6-based intraoperative fluorescence image-guided photodynamic immunotherapy of cancer in mice, i.e. a combinatorial FIGS, PDT and PD-L1 blockade therapy of glioma, and an FIGPDT in combination with PD-L1 and CTLA-4 dual checkpoint blockade therapy of colon cancer. The combinatorial treatment elicits prolonged survival of the tumor-bearing mice as well as a long-term memory response, exhibiting translational potential for glioma or colon cancer therapy. MATERIALS AND METHODS Mice. Female C57BL/6 mice (6-8 weeks old) were ordered from Sino-British Sippr/BK Lab Animal Inc. Male BALB/c mice (6-8 weeks old) were purchased from Shanghai Linchang Biological Co. Ltd. The mice were allowed to adapt to the environment for one week before the experiments, and housed under specific pathogen free conditions with free access to food and water. All animal experiments were performed in compliance with the guidelines established by Institutional Animal Care and Use Committee (IACUC) of School of Pharmacy, Fudan University. Affinity measurement. Biacore T200 instrument (GE Healthcare, USA) was utilized to measure the kinetic parameters. Each protein was immobilized on the Series S Sensor Chip CM5 chip (GE Healthcare, Lot No.10239303) by using the amine coupling kit (GE Healthcare, USA). The surface without coupling protein was used as reference channel. All binding experiments were performed at a flow rate of 30 µL/min. After each cycle, the sensor surface was regenerated by Glycine-HCl (pH 3.0) for 30 s at the flow rate of 30 µL/min. For the measurement of the kinetic parameters of Ce6 (Frontier Scientific) to αPD-L1 (Clone: 10F.9G2, BioXCell), Control IgG (Meilun Biotech), HSA (Sigma) or MSA (Equitech-Bio), serial dilutions of Ce6 or Ce6 in the presence of PVP (at a mass ratio of 1:1) were injected to the immobilized proteins. The running buffer was HBS-EP+ (pH 7.4). For the test of binding stability of Ce6 to IgG, gradient dilutions


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of Ce6 were injected to the Control IgG-immobilized channel. The running buffer was HBS-EP+ (pH 7.4) in the presence of 1% HSA or 1% MS. For measuring the affinity of αPD-L1 to PD-L1, serial dilutions of αPD-L1 or the prepared αPD-L1 Chloringlobulin were injected to the immobilized mouse PD-L1 (Biolegend). The running buffer was HBS-EP+ (pH 7.4). All biosensor data were processed and analyzed with Biacore T200 Evaluation Software (Version 2.0, GE Healthcare, USA) and fitted by a 1:1 binding model. Molecular Docking. Ce6 was docked into the active sites of rat IgG (PDB code: 1I1C) using SYBYL 6.9 program. Before docking procedure, the protein structure retrieved from PDB site was treated by removing ligands. The molecular structures were generated with Sybyl/Sketch module and optimized using Powell’s method with the Tripos force field with convergence criterion setting at 0.005 kcal/(Åmol) and assigned charges with the Gasteiger-Hückel method. Molecular docking was carried out via the Sybyl/Surflex-Dock module. Other docking parameters were kept as default values. Thermodynamic analysis. Control IgG (Meilun Biotech) was immobilized on the Series S Sensor Chip CM5 chip as described before. Serial dilutions of Ce6 were injected to the immobilized proteins, with an association time of 240 s and a dissociation time of 480 s at the temperature of 13, 19 and 25 °C, respectively. All cycle performed in the running buffer of HBSEP+ (pH 7.4) at a flow rate of 30 µL/min and surface without coupling protein was used as reference channel. Data were fitted to obtain KD of each temperature and then used to calculate Van’t Hoff thermodynamic parameters.18 Preparation and characterization of αPD-L1 Chloringlobulin. Ce6 was dissolved in 0.1% NaOH at a concentration of 4 mg/mL. PVP (MW ≈ 10 kD) was dissolved in 0.01 M of PBS (pH


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7.4) at a concentration of 6.7 mg/mL. One milligram of αPD-L1 was dissolved in 300 µL deionized water. The PVP solution (75 µL) was dropwisely added to the αPD-L1 solution under stirring followed by stirring for another 5 min. The prepared Ce6 solution (125 µL) was dropwisely added to the above mixture. After another 5-min stirring, the αPD-L1 Chloringlobulin were prepared. αPD-L1-αCTLA-4 Chloringlobulin, Control Chloringlobulin or PVP-Ce6 was prepared using the same method except for replacing the αPD-L1 (1 mg) with αPD-L1 + αCTLA-4 (0.5 mg αPD-L1+ 0.5 mg αCTLA-4), the rat IgG control (1 mg) or deionized water. The morphology of αPD-L1 Chloringlobulin was observed via TEM (FEI Tecnai G2 20 TWIN) and the presence of antibody was confirmed through STEM with energy-dispersive X-ray spectroscopy (EDX, FEI Tecnai Osiris). In vitro release profiles of Ce6 from Control Chloringlobulin was performed in PBS buffer (pH 7.4, 20mM) in the presence or absence of 10% FBS. Briefly, 0.5 mL of Control Chloringlobulin was sealed in a dialysis bag (MWCO 3500), and immersed in 49.5 mL of PBS buffer at 37 °C shaking incubator with a shaking rate of 100 rpm. At the predetermined time points, 0.5 mL of aliquots were taken from the solution and replaced with the same volume of the fresh corresponding buffer. The removed aliquot was determined by a fluorescence microplate reader (Bio-Tek) at λex = 400 nm ± 30 nm, λem = 680 ± 30 nm. For the in vitro stability assay, 100 μL of Control Chloringlobulin or PVP-Ce6 was added to 10 mL 10% FBS 37 °C. The size distribution of the solution at different time points was measured by DLS. PDT in vitro. GL261 cells were seeded in six-well plates at a density of 5 × 105 cells per well and incubated for 24 h to obtain approximately 80% cell confluency. Fresh complete medium was used to replace the serum free RPMI 1640 with Control Chloringlobulin containing 2 µM of Ce6 at 1 h after incubation at 37 °C. The cells were irradiated with a 660-nm diode laser (Changchun


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New Industries Optoelectronics Technology Co., Ltd.) for 10 min at power density of 8.5 mW/cm2, 17 mW/cm2 and 34 mW/cm2, respectively. The cells were then incubated for 24 h after the irradiation. For detection of the apoptotic cells, both detached cells and adherent cells were harvested, washed twice with PBS, and stained with FITC-labeled Annexin V, according to the manufacturer’s protocol (Invitrogen). For measurement of the PD-L1 expression, cells were stained with anti-PD-L1-PE mAbs or rat IgG2α K-PE isotype control, and analyzed using the CytoFLEX flow cytometer (Beckman Coulter). Establishment of tumor models. For mouse bearing glioma orthotopic model, GL261 cells (2 × 105) were suspended in 5 μL of PBS and intracranially injected into the right hemisphere of the anesthetized mice at an injection rate of 5 μL/min using a mouse stereotactic apparatus (Stone) at the position of 1 mm lateral from the bregma and 4 mm depth from the surface of the brain. Following the injection, the needles were allowed to stay for 5 minutes. For the experiment of FIGS, the cells were injected 2 mm depth from the surface of the brain. For the orthotopic CT26 tumor model, mice were anesthetized and immobilized on a plate. The abdominal skin was sanitized using 0.5% (w/v) of iodophor. A small midline incision (3-5 mm) was cut in the skin of lower abdomen to expose cecum. CT26-Luc cells (4 × 106) in 25 μL of PBS were injected into the subserosal layer of the cecum with an insulin needle. When the injected cells were absorbed, the cecum was repositioned and the abdominal cavity was sutured. Pharmacokinetic analysis. αPD-L1 Chloringlobulin, Control Chloringlobulin or PVP-Ce6 was injected into female C57BL/6 mice (6-8 weeks old) through the tail veins at a dose of 5 mg/kg of Ce6. The blood was collected from the tail veins into heparin tubes at 0, 0.5, 1, 2, 4, 6, 8, 12 and 24 h, respectively. Each blood sample (20 µL) was accurately transferred to a PCR tube for IVIS


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imaging (Caliper Life Sciences). The fluorescence intensity of Ce6 in blood (% ID/mL) was calculated using the following equation by Living Image software (Perkin Elmer):

% ID/mL =

(Tblood ― Tblank) 0.02(Tcontrol ― Tblank)

where Tblood, Tblank and Tcontrol represents the total radiant efficiency values of the collected blood, the blank and the control (1% of the injected dose), respectively. Pharmacokinetic parameters were calculated using the noncompartmental analysis. The elimination rate constant (k) was calculated by the linear regression of the terminal three points on the semi-log plot of plasma concentration against time. The elimination half-life (t1/2) was calculated as 0.693/k. Area under the concentration-time curve to the least measurable concentration (AUC0→n) was calculated by the linear trapazoidol rule. Clearance (Cl) was calculated by dividing 100% ID by AUC0→n. The area under moment curve (AUMC) and the mean residence time (MRT) was obtained according the statistical moment theory. Steady state volume of distribution (VSS) was calculated by multiply Cl by MRT. Distribution of Ce6 in the tumor-bearing mice. In mouse bearing glioma orthotopic model, 10 d after the tumor cell inoculation the mice were divided into three groups (n = 3). The mice were i.v. administered with αPD-L1 Chloringlobulin, Control Chloringlobulin or PVP-Ce6 for dynamic imaging using an IVIS imaging system at 1 h, 2 h, 4 h, 8 h, 12 h or 24 h. Total radiant efficiency of interest (tumor) was collected at λex = 675 nm ± 30 nm, λem = 720 ± 20 nm. The total radiant efficiency acquired in an equal dimension in the back of the same mouse was used as the blank control. Further, brain tumors from 1 h and 24 h following the injection were collected, and the distribution of Ce6 in tumor was confirmed by immunofluorescence staining.


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In addition, the ex vivo imaging was conducted. At 1 h post-administration, 20 μL of blood was collected in heparin tube. The mice were then euthanized. Heart, liver, spleen, lung, kidney, skin, and the tumor-bearing brain were harvested for IVIS imaging. One percent dilution of the injected αPD-L1 Chloringlobulin, Control Chloringlobulin or PVP-Ce6 in 20 μL was used as standard control. The tissue uptake of Ce6 expressed as percentage of injected dose (% ID) was calculated using the following equation based on the quantification of the fluorescence imaging of the harvested tissue or blood:

% ID =

Atest ― Ablank Acontrol ― Ablank

where Atest, Ablank and Acontrol represent the average radiant efficiency values of the collected tissues or blood, the blank and the control, respectively. This ex vivo distribution of interest organs was also carried out in CT26-Luc model, 4 d after the tumor cell inoculation, the mice were divided into three groups (n = 4), and received i.v. injection of αPD-L1-αCTLA-4 Chloringlobulin, Control Chloringlobulin or PVP-Ce6 at a dose of 5 mg/kg of Ce6. At 1 h, 2 h and 4 h post-administration, blood, heart, liver, spleen, lung, kidney, skin, and the tumor-bearing cecum were harvested for IVIS imaging. Meanwhile, cecum tumors resected under the guidance of Ce6 fluorescence imaging were collected for the immunofluorescence staining. Tumor treatment. αPD-L1 Chloringlobulin mediated PDT in combination with PD-L1 blockade: Six days post the tumor cell inoculation, the C57BL/6 mice bearing GL261 orthotopic brain tumors were randomly divided into 4 groups: Control group, mice without treatment; PDT group, mice injected with Control Chloringlobulin plus laser treatment; αPD-L1 group, mice


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injected with αPD-L1 Chloringlobulin; and PDT + αPD-L1 group, mice injected with αPD-L1 Chloringlobulin plus laser treatment. The i.v. injection dose of αPD-L1 Chloringlobulin or Control Chloringlobulin was 10 mg/kg of the antibodies and 5 mg/kg of Ce6. At 1 h post i.v. injection, the groups of the mice with PDT received laser irradiation in a dose of 458 mJ (80 mW/cm2, 15 min, 660 nm). For the stereotactic PDT, an 18-G needle serving as a catheter needle was accurately positioned in the tumor through a stereotactic instrument. A quartz fiber optics (0.9 mm diameter) was then inserted through the needle core. The other end of the fiber was connected to the 660-nm diode laser through an SMA connector. The laser power density was measured by an optical power meter (PS-100, Changchun New Industries Optoelectronics Technology Co., Ltd.). In αPD-L1treated groups, the mice received additional i.p. injection of αPD-L1 (5 mg/kg) on Days 9 and 12, respectively. αPD-L1 Chloringlobulin-mediated combinatorial FIGS, PDT and αPD-L1 treatment: The home-made intraoperative fluorescence imaging system was set up specifically for Ce6 as a fluorescent contrast agent. The system included two separate apparatus, i.e. the illuminator and the scope. The illuminator used a 660-nm diode laser equipped with an SMA fiber optics. The distal end of the fiber optics was connected to a collimator with a short-pass filter (660SP RapidEdge, Omega Optical). Fluorescence emission was collected by a 1.0-m-long, 600-µm-diameter flexible coherent fiber bundle (IGN-08/30, Sumitomo Electric) with a distal lens providing 70o of field of view (FOV) in the air, through a 7× lens and a long-pass emission filter (670LP RapidEdge, Omega Optical), onto a scientific complementary metal-oxide semiconductor (sCMOS) camera (PCO. Edge 4.2, PCO AG). The intraoperative fluorescence imaging was recorded and processed using PCO. Camware V04.02/1898 software.


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Eight days post the tumor cell inoculation (2 mm depth from the surface of the brain), the tumorbearing GL261 mice were randomly divided into 5 groups (n = 5): Control group, mice without treatment; Surgery group, mice received surgery without fluorescence imaging guidance; FIGS groups, mice were injected with Control Chloringlobulin and received fluorescence image-guided surgery; Surgery + PDT + αPD-L1 group, mice were injected with αPD-L1 Chloringlobulin and received surgery without fluorescence image guidance plus laser treatment; FIGS + PDT + αPDL1 group, mice were injected with αPD-L1 Chloringlobulin and received fluorescence imageguided surgery plus laser treatment. The i.v. injection dose of αPD-L1 Chloringlobulin or Control Chloringlobulin was 10 mg/kg of the antibodies and 5 mg/kg of Ce6. In αPD-L1-treated groups, the mice received additional i.p. injection of αPD-L1 (5 mg/kg) on Days 11 and 14, respectively. In the groups with FIGS, the mice were anesthetized at 1 h post i.v. injection of αPD-L1 Chloringlobulin or Control Chloringlobulin. The mouse head was immobilized in the stereotactic instrument. The scalp was cut with a scalpel to expose the skull. Fluorescent imaging was recorded using the intraoperative fluorescence imaging system. The skull in the tumor area was opened with an electric drill. The tumor was resected with the help of the intraoperative fluorescence imaging. Surgery was considered to be terminated unless discernible fluorescence signal in the surgical bed. For the stereotactic PDT, a quartz illumination fiber optics (0.9 mm diameter) connected to a 660nm laser was placed on the top of the surgical bed to irradiate the surgical margin at a power density of 40 mW/cm2 for 10 min. Following the PDT treatment, the skin of the head was closed with suture. The mice were allowed to be recovered before placed in the cages. In mice treated with surgery groups, the tumor was removed without the help of the intraoperative fluorescence imaging. The end point for the surgery was assessed by visual inspection.


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Chloringlobulin-mediated combinatorial FIGPDT, αPD-L1 and αCTLA-4 dual blockade therapy: This regimen was tested in mouse CT26-Luc orthotopic colorectal cancer model with a spontaneous metastasis. Four days post the tumor cell inoculation, mice were divided into 4 groups: Control group, mice without treatment; FIGPDT group, mice injected with Control Chloringlobulin plus fluorescence image-guided PDT; αPD-L1-αCTLA-4 group, mice injected with αPD-L1-αCTLA-4 Chloringlobulin; and FIGPDT + αPD-L1-αCTLA-4 group, mice injected with αPD-L1-αCTLA-4 Chloringlobulin plus FIGPDT treatment. The i.v. injection dose of αPDL1-αCALA-4 Chloringlobulin or Control Chloringlobulin was 5 mg/kg of αPD-L1 and 5 mg/kg αCALA-4, or 10 mg/kg of control IgG. The dose of Ce6 was 5 mg/kg. For the FIGPDT therapy, the mice were anesthetized at 1 h post-injection of αPD-L1-αCTLA-4 Chloringlobulin or Control Chloringlobulin. The abdominal cavity was opened. The cecum found from the left lower abdomen was pulled out for the intraoperative fluorescence imaging to identify the tumor. A quartz fiber optics (0.9 mm diameter) was then placed on the top of the tumor site at 0.6-cm height for PDT. The other end of the fiber was connected to the 660-nm diode laser through an SMA connector. The laser power dose was 80 mW/cm2, 10 min for primary tumor or 5 min for metastatic tumor. After the laser treatment, the cecum was repositioned and the abdominal cavity was sutured. The mice were allowed to be recovered before placed in the cages. In αPD-L1-αCTLA-4 treated groups, the mice received additional i.v. injection of αPD-L1-αCTLA-4 Chloringlobulin (5 mg/kg of αPD-L1 and 5 mg/kg αCALA-4) on Days 7 and 10, respectively. The tumor burden was monitored by the bioluminescence signal using IVIS imaging after i.p. injection of D-luciferin (15 mg/mL, 200 µL). Assessment of immune response. Flow cytometry analysis was performed on samples including tumors, spleens or DLNs obtained from the tumor-bearing mice. These tissues were


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homogenized by mechanical disaggregation, filtered through a 40 μm nylon cell strainer (BD Falcon), centrifuged at 500 g for 5 min, and lysed by red blood cell lysis buffer (Roche). For cellular membrane staining, cells were incubated with the indicated combinations of fluorochrome-conjugated antibodies at the manufacturer’s recommended dilution for 30 min on ice in dark. For intracellular (nuclear) staining, cells were fixed and permeabilized with Intracellular Fixation & Permeabilization Buffer Set (Foxp3/Transcription Factor Buffer Set) and then incubated with intracellular antibody. For cytokine staining, cells were first stimulated with Cell Stimulation Cocktail (plus protein transport inhibitors) prior to undergoing staining. In mouse GL261 orthotopic glioma model, the tested immune cells included T regulatory cells (Treg) and macrophages in tumors, CD4+ T cells and CD8+ T cells in spleens, activated mDCs, CD4+ T cells and CD8+ T cells in CLNs at Day 2 and CTLs (CD8+IFN-γ+) in tumors and CLNs, CD4+ T cells and CD8+ T cells in CLNs at Day 6. And in mouse CT26-Luc orthotopic colorectal cancer model, the tested items included CD4+ T cells, CD8+ T cells, CD8+granzyme B+ T cells, CTLs, activated mDCs in DLNs and CD4+ T cells, CD8+ T cells, CD8+granzyme B+ T cells, CTLs, Treg cells and MDSC in spleens at Day 21 after the treatment. In addition, the memory phenotypes in spleen by staining CD62L and CD44 in CD8+ T cells of CT26-Luc model were analyzed. All antibodies were purchased from Invitrogen (Table S2). Acquisition was performed on CytoFLEX flow cytometer. Data were analyzed with CytExpert Software. Evaluation of antitumor efficacy in vivo. The Kaplan-Meier survival curves were recorded to assess the antitumor effects following different treatment. For mice bearing GL261 orthotopic tumors, the brain tissues collected at Day 6 were immunofluorescence stained with CD8+ T cells activated NK cells (NK1.1+CD49b+) and cleaved caspase-3. For cleaved caspase-3 staining, the slices were firstly blocked by 10% FBS followed by overnight incubation with rabbit anti-mouse


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cleaved caspase-3 primary antibody (Boster, 1:100) at 4 °C. After PBS washing, the samples were incubated with goat anti-rabbit Alexa Fluor 555 secondary antibody (Invitrogen, 1:500) for 1 h at room temperature, followed by counterstaining with DAPI. For the orthotopic CT26-Luc tumor model, bioluminescence images were collected every 2 or 4 days with the IVIS System. Tumor rechallenge. The CT26-Luc-rejected mice following FIGPDT treatment (n = 2), αPDL1-αCTLA-4 Chloringlobulin treatment (n = 5) and FIGPDT + αPD-L1-αCTLA-4 treatment (n = 9) were inoculated with 5 × 105 of CT26-Luc in the right flank of mice. Age-matched naive male BALB/c (15-17 weeks) (n = 5) were used as control. The tumor burden was monitored similarly as above. At 45 d after the rechallenge, the mice spleens were harvested for the analysis of T cell memory response. Statistical analysis. Statistical analysis was performed using GraphPad Prism 6 software (GraphPad). Data are presented as the means ± SD for all results. Statistical significance was determined by paired or unpaired, two-tailed t test, one-way ANOVA or two-way ANOVA with Tukey’s or Bonferroni’s multiple comparisons post-hoc test. Survival was analyzed using KaplanMeier survival curves. The curves were compared with the log-rank Mantel-Cox test. *P < 0.05, **P < 0.01, ***P < 0.001. AUTHOR INFORMATION Corresponding Author *Email: [email protected] *Email: [email protected] Author Contributions


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W.L. and Z.X. designed the study. W.L., Z.X., J.X. and S.Y. analyzed the data and prepared the manuscript. Z.C. and J.X. performed the kinetic binding assay and analyzed the affinity data. Y.Q., J.X. and S.Y. prepared the nanocomplexes. S.G. and W.F. performed the molecular docking experiment. X.W. and Y.Q. carried out characterization of the nanocomplexes. W.W. and W.L. set up the fluorescence imaging apparatus. J.X., W.L., S.Y. and G.W. performed the experiment of the intraoperative fluorescence imaging. J.X., S.Z., S.Y. and B.Z. carried out cell experiment, animal experiment and flow cytometry analysis. #J.X.

and S.Y. contributed equally to this work.

ACKNOWLEDGMENT This work was supported in part by grants from the National Natural Science Foundation of China (81673018, 91859110) (W.L.), (31671003) (Z.X.) and by Program of Shanghai Academic/Technology Research Leader (19XD1420200) (W.L.). The authors thank Michael Rooks from Yale Institute of Nanoscience and Quantum Engineering for assisting in scanning transmission electron microscopy-energy-dispersive X-ray spectroscopy studies. ASSOCIATED CONTENT Supporting Information Supporting experimental methods include cellular binding of αPD-L1, cellular uptake of the αPD-L1 Chloringlobulin in vitro, in vivo tumor distribution of Ce6 in subcutaneous CT26-Luc colon cancer model. Supporting figures include size distributions of PVP-Ce6 or Control Chloringlobulin in 10% FBS, pharmacokinetics of Ce6 in C57BL/6 mice, in vivo tumor distribution of Ce6 in GL261 orthotopic glioma model, ex vivo distribution of Ce6 in GL261 orthotopic glioma model, ex vivo distribution of Ce6 in mouse CT26-Luc orthotopic colorectal


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cancer model, cellular binding of αPD-L1, MFI of Ce6 in GL261 cells following uptake of αPDL1 Chloringlobulin in vitro, setup of the home-made intraoperative fluorescence imaging system, fluorescence spectrum of PVP-Ce6 or Control Chloringlobulin, in vivo bioluminescence imaging of CT26-Luc tumors in response to αPD-L1-αCTLA-4 Chloringlobulin treatment described in Figure 8B, bioluminescence imaging of the cured mice after subcutaneously rechallenged with the tumor cells at Day 55, representative flow cytometric plots of Figure 6, representative flow cytometric plots of Figure 9, in vivo tumor distribution of Ce6 in subcutaneous CT26-Luc colon cancer model. Supporting tables include comparison of kinetic parameters of αPD-L1 or the prepared αPD-L1 Chloringlobulin to mouse PD-L1, the antibodies used for the flow cytometry experiments. This material is available free of charge via the Internet at http://pubs.acs.org. J.X. and W.L. submitted a patent application related to the findings in the manuscript.

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Figures

Figure 1. High affinity of Ce6 to IgG. (A) The sensorgrams of kinetic analysis of Ce6 to αPD-L1, Control IgG, HSA or MSA. Gradient concentrations of Ce6 trisodium were injected through flow


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cells immobilized with αPD-L1, Control IgG, HSA or MSA. In Ce6 + PVP group, the ratio of Ce6 to PVP was 1:1 (w/w). αPD-L1, rat anti-PD-L1 mAb; Control IgG, rat IgG without PD-L1-binding effect; HSA, human serum albumin; MSA, mouse serum albumin; PVP, polyvinylpyrrolidone (MW ≈ 10 kD). (B) Biacore kinetics assay sensorgram of Ce6 to IgG in presence of 1% HSA or 1% MS. Gradient concentrations of Ce6 trisodium in 1% HSA or 1% MS HBS-EP+ (pH 7.4) were first injected over Control IgG-immobilized Biacore CM5 sensor chips and then dissociated using 1% HSA or 1% MS HBS-EP+ (pH 7.4). MS, mouse serum. ka, association rate constant; kd, dissociation rate constant; KD, equilibrium dissociation constant (KD=kd/ka); KB, binding constant (KB=ka/kd).


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Figure 2. Binding characteristic of Ce6 to rat IgG. (A) Binding pockets of IgG (PDB code: 1I1C) obtained using SYBYL 6.9 program. (B-C) Overview of the molecular modeling of Ce6 (grey) bound to the active sites of rat IgG, P4 (B) or P5 (C). The inside binding residues of P4 are Lys246 (red), Ile377 (orange) and Asn393 (green). The inside binding residues of P5 are Lys370 (green), Asp401 (orange) and Lys409 (red). Yellow dashed lines, hydrogen bonds between IgG and Ce6. (D-F) Thermodynamic analysis of Ce6 to rat IgG. (D) The sensorgrams of kinetic analysis of Ce6 to Control IgG at 286 K, 292 K and 298 K, respectively. Gradient concentrations of Ce6 trisodium were injected through flow cells immobilized with Control IgG. (E) Van’t Hoff plot for the interaction of Ce6 and Control IgG in HBS-EP+ buffer (pH 7.4). (F) Kinetic and thermodynamic parameters of the Ce6-Control IgG system. ka, association rate constant; kd, dissociation rate constant; KD, equilibrium dissociation constant (KD=kd/ka); ΔG, free energy change; ΔH, enthalpy change; ΔS, entropy change.


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Figure 3. Characterization of αPD-L1 Chloringlobulin. (A) Diagrams of preparation of Chloringlobulin through simply mixing Ce6 trisodium, IgG (e.g. αPD-L1, αCTLA-4) and PVP. (B) TEM of αPD-L1 Chloringlobulin in PBS. Upper bar, 200 nm; lower bar, 50 nm. (C) STEM of αPD-L1 Chloringlobulin in PBS with EDS of the selected area in the image. Bars, 50 nm. Arrow, S element. (D) Photographs of αPD-L1 Chloringlobulin in 10% of mannitol (1) before lyophilization, (2) lyophilized powder, or (3) reconstitution after lyophilization. Dynamic light


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scattering analysis of particle sizes of αPD-L1 Chloringlobulin before and after lyophilization. (E) Biacore sensorgram of αPD-L1 or αPD-L1 Chloringlobulin binding to the immobilized mouse PDL1. (F) Cumulative release of Ce6 from Control Chloringlobulin (prepared with Control IgG) in PBS or PBS with 10% FBS over time. Data are means ± SD (n = 4). Statistical significance was calculated by two-way ANOVA with Bonferroni's post-hoc test. ***P < 0.001. (G) Representative size distribution of PVP-Ce6 or Control Chloringlobulin in PBS with 10% FBS after mixture for 2 h. Control, 10% FBS only.


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Figure 4. Pharmacokinetics and distribution of Ce6 from Chloringlobulin. (A) Blood Ce6 concentration-time curve following i.v. injection of PVP-Ce6, Control Chloringlobulin or αPD-L1 Chloringlobulin in C57BL/6 mice. Ce6 concentration was calculated from the fluorescence imaging (Figure S2). % ID, percentage injected dose. Data are means ± SD (n = 3). Statistical significance was calculated by two-way ANOVA with Tukey’s post-hoc test. **P < 0.001; ***P < 0.001 between Control Chloringlobulin group and PVP-Ce6 group.

##P

< 0.01; ###P < 0.001

between αPD-L1 Chloringlobulin group and PVP-Ce6 group. (B and C) Tumor distribution of Ce6 in C57BL/6 mice bearing GL261 orthotopic glioma model. (B) Distribution of Ce6 in the tumor of mice after the i.v. injection of PVP-Ce6, Control Chloringlobulin or αPD-L1 Chloringlobulin measured by the live fluorescence imaging (Figure S3). Data are means ± SD (n = 3). Statistical significance was calculated by two-way ANOVA with Tukey’s post-hoc test. *P < 0.05; ***P < 0.001 compared with the PVP-Ce6 group. (C) Representative fluorescence micrographs of tumor at 1 h or 24 h following the i.v. injection. DAPI, 4′,6-diamidino-2-phenylindole. Bar, 50 µm. (D and E) Distribution of Ce6 in BALB/c mice bearing CT26-Luc orthotopic colon tumor. (D) Tissue distribution of Ce6 calculated from the fluorescence imaging at 1 h, 2 h or 4 h after i.v. injection of PVP-Ce6, Control Chloringlobulin or αPD-L1-αCTLA-4 Chloringlobulin. Fluorescence images are shown in Figure S5. Data are means ± SD (n = 4). Statistical significance was calculated by two-way ANOVA with Tukey’s post-hoc test. *P < 0.05; **P < 0.01; ***P < 0.001 compared with PVP-Ce6 group. #P < 0.05;

###P

< 0.001 compared between the αPD-L1-αCTLA-4

Chloringlobulin group and the Control Chloringlobulin group. (E) Representative fluorescence micrographs of tumors at 1 h, 2 h or 4 h following the i.v. injection. Bar, 50 µm.


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Figure 5. Cellular responses to PDT illustrating the PD-L1 expression remaining in the residue tumor cells. (A) Representative bright field microscopic images (left) and flow cytometry histograms (right) of GL261 cells treated with Control Chloringlobulin containing 2 µM of Ce6 for 1 h, followed by a 660-nm laser irradiation for 10 min at different dose. Bar, 50 μm. FSC, forward scatter; SSC, side scatter. (B) Quantitative analysis of apoptotic GL261 cells (Annexin V+) in different treatment groups. (C) Mean fluorescence intensity (MFI) of PD-L1 in live GL261 cells (Annexin V-) in P1 and P2 population gated from the flow cytometry histograms of (A), respectively. Data are expressed as means ± SD (n = 4). In P1-Annexin V- group, statistical significance was calculated by one-way ANOVA with Tukey’s post-hoc test. **P < 0.01; ***P < 0.001 compared with the non-treatment group. Statistical significance between the group with the same laser dosage was calculated by paired, two-tailed t test. ###P < 0.001 compared with the P1Annexin V- group.


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Figure 6. αPD-L1 Chloringlobulin-mediated PDT in combination with PD-L1 blockade for treatment of GL261 glioma. GL261 orthotopic tumor-bearing mice were i.v. injected with Control Chloringlobulin or αPD-L1 Chloringlobulin containing 10 mg/kg of antibodies followed by the laser treatment (80 mW/cm2, 660 nm) through a quartz fiber optics in 0.9-mm diameter under the guidance of the stereotactic instrument for 15 min at Day 0. Control group, mice without treatment. PDT group, mice injected with Control Chloringlobulin plus laser treatment. αPD-L1 group, mice injected with αPD-L1 Chloringlobulin. PDT + αPD-L1 group, mice injected with αPD-L1 Chloringlobulin plus the laser treatment. (A) Scheme (left) and photograph (right) of stereotactic laser treatment of C57BL/6 mice bearing GL261 orthotopic glioma through a fiber optics. (B) Quantitative analysis of Treg cells (CD4+CD25+Foxp3+) or ratio of classic macrophage (M1, CD11b+F4/80+CD206-) to alternative macrophage (M2, CD11b+F4/80+CD206+) in tumor; CD4+ or CD8+ T cells in spleen; activated dendritic cells (mDC, CD11c+CD86+), CD4+ or CD8+ T cells in cervical lymph nodes (CLNs) at Day 2. Representative flow cytometry gating strategies plots are shown in Figure S12A. Data are means ± SD (n = 3-5). Statistical significance was calculated by one-way ANOVA with Tukey’s post-hoc test. *P < 0.05; **P < 0.01; ***P < 0.001 compared with Control group. #P < 0.05; ##P < 0.01; ###P < 0.001 compared with PDT group. &P < 0.05; &&P < 0.01;

&&&P

< 0.001 compared between PDT + αPD-L1 group and αPD-L1 group. (C) Flow

cytometry analysis of cytotoxic T lymphocytes (CTL, CD8+IFNγ+) in tumor or CLN and CD4+, CD8+ T cells in CLNs at Day 6. Representative flow cytometry gating strategies are shown in Figure S12B. Data are means ± SD (n = 3). Statistical significance was calculated by one-way ANOVA with Tukey’s post-hoc test. *P < 0.05; **P < 0.01; ***P < 0.001 compared with Control group. ###P < 0.001 compared with PDT group. &&&P < 0.001 compared between PDT + αPD-L1 group and αPD-L1 group. (D) Immunofluorescence imaging analysis of CD8+ T cells (left


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column), activated NK cells (middle column) or active Caspase 3 expression (right column) in the tumors at Day 6. Bar, 50 μm. (E) Kaplan-Meier survival curve of mice bearing GL261 tumor following different treatment regimen illustrated in the schematic diagram (n = 10). Significance was determined by log-rank analysis. *P < 0.05; **P < 0.01; ***P < 0.001 compared with Control (log-rank analysis). #P < 0.05, compared with PD-L1 group (log-rank analysis).


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Figure 7.

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αPD-L1 Chloringlobulin for combinatorial FIGS, stereotactic PDT and PD-L1

blockade. (A) FIGS of glioma in mice at 1 h after i.v. injection with αPD-L1 Chloringlobulin. Living tumor-bearing mice underwent craniotomy under general anesthesia. Photograph (upper) and intraoperative fluorescence imaging (lower) of mouse head after skull exposure before craniotomy, the resected tumor, and the brain after FIGS, respectively. Arrowheads, tumor with high fluorescence intensity. (B) H&E staining and fluorescence imaging of the resected tumor


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(upper) or the brain after FIGS (lower). Arrow, tumor residual. (C) H&E staining and fluorescence imaging of the resected tumor (upper) or the brain after conventional surgery (lower) through which the tumor resection was assessed by visual inspection. (D) Kaplan-Meier survival curve of mice bearing orthotopic GL261 tumor following different treatment regimen illustrated in the schematic diagram (n = 5). In groups with Surgery, the tumor resection was assessed by visual inspection. In groups with FIGS, the tumor-bearing mice received i.v. of Control or αPD-L1 Chloringlobulin, and the tumor removal was assessed by intraoperative fluorescence imaging at 1 h postinjection. In groups with PDT, a 660-nm laser at total dose of 153 mJ was applied to the surgical bed through a quartz fiber optics in 0.9-mm diameter under the guidance of the stereotactic instrument following surgery or FIGS. Significance was determined by log-rank analysis. *P < 0.05; **P < 0.01 compared with Control group (log-rank analysis). #P < 0.05 compared with FIGS group (log-rank analysis).


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Figure 8. αPD-L1-αCTLA-4 Chloringlobulin for intraoperative FIGPDT in combination with dual checkpoint blockade. (A) Intraoperative FIGPDT of orthotopic CT26-Luc colon tumor in mice 1 h after i.v. injection with αPD-L1-αCTLA-4 Chloringlobulin. Left two columns, intraoperative photograph and fluorescence imaging of the exposed mouse cecum with primary or metastatic tumor and the resected tumor, respectively. Arrowheads, tumor with high fluorescence intensity. Right two columns, H&E staining and fluorescence imaging of the resected tumor. (B) Scheme of therapeutic regimen. Control group, mice without treatment. FIGPDT group, mice injected with Control Chloringlobulin plus FIGPDT. αPD-L1-αCTLA-4 group, mice injected with αPD-L1-αCTLA-4 Chloringlobulin. FIGPDT + αPD-L1-αCTLA-4 group, mice injected with αPD-L1-αCTLA-4 Chloringlobulin plus FIGPDT treatment. Laser dose, 80 mW/cm2, 660 nm, 10 min for primary tumor and 5 min for metastatic tumor. Completely cured mice at Day 55 were rechallenged with CT26-Luc cells (5 × 105) subcutaneously and monitored for another 45 d. (C) Kaplan-Meier survival curve of mice bearing orthotopic CT26-Luc tumor following different treatment regimen illustrated in (B). Significance was determined by log-rank analysis (n = 10). ***P < 0.001 compared with Control group (log-rank analysis).

##P

< 0.01, compared with


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FIGPDT group (log-rank analysis). &P < 0.05, compared between FIGPDT + αPD-L1-αCTLA-4 group and αPD-L1-αCTLA-4 group (log-rank analysis).


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Figure 9. Combination of FIGPDT and dual checkpoint inhibition eliciting systemic anticancer immunity and T cell memory response. CT26-Luc orthotopic tumor-bearing mice were i.v. injected with Control Chloringlobulin or αPD-L1-αCTLA-4 Chloringlobulin containing 10 mg/kg of the antibodies (5 mg/kg αPD-L1 + 5 mg/kg αCTLA-4) followed by the laser treatment (80 mW/cm2, 660 nm) for 10 min for primary tumor or 5 min for metastatic tumor at Day 4. Control group, mice without treatment. FIGPDT group, mice injected with Control Chloringlobulin plus fluorescence image-guided PDT. αPD-L1-αCTLA-4 group, mice injected with αPD-L1-αCTLA4 Chloringlobulin. FIGPDT + αPD-L1-αCTLA-4 group, mice injected with αPD-L1-αCTLA-4 Chloringlobulin plus FIGPDT treatment. (A) Flow cytometry analysis of CD4+ T cells, CD8+ T cells, activated mDCs (CD11c+CD86+), granzyme B-positive CD8+ T cells (CD8+GranB+) or CTLs (CD8+IFN-γ+) in DLN; CD4+ T cells, CD8+ T cells, CD8+GranB+, CTLs or Treg cells (CD4+CD25+Foxp3+) in spleen at Day 21 after treatment. Representative flow cytometry gating strategies are shown in Figure S13. Data are means ± SD (n = 3-5). Statistical significance was calculated by one-way ANOVA with Tukey’s post-hoc test. *P < 0.05; **P < 0.01; ***P < 0.001 compared with Control group. #P < 0.05; ##P < 0.01; ###P < 0.001 compared with FIGPDT group. &P

< 0.05; &&P < 0.01; &&&P < 0.001 compared between FIGPDT + αPD-L1-αCTLA-4 group and

αPD-L1-αCTLA-4 group. (B) Flow cytometry analysis of CD11b+Gr-1+ MDSC cells at Day 21 in spleen, including Gr-1highCD11b+ granulocytic (G-MDSC) and Gr-1intCD11b+ monocytic (MMDSC) MDSC subsets. Data are means ± SD (n = 3-5). Statistical significance was calculated by one-way ANOVA with Tukey’s post-hoc test. ***P < 0.001 compared with Control group. #P < 0.05;

##P

< 0.01 compared with FIGPDT group. (C) Flow cytometric histogram analysis and

representative plots of CD44-CD62L+ (naïve), CD44+CD62L+ (central memory) and CD44+CD62L- (effector memory) of CD8+ T cells in spleen of tumor-bearing mice at Day 21 after


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tumor inoculation. Data are means ± SD (n = 3-5). Statistical significance was calculated by oneway ANOVA with Tukey’s post-hoc test. *P < 0.05; **P < 0.01; ***P < 0.001 compared with Control group. #P < 0.05 compared with FIGPDT group. &P < 0.05; &&P < 0.01 compared between FIGPDT + αPD-L1-αCTLA-4 group and αPD-L1-αCTLA-4 group. Pie charts depict the relative percentages of each subset in treated groups. (D) Flow cytometry analysis of memory populations at Day 45 after tumor rechallenge. Naïve mice were used as control. Data are means ± SD (n = 5 for naïve control, n = 2 for FIGPDT group, n = 5 for αPD-L1-αCTLA-4 group and n = 8 for FIGPDT + αPD-L1-αCTLA-4 group). Statistical significance was calculated by one-way ANOVA with Tukey’s post-hoc test. ***P < 0.001 compared with Control group. ##P < 0.01; ###P < 0.001 compared with FIGPDT group. &P < 0.05;

&&P

< 0.01 compared between FIGPDT + αPD-L1-

αCTLA-4 group and αPD-L1-αCTLA-4 group. Pie charts depict the relative percentages of each subset in treated groups.


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Table 1. Pharmacokinetic parameters of Ce6 after i.v. administration of αPD-L1 Chloringlobulin, Control Chloringlobulin or PVP-Ce6 (5 mg/kg of Ce6) to C57BL/6 mice.

Parameters

PVP-Ce6

AUC0→n (% ID mL-1 155 ± 14 h) t1/2 (h) 2.20 ± 0.26 Cl (mL h-1) 0.647 ± 0.059 AUMC (% ID mL-1 h2) 691 ± 79 MRT (h) 4.44 ± 0.17 VSS (mL) 2.87 ± 0.22

Control αPD-L1 Chloringlobulin Chloringlobulin 261 ± 7**

253 ± 11**

2.32 ± 0.55 0.383 ± 0.010** 1141 ± 83** 4.37 ± 0.32 1.67 ± 0.14**

2.92 ± 0.60 0.395 ± 0.017** 1161 ± 122** 4.58 ± 0.37 1.81 ± 0.13**

Data are presented as means ± SD (n = 3). **P

High Affinity of Chlorin e6 to Immunoglobulin G for Intraoperative

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