Protein Nanocage Mediated Fibroblast-Activation Protein Targeted

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Protein Nanocage Mediated FAP-targeted Photo-immunotherapy to Enhance Cytotoxic T Cell Infiltration and Tumor Control Zipeng Zhen, Wei Tang, Mengzhe Wang, Shiyi Zhou, Hui Wang, Zhanhong Wu, Zhonglin Hao, Zi-Bo Li, Lin Liu, and Jin Xie Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.6b04150 • Publication Date (Web): 28 Dec 2016 Downloaded from http://pubs.acs.org on December 29, 2016

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Protein Nanocage Mediated FAP-targeted Photo-immunotherapy to Enhance Cytotoxic T Cell Infiltration and Tumor Control

Zipeng Zhen1,2,§, Wei Tang2, §, Mengzhe Wang3, Shiyi Zhou2, Hui Wang3, Zhanhong Wu,3 Zhonglin Hao4, Zibo Li3, Lin Liu2,*, Jin Xie2,* 1. Department of Radiology, China-Japan Union Hospital, Jilin University, Changchun 130033, China 2. Department of Chemistry, Bio-Imaging Research Center, University of Georgia, Athens, Georgia 30602, USA 3. Department of Radiology and Biomedical Research Imaging Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA 4. Department of Internal Medicine, Medical College of Georgia, Georgia Regents University, Augusta, Georgia 30912, USA

* Corresponding authors: [email protected], [email protected] §

These authors contribute equally to this work.

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Abstract Carcinoma-associated fibroblasts (CAFs) are found in many types of cancer and play an important role in tumor growth and metastasis. Fibroblast-activation protein (FAP), which is overexpressed on the surface of CAFs, has been proposed as a universal tumor targeting antigen. However, recent studies show that FAP is also expressed on multipotent bone marrow stem cells. A systematic anti-FAP therapy may lead to severe side effects and even death. Hence, there is an urgent need of a therapy that can selectively kill CAFs without causing systemic toxicity. Herein we report a nanoparticle based photo-immunotherapy (nano-PIT) approach that addresses the need. Specifically, we exploit ferritin, a compact nanoparticle protein cage, as a photosensitizer carrier and we conjugate to the surface of ferritin a FAP-specific single chain variable fragment (scFv). With photo-irradiation, the enabled nano-PIT efficiently eliminates CAFs in tumors but causes little damage to healthy tissues due to the localized nature of the treatment. Interestingly, while not directly killing cancer cells, the nano-PIT caused efficient tumor suppression in tumor bearing immunocompetent mice. Further investigations found that the nano-PIT led to suppressed C-X-C motif chemokine ligand 12 (CXCL12) secretion and extracellular matrix (ECM) deposition, both of which are regulated by CAFs in untreated tumors and mediate T cell exclusion that prevents physical contact between T cells and cancer cells. By selective killing of CAFs, the nano-PIT reversed the effect, leading to significantly enhanced T cell infiltration, followed by efficient tumor suppression. Our study suggests a new and safe CAF-targeted therapy and a novel strategy to modulate tumor microenvironment (TME) for enhanced immunity against cancer. Keywords: photodynamic therapy, immunotherapy, cytotoxic T cells, carcinoma-associated fibroblast, fibroblast-activation protein


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1. Introduction Carcinoma-associated fibroblasts (CAFs) refer to a subset of fibroblasts that are perpetually active in tumors.1 CAFs are found in many types of cancer (e.g. breast, colorectal, and ovarian cancer1) and often account for a major portion of the tumor stromal cell population.2 It is increasingly clear that CAFs play important roles in cancer cell survival, proliferation, and metastasis.3,

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These include causing accelerated extracellular matrix (ECM) turnover by

excessive secretion of ECM components and ECM-degrading proteases;3, 5 promoting cancer cell proliferation and epithelial cell transformation by secreting high levels of growth factors such as hepatocyte growth factor (HGF), epidermal growth factor (EGF), and insulin-like growth factor (IGF);3 and inducing an immunosuppressive tumor microenvironment (TME) by releasing cytokines such as vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), interleukin-10 (IL-10), and transforming growth factor beta 1 (TGF-β1).6-9 In addition, CAFs also mediate T cell exclusion that prevents cancer cells from physical contact with cytotoxic T cells (CTLs).10 This may explain ineffective anti-cancer immune response seen in many patients who have cancerspecific T cells in their bodies.11, 12 T cell exclusion is achieved by CAFs depositing a dense layer of ECM surrounding tumor nests that physically traps T cells.12 Moreover, recent studies found a positive correlation between C-X-C motif chemokine ligand 12 (CXCL12) secretion by CAFs and T cell exclusion in tumors.10 T cell exclusion can be a major obstacle in cancer therapy. This includes the emerging adoptive T cell therapy and anti-programmed cell death protein-1/anti-programmed death-ligand 1 (anti-PD-1/anti-PD-L1) therapy,10 which augments the amount or activity of T cells in patients but does not address infiltration issue.


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There have been extensive efforts on exploring CAFs as a potential cancer therapy target. In particular, fibroblast-activation protein (FAP), which is overexpressed on CAFs in over 90% of epithelial cancers1 and a significant portion of melanomas,13 was proposed as a universal tumor target antigen. To this end, anti-FAP antibody F19 and its humanized version sibrotuzumab were developed and evaluated in the clinic.14,

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FAP-targeting vaccines,16,

17

antibody-drug

conjugates,18 and chimeric antigen receptor (CAR) T cells,19 have also been produced and investigated in preclinical studies. But contrary to the initial thought that the expression of FAP is negligible in normal tissues, recent studies found that FAP+ cells also exist in placenta,20 uterus,20, embryo,21 and bone marrow.22 Systematic therapy against FAP+ cells may lead to severe cachexia,19,

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muscle loss,23 bone toxicities,19 and even death.19 These findings cast

doubts on anti-FAP based therapies and have largely impeded the related developments. Hence, there is an urgent need of a novel therapy that can selectively kill CAFs in tumors while not causing systemic toxicity. We herein report a novel nanoparticle-based photo-immunotherapy (nano-PIT) approach that can address the need. Briefly, we employ apoferritin, a nanoparticle protein cage, as a photosensitizer (in this case ZnF16Pc) carrier and we conjugate to the surface of ferritin a FAPtargeted single chain variable fragment (scFv). Compared to the conventional, antibody based PIT agents, ferritin affords comparable size but much greater photosensitizer loading rates (~40 wt%), allowing for efficient and precise treatment. We found that the resulting nanoconjugate, Z@FRT-scFv, can selectively home to CAFs in tumors after systemic injection. Subsequent photo-irradiation led to elimination of CAFs, but minimally affected healthy tissues due to the confined photo-irradiation. Interestingly, while not directly killing cancer cells, the FAP-targeted PIT led to efficient tumor suppression. Further investigations revealed that treatment destructed


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ECM and suppressed CXCL12 secretion, leading to significantly improved infiltration of CD8+ T cells and an increased frequency of them (Scheme 1). The present study offers a novel nanoPIT approach that can selectively kill CAFs and modulate TME in favor of an anti-cancer immune response. It is expected to find wide applications in modern oncology, for instance to facilitate adoptive T cell therapy and anti-PD-1/anti-PD-L1 therapy for optimal tumor control.

2. Results Preparation and Characterization of ZnF16Pc Loaded Ferritin (Z@FRT) Mouse ferritin (FRT) was prepared by following a published protocol and was purified by size-exclusion chromatography (SEC).24 Photosensitizer was physically entrapped within FRT through a pH-mediated disassembly-and-reassembly approach.25 Briefly, ZnF16Pc in DMSO was mixed with FRT at pH 2.0, and the pH of the solution was slowly tuned back to neutral. A similar method has been exploited by us and others to load different therapeutics.24-26 The raw products were purified on a NAP-5 column. The ZnF16Pc loading rate of the conjugate (Z@FRT) was characterized through UV-vis spectroscopy by comparing to a pre-established standard curve. A formulation with a 41.2 wt% ZnF16Pc loading rate was used for subsequent studies.25 Transmission electron microscopy (TEM) analysis confirmed the cage-like structure of ZnF16Pc-loaded FRT, or Z@FRT, finding an external diameter of ~12 nm for the nanoparticle (Figure S1). This data corroborates with the dynamic light scattering (DLS) results, observing a hydrodynamic size of 12.25 ± 1.51 nm for Z@FRT (Figure 1a). The results were also comparable to those obtained with unloaded FRT (Figure 1a), suggesting minimal impact of ZnF16Pc loading on the particle size and colloidal stability. The particle stability was also


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confirmed by SEC, finding almost identical retention times (tR) for FRT and Z@FRT (Figure 1b, 22.423 min and 22.439 min for FRT and Z@FRT, respectively). Notably, free ZnF16Pc has poor water solubility and was quickly precipitated out in aqueous solutions (Figure 1c); as a comparison, Z@FRT was very stable in PBS (pH 7.4) and the solution can be kept for one week without showing visible precipitation (Figure 1c). Also noted is that when the pH was reduced to 2, ZnF16Pc precipitation started to form in Z@FRT solutions (Figure 1c). This is attributed to the breakdown of the nanocage structure and the release of the payloads from the nanocages.27 The capacity of 1O2 generation was studied by singlet oxygen sensor green (SOSG) assay.28 Under 671-nm laser irradiation (0.1 W/cm2), Z@FRT was able to efficiently produce 1O2, manifested in an increase of SOSG fluorescence at 525 nm (Figure S1). The 1O2 generation was comparable with ZnF16Pc-only (dispersed in PBS containing 1% Tween, Figure S1) at the same concentrations, suggesting minimal self-quenching among ZnF16Pc molecules despite the high loading.

Preparation and Characterization of Anti-FAP scFv The anti-FAP scFv was constructed by linking the variable regions of light chain and heavy chain with a 4× GGGGS linker (Figure S2). The sequence was first published by Brocks et al.29 (Figure S2). The scFv sequence was inserted into plasmid pOPE101 and expressed by E.coli. The raw product was purified on a Ni-NTA cartridge. The scFv molecule has a molecular weight of ~26 kDa, which was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, Figure S3).


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The binding of scFv against FAP was investigated by immunofluorescence (IF) microscopy using dye labeled scFv (coupled with IRDye800, ex/em: 780 nm/800 nm). The scFv was incubated with HTB-135 (gastric cancer) and 3T3 (fibroblast) cells, which are high and low in FAP expression, respectively.30,

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Compared to 3T3 cells, much more positive staining was

observed with HTB-135 cells (Figure 2a). Meanwhile, when HTB-135 cells were pre-incubated with unlabeled scFv (20 ×), the positive staining was effectively suppressed. We also investigated the binding with tumor tissues taken from different tumor models. These include primary tumors dissected from subcutaneous 4T1, U-87MG, and PC-3 models, and colonies established in the lung, liver, and kidney taken from a LL/2 metastasis tumor model.32 In all cases, we observed extensive positive staining with IRDye800 labeled anti-FAP scFv, and negligible staining if unlabeled scFv was co-applied as a blocking agent (Figure S4 and Figure 2b). Meanwhile, negative staining was found with normal tissues (e.g. heart, liver, kidney, muscle, and brain, Figure S5). These observations corroborate with previous studies33 and confirmed the good selectivity of the anti-FAP scFv.

Z@FRT-scFv Preparation and In Vivo Targeting Next,

we

conjugated

the

anti-FAP

scFv

to

the

surface

of

Z@FRT

using

bis(sulfosuccinimidyl)suberate (or BS3) as a crosslinker.34, 35 The conjugation led to an increased hydrodynamic size (to 14.58 ± 2.01 nm according to DLS, Figure 1a) and a shortened retention time on SEC (Figure 1b, to 20.25 min). In vivo tumor targeting was investigated in 4T1 tumor bearing balb/c mice by positron emission tomography (PET). To minimize impact from tracer uptake in the abdomen, the tumors were inoculated to the right shoulders of mice. Z@FRT-scFv


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was conjugated with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and was labeled with 64Cu before injection. Region of interest (ROI) analysis found clear accumulation of the probes in tumors (Figure 3a&b), with the standardized uptake values (SUVs) raised from 1.81 ± 0.03 %ID/g at 1 h, to 3.62 ± 0.08 %ID/g and 3.27 ± 0.06 %ID/g, respectively, at 5 h and 24 h (n = 3). When 30× free scFv was injected as a blocking agent, the SUVs were significantly decreased to 1.62, 1.78, and 1.74 %ID/g at 1 h, 5 h, and 24 h (Figure 3a&b), suggesting that the tumor uptake was mediated by scFv-FAP interaction. The relative small difference in SUV in the early time point was attributed to the long circulation half-life of ferritins.24 Meanwhile, the uptake in normal organs, such as in the liver, kidney, and muscle, was barely changed when scFv was co-injected (Figure 3c&d). The tumor uptake was confirmed in a separate study where we i.v. injected IRDye800-Z@FRT-scFv or IRDye800-Z@FRT-scFv along with free scFv (30×) into the tumor models. We then analyzed the probe distribution in tumors at 24 h by IF microscopy (Figure 3e). Extensive FRT-scFv distribution was observed in tumors, and the distribution was overall correlated with positive anti-alpha smooth muscle actin (α-SMA) staining (which is a CAF biomarker3). In the control animals, on the contrary, very low Z@FRTscFv signal was observed (Figure 3e).

CAF-targeted PIT Leads to Tumor Suppression and Improved Survival Therapy studies were conducted with the same 4T1 tumor model (n = 5). Briefly, Z@FRTscFv (1.5 mg ZnF16Pc/kg) was i.v. administered, followed by photo-irradiation (671 nm, 300 mW/cm2 for 15 min) to tumor areas at 24 hr (1PIT group). In a separate group, a second PIT procedure was applied three days after the first PIT (2PIT). For controls, animals were injected with PBS and received no photo-irradiation. A detailed injection plan was shown in Figure S6.


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The PIT treatment led to efficient tumor suppression (Figure 4a). The 2PIT group, in particular, showed almost complete growth arrest in the first two weeks (88.60% tumor growth inhibition rate on Day 12), with 80% of the tumor showing size reduction. The treatment led to significantly improved animal survival. Specifically, the mean animal survival was 12.0 days for the control group, but was extended to 18.8 days for the 1PIT group and 29.2 days for the 2PIT group (Figure 4b). Meanwhile, there was no animal body weight drop during the course of the treatment (Figure 4c). Hematoxylin and eosin (H&E) staining also found no pathological abnormalities in the major organs (Figure 4d), suggesting minimal collateral damage by the PIT.

Impact of CAF-targeted PIT to Cancer Cells To further understand the therapeutic effects, in a separate study, we euthanized mice on Day 1, 2, 3, and 7 after PIT treatments and analyzed the tissue and blood samples (Figure S6). Compared to the control group, anti-FAP PIT efficiently killed CAFs in tumors, manifesting in a significantly reduced level of anti-α-SMA positive staining (Figure 5a & S7). In particular, only a background level of anti-α-SMA positive staining was observed after two PIT treatments, suggesting efficient elimination of CAFs (Figure 5a). The efficient CAF killing was also confirmed by significantly reduced serum levels of EGF (Figure 5b) and IL-6 (Figure 5c), to which CAFs are a major source of secretion.36 Notably, in the 1PIT group, IL-6 level was increased at early time points (Figure 5c). This is likely attributed to an increased secretion by T cells and macrophages in response to tissue damage.37 While efficiently killing CAFs, the direct impact of PIT on cancer cell viability was small. This was shown in a therapy study conducted with 4T1-luc inoculated animals, where we


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monitored viable cancer cells by bioluminescence imaging (BLI). In both 1PIT and 2PIT groups, there was a relatively low level of cancer cell death compared to the control group on Day 1 and 2 (Figure 5d). Specifically, ROI analysis showed that the total flux of photons on Day 2 were (6.80 ± 1.78) × 105 p/s and (4.29 ± 2.07) × 105 p/s for the 1PIT and 2PIT groups, respectively, compared to that of (6.35 ± 0.84) × 105 p/s for the control group. This suggests that the PIT selectively killed CAFs, but left behind most cancer cells. Despite, significant difference in cancer cell numbers appeared after Day 4. In particular, the BLI intensity started to drop in the 2PIT group after Day 5 (Figure 5d & 5e). These corroborated with terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling (TUNEL) staining, finding extensive apoptotic cells in both treatment groups on Day 7, especially in the 2PIT group (Figure 5f). These results suggest that the photodynamic therapy (PDT), which often causes immediate cell necrosis, was not the direct cause of the cancer cell death. Meanwhile, IF microscopy found that the amount of CD8+ T cells in tumors was significantly increased after either one dose or two doses of PIT (Figure 6a). By analyzing multiple slices, it was determined that relative to the control, positive staining in tumors was increased by 6.13 times in the 1PIT group and 19.0 times in the 2PIT group (Figure 6b). These results were in accord with flow cytometry data (Figure 6c&d), which found that the CD8+ T cell frequency in tumors was increased from 9.5 ± 1.8 % to 26.2 ± 4.9 % and 37.4 ± 7.3 %, respectively, on Day 7 after 1PIT and 2PIT (Figure 6c). It is thus postulated that it was tumorinfiltrating lymphocytes (TILs) which were responsible for the cancer cell apoptosis. Notably, enrichment of CD8+ T cells in tumor areas does not necessarily warrant cancer cell killing.38 Indeed, in the control group, CD8+ T cells were also spotted in tumors, but they were accumulated almost exclusively at the peripheral region (Figure 6a). This is attributed to stroma-


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mediated T cell exclusion, which was also observed in other types of primary tumors.10, 39 As a comparison, T cell infiltration was significantly improved after PIT treatment, manifested as extensive positive CD8+ staining in the central areas of tumors (Figure 6a). In parallel, an increased infiltration of neutrophils was also observed on H&E images (Figure 6e). These patterns correlate with the TUNEL staining (Figure 5f), indicating that in addition to an increase in T cell frequency, T cell infiltration enhancement is also critical to the tumor control. The reversed T cell exclusion is intriguing. As mentioned earlier, tumor stroma mediates T cell exclusion mainly through two means: one is to deposit a dense ECM layer surrounding tumors and the other is to secret CXCL12 to TME.10 To investigate, we performed Masson’s trichrome staining on tumor tissues (Figure 6f). In the control group, we observed many cell islets across tumor sections that were separated by a thick layer of collagen. After PIT treatment, we observed significantly destructed ECM wrapping (positive collagen staining) and less obvious cell colonies. In particular, for the 2PIT group, such nests were almost completely disappeared on Day 7, accompanied with a drastically reduced number of cancer cells (Figure 6f). Meanwhile, ELISA assay found that the serum concentration of CXCL12 was reduced from 37.78 ± 3.14 pg/mL in the control to 16.07 ± 5.90 pg/mL in 2PIT group on Day 7 (Figure 6g). Moreover, IL-10, which mediates activation and expansion of CD8+ T cells in tumors,40 was remarkably increased from 35.08 ± 5.62 pg/mL in the control to 165.81 ± 30.69 pg/mL and 347.61 ± 39.34 pg/mL, respectively, in the 1PIT and 2PIT groups on Day 3 (Figure 6h). Taken together, the studies suggest that by killing CAFs, PIT overcomes the T cell exclusion mechanisms mediated by tumor stroma, leading to improved T cell infiltration and expansion, eventually causing cancer cell death.


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3. Discussion The wide participation of CAFs in cancer development and the fact that FAP is expressed highly in CAFs but not in most normal tissues had made FAP a promising tumor targeting antigen.3 Previously, anti-FAP monoclonal antibody F19 and sibrotuzumab were developed and tested in the clinic. But while showing overall good tumor targeting,14 the antibody treatment showed no clinical efficacy,14, 15 which is probably due to the fact that blocking FAP alone is insufficient to impact CAF functions.41 Recently, Rosenberg et al. produced FAP-reactive CAR T cells and explored the immunotherapy with T cells.19 However, severe cachexia and lethal bone toxicities were observed in their studies;19 subsequent investigation found that the morbidity was attributed to the expression of FAP on multipotent bone marrow stromal cells.19 This corroborated with observation by Fearon et al., who found that experimental ablation of FAP+ cells in mice led to a loss of muscle mass and decreased B-lymphopoiesis and erythropoiesis.23 These new findings raised questions on using a systemic therapy against FAP+ cells. Unlike the previous approaches, the present method combines FAP-targeted photosensitizer delivery and localized photo-irradiation to achieve selective CAF elimination while minimally affecting normal tissues. Such an approach is novel and should be able to be used in conjugation with conventional chemotherapies to achieve enhanced tumor control. CAFs produce many of the main components of ECM such as type I, type III, and type V collagen and fibronectin,3, 4 and the secretion levels are found to be inversely correlated with tumor uptake of therapeutic molecules.42-44 Moreover, CAFs express a high level of α-SMA and as such acquire contractile properties;3 this leads to an increased interstitial fluid pressure (IFP)45 which poses an obstacle for delivery of therapeutics to tumors.9 It is highly plausible that killing CAFs may lead to enhanced efficiency of drug delivery. In fact, we and others have exploited


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PIT to target tumor vasculature46, 47 or perivascular cancer cells,48 and as such enhance tumor uptake of therapeutics. The approaches, however, have limitations such as narrow windows of effective irradiation doses,46, 47 lack of a unique cancer cell biomarker, and inefficient penetration of the PIT agents themselves. CAF-targeting PIT holds clear advantages in this context. A more exciting opportunity comes from the capacity of CAF-targeted PIT to modulate TME to promote anti-cancer immunity. It has long been observed that cancer-specific T cells can exist in a cancer patient while not efficiently control cancer growth.49 One major mechanism this is CAF-mediated T cell exclusion.10 There have been recent developments in immunotherapy that aims to augment the numbers and functions of cytotoxic T cells, including anti-CTLA-4 therapy,50 anti-PD-1/PD-L1 therapy,51 and CAR T-cell therapy.52 These approaches, however, may still be limited by the immune privilege induced by CAFs which exclude T cells from the vicinity of cancer cells.10 In the present study, we showed after CAF-targeting PIT, ECM surrounding tumors were destructed and the level of CXCL12 was decreased, followed by enhanced frequency and infiltration of CD8+ T cells in tumors. These results are intriguing, suggesting the potential of the PIT approach to modulate TME and to sensitize cancer cells to immunotherapy. It will be interesting to investigate combining the PIT with other immunotherapies for optimal cancer control. From the perspective of PDT development, the current study also represents an advance. Conventional PDT often uses non-targeting photosensitizer molecules and inflicts damage on both cancer cells and tumor microvessels.53 PIT, often achieved with antibody-photosensitizer conjugates, was recently developed and investigated in both pre-clinical and clinical studies by Kobayashi et al.54-57 Meanwhile, photodynamic therapy technologies utilizing artificial carriers such as polyester and polyacrylamide based nanoparticles, liposomes, silica particles, and


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magnetic nanoparticles, have also been reported.58-60 So far, however, the efforts have been focused on targeting cancer cells or endothelial cells. In the present study, we employed antiFAP scFv as a targeting ligand to navigate photodynamic damage to CAFs in tumors. We also used ferritin, a natural protein nanocage, as a photosensitizer carrier, which affords multiple advantages such as excellent biocompatibility, high photosensitizer loading capacity, and a compact size (~12 nm). These attempts are novel to PDT and the method holds great potential in clinical translation.

4. Conclusion Overall, we have developed a novel PIT technology that exploits ferritin as a photosensitizer carrier and anti-FAP scFv as a targeting ligand. Such FAP-targeted PIT effectively and selectively eliminates CAFs in tumors while not inducing systemic toxicity. The CAF elimination is followed by ECM destruction and CXCL12 secretion reduction, both of which contribute to a significantly enhanced CD8+ T cell infiltration, eventually leading to efficient cancer cell death. Our strategy suggests an alternative strategy for CAF-targeted cancer therapies, whose developments have been largely impeded due to toxicity issues observed with systematic anti-FAP treatments. Our observation that CAF elimination can promote T cell infiltration is novel, suggesting the great potential of the approach in combing with conventional chemo- and immune-therapy for optimal tumor management.


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Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Materials and Methods, Figures S1-S7. Corresponding author *E-mail: [email protected], [email protected] Author Contributions (Z.Z. and W.T.) These authors contributed equally to this paper. Notes The authors declare no competing financial interest. Acknowledgements The lung, liver and kidney tumor slices were a courtesy from professor Dexi Liu at the Pharmacy College of UGA. This research was supported by a DoD CDMRP grant (CA140666, J.X.), an NSF CAREER grant (NSF1552617, J.X.), a UGA-Augusta seed grant (J.X.), and an American Cancer Society grant (MSRG-12-034-01-CCE).


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References 1. Garin-Chesa, P.; Old, L. J.; Rettig, W. J. Proc. Natl. Acad. Sci. U S A 1990, 87, 72357239. 2. Sappino, A. P.; Skalli, O.; Jackson, B.; Schurch, W.; Gabbiani, G. Int. J. Cancer 1988, 41, 707-712. 3. Kalluri, R.; Zeisberg, M. Nat. Rev. Cancer 2006, 6, 392-401. 4. Tomasek, J. J.; Gabbiani, G.; Hinz, B.; Chaponnier, C.; Brown, R. A. Nat. Rev. Mol. Cell Biol. 2002, 3, 349-363. 5. Chang, H. Y.; Chi, J. T.; Dudoit, S.; Bondre, C.; van de Rijn, M.; Botstein, D.; Brown, P. O. Proc. Natl. Acad. Sci. U S A 2002, 99, 12877-12882. 6. Gabrilovich, D. I.; Chen, H. L.; Girgis, K. R.; Cunningham, H. T.; Meny, G. M.; Nadaf, S.; Kavanaugh, D.; Carbone, D. P. Nat. Med. 1996, 2, 1096-1103. 7. Elgert, K. D.; Alleva, D. G.; Mullins, D. W. J. Leukoc. Biol. 1998, 64, 275-290. 8. Muraoka, R. S.; Dumont, N.; Ritter, C. A.; Dugger, T. C.; Brantley, D. M.; Chen, J.; Easterly, E.; Roebuck, L. R.; Ryan, S.; Gotwals, P. J.; Koteliansky, V.; Arteaga, C. L. J. Clin. Invest. 2002, 109, 1551-1559. 9. Loeffler, M.; Kruger, J. A.; Niethammer, A. G.; Reisfeld, R. A. J. Clin. Invest. 2006, 116, 1955-1962. 10. Joyce, J. A.; Fearon, D. T. Science 2015, 348, 74-80. 11. Mukai, S.; Kjaergaard, J.; Shu, S. Y.; Plautz, G. E. Cancer Res. 1999, 59, 5245-5249. 12. Salmon, H.; Franciszkiewicz, K.; Damotte, D.; Dieu-Nosjean, M. C.; Validire, P.; Trautmann, A.; Mami-Chouaib, F.; Donnadieu, E. J. Clin. Invest. 2012, 122, 899-910. 13. Huber, M. A.; Kraut, N.; Park, J. E.; Schubert, R. D.; Rettig, W. J.; Peter, R. U.; GarinChesa, P. J. Invest. Dermatol. 2003, 120, 182-188. 14. Welt, S.; Divgi, C. R.; Scott, A. M.; Garin-Chesa, P.; Finn, R. D.; Graham, M.; Carswell, E. A.; Cohen, A.; Larson, S. M.; Old, L. J.; et al. J. Clin. Oncol. 1994, 12, 1193-1203. 15. Hofheinz, R. D.; al-Batran, S. E.; Hartmann, F.; Hartung, G.; Jager, D.; Renner, C.; Tanswell, P.; Kunz, U.; Amelsberg, A.; Kuthan, H.; Stehle, G. Onkologie 2003, 26, 44-48. 16. Wen, Y.; Wang, C. T.; Ma, T. T.; Li, Z. Y.; Zhou, L. N.; Mu, B.; Leng, F.; Shi, H. S.; Li, Y. O.; Wei, Y. Q. Cancer Sci. 2010, 101, 2325-2332. 17. Liao, D.; Luo, Y.; Markowitz, D.; Xiang, R.; Reisfeld, R. A. PLoS One 2009, 4, e7965. 18. Ostermann, E.; Garin-Chesa, P.; Heider, K. H.; Kalat, M.; Lamche, H.; Puri, C.; Kerjaschki, D.; Rettig, W. J.; Adolf, G. R. Clin. Cancer Res. 2008, 14, 4584-4592. 19. Tran, E.; Chinnasamy, D.; Yu, Z.; Morgan, R. A.; Lee, C. C.; Restifo, N. P.; Rosenberg, S. A. J. Exp. Med. 2013, 210, 1125-1135. 20. Dolznig, H.; Schweifer, N.; Puri, C.; Kraut, N.; Rettig, W. J.; Kerjaschki, D.; GarinChesa, P. Cancer Immun. 2005, 5, 10. 21. Niedermeyer, J.; Garin-Chesa, P.; Kriz, M.; Hilberg, F.; Mueller, E.; Bamberger, U.; Rettig, W. J.; Schnapp, A. Int. J. Dev. Biol. 2001, 45, 445-447. 22. Kraman, M.; Bambrough, P. J.; Arnold, J. N.; Roberts, E. W.; Magiera, L.; Jones, J. O.; Gopinathan, A.; Tuveson, D. A.; Fearon, D. T. Science 2010, 330, 827-830. 23. Roberts, E. W.; Deonarine, A.; Jones, J. O.; Denton, A. E.; Feig, C.; Lyons, S. K.; Espeli, M.; Kraman, M.; McKenna, B.; Wells, R. J.; et al. J. Exp. Med. 2013, 210, 1137-1151. 24. Zhen, Z.; Tang, W.; Chen, H.; Lin, X.; Todd, T.; Wang, G.; Cowger, T.; Chen, X.; Xie, J. ACS Nano 2013, 7, 4830-4837.


Page 17 of 26

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25. Zhen, Z.; Tang, W.; Guo, C.; Chen, H.; Lin, X.; Liu, G.; Fei, B.; Chen, X.; Xu, B.; Xie, J. ACS Nano 2013, 7, 6988-6996. 26. Falvo, E.; Tremante, E.; Arcovito, A.; Papi, M.; Elad, N.; Boffi, A.; Morea, V.; Conti, G.; Toffoli, G.; Fracasso, G.; et al. Biomacromolecules 2016, 17, 514-522. 27. Lin, X.; Xie, J.; Niu, G.; Zhang, F.; Gao, H.; Yang, M.; Quan, Q.; Aronova, M. A.; Zhang, G.; Lee, S.; et al. Nano Lett. 2011, 11, 814-819. 28. Rota, C.; Chignell, C. F.; Mason, R. P. Free Radic. Biol. Med. 1999, 27, 873-881. 29. Brocks, B.; Garin-Chesa, P.; Behrle, E.; Park, J. E.; Rettig, W. J.; Pfizenmaier, K.; Moosmayer, D. Mol. Med. 2001, 7, 461-469. 30. de Paula, C. A.; Coulson-Thomas, V. J.; Ferreira, J. G.; Maza, P. K.; Suzuki, E.; Nakahata, A. M.; Nader, H. B.; Sampaio, M. U.; Oliva, M. L. J. Biol. Chem. 2012, 287, 170-182. 31. Hossain, M. I.; Switalska, M.; Peng, W.; Takashima, M.; Wang, N.; Kaiser, M.; Wietrzyk, J.; Dan, S.; Yamori, T.; Inokuchi, T. Eur. J. Med. Chem. 2013, 69, 294-309. 32. Sun, H.; Liu, D. Cancer Gene Ther. 2016, 23, 54-60. 33. Niedermeyer, J.; Scanlan, M. J.; Garin-Chesa, P.; Daiber, C.; Fiebig, H. H.; Old, L. J.; Rettig, W. J.; Schnapp, A. Int. J. Cancer 1997, 71, 383-389. 34. Di Marco, M.; Shamsuddin, S.; Razak, K. A.; Aziz, A. A.; Devaux, C.; Borghi, E.; Levy, L.; Sadun, C. Int. J. Nanomed. 2010, 5, 37-49. 35. Thamake, S. I.; Raut, S. L.; Ranjan, A. P.; Gryczynski, Z.; Vishwanatha, J. K. Nanotechnology 2011, 22, 035101. 36. Bhowmick, N. A.; Neilson, E. G.; Moses, H. L. Nature 2004, 432, 332-337. 37. Kopf, M.; Baumann, H.; Freer, G.; Freudenberg, M.; Lamers, M.; Kishimoto, T.; Zinkernagel, R.; Bluethmann, H.; Kohler, G. Nature 1994, 368, 339-342. 38. McGray, A. J.; Hallett, R.; Bernard, D.; Swift, S. L.; Zhu, Z.; Teoderascu, F.; Vanseggelen, H.; Hassell, J. A.; Hurwitz, A. A.; Wan, Y.; et al. Mol. Ther. 2014, 22, 206-218. 39. Chiba, T.; Ohtani, H.; Mizoi, T.; Naito, Y.; Sato, E.; Nagura, H.; Ohuchi, A.; Ohuchi, K.; Shiiba, K.; Kurokawa, Y.; et al. Br. J. Cancer 2004, 91, 1711-1717. 40. Emmerich, J.; Mumm, J. B.; Chan, I. H.; LaFace, D.; Truong, H.; McClanahan, T.; Gorman, D. M.; Oft, M. Cancer Res. 2012, 72, 3570-3581. 41. Kelly, T.; Huang, Y.; Simms, A. E.; Mazur, A. Int. Rev. Cell Mol. Biol. 2012, 297, 83116. 42. Netti, P. A.; Berk, D. A.; Swartz, M. A.; Grodzinsky, A. J.; Jain, R. K. Cancer Res. 2000, 60, 2497-2503. 43. Boucher, Y.; Brekken, C.; Netti, P. A.; Baxter, L. T.; Jain, R. K. Br. J. Cancer 1998, 78, 1442-1448. 44. Heldin, C. H.; Rubin, K.; Pietras, K.; Ostman, A. Nat. Rev. Cancer 2004, 4, 806-813. 45. Gabbiani, G. J. Pathol. 2003, 200, 500-503. 46. Snyder, J. W.; Greco, W. R.; Bellnier, D. A.; Vaughan, L.; Henderson, B. W. Cancer Res. 2003, 63, 8126-8131. 47. Zhen, Z.; Tang, W.; Chuang, Y. J.; Todd, T.; Zhang, W.; Lin, X.; Niu, G.; Liu, G.; Wang, L.; Pan, Z.; Chen, X.; Xie, J. ACS Nano 2014, 8, 6004-6013. 48. Sano, K.; Nakajima, T.; Choyke, P. L.; Kobayashi, H. ACS Nano 2013, 7, 717-724. 49. Gajewski, T. F.; Schreiber, H.; Fu, Y. X. Nat. Immunol. 2013, 14, 1014-1022. 50. Weber, J. S.; D'Angelo, S. P.; Minor, D.; Hodi, F. S.; Gutzmer, R.; Neyns, B.; Hoeller, C.; Khushalani, N. I.; Miller, W. H., Jr.; Lao, C. D.; et al. Lancet Oncol. 2015, 16, 375-384.


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51. Topalian, S. L.; Hodi, F. S.; Brahmer, J. R.; Gettinger, S. N.; Smith, D. C.; McDermott, D. F.; Powderly, J. D.; Carvajal, R. D.; Sosman, J. A.; Atkins, M. B.; et al. N. Engl. J. Med. 2012, 366, 2443-2454. 52. Gross, G.; Eshhar, Z. Annu. Rev. Pharmacol. Toxicol. 2016, 56, 59-83. 53. Agostinis, P.; Berg, K.; Cengel, K. A.; Foster, T. H.; Girotti, A. W.; Gollnick, S. O.; Hahn, S. M.; Hamblin, M. R.; Juzeniene, A.; Kessel, D.; et al. CA Cancer J. Clin. 2011, 61, 250281. 54. Kobayashi, H.; Choyke, P. L.; Ogawa, M. Curr. Opin. Chem. Biol. 2016, 33, 32-38. 55. Nagaya, T.; Nakamura, Y.; Sato, K.; Zhang, Y. F.; Ni, M.; Choyke, P. L.; Ho, M.; Kobayashi, H. Oncotarget 2016, 7, 23361-23369. 56. Sato, K.; Nagaya, T.; Mitsunaga, M.; Choyke, P. L.; Kobayashi, H. Cancer Lett. 2015, 365, 112-121. 57. Mitsunaga, M.; Ogawa, M.; Kosaka, N.; Rosenblum, L. T.; Choyke, P. L.; Kobayashi, H. Nat. Med. 2011, 17, 1685-1691. 58. Lucky, S. S.; Soo, K. C.; Zhang, Y. Chem. Rev. 2015, 115, 1990-2042. 59. Chatterjee, D. K.; Fong, L. S.; Zhang, Y. Adv. Drug Deliv. Rev. 2008, 60, 1627-1637. 60. Calixto, G. M.; Bernegossi, J.; de Freitas, L. M.; Fontana, C. R.; Chorilli, M. Molecules 2016, 21, 342. 61. Zhou, B.; Wang, H.; Liu, R.; Wang, M.; Deng, H.; Giglio, B. C.; Gill, P. S.; Shan, H.; Li, Z. Mol. Pharm. 2015, 12, 3527-3534.


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Figures

Scheme 1. Schematic illustration showing the working mechanism of FAP-targeted PIT. After systemic injection, scFv-Z@FRT home to CAFs in tumors and, following photo-irradiation, selectively kill CAFs. This causes destruction of ECM and reduced secretion of CXCL12, promoting infiltration of CD8+ T cells that mediate cancer cell death.


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Figure 1. Nanocage characterization. (a) DLS analysis of FRT, Z@FRT, and scFv-Z@FRT. (b) SEC analysis of FRT, Z@FRT, scFv, and scFv-Z@FRT. The retention times were 22.423, 22.439, 29.627, and 20.247 min, respectively. (c) Photographs of scFv-Z@FRT and free ZnF16Pc in aqueous solutions at different conditions.


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Figure 2. Selective binding against FAP+ cells. (a) IF microscopy with HTB-135 and 3T3 cells. Red, IRDye800 (anti-FAP scFv); blue, DAPI. Scale bar, 50 µm. (b) IF microscopy with tumor tissues taken from lung, liver, and kidney metastases. Green, FITC; red, IRDye800; blue, DAPI. Scale bars, 100 µm.


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Figure 3. In vivo tumor targeting with Z@FRT-scFv, evaluated in a 4T1 tumor bearing balb/c mice. (a) PET imaging results. Z@FRT-scFv-DOTA-64Cu was i.v. injected. Images were acquired at 1, 5, and 24 h post injection. In a control group, free anti-FAP scFv (30x) was administered as a blocking agent. (b) Tumor uptake at different time points, calculated based on decay-corrected coronal imaging results. (c,d) Distribution of Z@FRT-scFv-DOTA-64Cu in the liver, kidney and muscle, with and without scFv blocking. e) IF microscopy results with IRDye800 labeled Z@FRT, with and without scFv blocking.


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Figure 4. Impact of FAP-targeted PIT on tumor growth and survival. (a) Tumor growth curves. Relative to the control, tumor growth was significantly suppressed after PIT, especially in the 2PIT group. (b) Kaplan-Meier plot of animal survival. (c) Animal body weight changes. No detectable impact from PIT was observed. (d) H&E staining of tissues taken from major organs after therapy. Scale bar: 50 µm.


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Figure 5. Impact of FAP-targeted PIT on cancer cell proliferation. (a) IF microscopy analysis. Green, α-SMA, a common CAF bio-marker; blue, DAPI. Scale bar, 100 µm. (b) Plasma EGF concentration changes over PIT treatment. (c) Plasma IL-6 concentration changes over PIT treatment. (d) BLI to track tumor growth after PIT. (e) Viable cancer cell number changes, based on ROI analysis on images from (d). (f) TUNEL staining, conducted with tumor tissues taken on Day 7 from different treatment groups. Green, TUNEL; blue, DAPI; scale bar, 50 µm.


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Figure 6. PIT induced T cell infiltration enhancement. (a) anti-CD8a staining. Tumor tissues were taken on Day 3 post treatment. “C” and “P” indicate the central and peripheral regions of a tumor, respectively. Red, CD8+ T cells; blue, DAPI. Scale bar, 50 µm. (b) CD8+ cell frequency in tumors, based on fluorescence intensity in (a). **, P

Protein Nanocage Mediated Fibroblast-Activation Protein Targeted

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