Bifunctional fusion proteins derived from Tumstatin and 4-1BBL for

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Bifunctional fusion proteins derived from Tumstatin and 4-1BBL for targeted cancer therapy Chao Sun, Dongyang He, Chao Ma, Zhenyue Gao, Yijun Chen, and Shuzen Wang Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/ acs.molpharmaceut.8b01190 • Publication Date (Web): 19 Dec 2018 Downloaded from http://pubs.acs.org on December 21, 2018

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Molecular Pharmaceutics

Bifunctional Fusion Proteins Derived from Tumstatin and 4-1BBL for Targeted Cancer Therapy

Chao Sun, Dongyang He, Chao Ma, Zhenyue Gao, Yijun Chen*, and Shuzhen Wang*

State Key Laboratory of Natural Medicines and Laboratory of Chemical Biology, China Pharmaceutical University, Nanjing 210009, China

C. S. and D. H. contributed equally to this work.

*Corresponding author: Yijun Chen: #24 Tongjiaxiang, Nanjing 210009, China; Tel: +86 25 83271045; E-mail: [email protected]; Shuzhen Wang: #24 Tongjiaxiang, Nanjing 210009, China; Tel: +86 25 83271031; E-mail: [email protected].

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ABSTRACT: The therapeutic utilities of antiangiogenesis and immunotherapy have been proven in clinics, and cancer patients have benefited from respective therapy. Given that the combination of both therapeutic strategies may further improve the effectiveness, a recombinant human 4-1BBL/Tumstatin fusion protein (rh4TFP) library was constructed in the present study to target both angiogenesis and T lymphocyte activation, in which the fragments of an endogenous angiogenesis inhibitor Tumstatin and a T lymphocyte co-stimulatory 4-1BBL are coupled with different linkers. After comparison of different combinations, rh4TFP-2 was found to show a promise on potential antiangiogenic immunotherapy. On one hand, rh4TFP-2 inhibited proliferation and migration of human umbilical vein endothelial cells, exhibiting the antiangiogenic activity similar to Tumstatin. On the other hand, rh4TFP-2 led to significant increase of T lymphocyte activation for the release of IL-2 and IFN-γ, showing the T lymphocyte activation by 4-1BBL. Moreover, administration of rh4TFP-2 suppressed tumor growth and prolonged survival in a B16F10 melanoma-bearing mouse model. Taken together, the present study provides a new approach of using bifunctional fusion proteins to target both angiogenesis and T lymphocyte activation for cancer therapy.

KEYWORDS: antiangiogenesis, bifunctional fusion proteins, cancer immunotherapy, Tumstatin, 4-1BBL


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1. INTRODUCTION As one of the hallmarks of cancer, angiogenesis has long been recognized as an attractive therapeutic approach for suppressing tumor growth.1 Since the approval of the first antiangiogenic agent fourteen years ago, a large number of cancer patients have benefited from the therapies against angiogenesis. However, continued investigations have identified major limitations associated with this anticancer strategy, including insufficient clinical efficacy, intrinsic refractoriness and drug resistance.2 Thus, more effective ways are needed for the development through the combination of antiangiogenic therapy with other established therapies or new treatment options. Meanwhile, cancer immunotherapy, named as 2013’s Breakthrough of the Year by Science, is therefore an ideal choice for such a partner to combine.3 By modulating or harnessing the immune system against tumors, cancer immunotherapy has demonstrated to exhibit great potential for the control of cancers. However, it is still challenging from a number of aspects regarding drug development, particularly the serious adverse reactions.4 A series of clinical successes of immunotherapy by blocking antibodies against two co-inhibitory receptors that suppress T lymphocyte activities, cytotoxic T-lymphocyte antigen 4 (CTLA-4) and programmed cell death protein 1 (PD-1), have been reported in recent years and restored the confidence of numerous researchers and enterprises in cancer immunotherapy worldwide.4 This has also endorsed impetus for therapeutically



targeting other co-stimulatory and co-inhibitory molecules, including 4-1BB (also known as TNFRSF9 or CD137) and lymphocyte activation gene 3 protein (LAG3).5 Further analysis of the strengths and weaknesses of antiangiogenic therapy and immunotherapy has suggested that two strategies could be complementary in cancer treatment. Antiangiogenesis can induce rapid tumor regressions by sequestering cancerous cells from an adequate blood supply, while immunotherapy is especially useful for attacking small metastases by activation of cancer specific T cell-mediated immune responses.6 This warrants the feasibility and potential benefits of combining these strategies. Meanwhile, the synergetic effect has already been observed when antiangiogenic molecules and immunotherapeutic protocols are combined in several clinical studies.7 Given that the combination of both strategies may potentially further improve the effectiveness of antitumor response and reduce respective adverse reactions, we herein describe the construction and functional analysis of a bifunctional fusion protein library that simultaneously targets angiogenesis and T lymphocyte activation, in which the fragments of an endogenous angiogenesis inhibitor Tumstatin (the NC1 domain of α3 chain of type IV collagen) 8-12 and a co-stimulatory molecule of T-cells, 4-1BB ligand (4-1BBL),13-18 are fused with different linkers. After comparison of various combinations, a recombinant human 4-1BBL/Tumstatin fusion protein (rh4TFP-2) was found to show favorable antitumor responses against melanoma both in vitro and in a B16F10-bearing mouse model. Our results indicate that the combination of Tumstatin and 4-1BBL could be a promising approach for


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antiangiogenic cancer immunotherapy.

2. EXPERIMENTAL SECTION 2.1. Reagents and strains. The indoleamine 2,3-dioxygenase (IDO) inhibitor NLG919 (CAS No. 1402836-58-1) was purchased from ApexBio. The active fragment of Tumstatin, namely T7 peptide, exhibiting an equivalent antiproliferative effect on endothelial cells as full length of Tumstatin protein in vitro,11 was synthesized by ChinaPeptides Co., Ltd (Suzhou, China). RPMI1640 medium and fetal bovine serum (FBS) was purchased from Gibco Life Technologies (Carlsbad, CA, USA). RBC Lysis Buffer (#FMS-RBC100) was from Fcmacs Biotech Co., Ltd (Nanjing, China). Anti-CD3e (#553057) and anti-CD28 (#553294) were purchased from BD Biosciences (New Jersey, USA). Anti-CD31 mAb (#ab24590) was purchased from Abcam (Cambridge, UK). Fluorochrome-labeled antibodies including CD3e-FITC (clone 145-2C11, #11-0031-81), CD4-PE (clone GK1.5, #12-0041-81) and CD8a-APC (clone 53-6.7, #17-0081-81) were obtained from Thermo Fisher Scientific Inc. (Waltham, MA, USA). CD45-VIO-PE770 (clone 30F11, # 130-117-529) was from Miltenyi Biotec (Bergisch Gladbach, Germany). E. coli Top10, E. coli BL21 (DE3) and pET11a (+) vector were from Novagen (Madison, WI, USA). Plasmid pMD18T was purchased from Takara (Dalian, China). 2.2. Cell culture and animals. Mouse melanoma B16F10 cells and human umbilical vein endothelial cells (HUVECs) originated from ATCC were obtained from Nanjing KeyGen Biotech. Co. Ltd. (Nanjing, China), authenticated by short tandem repeat



(STR) matching analysis, and cultured in RPMI 1640 and F12K medium, respectively. Both medium are supplemented with 10% fetal bovine serum, 2 mM L-glutamine, penicillin (50 IU/mL) and streptomycin (50 mg/mL) at 37℃ in a humidified atmosphere 5% CO2. No mycoplasma contamination was detected. C57BL/6 male mice provided by Model Animal Research Center of Nanjing University (Nanjing, China) were bred under pathogen-free conditions and used for the experiments at 6-8 weeks of age. All animal experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Animal Experimentation Ethics Committee of China Pharmaceutical University. 2.3. Construction of plasmids. Genes encoding the entire extracellular domain of human 4-1BBL (Ex4-1BBL, GenBank No.: NP_003802.1, amino acids 49-254) was subcloned from pET-22b-4-1BBL constructed in our laboratory previously13, 14 to pET-11a (+) between Nde I and Nhe I to produce plasmid pET-11a-Ex4-1BBL. Genes encoding 14 types of combinations of Tumstain (GenBank No.: AAF72632.1) and 4-1BBL fragments through different linkers (Table 1) were synthesized and subcloned into pET-11a (+) between Nde I and Nhe I by Shanghai Generay Biotech Co., Ltd (Shanghai, China) to yield 14 pET11a-rh4TFP plasmids. The positive recombinant plasmids were confirmed by sequencing and transformed into E. coli BL21 (DE3) for protein expression. 2.4. Expression and preparation of rh4TFP proteins. E. coli BL21 (DE3) harboring the plasmid pET11a-Ex4-1BBL or pET11a-rh4TFPs was screened in a LB


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agar plate with 100 μg ampicillin/mL. Single colonies of transformed E. coli BL21 (DE3) strain were inoculated into LB medium and cultured at 37 °C in I26R stackable incubator shakers (New Brunswick, USA) at 220 rpm. The induction of target proteins was initiated at an OD600 of 0.7 by adding 1 mM IPTG. After 4 h of induction, the cells were harvested by centrifugation and disrupted at 4 °C with a high-pressure cell-disruption system (Constant Systems, Daventry, UK). The inclusion bodies were isolated and solubilized by denaturation buffer containing 50 mM Tris-HCl, 6 M GdHCl, 5 mM DTT and 1 mM EDTA, pH 8.2. The renaturation was carried out by slowly dropping the solubilized proteins (20 µg/mL) into renaturation buffer (0.3 M GdHCl, 0.8 M L-Arginine, 0.1 mM GSSG, 0.1 mM GSH, 20 mM NaCl, 0.8 mM KCl and 1 mM EDTA) at a constant stirring speed at 4 ℃ for 24 h. The refolded proteins were concentrated and dialysised in a buffer containing 50 mM Tris-HCl, 1mM NaCl and 1 mM EDTA at 4 ℃ for 12 h with a centrifugal filter device (Millipore, USA). Target proteins were examined by SDS-PAGE and protein concentrations were quantified by BCA assay (KeyGen, China). 2.5. HUVEC proliferation assay. The HUVEC proliferation was determined using a Cell Counting Kit‑8 (CCK‑8) assay according to the manufacturer’s instructions. Cells were seeded in 96-well plate at a density of 1×104 cells/well. After overnight incubation, solutions of rh4TFP proteins or T7 peptide with incremental concentrations (0-32 μM) were added to the cells and further incubated for 48 h. Then, CCK-8 solution was added to each well with a final volume of 10% and the cells were cultured for another 1 h. The absorbance at 450 nm was measured using a microplate



reader (BioTek, USA). IC50 values were calculated by four-parameter logistic regression using PRISM 5.0 (GraphPad). 2.6. Stimulation of T lymphocyte in vitro. Mouse T lymphocytes were purified according to the instructions of Mouse T Cell Enrichment Kit (Stem Cell) and cultured as described in our previous study.13 Purified T lymphocytes (1.5×104 cells/well) were seeded into flat bottom 96-well plates and stimulated by 8 μM Ex4-1BBL or incremental concentrations of rh4TFP proteins (0-32 μM) in the presence of 2 μg/mL coated anti-CD3 mAb and 2 μg/mL soluble anti-CD28 mAb. The T lymphocytes stimulated by anti-CD3 plus anti-CD28 mAb were used as negative controls. After co-stimulation for 72 h, T lymphocyte proliferation was measured by CCK‑8 assay and EC50 values were calculated. 2.7. Adhesion assay of HUVEC. Assay for HUVECs adhesion to Matrigel was performed as described previously with some modifications.19 Briefly, HUVECs (5×104 cells/well) were seeded into 50% Matrigel (BD Biosciences, USA) pre-coated flat bottom 96-well plate. Incremental concentrations of rh4TFP-2 or T7 peptide (0-8 μM) were added to each well. Plates were incubated for 90 min at 37 °C. After two rinses with PBS, adherent cells were quantified using CCK-8 assay and cell adhesion rates were calculated. 2.8. Wound-healing scratching assay of HUVEC. To analyze two-dimensional migration, a wound-healing scratching assay was performed.19 HUVECs (5×105 cells/well) were seeded onto 6-well plates. After the formation of confluent monolayers, wounds were created with a sterile pipet tip. After washing away


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suspended cells, cells were incubated in culture medium supplemented with or without rh4TFP-2 or T7 peptide (0-2 μM). Three parallel wounds were created in each well, and their locations were marked on the bottom of the plate. The areas newly occupied as a result of cell migration were measured at 24 h after scratching by photographing them under a microscope. The migration distances were determined using Image J software and relative migration rates were then calculated. 2.9. Tube formation assay of HUVEC. For tube formation assay, HUVECs (4×104 cells/well) were seeded into 50% Matrigel (BD Biosciences, USA) pre-coated flat bottom 24-well plate in the presence of incremental concentrations of rh4TFP-2 or T7 peptide (0-16 μM). After incubation for 12 h at 37℃, tube formation was evaluated in five randomly chosen fields at 200 × magnification using a microscope.19 2.10. Cytotoxicity assay of T lymphocytes. To examine the cell killing activities of T cells against tumor cells, a T lymphocyte-B16F10 cell co-culture study was conducted as described previously with some modifications.20 Purified T lymphocytes (2×105 cells/well) and B16F10 cells (2×104 cells/well) at an effector-to-target ratio of 10:1 were seeded into 2 μg/mL anti-CD3 mAb pre-coated flat bottom 96-well plates and cultured in the presence of 2 μg/mL soluble anti-CD28 mAb and incremental concentrations of rh4TFP-2 (0-16 μM) for 3 or 5 days. T lymphocytes co-stimulated by anti-CD3 mAb, anti-CD28 mAb and different concentrations of rh4TFP-2 were used as the control of effector groups, and B16F10 cells without any treatments were the control of target group. The cell killing effects on B16F10 cells by T lymphocytes were determined by CCK-8 assay. The killing rates were calculated according to



following formula: [1-(OD450/experimental-OD450/effector)/OD450/target] × 100%. 2.11. Antitumor effects in tumor xenograft in C57BL/6 mice. Tumors were established by subcutaneously injection of 1×106 B16F10 cells into the right flank of C57BL/6 mice. Tumor growth was monitored once every two days. Tumor volume was calculated as (L × W2)/2, where L is the length and W is the width of tumor. Treatments were initiated when tumors reached about 20 mm3. rh4TFP-2 (10 mg/kg) or Tris-HCl solution (pH 8.0, blank control) was administered via i.v. once every two days for a total of 11 days, NLG919 (50 mg/kg, positive control) was administered via i.g. once per day.21 At the end of the experiments, tumors were dissected and photographed, and wet weights of each tumor were measured. 2.12. Detection of cytokine release. Purified T lymphocytes were co-stimulated as described above in flat bottom 96-well plates. The levels of cytokines released in the supernatants of T lymphocyte cultures were determined on day 3 and 5 by ELISA methods according to the manufacturer’s instructions using mouse IL-2 and IFN-γ ELISA Ready-SET-Go kits (eBioscience, USA). In the in vivo experiments, the mice were sacrificed on day 11 after tumor challenge, and the serum from each mouse was gathered. Then, the concentration of cytokine in each sample was determined by ELISA assay as described above. 2.13. Flow cytometry analysis of lymphocyte subsets. Peripheral blood samples from mice were harvested one day following the last treatment and lysed by RBC Lysis Buffer. Single cell suspensions were co-stained by fluorochrome-labeled antibodies against CD45, CD3, CD4 and CD8. The T lymphocyte subpopulations


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were analyzed by flow cytometry. Stained cells were acquired using a BD FACS Canto II flow cytometer, and data were analyzed by FACS Diva and FlowJo software. 2.14. Immunohistochemistical analysis. Six tumors were randomly selected from rh4TFP-2, NLG919 or control groups. Tissues were fixed in 10% formaldehyde for 12 h, paraffin-embedded, sectioned, and placed on slides. The tissues were immunostained with anti-CD31 mAb or counterstained with diluted Harris hematoxylin and eosin (H&E). After staining, microvessel density (MVD) was determined by calculating the average number of microvessels from five highly vascular fields in each section (at a magnification of 200×). 2.15. Statistical analysis. Each experiment was repeated for more than three times. Results were expressed as the mean ± SD. All statistical analysis was conducted using Prism 5.0 (Graph Pad software, Inc) and PASW Statistics18.0 software package. Statistical analysis of multiple-group comparisons was performed by one-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test. Comparisons between 2 groups were analyzed using 2-tailed Student t-tests. The survival of C57BL/6 mice was evaluated by Kaplan-Meier analysis using the log-rank test to compare the difference. A P value < 0.05 was considered statistically significant, a value of < 0.01 as very significant and a value of < 0.001 as highly significant.

3. RESULTS 3.1. Design of rh4TFP proteins derived from Tumstatin and 4-1BBL. Since linker



has become an indispensable component of recombinant fusion proteins,22, 23 two sets of linkers composed of four flexible linkers (F1-F4) and four rigid linkers (R1-R4) were chosen to fuse T7 peptide with Ex4-1BBL, yielding fusion proteins rh4TFP 1-8 (Table 1). Then, two commonly used empirical linker F1 and R1 were utilized to ligate three fragments of Tumstatin (amino acids 45-98, 60-132, 60-98) with a fragment of Ex4-1BBL (amino acids 50-240), producing fusion proteins rh4TFP 9-14. As a result, the bifunctional fusion protein library consisting of 14 rh4TFP proteins in total were designed. In this library, different lengths of Tumstatin to exhibit possible variations on activity were combined with Ex4-1BBL based on previous reports and our attempt.8, 15 3.2. Production and purification of rh4TFP proteins. Although different fermentation conditions (such as pH, temperature, inducer concentration, medium composition, inoculum volume, and co-expression with chaperones) were compared, rh4TFP proteins were mainly expressed in the form of conclusion body in E. coli.14 Therefore, we chose inclusion body as the form for expression in order to obtain sufficient amount of recombinant proteins. The yield of inclusion bodies for all rh4TFP proteins with different linkers or fusions showed no significant differences, and an average of 740 mg of protein per litre of cell cultures was achieved. All refolded rh4TFP proteins reached a purity of greater than 90% by SDS-PAGE with a final yield of 4.9 mg from 100 mg of inclusion bodies (Figure 1A and B). Different from the previously reported multimeric forms of AviTag-CD137L,24 all purified rh4TFP proteins exhibited a clear single peak on


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Superdex-200 column (data not shown), and their molecular weights were estimated to be around 400 kDa, suggesting that rh4TFP proteins refolded from the inclusion bodies are in the polymeric states. 3.3. Inhibition the proliferation of HUVECs by rh4TFP proteins. To investigate the antiangiogenic activity in vitro, rh4TFP proteins were evaluated on the proliferation of HUVECs. Cell numbers relative to Tris-HCl solution treated controls were determined using CCK-8 assay. As expected, all rh4TFP proteins effectively inhibited the proliferation of the endothelial cells. Overall population mean IC50 value (rh4TFP concentration inhibiting cell growth by ~50% relative to Tris-HCl control) was between 4.05±0.22 µM (rh4TFP-12) and 9.42±0.10 µM (rh4TFP-5) (Table 1). However, all rh4TFP proteins exhibited higher IC50 vales than T7 peptide (4.02±0.20 µM), suggesting that fusion with other fragments would interfere the antiangiogenic activity of T7 peptide in a small degree. 3.4. Stimulation of T lymphocyte proliferation by rh4TFP proteins. To investigate the co-stimulation function of rh4TFP proteins for T cell activation, we next measured the effects of rh4TFP proteins on T lymphocyte proliferation. All rh4TFP proteins except for rh4TFP-4 and rh4TFP-10 could markedly promote T lymphocytes (about 99% purity) growth without substantial difference compared to Ex4-1BBL (Table 1). The EC50 values (rh4TFP protein concentration required to increase cell growth by ~50% relative to anti-CD3 plus anti-CD28 control) for the enhancement of T lymphocyte growth by rh4TFP proteins ranged from 6.45±0.26 µM (rh4TFP-7) to 11.45±0.18 µM (rh4TFP-10), while the EC50 value of non-fused Ex4-1BBL was



7.30±0.44 µM (Table 1). After a comprehensive assessment of the expression, purification as well as the values of IC50 against HUVECs and EC50 against T lymphocytes of all rh4TFP proteins, rh4TFP-2 was chosen to further investigate its antiangiogenic activities and T cell co-stimulatory activities in the following study. 3.5. Inhibition of adhesion, migration and tube formation of HUVCEs by rh4TFP-2. To examine the antiangiogenic activity of rh4TFP-2, the effects of rh4TFP-2 on HUVEC adhesion to Matrigel were firstly investigated. As shown in Figure 2A, both rh4TFP-2 and T7 peptide treated cells showed a significant reduction in adhesive ability compared to untreated cells. Next, the antimigratory effects of rh4TFP-2 were assessed using a wound-healing scratching assay. The results showed that both rh4TFP-2 and T7 peptide markedly delayed wound closure in a dose-dependent manner (Figure 2B). The relative migration rate of HUVEC treated with 2 μM rh4TFP-2 was only about 8.89±4.97% at 24 h (Figure 2C). In addition, the effects of rh4TFP-2 on the endothelial cell tube formation were explored, showing that both rh4TFP-2 and T7 peptide significantly inhibited capillary-like tube formation (Figure 2D). rh4TFP-2 also exhibited superior inhibitory effects than T7 peptide at 4 µM and higher concentrations (Figure 2E). Collectively, these results suggest that rh4TFP-2 could significantly inhibit the adhesion, migration and tube formation of HUVEC in vitro. 3.6. Enhancement of the cytotoxicity of activated T lymphocytes by rh4TFP-2. To evaluate the 4-1BBL co-stimulatory activity of rh4TFP-2, we stimulated purified T cells with incremental concentrations of rh4TFP-2 and analysed the cytotoxicity of


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activated T lymphocytes against B16F10 melanoma cells by co-culturing at an effector-to-target ratio of 10:1. As shown in Figure 3A, rh4TFP-2 significantly enhanced the cytotoxicity of T lymphocytes in a dose- and time-dependent manner. After cultured for 5 days, the killing rate of 16 μM rh4TFP-2 reached 83.41±5.84%. The potent ability of T lymphocytes to kill B16F10 cells was mediated by the enhanced release of cytokines. As shown in Figure 3B, stimulation of T lymphocytes by rh4TFP-2 effectively induced elevated production of IFN-γ and IL-2 within the same time-frame. 3.7. Antitumor efficacy of rh4TFP-2 in B16F10 melanoma xenograft mice. The in vivo antitumor efficacy of rh4TFP-2 was further evaluated with B16F10 melanoma tumor bearing C57BL/6 mice (12/group) using NLG919 as a positive control. The rh4TFP-2 treatment was started after subcutaneous lumps became palpable. Statistical analyses indicated that tumor growth was significantly inhibited by rh4TFP-2 (Figure 4). The final tumor volume and weight were markedly lower from the treatments by rh4TFP-2 and NLG919 compared to control (Figure 4A-C). Cell apoptosis was also observed by HE staining in the treatments by rh4TFP-2 and NLG919 (Figure 4D). The median survival time of mice xenografted B16F10 cells was prolonged to 20 and 18 days by rh4TFP-2 and NLG919 respectively, compared to 14 days for control (Figure 4E). No significant changes on body weight, thymus index and spleen index were observed in these experiments (Figure S1). Moreover, degeneration or necrosis was not found in major tissues including lung, liver, spleen, kidney and heart under microscopic examination (Figure S2), indicating that rh4TFP-2 possesses a safer



pharmacological profile in addition to its favourable efficacy in suppressing melanoma cell growth in vivo. 3.8. Antiangiogenesis and immune activation of rh4TFP-2 in vivo. To ascertain the antiangiogenic activity of rh4TFP-2 in vivo, we examined the expression levels of CD31, a well-recognized marker of angiogenesis, in tumor tissues from the xenografted mice by immunohistochemical analysis. The mean MVD in CD31 stained tumor sections from the mice treated with rh4TFP-2 were significantly lower than that in control group (Figure 5A). To delineate the immunological mechanism of antitumor activity of rh4TFP-2, immune cell subpopulations and cytokines were analyzed after the B16F10 tumor bearing mice were treated with rh4TFP-2 for 11 days. The representative results of the T lymphocyte subpopulations identified by flow cytometry are shown in Figure 5B. A significant increase in the percentage of CD3+, CD3+CD4+ and CD3+CD8+ T cells was observed in rh4TFP-2 treated mice (Figure 5C). In addition, the cytokine secretion of IL-2/IFN-γ in serum was markedly increased by rh4TFP-2 treatment as anticipated (Figure 5D). Taken together, the antitumor activity of rh4TFP-2 was mediated through the synergetic effects of antiangiogenesis and immune activation.

4. DISCUSSION As the products of recombinant DNA technology, more than 130 therapeutic proteins have been widely used in the treatment of a broad range of diseases such as cancer, diabetes, anemia and infections.25 Fusion proteins, a class of macromolecules with


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multi-functional properties, have emerged as promising candidates for cancer drug discovery over the last decade.26 To construct an effective recombinant fusion protein combining antiangiogenesis and immunotherapy, Tumstatin and 4-1BBL were chosen in the present study as two major components based on the encouraging results from their individual activity.8-18 Moreover, it was recently evidenced that activation of 4-1BB signaling promotes angiogenesis, which also underlines the rationale for immunotherapy targeting 4-1BB in combination with antiangiogenesis.27 Our previous study revealed that rational design of a protein library is a necessary and feasible approach to screen and obtain a satisfactory drug candidate.12 Thus, a library consisting of 14 rh4TFP proteins were constructed in the present study. A recently reported crystal structure of the wild-type human 4-1BB/4-1BBL complex indicated that 4-1BB dimerization, in addition to trimerization via 4-1BBL binding, could result in cross-linking of individual ligand-receptor complexes to form a 2D network that stimulates strong 4-1BB signalling.28 Moreover, the binding of galectin-9 to N-linked glycans on 4-1BB was found to be critical for the functional activity of 4-1BB in controlling immune disease in vivo. Although 4-1BBL has also been predicted to possess N-linked and O-linked glycosylation sites, the necessity of post-translational modifications on 4-1BBL for its trimerization or aggregation and its engagement to 4-1BB remains to be determined.29 Given that prokaryotic expressed soluble and functional recombinant Tumstatin or 4-1BBL fragments have been previously reported,8, 11, 13, 14 E. coli expression system was employed in the present study based on its simple, efficient, and cost-effective characteristics. Meanwhile, the



effects of post-translation modifications on the influence of the activity of rh4TFP proteins can also be investigated to some extent. However, when the fragments of Tumstatin were combined with 4-1BBL fragments and expressed in E. coli in the present study, they were predominantly generated in an aggregate form under a variety of expression conditions. In view of the fact that the aggregation or cross-linking of 4-1BBL is essential to its T cell co-stimulation activity and the antiangiogenic activity of Tumstatin is determined by the primary sequence rather than molecular structure itself,8, 24 we hypothesize that, even though rh4TFP proteins refolded from the inclusion bodies would exhibit high degree of complexity, they may still have the desired bifunctional activity. Furthermore, a great deal of examples has demonstrated that proteins expressed as inclusion bodies in bacteria could still be biologically active when self-aggregation or fused to a tag containing an aggregation-prone part, which is also supportive to our study.30, 31 After solubilization and refolding, rh4TFP proteins were purified in this study. Not surprisingly, gel filtration analysis indicated that rh4TFP proteins are all in the form of polymers. The antiangiogenic activities and the co-stimulatory effects on T lymphocyte activation of rh4TFP proteins were initially assessed by the assays of HUVEC and T lymphocyte proliferation. Although T7 peptide is responsible for the antiangiogenic activity of Tumstatin, our results showed that most of the rh4TFP proteins exhibit a lower inhibition than T7 peptide. Similarly, all rh4TFP proteins except rh4TFP-7 showed a weaker co-stimulatory activity than non-fused Ex4-1BBL. These results


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indicated that the fusion of T7 peptide and 4-1BBL may reduce their original activity but to an acceptable level. Although it is becoming increasingly evident that “connector regions” are important components of the dynamic personality of protein structures, no significant differences in activity were found between the rh4TFP proteins containing flexible linkers and those containing rigid linkers.23 We reason that this may be caused by the structural integrity of the refolded rh4TFP proteins and the formation of polymeric structures that limit the conformational changes of rh4TFP proteins. After a ranking analysis of the bifunctionality of each rh4TFP, the potential of rh4TFP-2 as a candidate protein for antiangiogenic immunotherapy was further evaluated, suggesting that a flexible linker with 19 amino acids in length between T7 peptide and Ex4-1BBL could be optimal for the bifunctional activity. Three classic angiogenic assays including cell adhesion assay, scratch-wound healing assay and HUVEC tube formation were thus carried out and confirmed the inhibitory effects of rh4TFP-2 on the angiogenesis. Meanwhile, the co-culturing study of T lymphocyte-B16F10 cells further confirmed the co-stimulatory effects of rh4TFP-2 on the expansion of cytotoxic T lymphocytes in vitro, which may be mediated by an elevated production of cytokines. Given the encouraging results observed in vitro, evaluation of the antitumor effects of rh4TFP-2 in vivo was then conducted. Checkpoint molecule IDO is an enzyme overexpressed in the cytoplasm of various tumor types including melanoma, which catabolizes tryptophan into active metabolites that potently inhibit T- and NK-cell proliferation.32 The competitive inhibitors of IDO have attracted a great



interest among immune checkpoint therapies, and a number of IDO inhibitors are currently in the phase I/II clinical trials.32 Considering that multiple pathways including immunity and angiogenesis are involved in the IDO inhibition, NLG919 was chosen as a drug to compare in the present study based on its effectiveness in melanoma, easy administration and bioavailability.21 Although rh4TFP-2 showed comparable effects as NLG919 on tumor volume and weight of B16F10 melanoma-bearing mice, rh4TFP-2 exhibited a superior benefit on overall survival time of the mice. The subsequent immunohistochemical analysis of the markers of angiogenesis in tumor tissues and flow cytometry analysis of T lymphocyte subpopulations also verified the antitumor effects of rh4TFP-2 in vivo. In addition to the anti-tumor efficacy, the safety of costimulators is one of the most critical factors to be extensively addressed. Co-stimulation is a double-sided sword which may induce massive T cell proliferation and release of pro-inflammatory cytokines, producing severe side effects known as “cytokine storm”.33 For example, despite the initial signs of potent antitumor efficacy, the clinical development of the first agonistic anti-4-1BB mAb, urelumab (BMS-663513), has been halted by inflammatory liver toxicity.18 Although elevated secretion of IL-2/IFN-γ cytokines were also induced in sera of the mice treated by rh4TFP-2, no apparent signs of toxicity were observed in vivo, suggesting that the in vivo treatments by the fusion protein result in a synergetic effect of antiangiogenesis and immune activation. We postulate that the potential side effects may have been alleviated by the antiangiogenetic activity of rh4TFP-2 due to the fact that antiangiogenic molecules


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appear to have advantage to restore immunosuppressive Treg cell proportion to a physiological level to avoid autoimmune mediated side effects.34 Further cellular and molecular mechanisms for rh4TFP-2 exerting the bifunctional activity, such as, the tumor-immune cells infiltration by T cells, NK cells and Treg cells, the structure of tumor vessels, and the vascular perfusion, are currently under investigation in our laboratory.

5. CONCLUSIONS In summary, the present study constructed a fusion protein library to combine the fragments of Tumstatin and 4-1BBL. Activity assays of the fusion proteins revealed that they not only inhibit the proliferation of HUVECs, but also significantly increase T lymphocyte activation. After comparison of different combinations, a candidate protein rh4TFP-2 was identified and demonstrated to exhibit desired bifunctional activity both in vitro and in vivo. In addition, the fact that polymeric proteins recovered from inclusion bodies may be active was also confirmed. The present approach on constructing bifunctional fusion proteins could be an effective way to simultaneously target angiogenesis and T lymphocyte activation for cancer therapy.

ASSOCIATED CONTENT Supporting Information Immune response and histopathological data for rh4TFP-2 administration in vivo



(DOC).

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]; [email protected]. ORCID Yijun Chen: 0000-0002-4920-152X Shuzhen Wang: 0000-0003-3869-2463 Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was supported by grants from the “111 Project” from the Ministry of Education of China and the State Administration of Foreign Export Affairs of China (No: 111-2-07), National Science Foundation of China (No: 81473126) and the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

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Figure Legends Figure 1. Expression and purification of rh4TFP proteins. (A) Schematic representation of the production procedures of rh4TFP proteins. (B) SDS-PAGE of refolded Ex4-1BBL and rh4TFP proteins. Left panel: Lane 1, Ex4-1BBL; Lane 2-9: rh4TFP 1-rh4TFP 8. Right panel: Lane 10-15: rh4TFP 9-rh4TFP 14. M, molecular weight marker. Figure 2. In vitro antiangiogenic activity of rh4TFP-2. (A) rh4TFP-2 inhibited HUVCE adhesion to Matrigel. (B) Representative scratch-wound images showing the effects of rh4TFP-2 on healing ability of HUVCEs (magnification, ×200). (C) Percentage of HUVCEs that migrated into the wound following rh4TFP-2 or T7 peptide treatments relative to untreated cells. (D) The average number of HUVCE tubes with different treatments. (E) Representative images showing the effects of rh4TFP-2 on HUVCE tube formation (magnification, ×200). Data represents the average of three independent experiments. Results are presented as mean ± SD. * P < 0.05, ** P < 0.01, # P < 0.05, ## P < 0.01. Figure 3. The co-stimulatory activity of rh4TFP-2. (A) rh4TFP-2 induced cytotoxic activity of activated T lymphocytes against B16F10 melanoma cells. Incremental concentrations of rh4TFP-2 were incubated for 3 or 5 days with purified T lymphocytes (2×105 cells/well, effector) and B16F10 cells (2×104 cells/well, target). After CCK-8 assay, the killing rates were calculated. (B) Effects of rh4TFP-2 on the release of IFN-γ and IL-2 from activated T lymphocytes. Data represents the average of three independent experiments. Results are presented as mean ± SD. * P < 0.05, **


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P < 0.01. Figure 4. In vivo antitumor activity of rh4TFP-2. (A) Tumor volumes of B16F10 melanoma xenograft mice after treatments with rh4TFP-2 (10 mg/kg) or Tris-HCl solution (pH 8.0) via i.v. once every two days, or NLG919 (50 mg/kg) via i.g. once per day for a total of 11 days. (B) Final tumor mass from rh4TFP-2 treated and untreated B16F10 tumors. (C) Representative mice and tumors on day 11. (D) Representative histopathological sections of the B16F10 melanoma xenograft tumor. (H&E staining, ×200). (E) Survival curve of B16F10 melanoma xenograft mice. Data in a and b are presented as mean ± SD. * P < 0.05, ** P < 0.01. Figure 5. In vivo activity of antiangiogenesis and immune activation by rh4TFP-2. (A)Representative images showing anti-CD31 staining of the tumors sections from rh4TFP-2 treated or untreated mice (magnification, ×200). (B) Average MVD of CD31-stained sections in rh4TFP-2 treated or untreated mice. (C) Representative flow cytometry analysis of T cell subpopulations in rh4TFP-2 treated or untreated mice. (D) The relative abundance of CD3+, CD3+CD4+ and CD3+CD8+ T cells. (E) Serum concentration of IFN-γ and IL-2 in rh4TFP-2 treated or untreated mice. Results are presented as mean ± SD. * P < 0.05, *** P

Bifunctional fusion proteins derived from Tumstatin and 4-1BBL for

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