The therapeutic effect of a novel anti-TNF-α/IL-6R triple-specific fusion protein under experimental septic condition


 A novel anti-TNF-α/IL-6R triple-specific fusion protein, by linking 3 single domain chains, was designed and constructed in our lab. The high purity fusion proteins were obtained by our developed prokaryotic expression system process with high binding affinity with TNF-α (94.75 pM), Human Serum Albumin (1.83 nM) and IL-6R (2.29 nM). In this study, the anti-TNF-α/IL-6R triple-specific fusion protein protected the mouse fibroblast fibrosarcoma cell line (L929) from the apoptosis effects induced by TNF-α, establishing that the expressed fusion proteins can selectively combine with TNF-α in vitro. In vivo, the survival rate of cecal ligation and puncture (CLP) was notably increased in the group with anti-TNF-α/IL-6R triple-specific fusion protein treatment, and meaningfully higher compared with the single-targeted IL-6R and TNF-α fusion protein at the same dose. After the treatment with anti-TNF-α/IL-6R triple-specific fusion protein, the level of serum TNF-α, IL-1β and IL-6 were significantly decreased, and sepsis-induced pathological injuries in the kidney were remarkably attenuated. The anti-TNF-α/IL-6R triple-specific fusion protein can be the potential candidate for the development of new drug design against sepsis.


Introduction
Sepsis constitutes a major healthcare problem which is expected to increase due to an ageing population. In 2017, WHO recognized that sepsis was a global health priority [1].Sepsis is one of the most sever disease caused by a dysregulated host response to infection [2][3]. Recent studies have demonstrated that the extensive release of cytokines and other mediators play critical roles in the development of sepsis [4]. Tumor necrosis factor alpha (TNF-α) is the closest mediator of the cytokine cascade. Serum TNF-α levels increase within 1-2 hours after injection of endotoxin that may be involved in sepsis. [1,5,6]. The antibody or molecules against TNF-α have been developed to treat sepsis, though the clinical trial results were not ideal.
Interleukin-6 (IL-6) is produced as a prominent activator of the acute phase response with extensive biological activities [7,8]. The plasma half-life of IL-6 is longer than other in ammatory factors such as TNF-α or IL-1β [9], when the level of TNF-α in the plasma of patients with sepsis is signi cantly decreased, the concentration of IL-6 is signi cantly increased [10]. A link between high IL-6 level and mortality in septic patients [6] has been found in the previous studies. Therefore, IL-6, widely accepted as a valuable biomarker to evaluate the therapeutic response and the prognosis of sepsis [5], can be employed as potential therapeutic target in the clinical treatment of sepsis.
Hereby, a triple-speci c fusion protein, composed of anti-TNF-α, anti-Human Serum Albumin (HSA) and anti-IL-6R single domain chains, was designed and constructed by us. Different from other conventional single-chain Fv (ScFv) based antibodies from human IgGs, the single domain chain derived from the natural camel heavy-chain only antibodies (HCAbs) [11,12]presents various superior advantage features, such as lower molecular weight, increased stability, and better solubility [12,13]. Furthermore, a recombinant fusion protein can be easily constructed by different single domain chains allowing for target one or two antigens at the same time. The novel anti-TNF-α/HAS/IL-6R fusion protein are supposed to bind to TNF-α and IL-6 receptor, respectively, and also bind to human serum albumin (HSA) leading to signi cantly increase the half-life of fusion protein in vivo as well as block cytokines at particular anatomical sites [14]. The present study aimed to explore the potential associations of the   functions of TNF-α and IL-6 being blocked under experimental septic condition at the same time by a   novel anti-TNF-α/IL-6R triple-speci c fusion protein, and investigate its therapeutic effects in vitro and  vivo. 2 Material And Methods 2.1 The preparation of anti-TNF-α/IL-6R triple-speci c fusion protein The 100 ng plasmid of pET28a-anti-TNF-α/IL-6R designed and constructed by our lab was transformed into competent cell, E. Coli.BL21(DE3), (Beijing Quanshijin Biotechnology Company) by heat shock method. The recombinant cells from 50 ml culture were harvested by centrifugation at 10,000 g for 10 min and the pellet was resuspended in 5mL TGE buffer (50mM Tris pH 7.9, 0.5 mM EDTA, 50 mM NaCl, 5% glycerin), then ultrasonic crushed on the ice for 30min.The fusion protein fused with His-tag were expressed and puri ed using a BeaverBeads™ IDA-Nickel (Suzhou Beaver Bioengineering Company) and desalted with Äkta (GE Healthcare, USA). The puri ed fusion protein was analyzed by 10% SDS-PAGE.

Anti-TNF-α/IL-6R triple-speci c fusion protein speci city analysis
The plates were rst coated with IL-6R (1 µg·mL − 1 ), TNF-α (1 µg·mL − 1 ) and HSA (Human serum albumin, 1 µg·mL − 1 ) respectively at 4°C, overnight (diluted in 0.1 M Na 2 CO 3 -NaHCO 3 buffer, pH 9.6). The next day, the plates were washed 3 times with PBST (0.05% Tween-20 in PBS) and blocked for 2 h with 100 µL 1% BSA-PBS. The fusion protein diluent was added at various nal concentrations after 3 times washes and incubated for 1 h at 37°C. 50 µL anti-rabbit IgG with His-tag antibody (CST, USA) was added and incubated for another hour. The plates were washed 3 times and then 50 µL goat anti-rabbit IgG-HRP antibody (CST) was added and incubated for another hour. After washing, a total of 100 µL reaction buffer (5 mg o-Phenylenediamine, 10 µL H 2 O 2 , 5 mL Citric acid-Na 2 HPO 4 ) was added into the wells. The reaction was stopped by 2 M H 2 SO 4 after 10minutes. Absorbance was measured at 492 nm on an ELISA plate reader (Bio Tek, USA).

2.3
Cell-based functional assays of anti-TNF-α/IL-6R triplespeci c fusion protein in vitro Murine L929 broblast cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) were cultured in 1640 (Gibco, USA) supplemented with 10% FBS (Gibco, USA). Approximately 12,000 cells were seeded in each well of 96-well plate and incubated for 24 h at 37°C under a humidi ed atmosphere and 5% CO 2 .The reaction mixtures were prepared using TNF-α (0.15 nM) and various concentrations of puri ed anti-TNF-α/IL-6R triple-speci c fusion protein (ranging from 0.032 to 32 nM) in the presence of actionomycin (1 mg·mL − 1 ).The mixtures were incubated on ice for 10 min and then added onto the previously L929 seeded wells for 12 h treatment. 20 µL of MTT (5 mg/ml) was added to each well and the plate was kept in the incubator for further 4 h. The supernatant was discarded and 150 µL of dimethyl sulfoxide was added onto wells with incubation for 40 min at room temperature with agitation. The absorbance was measured at 570 nm and the achieved results were t into dose-response inhibition curve in Prism program (version 5.01, GraphPad Software Inc.).
For apoptotic cells analysis, L929 cells was treated with the same methods mentioned above. After incubation for 12 h, the supernatant was discarded. The plates were washed with PBS. 100 µL of PBS and 5µL of PI (Propidium Iodide) was added in each well and incubated for 45 min at 37°C. The L929 cells were xed in 4% paraformaldehyde for 15min.The plates were washed and 100 µL of PBS and 5 µL of DAPI(4',6-diamidino-2-phenylindol) were added for each wells at room temperature for 30 min. After staining, the cells were imaged and analyzed using an Operetta™ High Content Screening instrument with Harmony™ software version 3.5.2 (PerkinElmer, Waltham, MA, USA) and different excitation/emission channels.

The therapeutic effect of anti-TNF-α/IL-6R triple-speci c fusion protein in a vivo model of animal sepsis
Speci c pathogen free (SPF) male Sprague-Dawley rats, weighing 180-200 g, were supplied by Fujian Medical University Animal Center (Fuzhou, China). The rats were housed in Fujian Medical University Animal Center, with free access to food and water, under a light/dark (12 h/12 h cycle) at a temperature of 20-23°C. The rats were acclimatized for more than 7 days before the experiments.

Survival analysis and blood urea nitrogen (BUN) examinations
For Survival analysis, after surgery, the survival status of each group of rats was observed every 12 hours and recorded. The level of creatinine and BUN were examined using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions.

Hematoxylin-eosin (HE) staining
Kidney tissues were rinsed with ice-cold saline solution and xed with paraformaldehyde (4 %), embedded in para n, and cut into 5 mm sections. Then all sections were stained with HE and photographed under an optical microscope with the magni cation of 20×.

Statistical analysis
All data are expressed as means ± SD. One-way ANOVA followed by Bonferroni's Multiple Comparison Test using GraphPad software was used for comparisons among experiment groups. A p-value of less than 0.05 was considered statistically signi cant.

3.1
The preparation of anti-TNF-α/IL-6R triple-speci c fusion protein The cDNA encoding the recombinant anti-TNF-α/IL-6R triple-speci c fusion protein, was made synthetically based on the sequence information of patent US2010/0172894 and US2012/0093839. The 3D structure of an anti-TNF-α/IL-6R triple-speci c fusion protein was mimicked by SWISS-MODEL (Fig. 1a). The 39.27kb inserts were ligated into the multiple cloning sites region downstream of the pET28a + vector using the EcoRI/NotI restriction sites. Colony PCR results showed that the size of the ampli ed gene fragment was 1600 bp (Fig. 1b), where the plasmid of anti-TNF-α/IL-6R was transformed into competent cells BL21(DE3) successfully. The fusion protein was expressed and analyzed by 10 % SDS-PAGE showed in Fig. 1c, indicating that anti-TNF-α/IL-6R triple-speci c fusion protein is expressed in E.coli classical inclusion bodies. The inclusion bodies were puri ed by BeaverBeads™ IDA-Nickel (Fig. 1d) and desalted with Äkta (Fig. 1e). The purity of anti-TNF-α/IL-6R triple-speci c fusion protein reached 95%.

Binding capacity analysis of anti-TNF-α/IL-6R triplespeci c fusion protein
As shown in Fig. 1f, anti-TNF-α/IL-6R triple-speci c fusion proteins were able to bind TNF-α, HSA and IL-6R in a dose-dependent manner. The results indicate that the EC 50 value of anti-TNF-α/IL-6R triple-speci c fusion protein was 94.75 pM for TNF-α, 1.83 nM for HSA and 2.29 nM for IL-6R.

Inhibition of TNF-α by anti-TNF-α/IL-6R triple-speci c fusion protein in vitro
The MTT results ( Fig. 2A) presented that anti-TNF-α/IL-6R triple-speci c fusion protein can inhibit TNFcytotoxicity on L929 cells in a dose-dependent manner with IC 50 values of 19.6 pM. DAPI/PI staining was employed to observe the effect of anti-TNF-α/IL-6R trispeci c fusion protein on the apoptosis of L929 cells induced by TNF-α. Under the uorescence microscope, the negative control group showed uniform blue uorescence, and the nuclei treated with TNF-α were evenly colored red by PI (Fig. 2B). The results of cell death by High-content analysis exhibited that the control group had an apoptotic rate of (3.40 ± 0.77) %, which was signi cantly different from (79.45 ± 5.89) % of the TNF-α (2 ng/ml) group (P < 0.001). The apoptotic rates of the anti-TNF-α/IL-6R triple-speci c fusion protein administration group (32 nM, 3.2 nM, 0.32 nM) were (8.66 ± 4.93) %, (8.93 ± 2.16) % and (64.89 ± 4.69) %, respectively. These results indicate that TNF-α can induce apoptosis of L929 cells, whereas the anti-TNF-α/IL-6R triple-speci c fusion protein administration group can effectively reduce the apoptosis rate of TNF-α-induced apoptosis of L929 cells in a dose-dependent manner (Fig. 2C).

Anti-TNF-α/IL-6R triple-speci c fusion protein signi cantly alleviates CLP induced sepsis
As demonstrated in Fig. 3a, the survival rate in the sham was 100%, while survival rate of CLP decreased to 10% within 72 h. Different doses of anti-TNF-α/IL-6R triple-speci c fusion protein intervention group improved the survival rate of rats within 72 h. The survival rate was 60% in 5 mg·kg − 1 group and 30% in 1mg·kg − 1 group, where 10mg·kg − 1 group was the most signi cant with the 70% survival rate. Moreover, compared with the single-targeted IL-6R or TNF-α fusion protein, constructed and obtained by our lab as well, anti-TNF-α/IL-6R triple-speci c fusion protein displayed the higher survival rate at the same dose.
As shown in the Fig. 3b, compared with the sham group, the expression level of serum urea nitrogen (BUN) in the CLP group increased signi cantly (P < 0.001). The expression levels of BUN were meaningfully decrease in the single-targeted IL-6R or TNF-α fusion protein, and anti-TNF-α/IL-6R triplespeci c fusion protein treated groups (P < 0.05, P < 0.01 or P < 0.001), compared with the CLP group. The same dose of anti-TNF-α/IL-6R triple-speci c fusion protein down-regulated in BUN expression more prominently.

Anti-TNF-α/IL-6R triple-speci c fusion protein inhibits in ammatory mediators in the serum of septic rats
Cytokines, including IL-1β, IL-6 and TNF-α, have been identi ed as key mediators in sepsis. In our experiment, these biomarkers were selected to demonstrate the therapeutic effects of anti-TNF-α/IL-6R triple-speci c fusion protein against sepsis as shown in Fig. 3c-e. Satisfactorily, anti-TNF-α/IL-6R triplespeci c fusion protein treatment signi cantly reduced these in ammatory mediators in a dose dependent manner. The single-targeted IL-6R or TNF-α fusion protein also had effect on down regulating these cytokines, although the effects were signi cantly weaker than anti-TNF-α/IL-6R triple-speci c fusion protein at the same dose.
3.6 Anti-TNF-α/IL-6R triple-speci c fusion protein alleviates histopathological lesions in the kidney of CLP rats Acute kidney injury (AKI) is a serious complication of sepsis which occurs frequently, particularly in the elderly. The kidneys of different groups of rats were collected for histopathological analysis. In the sham operation group, the structure of the renal tubules and glomeruli was normal, with no damage. (Fig. 4a). The histopathological changes of the CLP group including marked interstitial hemorrhage and renal proximal tubular congestion, edema, necrosis and exfoliated cells are visible (Fig. 4b). In contrast, these lesions were obviously attenuated in the group with anti-TNF-α/IL-6R triple-speci c fusion protein treatment in a dose-dependent manner (Fig. 4c-e) and minimal lesions were seen in anti-TNF-α/IL-6R triple-speci c fusion protein (10 mg/kg) -treated CLP rat (Fig. 4e). The histologic features were less severe and extensive in the rats treated by the single-targeted TNF-α (Fig. 4f) or IL-6R (Fig. 4g) fusion protein, through their relieving effects were signi cantly weaker than that of anti-TNF-α/IL-6R triplespeci c fusion protein at the same dose. The bar graph showed that all the treatment groups had the therapeutic effect compared to CLP group.
Pro-in ammatory cytokine levels were also detected in kidney tissue. Compared with the sham operation group (P < 0.01), the levels of TNF-α, IL-1β and IL-6 in the CLP group were signi cantly increased, while the fusion protein group was signi cantly decreased (Fig. 4h). The anti-TNF-α/IL-6R triple-speci c fusion protein (10 mg·kg − 1 and 5 mg·kg − 1 ) administration markedly decreased all of the three cytokines (P < 0.01, P < 0.01 vs CLP). As shown in Fig. 4i, the mRNA transcription level of IL-1β, TNF-α and IL-6 in CLP group was signi cantly elevated, compared with the sham group. All fusion protein treated groups decreased the expression level of these in ammatory mediators, where the anti-TNF-α/IL-6R triple-speci c fusion protein remarkably inhibited these gene expressions in a dose-dependent manner.

Discussion
Sepsis caused by the host response to infection is a life-threatening organ dysfunction that can signi cantly extend the length of hospitalization of patients [15,16]. However, the underlying mechanism of sepsis is complicated and still unclear, thus the clinical treatment of sepsis is short of medicines. It is widely accepted that the extensive releasing of cytokines and other mediators like TNF-α and IL-6 are valuable biomarkers in septic condition, which can not only be employed to evaluate the prognosis of sepsis [6], but also be the therapeutic target to develop the medicines against sepsis.
In this study, we rstly constructed triple-speci c fusion protein by linking 3 single domain chain, anti-TNF-α/HAS/IL-6R, which can block the functions of TNF-α and IL-6 under the septic experimental condition at the same time. Anti-serum albumin (HSA) moiety single-domain chain involved in this anti-TNF-α/IL-6R triple -speci c fusion protein can extend the half-lives in vivo that its pharmacokinetic behavior can also be improved [14]. Based on the new prokaryotic expression system process developed in our lab, the triple-speci c fusion protein allows for e cient expression of the excellent soluble and stable molecule in E.coli in large quantities with high purity over 95%, and without compromising its properties. The obtained triple-speci c fusion protein displayed high binding a nity with TNF-α (94.75 nM), HSA (1.83 nM) and IL-6R (2.29 nM), analyzed by ELISA binding assay. In vitro, the anti-TNF-α/IL-6R triple-speci c fusion protein can high-e ciency protect L929 cells from the apoptosis effects induced by TNF-α.
In our CLP septic model rats, the level of serum TNF-α, IL-1β and IL-6, as promised biomarkers of sepsis, is signi cantly increased which indicated that sepsis had been initiated. After intervention treatment by the anti-TNF-α/IL-6R triple-speci c fusion protein alone, the survival rate of CLP is signi cantly increased compared with the 10% survival rate of CLP groups within 72-hour. To compare with the single-targeted IL-6R or TNF-α fusion protein, the same dose of anti-TNF-α/IL-6R triple -speci c fusion protein can increase the survival rate of CLP more signi cantly. Meanwhile, serum urea nitrogen (BUN), TNF-α, IL-1β and IL-6 collected from different CLP sepsis rat groups presented that triple-speci c fusion protein can decrease the cytokine mediators more notably than monospeci c fusion protein at the same concentration, supplying more evidences to support that anti-TNF-α/IL-6R triple-speci c fusion protein have more potential treatment effects on sepsis.
Sepsis is de ned as life-threatening organ dysfunction caused by a dysregulated host response to infection, where the important organs, speci cally heart, lung and kidney, can be seriously damaged under septic condition [2]. In our experiment, the rat kidneys collected from the CLP group displayed histopathological changes including marked interstitial hemorrhage and renal proximal tubular congestion, edema, necrosis and exfoliated cells are visible. After the treatment by anti-TNF-α/IL-6R triplespeci c fusion protein, the lesions appeared in untreated-CLP group were signi cantly attenuated by histopathological analysis. Levels of TNF-α, IL-1β and IL-6 in detected in the kidney tissue by ELISA assays is meaningfully decreased in anti-TNF-α/IL-6R triple-speci c fusion protein-treated CLP rat groups, and following the expression level of these in ammatory mediators were con rmed by RT-PCR in the gene level.
In conclusion, the triple-speci c fusion protein presented potent therapeutic effects on septic animal model in this study, indicating that blocking the bioactivities of important in ammatory mediators, TNF-α and IL-6, at the same time can be potential assistant treatment method co-worked with antibiotic drugs in the sepsis clinical treatment. Continued studies in this direction in animal experiments and clinical trial under septic condition have the potential to shed light on the treatment for sepsis.

Declarations
Ethics approval and consent to participate All procedures were performed in accordance with protocols approved by the Ethics Review Committee for Animal Experimentation of Fujian Medical University (No. 2018-104). All animals were raised in the Laboratory Animal Center of Fujian Medical University (Certi cate No. SCXK (Fujian) 2016-0006), where the animal work has taken place and animal handling procedures were performed in strict accordance with the care of laboratory animals, according to the Fujian Province Zoological Society.

Consent for publication
The manuscript is approved by all authors for publication.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.