key: cord-0735648-sapfn8ex
authors: Hayashi, Akiko; Hayashi, Hidetoshi; Chiba, Taku; Sasayama, Satoshi; Onozaki, Kikuo
title: N-acetylneuraminic acid coupled human recombinant TNFα exhibits enhanced anti-tumor activity against Meth-A fibrosarcoma and reduced toxicity
date: 2006-10-10
journal: Cancer Immunol Immunother
DOI: 10.1007/s00262-006-0210-2
sha: fd67923dc172aa2a9691608488c040ae7264b107
doc_id: 735648
cord_uid: sapfn8ex

In order to study the effect of glycosylation on its biological activities and to develop tumor necrosis factor α (TNFα) with less deleterious effects, N-acetylneuraminic acid (NeuAc) with a C9 spacer was chemically coupled to human recombinant TNFα. NeuAc-coupled TNFα (NeuAc-TNFα) exhibited reduced activities in vitro by about threefold compared to native TNFα. In this study, we examined a variety of TNFα activities in vivo. NeuAc-TNFα reduced activities in the up-regulation of serum levels of IL-6 and NOx, but comparable activity as native TNFα in the down-regulation of the serum level of glucose. However, NeuAc-TNFα was more potent than TNFα in the up-regulation of the serum level of serum amyloid A (SAA). NeuAc-TNFα was less toxic to mice. In addition, NeuAc-TNFα exhibited an augmented anti-tumor activity against Meth-A fibrosarcoma without hemorrhagic necrosis. These results indicate that coupling with NeuAc enabled us to develop neoglycoTNFα with selective activities in vivo, including enhanced anti-tumor activity but reduced toxicity.

Glycoproteins are widely distributed in animals, plants and microorganisms. Neoglycoproteins, proteins chemically or enzymatically coupled with carbohydrates, are quite useful to investigate the role of carbohydrates in the functions and physicochemical properties of glycoproteins. The conjugation of carbohydrates is also useful to modify the functions and stability of target proteins. The advantage to synthesize neoglycoproteins is that chemically synthesized carbohydrates, not only natural, but also unnatural carbohydrates, can be coupled to proteins [1] [2] [3] [4] .

Sialic acid is usually present at the non-reducing position of oligosaccharide in glycoproteins and glycolipids, and plays an important role in the function, stability and tissue distribution of glycoproteins [5] and as a ligand for viruses, including inXuenza viruses, paramyxoviruses, coronaviruses, polyomaviruses and retroviruses [6] . In glycoproteins, sialic acid is usually conjugated with galactose and plays an important role in preventing the clearance of glycoproteins from the serum because asialoglycoproteins are rapidly cleared through galactose binding lectins present in the liver [7] . Furthermore, sialic acid is also important as a ligand for selectins [8] and Siglecs [9] and is involved in cellular signaling events and the modulation of immune reactions. Therefore, it is expected that the coupling of sialic acid enables its conjugate to bind to a variety of cell types and prolong its serum level as well.

Tumor necrosis factor (TNF ) is a cytokine produced mainly by macrophages and monocytes. TNF exhibits a variety of biological activities and plays an important role in immunological and inXammatory reactions [10, 11] . Although TNF has been expected to be an eYcient therapeutic agent for certain tumor cells, its clinical application is limited because of its proinXammatory activity and toxicity. At high doses, TNF induces acute responses such as shock, tissue injury, catabolic hormone release, vascular leakage syndrome, gastrointestinal necrosis, acute renal tube necrosis, adrenal hemorrhage, fever, and at low doses, chronic responses such as weight loss, anorexia, protein catabolism, lipid depletion, hepatosplenomegaly, subendocardial inXammation, insulin resistance, acute protein release and endothelial activation [11] .

In the previous studies, we have synthesized neoglyco human IL-1 by conjugating human recombinant IL-1 a variety of carbohudrates, including D-Man (1-6)Man [Man 2 (1-6)], Gal or N-acetylneuraminic acid (NeuAc), a major constituent of sialic acid [12] [13] [14] [15] [16] [17] [18] [19] . Although these carbohydrate-conjugated IL-1 variants exhibited reduced activities in vitro, the activities in vivo were diVerent depending on the type of carbohydrates [12, 15, 18] . Man 2 (1-6)-IL-1 exhibited comparable activities to nonglycosylated IL-1 in the down-regulation of the serum level of glucose and the recovery of peripheral white blood cells from myelosuppression in 5-Xuorouracil-treated mice, irrespective of the decrease of all other activities in vivo [13] . In addition, the tissue distribution of Man 2 (1-6)-IL-1 in mice diVered from that of nonglycosylated IL-1 [14] . D-Gal-conjugated IL-1 exhibited a decrease in all the activities in vivo with a similar magnitude [16] . NeuAc coupled IL-1 exhibited selective activities in vivo as Man 2 (1-6)-conjugated IL-1 and enhanced tissue distribution [17] [18] [19] .

We also synthesized NeuAc conjugated human recombinant TNF . We obtained two glycosylated TNF s, L NeuAc-TNF and H NeuAc-TNF containing 1.0 and 1.5 molecules of NeuAc per molecule of TNF , respectively. L NeuAc-TNF and H NeuAc-TNF exhibited reduction in a variety of activities in vitro, including growth inhibitory and cytotoxic activities to tumor cells, a growth stimulatory eVect on normal Wbroblasts, induction of IL-6 and the activation of NF-B by about 1/3 and 1/10, respectively. As a major product was L NeuAc-TNF (the yields of L NeuAc-TNF and H Neu Ac-TNF were 28.6 and 4.87%, respectively), in this study we examined L NeuAc-TNF for a variety of activities in vivo including anti-tumor activity and toxicity.

Animals ICR female mice (6 weeks old) and Balb/c female mice (6 week old) were purchased from Charles River (Yokohama, Japan) and fed ad lib. and housed in temperature-and light-controlled (12 h/day) rooms. Mice were used in experiments after at least 1 week of acclimation. ICR mice were used for the induction of serum IL-6, serum amyloid A (SAA), NOx and the reduction of glucose. Balb/c mice were used for acute lethal toxicity and anti-tumor activity.

RPMI 1640 was purchased from Sigma Chemical Co. (St. Louis, MO). Fetal bovine serum (FBS) was from JRH Biosciences (Lenexa, KS). Human recombinant TNF (rhTNF ) was provided by Dainippon Pharmaceutical Co. (Osaka, Japan). The speciWc activity of rhTNF was 6 £ 10 7 U/mg based on the cytotoxic assay using L929 cells cultured with actinomycin D. Human recombinant IL-6 (rhIL-6) was provided by Ajinomoto Co. (Tokyo, Japan). The speciWc activity of rhIL-6 was 5 £ 10 6 U/mg based on the proliferative assay using MH60·BSF2 cells.

Murine hybridoma clone MH60·BSF2, provided from Dr. T. Hirano (University of Osaka), was maintained in culture medium (RPMI 1640, 100 U/ml of penicillin G, 100 g/ml of streptomycin, and 10% heat-inactivated FBS) containing 1 U/ml of rhIL-6 [20] .

An acyl azide derivative of NeuAc with a C9 spacer was synthesized and coupled to rhTNF . The NeuAc-TNF was puriWed by anion-exchange chromatography, and the NeuAc-coupling was conWrmed by blotting with NeuAc-speciWc LFA lectin, the increase in its molecular weight on SDS-PAGE and time of Xight mass spectroscopy (TOF-MS) analysis. Two glycosylated TNF were obtained and termed L NeuAc-TNF and H NeuAc-TNF , which contained 1.0 and 1.5 molecules NeuAc per molecule of TNF , respectively. As a major product was L NeuAc-TNF , we used it for in vivo experiments. For the sake of brevity, L NeuAc-TNF was termed NeuAc-TNF . With endotoxin test using a Limulus amoebocyte assay (sensitivity limit, 0.1 ng/ml), endotoxin contamination was negative in these TNF s.

Measurement of serum levels of IL-6, glucose, serum amyloid A, and NOx TNF s were diluted to the desired concentration with sterile PBS and intraperitoneally administered to mice.

Although the samples were endotoxin negative, to prevent the eVect of an undetectable amount of endotoxin, polymyxin B was added at 5 g/ml. Mice were fasted after the administration. At the times indicated for experiment, the mice were bled.

The IL-6 activity in the serum was measured by proliferation assay with IL-6-dependent MH60·BSF2 cells [20] . The amount of IL-6 was expressed as the equivalent amount of rhIL-6. The glucose level in the serum was determined by using a glucose B-test kit (Wako Pure Chemical Industries, Ltd, Osaka, Japan).

The concentration of SAA in the serum was measured by ELISA, as described previously [21] .

The serum nitrite/nitrate (NOx) level was measured by the method described by Misko et al. [22] . BrieXy, 30 l of each sample were incubated for 15 min at 37°C with 10 l of the nitrate reductase (2.5 U/ml; Boehringer Mannheim) and 10 l nicotinamide-adenine dinucleotide phosphate (2 mM; Sigma Chemical Co.). After incubation, 50 l of Griess reagent and 50 l of TCA (10% aqueous solution) were added. The protein precipitates were removed by centrifugation at 15,000 rpm for 5 min and 50 l of each supernatant was transferred to 96-well plate (Falcon) and the O.D. 595 nm was measured using an ELISA autoreader (Bio-Rad Laboratories, Richmond, CA).

Evaluation of the toxicity of TNF to mice D-galactosamine (18 mg/0.2 ml in PBS) was intraperitoneally injected into 9-week-old female Balb/c mice. Ten minutes after the injection, test samples were administered intraperitoneally into the mice. Every 2 h after the injection of the samples, the mortality of the mice was determined.

Evaluation of the anti-tumor eVect of TNF Meth-A Wbrosarcoma cells (4 £ 10 5 cells) were inoculated intradermally into the abdomen of 9-week-old female Balb/c mice. Eight days later after the conWrmation of tumor establishment (tumor size, 5-8 mm in diameter), test samples were intravenously administered twice a week for 2 weeks. The tumor size was determined based on the formula of Haranaka et al. [23] . At 24 h after the Wrst injection of TNF , a score of tumor hemorrhagic necrosis, from 1 to 4, was determined according to the method of Carswell et al. [24] .

The amount of protein was determined using a Protein Assay kit (Bio-rad, Richmond, CA) with bovine serum albumin as the standard.

DiVerences between group means were assessed using the t test.

Mice were injected intraperitoneally with native TNF (termed TNF ) or NeuAc-TNF and the serum IL-6 level was determined. Mice injected with TNF exhibited a sharp increase in the IL-6 level with a maximum level 2 h after TNF treatment (Fig. 1a) . The elevation of serum IL-6 at 2 h was not observed at the injection of 0.5 g TNF /mouse (Fig. 1b) . NeuAc-TNF exhibited a signiWcant decrease in its activity. Ability of TNF to induce SAA in mice

Mice were injected intraperitoneally with native TNF , and their ability to induce SAA, an acute phase protein produced by hepatocytes in response to TNF , was examined. TNF increased the SAA level after 4 h treatment, which exhibited a peak at 8 h and the level was sustained for up to 24 h. NeuAc-TNF exhibited a more potent activity in the induction of SAA (Fig. 2a) .

Although the data were not signiWcant, the doseresponse experiment at 8 h indicated that NeuAc-TNF at 2 g/mouse is more potent than TNF (Fig. 2b) .

EVect of TNF treatment on the serum glucose level in mice

The eVect of TNF on the serum glucose level was examined. The mice were injected intraperitoneally with native TNF or NeuAc-TNF , and then fasted. In the control mice the serum glucose level decreased with the duration of fasting up to 24 h (Fig. 3a) . TNF caused a signiWcant reduction at 2 h after treatment, and the decrease continued to 24 h. NeuAc-TNF also caused a reduction for up to 24 h after treatment. A dose-response experiment at 8 h indicated that NeuAc-TNF exhibited comparable activity to TNF (Fig. 3b) .

Ability of TNF to induce serum NOx in mice TNF induces nitric oxide (NO) synthesis in a variety of cell types. The generated NO reacts with molecular oxygen and water, and subsequently, nitrite and nitrate (NOx) were accumulated in the biological Xuids. To examine the ability of TNF to induce NO, serum nitrate was converted to nitrite by nitrate reductase, and then the amount of total nitrite was determined. Native TNF injected mice exhibited an increase in the serum NOx level after 4 h treatment with a maximum level at 8 h, and the level decreased, but still remained high as compared to control at 24 h (Fig. 4a) . NeuAc-TNF also up-regulated the serum NOx level. The time course experiment * * * * and the dose-response experiment at 8 h showed that NeuAc-TNF is less active than TNF (Fig. 4b) .

Acute lethal toxicity of TNF to mice We determined the lethal toxicity of TNF s to mice. Mice were sensitized to LPS by the injection of D-galactosamine intraperitoneally, and then the TNF s (0.3 g/ mouse) were administered intraperitoneally. As shown in Fig. 5 , all the mice injected with native TNF died after up to 12 h. In contrast, 4/5 of the mice injected with NeuAc-TNF survived up to 24 h. Three out of Wve and two out of Wve of the mice were still survived after 48 and 72 h injection, respectively. The dose-response experiment showed that the toxicity of NeuAc-TNF decreased to 1/3, as compared to TNF (Table 1) .

In order to compare the anti-tumor activity of TNF s, TNF s were injected intravenously into the mice twice a week 8 days after the inoculation with Meth-A Wbrosarcoma, at this time, tumor establishment was conWrmed. As shown in Fig. 6a , tumor growth was signiWcantly reduced by the injection of TNF s at either 2.0 or 0.5 g per injection. However, the anti-tumor eVect of NeuAc-TNF was more potent as compared to native TNF (Fig. 6b) . In another experiment, NeuAc-TNF at 2 g per injection resulted in the complete disappearance of tumors in 1, 2, 3 and 4 per 7 mice on day 13, 18, 20 and 32, respectively, and NeuAc-TNF at 0.5 g per injection did so in 2 and 3 per 7 mice on day 19 and 21, respectively, while native TNF at either 2 or 0.5 g per injection could not up to 32 days, though it inhibited the tumor growth. In addition, while native TNF induced hemorrhagic necrosis, NeuAc-TNF induced necrosis without hemorrhage (Fig. 7) . During the course of the treatment up to 32 days, no signiWcant body weight loss was observed in either the native TNF -or NeuAc-TNF treated control and tumor bearing mice, as compared to the PBS control (data not shown).

In the present study, we demonstrated that NeuAc-TNF exhibits selective activities in vivo. This is in contrast to its in vitro activities, in all the assays performed in vitro, including antiproliferative or cytotoxic activity to tumor cells, a proliferative eVect on Wbroblast cells, a stimulatory eVect on IL-6 production by melanoma cells and NF-B activation in hepatocytes, NeuAc-TNF exhibited similarly reduced activities by about 1/ 3, as compared to native TNF . It is interesting as to why the eVects of NeuAc coupling to TNF activities in vivo diVered according to the activities. The activities of TNF in vitro could be mainly determined by its aYnity for the receptor. In contrast, the in vivo system is more complex, and the potency of TNF activity cannot be determined solely by the aYnity to its receptor. In our previous studies, NeuAc-IL-1 similarly exhibited reduced activities in all the assays performed in vitro, including its receptor binding aYnity, as compared to native IL-1 , but in vivo, it exhibited selective activities, such as reduction in the activity to induce serum IL-6, moderate reduction in the activities to induce serum SAA and NOx, comparable activity to reduce the serum glucose level and augmented activity to improve the recovery of peripheral white blood cells from myelosuppression in 5-FU treated mice [18, 19] . There are many proteins or glycoproteins interacting with sialic acid in the serum, tissues and on the cell surface, thus they will inXuence NeuAc-TNF in its distribution into tissues, accessibility to target cells, retardation in the serum and receptor binding activity.

TNF exerts pleiotropic eVects in vivo by acting on many cell types. IL-6 is produced from many cell types by TNF alone and in synergy with IL-1. IL-6 plays an important role in acute and chronic inXammatory reactions, including diVerentiation and proliferation of T and B cells and the induction of acute phase proteins from hepatocytes [25] . NeuAc-TNF exhibited a sig-niWcant decrease in the IL-6 induction activity. (µg/mouse) * Necrotic score TNF alone or in synergy with IL-1, IL-6 or glucocorticoid induces the synthesis of acute phase proteins by hepatocytes, and the acute phase proteins are implicated in the reduction of inXammation and the recovery of damaged tissues. SAA is the representative produced by hepatocytes in response to cytokines [26] . NeuAc-TNF exhibited a more potent activity in the induction of SAA.

In response to TNF or IL-1, the serum level of glucose decreases due to insulin-dependent and independent manners, the latter mechanism involves the reduction of glucogenesis in the liver and glucoseuptake by tissues [27] . NeuAc-TNF exhibited a comparable activity to native TNF .

NO is an important eVecter molecule in neurotransmission, vasodilatation and host defense against microorganisms and tumor cells. TNF alone or in synergy with IL-1 and interferon augments the production of NO from many cell types, including macrophages, hepatocytes, vascular endothelial cells and smooth muscle cells [28] . In particular, NO produced by smooth muscle cells is implicated in the hypotension caused by TNF , which is a serious deleterious eVect in the application of TNF to patients [11] . NeuAc-TNF was weak in the induction of serum NOx level. Therefore, it is beneWcial for the therapeutic use of NeuAc-TNF . A disadvantageous aspect is that Neu-TNF may be weak in augmenting the host defense against infection of microorganisms and tumors.

The toxic eVect of TNF contributes to endotoxin shock [24] , which is also a major factor for the limited use of TNF to patients. NeuAc-TNF exhibited reduced acute lethal toxicity in D-galactosamine sensitized mice. This is also beneWcial for the therapeutic use of NeuAc-TNF . When native TNF or NeuAc-TNF was injected into the control or tumor-bearing mice that were not sensitized with D-galactosamine, there was no obvious toxicity.

It is well established that TNF exerts its anti-tumor eVect on the Wbrosarcoma Meth-A in mice [10, 11, 29] . NeuAc-TNF inhibited the tumor growth more rapidly and eYciently than native TNF . The rate of complete disappearance of the tumor was also higher in NeuAc-TNF as compared to native TNF . As has been known, TNF caused hemorrhagic necrosis. Quite interestingly, however, NeuAc-TNF caused necrosis without hemorrhage. The tumor lesion was pale; subsequently, the tumor regressed and disappeared. It is of note that when the necrotic tumor lesion was touched by hand, the TNF -injected mice, but not the NeuAc-TNF injected mice, became rowdy. Probably the TNF -injected, but not the NeuAc-TNF injected mice, felt pain. TNF exerts its anti-tumor eVect through its direct cytotoxic eVect, necrosis or apoptosis, on tumor cells and also indirectly through host cells. TNF induces inXammatory exudates by inducing chemokines and cytokines, and augments the tumor cell killing activity of monocytes, macrophages, neutrophils and NK cells [10, 11] . It is also reported that TNF selectively stems blood Xow in newly formed microcapillaries in the tumor lesion by inducing thrombus formation, leading to the autolysis of tumors [29] . Although the mechanism by which NeuAc-TNF caused necrosis without hemorrhage remained to be elucidated, our study indicated that conjugation of NeuAc to TNF rendered the conjugate more potent in anti-tumor eVect and less toxic. The study also suggests that the site-directed conjugation of NeuAc will enable us to develop a more ideal TNF in future.

Chemo-immunological studies on conjugated carbohydrated-protiens: I. The synthesis of p-aminophenol -glucoside, p-aminophenol -galactoside, and their coupling with serum globulin

In the speciWcity of serological reactions

Immunological responses to Salmonella R antigens. The bacterial cell and the protein edestin as carriers for R oligosaccharide determinants

Neoglycoproteins: the preparation and application of synthetic glycoproteins

Sialic acids: fascinating sugars in higher animals and man

The recognition event between virus and host cell receptor: a target for antiviral agents

Binding site of the rabbit liver lectin speciWc for galactose/nacetylgalactosamine

Siglecs: sialic-acid-binding immunoglobulin-like lectins in cell-cell interactions and signaling

Siglecs in innate immunity

Tumor necrosis factor and interleukin 1: cytokines with multiple overlapping biological activities

Tumor necrosis factor: a pleiotropic cytokine and therapeutic target

Development of glycosylated human interleukin-1 alpha, neoglyco IL-1 alpha, coupled with D-mannose dimmer: synthesis and biological activities in vitro

Glycosylated human recombinant interleukin-1 alpha, neo interleukin-1 alpha, with D-mannose dimmer exhibits selective activities in vivo

D-mannose dimmer introduced human recombinant interleukin-1 alpha, neo IL-1 alpha, exhibits altered tissue distribution in mice

Development of glycosylated human interleukin-1alpha, neoglyco IL-1alpha, by coupling with D-galactose monosaccharide: biological activities in vitro

Development of glycosylated human interleukin-1alpha, neoglyco IL-1alpha, coupled with D-galactose monosaccharide: biological activities in vivo

Synthesis of glycosylated human interleukin-1alpha, neoglyco IL-1alpha, coupled with N-acetylneuraminic acid

In vitro biological activities of glycosylated human interleukin-1alpha, neoglyco IL-1alpha, coupled with N-acetylneuraminic acid

Glycosylated human interleukin-1alpha, neoglyco IL-1alpha, coupled with N-acetylneuraminic acid exhibits selective activities in vivo and altered tissue distribution

Establishment of an interleukin 6 (IL 6)/B cell stimulatory factor 2-dependent cell line and preparation of anti-IL 6 monoclonal antibodies

Augmentation of type I IL-1 receptor expression and IL-1 signaling by IL-6 and glucocorticoid in murine hepatocytes

A Xuorometric assay for the measurement of nitrite in biological samples

Antitumor activity of murine tumor necrosis factor (TNF) against transplanted murine tumors and heterotransplanted human tumors in nude mice

An endotoxin-induced serum factor that causes necrosis of tumors

Biology of multifunctional cytokines: IL 6 and related molecules (IL 1 and TNF)

The acute phase response

Tumor necrosis factor enhances glucose uptake by peripheral tissues

Nitric oxide as a secretory product of mammalian cells

Recombinant human tumor necrosis factor-alpha: thrombus formatin is a cause of anti-tumor activity

Acknowledgments This work was supported in part by Grant-in Aid for Grant-in-Aids for ScientiWc Research on Priority Areas (C) from The Ministry of Education, Science, Sports and Culture.