JOURNAL OF INTERFERON & CYTOKINE RESEARCH 25:414–417 (2005)© Mary Ann Liebert, Inc.

Antiproliferative Properties of Chemically ModifiedRecombinant IFN-�2b

A.V. MARTYNOV and M.V. SMELYANSKAYA

ABSTRACT

The antiproliferative activity of aconitylated (AIFN) and succinylated (SIFN) derivatives of recombinant in-terferon-�2b (IFN-�2b) was examined. Acylation of IFN-�2b was performed by succinic and cis-aconitic an-hydrides. Antiproliferative properties of AIFN and SIFN were studied in vitro on the CaOv cell line, highlysensitive to IFN, and on the SW-480 cell line, with low sensitivity to IFN-�2b. Acylation of one lysine in theIFN-�2b molecule with cis-aconitic or succinic anhydride resulted in a 3–3.5-fold increase of its antiprolifer-ative activity on CaOv cells. The highest antiproliferative activity of acylated IFN-�2b on SW-480 cells wasobserved for both AIFNs and SIFNs with three modified lysine residues. In conclusion, aconitylated and suc-cinylated IFNs may be useful antiproliferative agents for cancer treatment.

414

INTRODUCTION

INTERFERONS (IFNS) HAVE BEEN USED in anticancer therapy fora long time and often possess relatively high antitumor ac-

tivity. IFN-�2b is the most studied IFN and has been used suc-cessfully for therapy of a variety of cancers, for example, ma-lignant melanoma, hairy cell leukemia, chronic myelogenousleukemia, and Kaposi’s sarcoma.(1) Some cases of complete tu-mor regression after IFN-�2b monotherapy (renal and bladdercancer) have been observed. In many cases, however, a thera-peutic effect was not seen, and the reasons remained unknown.

One potential way to increase the biologic activity of IFNsis chemical modification by acylation or alkylation. For exam-ple, treatment of human albumin by succinic or aconitic anhy-dride significantly alters its properties in that it acquires strongantiviral properties due to increased affinity to viral surface pro-teins.(2) It has been shown that the affinity of modified proteinto its receptor is dependent on the acylation level; for example,partial acylation of immunoglobulins with succinic anhydridecauses a 4–8-fold increase in their specificity and sensitivity.(3,4)

By virtue of these data, a study of the influence of acylation ofIFN-�2b on its antiproliferative activity was performed. For thispurpose, acylation of IFN-�2b with cis-aconitic anhydride andsuccinic anhydride was performed, and the activity of modifiedIFN-�2b was studied in vitro on CaOv (human ovarian adeno-carcinoma) and SW-480 (human rectal cancer) cell lines.

MATERIALS AND METHODS

Cis-aconitylation and succinylation procedure

To synthesize acylated derivatives of recombinant IFN-�2b(Biolek, Kharkiv, Ukraine), the anhydrides of succinic andaconitic acids (Aldrich-Sigma, Milwaukee, WI) were used.Acylation of IFN-�2b was carried out in slightly alkalinemedium by the addition of different anhydride dilutions (indioxane) to an aqueous solution of IFN-�2b according to thecalculations shown in Tables 1 and 2. The reaction was termi-nated by means of pulse electrophoresis in 30% polyacrylamidegel (PAAG) (Bio-Rad, Hercules, CA). Nonacetylated controlpreparations were run in parallel.(5) Gels were silver stained toobserve the acylated IFNs.(6) Modified IFN-�2b was purified bychromatography on Sephadex G-25 and then stored at �20°C.The antiproliferative activity was studied on a CaOv cell linehighly sensitive to IFN-�2b in vitro (obtained from the Mu-seum of Cultures of the I.I. Mechnikov Institute of Microbiol-ogy and Immunology AMS of Ukraine, Kharkiv, Ukraine) andon an SW-480 cell line that exhibits low sensitivity to IFN-�2bin vitro (obtained from the National Cancer Institute, Lyon,France). Cells were routinely cultured in DMEM (Aldrich-Sigma) supplemented with 10% fetal bovine serum (FBS) (In-stitute of Experimental and Clinical Veterinary Medicine,Kharkiv, Ukraine) and 50 �g/ml gentamicin (Aldrich-Sigma)

I.I. Mechnikov Institute of Microbiology and Immunology AMS of Ukraine, Kharkiv 61057, Ukraine.

at a density of 104 cells/cm2 in 96-well plates (Bayer, Lev-erkusen, Germany) at 37°C in an atmosphere of 5% CO2. Thesensitivity of cell cultures to IFN-�2b was evaluated by exam-ining the cell growth inhibition after treatment by IFN-�2bcompared with control (untreated) cells.

The index of antiproliferative activity (IAPA) of IFN (%)was calculated by the formula:

IAPA � [1 � (cells in experimental well/cells in controlwell)] � 100.

The nonspecific cytotoxic effect was evaluated by trypan bluestaining of cells incubated for 24 h with the IFN derivatives.None of derivatives exhibited nonspecific cytotoxicity. The ini-tial titer of unmodified IFN-�2b was 2.5 � 103 IU/ml. IFN-�2band its derivatives were added to the medium at a dose of 1000IU/ml (0.05 ml/well) by calculation of the protein concentra-tion (2.5 � 108 IU IFN-�2b is equivalent to 1 mg protein). Cellswere incubated with IFN-�2b for 72 h, and the number of cellswas determined using an inverted microscope (Leica, Wetzlar,Germany) every 12 h.

RESULTS

IFN-�2b is a protein with a molecular weight of approxi-mately 18 kDa. It contains 8 lysine and 3 histidine residues,(7)

that may be acylated by succinic and aconitic anhydrides. Sevenfree lysines (Lys31, 70, 83, 121, 131, 133, and 134) are foundon the surface of the molecule (Fig. 1). Based on the buffer sys-tem and the more potent nucleophilic properties of the lysineresidues, the lysine residues are derivatized. The region that in-teracts with the receptor (lower part of IFN on Fig. 1) containsonly one lysine (Lys49) and one histidine residue, which areinaccessible for acylation. Histidine residues from the regula-tory domain of IFN-�2b (upper part of Fig. 1) form an innermolecule structure that is inaccessible for acylation because ofsteric hindrance. Although the hydroxyl groups of threonine andserine, as well as sulfhydryl amino acids, are accessible to acy-lation, this possibility was prevented by conducting the reac-tion in a slightly alkalinized medium. The deprotonation of ly-sine amino groups occurs at pH � 7.5, and lysines acquire morepotent nucleophilic properties than hydroxyl and sulfo groups.Based on the ratio of IFN-�2b/anhydride, seven derivatives,which contain one to seven acylated lysine amino groups, maybe produced. The reagents ratio and properties of the synthe-sized succinylated and aconitylated IFN-�2b derivatives arepresented in Tables 1 and 2, respectively. Data suggest that thecalculated and the actual weights of the IFN-�2b derivativesare similar for one through six acylated derivatives, thus beingan indication of complete acylation reaction for both types ofIFN derivatives.

The antiproliferative activity of aconitylated IFN (AIFN) andsuccinylated IFN (SIFN) was studied in vitro in cell cultures of

ANTIPROLIFERATIVE ACTIVITY OF ACYLATED IFN 415

TABLE 1. REAGENTS RATIO AND PROPERTIES OF SUCCINYLATED IFN-�2B DERIVATIVES

IFN/anhydride(mol/mol) Rf Calculated Determined

1�1 0.490 � 0.005 18359 � 10 18360 � 501�2 0.462 � 0.005 18458 � 10 18460 � 501�3 0.460 � 0.005 18557 � 10 18560 � 501�4 0.456 � 0.005 18656 � 10 18655 � 501�5 0.442 � 0.005 18755 � 10 18750 � 501�6 0.440 � 0.005 18854 � 10 18855 � 501�7 0.436 � 0.005 18953 � 10 18920 � 500 (Control, IFN) 0.492 � 0.005 18260 � 10 18250 � 50

*Molecular weight.

Mr, Daa

TABLE 2. REAGENTS RATIO AND PROPERTIES OF ACONITYLATED IFN-�2B DERIVATIVES

IFN/anhydride(mol/mol) Rf Calculated Determined

1�1 0.482 � 0.005 18417 � 10 18400 � 501�2 0.472 � 0.005 18574 � 10 18570 � 501�3 0.462 � 0.005 18731 � 10 18740 � 501�4 0.453 � 0.005 18888 � 10 18860 � 501�5 0.446 � 0.005 19045 � 10 19060 � 501�6 0.441 � 0.005 19202 � 10 19200 � 501�7 0.438 � 0.005 19539 � 10 19320 � 500 (Control, IFN) 0.492 � 0.005 18260 � 10 18250 � 50

*Molecular weight.

Mr, Daa

the CaOv and SW-480 cell lines (Figs. 2 and 3). CaOv cells arevery sensitive to IFN-�2b. Acylation of one lysine residue bysuccinic anhydride caused a 3.2-fold increase in the IAPA, andacylation of two to five lysine residues caused a gradual de-crease in IAPA., Finally, acylation of six lysine residues led tothe elimination of antiproliferative activity. Acylation of one

lysine residue by aconitic anhydride caused a 3.6-fold increasein IAPA, but antiproliferative activity steadily decreased there-after with the increase in the number of acylated residues. Inthe case of less sensitive SW-480 cells, unmodified IFN-�2bpossessed no antiproliferative activity. The highest activity wasdetected for derivatives with three acylated lysine residues. TheIAPA of AIFN with three acylated lysine residues was 16%higher than that of SIFN with three modified lysine residues.The IAPA of SIFN with three to five modified residues wasconstant, but in the case of AIFN, the index decreased gradu-ally with increasing acylation. The antiproliferative effect ofacylated IFN-�2b reached the maximum at 12 h incubation anddid not change in the subsequent 60 h.

DISCUSSION

In conclusion, acylation of one lysine residue IFN-�2b mole-cule by cis-aconitic or succinic anhydride causes a 3–3.5-fold in-crease in its antiproliferative activity on the CaOv cell line. IFN-�2b acylation extends the spectrum of its antiproliferative activityand allows it to reach its maximal value at 12 h postincubationwith both IFN-sensitive and IFN-insensitive cultured cells. Thehighest antiproliferative activity against SW-480 cells was ob-tained for IFN-�2b with three modified lysine residues by aconity-lation or by succinylation. We conclude that aconitylated and suc-cinylated IFN-�2b may be considered potential antiproliferativeagents, although their properties should be tested in nonclinicalstudies, for example, animal models, and, if appropriate, in clin-ical studies to evaluate their anticancer and pharmacokinetic prop-erties and their ability to induce antibodies in the treated host. Wehypothize that acylated IFN-�2b may have enhanced antiprolif-erative activity as a result of its interaction with its receptor, thatis, an increased affinity to its receptor.(2–4)

MARTYNOV AND SMELYANSKAYA416

FIG. 1. Structure of IFN-�2b by X-ray structural analysis (online database of U.S. National Library of Medicine: www.ncbi.nlm.nih.gov/Structure/mmdb/mmdbsrv.cgi?form�6&db�t&Dopt�s&uid�6520). Lysine, histidine and arginine residuesare depicted.

FIG. 2. Index of antiproliferative activity (IAPA) of IFN-�2b derivatives on CaOv cells.

ACKNOWLEDGMENTS

We express sincere gratitude to Professor Dr. Yu. M.Krasnopolskiy (Cas “Biolek”) for kindly providing IFN andother reagents. We thank I.V. Korobkova, Ph.D., M.D. (Labo-ratory of Viral Infections), for the tissue culture. We acknowl-edge “Sinbias” (Donetsk) for providing the Sigma-Aldrichreagents.

REFERENCES

1. Lauta VM. Update on the use of interferons in clinical practice. Clin.Ther. 1995;146:393–448.

2. Kuipers ME, vd Berg M, Swart PJ, Laman JD, Meijer DK, Kop-pelman MH, Huisman H. Mechanism of anti-HIV activity of suc-cinylated human serum albumin. Biochem. Pharmacol. 1999;57:889–898.

3. Zaghloul M, Prakash V. Effect of succinylation on the functionaland physicochemical properties of alpha-globulin, the major proteinfraction from Sesamum indicum L. Nahrung 2002;46:364–369.

4. Martynov AV, Chernykh VP. Synthesis and biological properties ofimmunoglobulins acylated with succinic anhydride. News Pharm.2002;3:13–16.

5. Merril CR, Dunau ML, Goldman D. A rapid sensitive silver stainfor polypeptides in polyacrylamide gels. Anal. Biochem. 1981;1:201–207.

6. Butcher LA, Tomkins JK. A comparison of silver staining meth-ods for detecting proteins in ultrathin polyacrylamide gels on support film after isoelectric focusing. Anal. Biochem. 1985;1:384–388.

7. Melen K, Ronni T, Broni B, Krug RM, von Bonsdorff CH, JulkunenI. Interferon-induced Mx proteins form oligomers contain a putativeleucine zipper. J. Biol. Chem. 1992;267:25898–25907.

Address reprint requests or correspondence to:Dr. A.V. Martynov

I.I. Mechnikov Institute of Microbiology and ImmunologyAMS of Ukraine

14-Puskhinskaya StKharkiv 61057

Ukraine

Tel: 38 0577313151Fax: 38 0577142785

E-mail: imiamn@mail.ru

Received 24 September 2004/Accepted 1 March 2005

ANTIPROLIFERATIVE ACTIVITY OF ACYLATED IFN 417

FIG. 3. Index of antiproliferative activity (IAPA) of IFN-�2b derivatives on SW-480 cells.