Application of Cytokine Drug Delivery Systems t o the lmmunotherapy of Renal Cell Carcinoma in Mice Ken Marumo,* Mototsugu Oya, Masaru Murai, and Hiroshi Tazaki Department of Urology, School of Medicine, Keio University, Tokyo, Japan

We have investigated the antitumor effects of human lynnphoblastoid interferon (HLBI) mini-pellets, interleukin-2 (ILL ) entrapped in liposome (IL-2 liposome) and an immune complex of IL-2 and monoclonal c>ntillotlyagainst IL-2 (IC1 ) . The HLBI mini-pellets were administered to nude mice bearing a human renal cant.er ( e l l line (KCI-2). HLBI levals remained detectable both in the tumor tissue and the serum up to 10 days after peritumor injection. The HLBl minipellet significantly suppressed tumor growth by peritumor administration. The antitumor eftect of IL-2 liposonne on Renca, a niurine renal cancer, resulted in the inhibition of tumor growth. An accumulation of Lyt-2- a n d L3T4 lymphocytes was seen in the tumor tissue which was treated with IL-2 liposomes. The IC-1 was preli.ired b y mixing IL-2 and anti-lL-2 monoclonal antibody at a molar ratio of ? : I , Plasma IL-2 levels were sustained longer in mice given the IC-1 than in mice given IL-2 alone. The IC-l complex exerted a inore signiticdnt antitumur eftect by locc~l administration in Renca-bearing mice than the administration of IL-2 alone. We spec ulatetl that these effects wrre a result of sustained tumor IL-2 levels due to the increase in molecular weight.The results we olit,i.ined indicate that the cytokine drug delivery system has a long-acting cytotoxicity hy administration to the tumor sites thl-ougln efficient stimulation of the local immune response, and thus provides a useful tool for treatment of renal cell carcinoma. I n t j Urol 1996;3(suppl l):S24-S27 Key words: interferon, interleukin-2, renal cell carcinoma, drug delivery system

INTRODUCTION

Renal cell carcinoma (RCC) has a poor prognosis once the disease becomes metastatic or recurrent. Recent progress in tumor immunology and biotechnology has offered a new approach in the treatment of RCC utilizing interferon (IFN)',? and interleukin-2 (IL-2).3.4 However, the conventional therapy of systemic administration of IFN or IL-2 has limited antitumor action. Additionally, there are problems with the clinical use of these cytokines, in that they are cleared rapidly from the blood. In order to circumvent these problems, the cytokines should act on the tumor for longer periods in higher concentrations. Therefore we considered administering the cytokines directly to the tumor site with the use of sustained-release agents. We investigated the antitumor effects and biological properties of human lymphoblastoid interferon (HLBI) mini-pellets, IL-2 entrapped in liposomes (IL-2 liposomes) and an immune complex of IL-2 and monoclonal antibody against IL-2 (IC-1).

Tumor Cells

The human renal cancer cell line, KU-2, was derived from a pulmonary metastatic lesion of RCC capable of producing erythropoietin. After being transplanted to the nude mice, it was established as a cell 1ine.j Renca, which is a transplantable murine RCC of spontaneous origin, was established at the National Cancer Institute, USA.b Interferon

The human lymphoblastoid IFN (HLBI), a human natural alpha IFN, was a product of Sumitomo Pharmaceutical Co, Ltd (Japan). The HLBI was derived from the Namalwa strain of Burkitt lymphoma, and its specific activity was more than 1 x lo8 Uimg protein.

MATERIALS A N D METHODS

Animals Male athymic nude mice (nuinu, BALBic background) were purchased from the Shizuoka Experimental Animal Center (Japan). Female BALBic mice were purchased from Charles River Japan Inc (Japan). The animals were kept in established pathogen-free-conditioned animal cages. For all experiments, 6-week-old mice were used.

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*C orre\ponden( e m d request5 tor reprints to: Department of Urology, School ( I t Mrtlicine, Keto University, 35 Shinaiiomaclit, Shinluku-ku. Tokyo 160, )'lp'ln

S24

Fig. 1 . The human lynnphoblastoid interferon (HLBI) mini-pellet is 6 inn1 in length and 1 nnni in diameter. The pellet cont'iins 4 x 10' U or 3 x 10" U of HLBI. It can he administered by standard injection, and is biocoinpatihle and biodegradable.

0919-8172/1996/0351-524/US$3.00

0 JUAICLJ

Cytokine Drug Delivery System in Renal Cancer

K. Marumo el al.

HLBl Mini-Pellets Each HLBI mini-pellet was 6 mm in length, 1 mm in diameter, and contained 4 x lo5 units or 4 x 10b units of HLBI (Fig. 1 ) . Atelocollagen, supplied by Koken Co Ixd (Japan), was used as the carrier material. This collagen has undergone protease treatment in order to remove the telopeptides expressing its major immunogcnicity. The HLBI mini-pellets were produced by Sumitomo Pharmaceutical Co Ltd as previously described.'

HRLl-52, prepared by Takeda Chemical Industries, as prcviously described,8 in 1.2 mL of phosphate-buffered saline (I'BS) and the reaction mixture was incubated at room temperature for 10 minutes. MoAb-HRL1-52 had been shown not to neutralize the biological properties of IL-2 in immune complex formation.x RESULTS Pharmacokinetics of HLBl Mini-Pellets

Interleukin-2

The recombinant human IL-2 produced by Shionogi C o Ltd (Japan) was used in the experiments for the IL-2 liposomes. The IL-2 produced by Takeda Chemical Industries Ltd (Japan) was used for the experiments as an immune complex of IL-2 and monoclonal antibody against IL-2. Titer of IL-2 produced by Shionogi was expressed in units, and titer of IL-2 produced by Takeda Chemical Industries Ltd was expressed in pg. One microgram of 1L-2 had proved to be equivalent to approximately 10' units of 1172. 11-2 Liposomes

The IL-2 preparation was entrapped in liposomes in the following way. Egg phospharydyl choline (Nippon Oil & Fats Co, Japan) and cholesterol (Sigma Chemical Co, USA) were mixed in a ratio of 2: 1 in a pear-shaped container with chloroform and methanol (2:l). The organic solvent was removed using a revolving evaporator in a basin at 42°C for 30 minutes. A thin lipid film formed in the container. One hundred thousand units of 11.-2 were dissolved in 1 mL of distilled water and added to the container. IL-2 liposomes were prepared by vortex dispersion for 10 minutes. The I I 2 liposomes were separated from the unentrapped IL-2 by centrifuging at 15,000 g for 30 minutes. immune Complex

An immune complex of IL-2 and a monoclonal antibody (MoAb) against IL-2 (IC-1) was prepared by mixing IL-2 with anti-IL-2 MoAb at a molar ratio of 2:1 (Fig. 2). Briefly, 0.2 mg of 11,-2 was mixed with 1.0 mg of MoAb-

Following the peritumor injection of mini-pellets containing 4 x 10' units of HLRI, HLBl concentrations in the serum and tumor tissue were determined using radioimmunoassays (RIA), at various times after administration. After administration of the mini-pellet, the serum HLBI concentrations reached their peak levels 1 day after treatment, subsequently declined, and were still detectable up to 10 days after administration. The tumor nodules were divided into a proximal half portion adjacent to the minipellet and a distal half portion adjacent to the skin, homogenized in PBS, and then subjected to RIA to determine the HLBI concentration. HLBI levels in tumor tissue were shown to be 18,710 Uig in the proximal portion and 4835 Uig in the distal portion 1 day following administration of the mini-pellets, and 400 Uig or higher HLBI levels were sustained in tumor tissue up to 1.0 days after administration of the mini-pellets (Fig. 3). Antitumor Activity of the HLBl Mini-Pellets

KU-2 tumors were subcutaneously implanted into nude mice on day 0 and therapy was then given on days 20 and 30. Peritumor injection of HLRI mini-pellets in the treated mice produced significant inhibition of tumor growth compared to the untreated mice, the mice treated by HLBI alone, and the mice treated with subcutaneous injections of HLBI mini-pellet ( P