ICANCER RESEARCH56. 5205-5210, November 15. 9961 ABSTRACT A marked effect ofcharge modification on the uptake and phototoxicity of a photoimmunoconjugate (PlC) was demonstrated. A site-specific con jugation strategy was developed to attach the photosensitizer chlorin e6 (ce6)to the F(ab')2 fragment of the murine antiovarian cancer monoclonal antibody 0C125. Poly-L-lysine linkers carrying ce6with a cationic charge or by polysuccinylation with an anionic charge were used and covalently attached to partially reduced antibody via a heterobifunctional reagent. PICs were purified by column chromatography and were also radiola beled with 1251.PlC binding and uptake were studied with a human ovarian cancer cell line, NIH-OVCAR-5, and a nonantigen-expressing colon cancer cell line, SW1116, and the data were compared with the binding and uptake of nonspecific rabbit IgG PICs. PICs with both cationic and anionic charges preserved antigen binding as shown by competition studies with native antibody, but the cationic PlC had up to 17 times higher cellular uptake of ceo, probably due to enhanced inter nalization. The ratio of ce, to ‘@I retained by the cells varied with the likelihood ofinternalization and lysosomal degradation. The phototoxicity ofthe PICs generally varied with their uptake, but a correlation was found between lysosomal hydrolysis as measured by an increased cellular ratio ofce,:'251 and increased relative phototoxicity. These data suggest cationic PICs may have advantages for photoimmunotherapy of disseminated intracavity cancer following local administration. INTRODUCTION Mabs3 directed against tumor-associated antigens are used to target drugs or radioisotopes to tumors for both diagnosis and therapy (1). Photoimmunotherapy, in which PSs are conjugated to Mabs has recently been explored (2—5).These PSs produce cytotoxic species (singlet oxygen or radicals) when irradiated with red light. This approach using PICs has inherent dual selectivity: in addition to the tumor-targeting properties of the Mab, tissue destruction is restricted to the field of illumination. This modality may be suitable for treat ment of small diffuse malignancies present in a cavity such as the peritoneum or bladder (6). Therapeutic success using Mab conjugates has been modest due to (a) limited uptake and internalization of conjugates and (b) lack of specificity. It is well known that the introduction of positive charges onto macromolecules increases the rate of endocytosis (7, 8), and we hypothesized that this would apply to PICs and that a positively charged PlC would have greater tumor uptake when administered intracavitary. Uptake of the cationized PICs could be further enhanced by the use of F(ab')2 fragments, which have the advantages of greater penetration into the tumor and faster clearance (9). This study tests the above hypothesis and is a first report on (a) the synthesis of site-specific PICs using a strategy which is equally Received 5/8/96; accepted 9/16/96. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. I This work was supported by NIH Grant RO1 AR40352 and in part by the Department of Defense Free Electron Laser Program facilities. 2 To whom requests for reprints should be addressed. Phone: (617) 726-6996; Fax: (617) 726-3192. 3 The abbreviations used are: Mab, monoclonal antibody; PS, photosensitizer; PlC. photoimmunoconjugate; NHS, N-hydroxysuccinimide; ce,, chlorin ,.,,; p1, poly-L-lysine; succ, succuccinylated; SPDP, 2-pyridyl-dithiopropionic acid amide. applicable to intact IgG or to F(ab')2 fragments and which has the option of preparing PICs bearing either cationic or anionic charges and (b) the effect of charge modification of PICs constructed from the F(ab')2 fragment of the Mab OCl25 on their interaction with a human ovarian cancer cell line, NIH:OVCAR-5, expressing the target antigen CA125 (10). MATERIALS AND METHODS Cell Line and Mab. NIH:OVCAR-5(OVCAR-5) cells were purchased from Dr. T. Hamilton (Fox Chase Cancer Institute, Philadelphia, PA) and SW1116 human colon cancer cells were purchased from American Type Culture Collection (Rockville, MD). Cells were grown in RPM! 1640 contain ing HEPES, glutamine, 10% heat-inactivated fetal bovine serum, 100 units/ml penicillin, and 100 pg/mI streptomycin and maintained in an incubator at 37°C in an atmosphere of 5% carbon dioxide. OC125F(ab')2 murine Mab was a generous gift from Centocor (Malvern, PA). Rabbit lgG was obtained from Sigma (St. Louis, MO). Radiolabeling. OC125F(ab')2 and rabbit IgG were radiolabeled with 1251 using the lodo-Gen procedure (I 1). ‘251-labeled OC125F(ab')2 had a specific activity of 8.7 MBq/mg whereas ‘251-labeled rabbit IgG had a specific activity of2.l MBq/mg. Preparation of Poly-L-lysine Ce6 Conjugates. All reactions were carried out in the dark at room temperature. The NHS ester of ceo was prepared by reacting 1.5 equivalents of dicyclohexylcarbodiimide and 1.5 equivalents of NHS with 1 equivalent of ceo in dry DMSO for 24 h and was frozen in aliquots for further use. p1 hydrobromide (average Mr 25,000 50 mg) was dissolved in dry DMSO (50 ml) and N-ethylmorpholine (1 ml) was added followed by ceo-NHS ester (25 mg) and reacted for 24 h. The solution of pl-c@6 obtained was placed into a DMSO-resistant dialysis membrane of Mr 3500 cutoff (Spectrum Medical Industries, Los Angeles, CA) and dialyzed twice against 5 liters of I mM potassium phosphate buffer (pH 9). The pl-c,@ dissolved in DMSO was treated with an excess of succinic anhydride (100 mg dissolved in 0.5 ml of dry DMSO, 4-fold excess for each primary amino group) for 24 h to afford the anionic pl-ceo-succ which was dialyzed as above. Preparation of Site-specific PICs with OC12SF(ab')2 and Rabbit IgG. The DMSO solution of p1-c@6 was treated with a solution of pyridyldithiopro pionic acid-NHS ester in DMSO (3 equivalents/pI chain). After reacting for 24 h, it was dialyzed twice against 5 liters of 1 mist acetic acid. OC125F(ab')2 fragment or rabbit IgG (5 mg) was dissolved in S ml of 10 miss phosphate buffer (pH 7.5) containing I msi EDTA and 5 mM mercaptoethylamine hydrochloride, and the solution was purged with nitrogen and shaken in the dark for 2 h. The solution was then dialyzed three times against 5 liters of 10 mM phosphate buffer (pH 7.5); 1 msi EDTA. To this solution was added 3 equivalents of pl-ceo-SPDP and left 24 h before purification. To the pl-cen-SPDPsolution described above was added a 4-fold excess of succinic anhydride. After 24 h the resulting p1-c@6-SPDP-succ was dialyzed (three times) against 5 liters of 1 mMsodium acetate (pH 9) and then added to the partially reduced Mab as described above. PlCs were purified on Sephadex G200 columns and characterized by SDS-PAGE (12). Quantities of PICs were calculated by measuring absorption spectra in 0.1 MNaOHI1% SDS solution and assuming the € (400 nm) of c,.,,, 1.5 X l0@,remained unchanged by conjugation. Fluorescence Localization in Living Cells. OVCAR-5 cells were grown (60% confluent) in P35 dishes containing 4 ml of medium and a 22-mm square glass coverslip. PICs were added at a final concentration of 2 pM cen equivalent in serum-containing medium and allowed to incubate for 3 h. The coverslips were washed three times with PBS and put on histological slides in PBS and examined with an epiillumination fluorescence microscope (Axiophot; ZeiSS, 5205 Effect of Charge on the Interaction of Site-specific Photoimmunoconjugates with Human Ovarian Cancer Cells' Michael R. Hamblin, Jaimie L. Miller, and Tayyaba Hasan2 Wellman Laboratories of Photomedicine, Department of Dermatology, Massachusetts Genera! Hospital. WEL224, Harvard Medical School. Boston, Massachusetts 02114 on July 13, 2017. © 1996 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from