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Use of Immunotoxin to Inhibit Proliferating Human Corneal Endothelium

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Use of Immunotoxin to Inhibit Proliferating Human Corneal Endothelium Investigative Ophthalmology & Visual Science, Vol. 29, No. 5. May 1988 Copyright © Association for Research in Vision and Ophthalmology Use of Immunotoxin to Inhibit Proliferating Human Corneal Endothelium Samuel Fulcher,* Geming Lui,f L. L. Houston,£ 5. Romokrishnon,^: Terry Burris,§ Jon Polansky,t ond Jorge Alvaradof Transferrin plays a central role in cellular proliferation and proliferating ceils have been shown to express transferrin receptors with increased density. We examined the effect of an immunotoxin consisting of anti-transferrin receptor monoclonal antibody (454A12) conjugated to recombinant ricin A chain (rRTA) on the proliferation of human corneal endothelium (HCE) in vitro. In proliferating cultures an immunotoxin (454A12-rRTA) concentration of 50 ng/mL reduced cell counts at day 7 by at least 89%, with no effect observed at 0.01 ng/ml. In contrast, cell counts were only minimally reduced in confluent cultures, even after 7 days' exposure to high concentrations of immunotoxin. These data suggest that 454A12-rRTA may be used to prevent growth of human corneal endothelium in patholog- ical conditions such as the iridocorneal endothelial (ICE) syndrome. Invest Ophthalmol Vis Sci 29:755-759,1988 The hyperproliferation of nonmalignant tissue munotoxins is the development of monoclonal anti- causes a wide spectrum of clinically significant ocular bodies that specifically react with antigens on the sur- disease such as proliferative vitreoretinopathy, the ir- faces of cells and that can be used to carry potent idocorneal endothelial syndromes, fibrous or epithe- cellular toxins to target cells. Several toxins or frag- lial downgrowth and posterior capsular fibrosis. ments of toxins such as diphtheria toxin, ricin and Methods currently used to treat these conditions in- ricin A chain have been coupled chemically to anti- clude surgery, laser surgery, cryotherapy and chemo- bodies to form immunotoxins.12 Ricin A chain is an therapy using 5-fluorouracil. These methods often enzyme that inhibits protein synthesis by chemically fail completely or are unsatisfactory because they do modifying 28 S rRNA on the 60 S ribosomal subunit not successfully differentiate between unwanted, pro- to prevent protein synthesis.3 Because of the presence liferating cells from normal tissue. of the antibody in the conjugate, immunotoxins rec- The use of immunotoxins offers a way to selec- ognize the surface of specific cells. If internalization tively deliver drugs to proliferating cells for treatment occurs, the target cell may be killed selectively. We of some ocular diseases. Central to the use of im- tested the ability of such an immunotoxin directed against transferrin receptor to selectively inhibit the growth of proliferating human corneal endothelial From the *Scott and White Clinic, Scott and White Memorial Hospital, Scott, Sherwood and Brindley Foundation, Texas A&M cells in vitro. University College of Medicine, Temple, Texas, the fUniversity of Transferrin is a growth factor that is required for California Medical Center, San Francisco, California, the |CETUS the growth of all cells, and through endocytosis using Corporation, Emeryville, California, and the §Departments of the transferrin receptor contributes to the maximal Clinical Investigation and Ophthalmology, U.S. Naval Hospital, 45 Oakland, California. proliferation of cells in vitro. Other growth factors Work performed at the U.S. Naval Hospital, Oakland, was such as erythropoietin and epidermal growth factor sponsored under the United States Navy Clinical Investigation Pro- influence cell proliferation and the expression of gram, Study Number 85-48-2098. Work sponsored at the Univer- transferrin receptors.6'7 Because transferrin receptor sity of California, San Francisco, was supported by grants is located at the cell surface, is present in increased EY-02068, EY-02477, and EY-02162. amounts on the surface of proliferating cells,8"10 and The opinions or assertions expressed herein are those of the 810 authors and are not to be construed as official or as necessarily is rapidly internalized after it binds to transferrin, reflecting the views of the U.S. Department of the Navy or the it has been an effective target for the inhibition of Naval Service at large. malignant cells by immunotoxins.1'211"13 Because ac- Submitted for publication: March 31, 1987; accepted December tively proliferating cells express a higher number of 4, 1987. transferrin receptors, we think that proliferating cells Reprint requests: Samuel Fulcher, MD, Department of Ophthal- mology, Scott and White Clinic, Scott and White Hospital, 2401 may be killed preferentially over resting cells by an South 31st Street, Temple, TX 76508. immunotoxin directed toward transferrin receptors. 755 Downloaded from iovs.arvojournals.org on 09/26/2021 756 INVESTIGATIVE OPHTHALMOLOGY 6 VISUAL SCIENCE / May 1988 Vol. 29 In this study, we show that an immunotoxin that Assay of Immunotoxin Activity contains recombinant ricin A chain and an antibody The effect of 454A12-rRTA on cellular prolifera- directed against transferrin receptor selectively in- tion was assayed using two sets of duplicate plates hibits proliferation of dividing human corneal endo- containing serial dilutions of immunotoxin. Four sets thelial cells compared to confluent cell cultures. of controls were used, including plates with unconju- gated rRTA, with purified unconjugated 454A12 an- Materials and Methods tibody, with rRTA conjugated to MOPC21 (an irrele- vant immunotoxin) or without any additives. Prolif- Production and Characterization erating cells were exposed continuously to a single of Monoclonal Antibodies dose of 454A12-rRTA or control protein for all 7 Murine monoclonal antibody 454A12 (IgG2a iso- days of culture, whereas confluent cultures were ex- tope) is specific for human transferrin receptor and posed for 7 days after achieving confluence. Dead was produced by standard fusion techniques de- cells were observed to detach from the dishes and scribed previously.2 This antibody binds to cell sur- were aspirated prior to counting; unexposed plates face transferrin receptors, immunoprecipitates the did not contain detached nonviable cells. No de- 95K. MW subunit transferrin receptor, recognizes an tached cells were viable as assessed by trypan blue antigenic site distant to the actual transferrin binding exclusion. The number of live cells was determined site and does not interfere with transferrin binding to with a Coulter counter after 7 days of incubation with its receptor or to the internalization of transferrin. immunotoxin or control protein. Ammonium sulfate precipitation, DEAE-Sepharose The effect of immunotoxin on amino acid incorpo- chromatography, and Sephacryl S-300 gel filtration ration was assayed in duplicate with 3 X 104 cells in were used to purify the antibody contained in ascites 0.5 mL of medium seeded in borosilicate glass vials fluid from Balb/c mice. (ICN Radiochemicals, Irvine, CA) coated with 0.2% gelatin. After 48 hr of proliferation, cells were incu- Immunotoxin Synthesis bated in duplicate for 18 hr in the presence or absence of 454A12-rRTA. The cells were then rinsed three Recombinant ricin A chain was obtained from E. times with phosphate-buffered saline (PBS) and incu- co/i and purified to over 99% homogeneity. Imino- bated with 0.2 ml of leucine-free 1640 RPMI thiolane was used to introduce a thiol group onto the (GIBCO) containing 2.0 ^Ci of [H3H]leucine (Du- surface of 454A12 and a mixed disulfide bond was pont NEN Research Products, Boston, MA). Fetal made using Ellman's reagent [5,5'-dithiobis(2-nitro- calf serum (10%) was added to maintain cellular ad- benzoic acid), DTNB]. Disulfide exchange between herence during the incubation with [H3H]leucine. the mixed disulfide bond and the free thiol group of After 3 hr of incubation, the cells were washed three rRTA formed a disulfide bond between 454A12 and times with PBS and the protein was precipitated with rRTA. The immunotoxin was purified by a combina- 5% trichloroacetic acid (GIBCO). Counts were mea- tion of chromatography and gel filtration; the final sured in a Tricarb® liquid spectrometer scintillation product contained no detectable unconjugated rRTA counter with 5 ml of scintillation fluid (Research or454A12. Products International, Elk Grove, IL). Cell Cultures Data Analysis Under sterile conditions, human corneal endothe- The effects of 454A12-rRTA on cellular prolifera- lial cells were scraped from donor corneas within 2 hr tion and [H3H]Ieucine incorporation were deter- of harvesting. Stock cultures were seeded on gelati- mined by comparing the growth or incorporation in nized 35 mm dishes (Becton/Dickenson, Oxnard, plates or vials containing immunotoxin with control CA) in medium 199 (GIBCO, Grand Island, NY) plates or vials from the same stock to which no containing 15% fetal calf serum (Sterile Systems, 454A12-rRTA was added. Results from plates or Logan, UT), Earle's balanced salts (GIBCO), and 1% vials without additives were defined as representing of 200 mM glutamine (Sigma, St. Louis, MO). The 100% of control numbers for that experiment and cells were maintained in 5% CO2 at 37°C with fibro- were compared to results from plates or vials with blast growth factor (University of California, San 454A12-rRTA, rRTA or 454A12 alone. Percent cell Francisco) added every other day. Once confluent, survival and [H3H]leucine incorporation were calcu- cells were trypsinized and seeded in 2 ml of medium lated for plates or vials containing additives as com- on gelatinized 35 mm dishes at 2 X 104 cells/ml for pared to negative controls. These data were plotted individual experiments. against the log dose (ng/ml) of 454A12-rRTA. Downloaded from iovs.arvojournals.org on 09/26/2021 No. 5 IMMUNOTOXIN INHIBITS HUMAN CORNEAL ENDOTHELIUM / Fulcher er ol. 757 6 100 10'' 10° 10' 10' IMMUNOTOXIN CONCENTRATION (ng/ml) IMMUNOTOXIN CONCENTRATION (ng/ml) Fig. 1. Percentage of surviving cells recovered as compared to Fig. 2. Percentage of surviving cells in plates with 454A12-rRTA controls plotted against immunotoxin level in confluent and prolif- (solid squares) is lower than in plates with 454A 12-rRTA and 2500 erating HCE cells.
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