Inhibition of Antibody Response to Pseudomonas Exotoxin and an Immunotoxin Containing Pseudomonas Exotoxin by 15-Deoxyspergualin in Mice Lee H
Total Page：16
File Type：pdf, Size：1020Kb
(CANCER RESEARCH 50. 7750-7753, December 15, 1990] Inhibition of Antibody Response to Pseudomonas Exotoxin and an Immunotoxin Containing Pseudomonas Exotoxin by 15-Deoxyspergualin in Mice Lee H. Pai, David J. FitzGerald, Mark Tepper, Bernice Schacter, George Spitalny, and Ira Pastan1 Laboratory of Molecular Biology, National Cancer Institute, N1H, Bethesda, Maryland 20892 [L. H. P., D. J. F., I. P.], and Bristol-Myers Squibb Co., Wallingford, Connecticut 06942 fM. T., B. S., G. S.J ABSTRACT has antitumor activity against murine leukemias (10). It has also been shown to possess immunosuppressive activities in Immunotoxins are potent cell-killing agents that may be useful in the experimental animal models (11, 12). In this study, we assessed treatment of cancer. The early production of neutralizing antibodies to the ability of DSG to suppress the antibody response to PE and immunotoxins is one of the major limiting factors for their use in humans. a PE-containing immunotoxin. The antibody formation against 15-Deoxyspergualin (DSC), a derivative of spergualin, which is a metab olite of Bacillus laterosporus, has been found to have immunosuppressive PE was measured either by an ELISA or by the ability of serum from immunized mice to block the cytotoxic action of anti-Tac- activity in rodents, dogs, and primates. We examined the suppressive activity of DSG on the antibody response to Pseudomonas exotoxin in LysPE40 in human T-cell lymphoma cells (HUT 102). mice by enzyme-linked immunosorbent assay. Male UDÃŒ,mice were immunized with a single dose of a nontoxic mutant of Pseudomonas MATERIALS AND METHODS exotoxin (40 *jg) and then treated with i.p. injections of DSG at a dose of 10 mg/kg for 3 days. Although antibodies to Pseudomonas exotoxin Animals were observed within 7 days in the control group, there was complete suppression of antibody production in the DSG-treated group. Immuno- Male BOP, mice (20-25 g), 6-8 wk of age, were obtained from the suppression has also been observed in animals immunized with multiple Frederick Cancer Research Facility, Frederick, MD, and kept in an doses (10 mg x 7 d) of Pseudomonas exotoxin and treated with DSG at approved animal facility. Animals were fed a standard diet with drinking a dose of 5 mg/kg for 21 days. Similar immunosuppression was observed water ad libitum. in mice given multiple doses of the immunotoxin, anti-Tac-LysPE40. We Drug conclude that the immunosuppressive activity of DSG may be useful in increasing the duration of immunotoxin treatment. DSG was supplied by Bristol-Myers Squibb Co., Wallingford, CT. It was dissolved in sterile PBS, pH 7.2, just prior to use each day. INTRODUCTION Toxins Over the past decade, advances in protein chemistry and PE553D. PE553D is a recombinant mutant form of PE expressed in hybridoma technology have led to the development of immu Escherichia coli in which the glutamic acid at position 553 is deleted, notoxins consisting of monoclonal antibodies conjugated to resulting in loss of ADP ribosylation activity but not its immunogenicity protein toxins made by bacteria and plants: diphtheria toxin, (13). It was chosen for these experiments because of its lack of toxicity PE2, and ricin have been widely used for this purpose (1-3). in animals. Anti-Tac-LysPE40. Construction of this immunotoxin has been pre These toxin conjugates are potent cell-killing agents, and their viously described (14). LysPE40 is a recombinant form of PE with M, possible use in the treatment of cancer is being explored (4, 5). 40,000 that lacks domain I of PE and has very low liver toxicity because Results from the first clinical trials with these hybrid molecules it no longer binds to PE receptor present on liver cells. A lysine residue have shown several problem areas that must be addressed for has been included near the amino end, which makes it readily coupled immunotoxins to become broadly effective therapeutics. One to antibodies. LysPE40 was purified from the culture medium of BL21 of these problems is the early production of antibodies against (XDE3) cells containing plasmid pJYSSL (15). After purification, immunotoxins, which may neutralize the toxic activity of these LysPE40 was chemically coupled to anti-Tac antibody, which binds to conjugates, limiting their therapeutic efficacy (6, 7). The for the M, 55,000 subunit of human interleukin 2 receptor (16). After the conjugation, 1:1 forms of the immunotoxin were purified on Mono Q mation of human antibodies against murine monoclonal anti and TSK-250 columns. Purified conjugates were found to contain bodies is well described and has been shown to alter their LysPE40 coupled to either the light or the heavy chain of the antibody. pharmacodynamics (8) and pharmacokinetics (9). It is clear that, to improve the efficacy of the immunotoxins, strategies to Immunoassay inhibit the immune response to foreign proteins need to be ELISA was used to determine the presence of mouse anti-PE anti explored. bodies in the serum of treated and control animals. Microtiter with Cyclophosphamide, a widely used antitumor alkylating agent, plates 96 wells (Dynatech Laboratories, Alexandria, VA) were coated together with prednisone has been shown to inhibit the forma with whole PE or LysPE40 (50 ng/well), incubated overnight at 4°C, tion of antibodies against abrin and ricin in mice (7); however, and then thoroughly washed. Wells were blocked with PBS-bovine these agents are not without toxic side effects. In this study, we serum albumin 2.5% and incubated for 30 min. Serum samples were examined the suppressive activity of DSG. This compound is a first diluted 1:10 in 1% bovine serum albumin in PBS and then serially derivative of spergualin, a metabolite of B. laterosporus, and diluted for titration of binding activity. Samples were added to wells in triplicate and incubated for l h at 37°C.Wells were again washed Received 5/14/90; accepted 9/17/90. thoroughly and to each well was added 50 n\ of peroxidase-conjugated The costs of publication of this article were defrayed in part by the payment AffiniPure goat anti-mouse IgG+IgM (H + L) or IgM or IgG alone 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. (1:1000 dilution) (Jackson Immunoresearch Laboratories, Inc., West ' To whom requests for reprints should be addressed, at National Cancer Grove, PA). A 30-min incubation was followed by thorough washing Institute, NIH, 9000 Rockville Pike, Building 37. Room 4E16, Bethesda, MD with PBS. Substrate [100 ^I/well of 1 mg/ml 2,2-azinodi(3-ethylbenzth- 20892. 2The abbreviations used are: PE. Pseudomonas exotoxin; DSG. 15-deoxysper- iazolinsulfonate) (6) (Boehringer Mannheim)] with 1 ^1/ml 30% H2O2 gualin; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered in citric acid and sodium phosphate buffer, pH 4.4, was added to each saline. well. Reaction was stopped with 100 p\/vte\\ of 10% sodium dodecyl 7750 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1990 American Association for Cancer Research. SUPPRESSION OF ANTIBODY RESPONSE TO PSEUDOMONAS EXOTOXIN sulfate. Binding activity was measured at dilution levels ranging from 1:10 to 1:2000. Results were read on a Microelisa Autoreader at 405 nm. Final results are expressed in absorbance units after subtracting a prebleed sample as background. Cells E c m HUT 102 is a human cutaneous T-cell lymphoma cell line purchased o from the American Type Culture Collection (Rockville, MD). The cells were grown in RPMI 1640, with 10% fetal bovine serum, supplemented q with penicillin-streptomycin (Hazelton Biologies, Inc.). Cells were cul O tivated at 37°Cina humidified atmosphere containing 5% CO2. Immunotoxin Neutralization by Serum Containing Antibody Anti-PE Serum from eight animals immunized with multiple does of anti- Tac-LysPE40, four treated with DSG, and four controls was diluted 1:1, 1:10, 1:100, and 1:1000 with anti-Tac-LysPE40 at a final concen tration of 80 ng/ml for l h at 37°Cprior to protein synthesis assay. time Fig. 1. Histogram of mouse IgM response to PE following a single dose of Protein Synthesis Assay PE553D. Mice were treated with DSG, 10 mg/kg for 3 days. Antibody response was measured on days 4, 7, 14, and 21 by ELISA. Results are reported as The activity of anti-Tac-LysPE40 after incubation with sera was absorbance (O.D.) values of serum specimens diluted 1:10. Results demonstrated a significant effect of the DSG (P2 = 0.009). Bars, SD. m, DSG; •PBS. determined by measuring its effect on protein synthesis on HUT 102 cells. Cells were washed 3 times with appropriate media and planted at a density of 2 x IO4cells/well. Serum (10 M')incubated with anti-Tac- LysPE40 was added to each well in triplicate and incubated at 37°Cfor 1.4 16-20 h, after which [3H]leucine (5 ^Ci/ml) was added for 90-120 min. 1.2 The cells were then harvested using a 96-well Skatron harvester (Ska- 1.0- tron, Inc., Sterling, VA). The incorporated radioactivity was determined E by scintillation spectroscopy. c in 0.8- o Statistical Analyses Q Statistical comparisons were made with the Wilcoxon rank sum test. O RESULTS Suppression of Antibody Response to a Single Dose of PE553D by DSG To measure the antibody response to a single dose of toxin, time groups of 5 mice received a single dose of PE553D (40 /¿g)i.p. Fig. 2. Histogram of mouse IgG response to PE following multiple doses (7) of PE553D. One-half of the mice were treated with DSG, 5 mg/kg for 21 days.