Immunogenicity in Rabbits and Mice of an Antibody-Chelate Conjugate: Comparison of (S) and (R) Macrocyclic Enantiomers and an Acyclic Chelating Agent
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[CANCER RESEARCH 54, 1049-1054, February 15, 1994] Immunogenicity in Rabbits and Mice of an Antibody-Chelate Conjugate: Comparison of (S) and (R) Macrocyclic Enantiomers and an Acyclic Chelating Agent Naoto Watanabe, David A. Goodwin, 2 Claude F. Meares, Maureen McTigue, Warak Chaovapong, Charles McK. Ransone, and Oliver Renn Nuclear Medicine Service, VA Medical Center, Palo Alto 94304, and Stanford University School of Medicine, Stanford 94305 IN. W., D. A. G., M. McT., W. C., C. M. R.], and Department of Chemistry, University of California, Davis 95616, California [C. F. M., O. R.] ABSTRACT antibody response reported from the use of mAb conjugates in hu- mans, but anti-chelate-conjugate responses have not been sought. This The macrocyclic bifunctional chelating agent 2-(p-bromoacetamido- report of a severe reaction to the chelate moiety itself prompted us to benzyl)-l,4,7,10-tetraazacyclododecanetetraacetic acid (BAD), forms inert study the immunogenicity of a variety of chelate conjugates in animals metal complexes ideal for radioimmunotherapy. Kosmas et al. (Cancer (2). Here we report the effects of chelate structure, protein carrier, Res., 52: 904-911, 1992) found 2-imminothiolane linker-(S)-BAD mono- clonal antibody HMFGI highly immunogenic in patients. We studied the amount of conjugate injected, and timing of administration to show immunogenicity of (S) and (R) enantiomers of 2-imminothiolane linker- that chelates behave like other haptens in eliciting a specific antibody BAD rabbit IgG, monoclonal antibody Lym-l, and Lym-1 2-imminothio- response when injected as foreign protein conjugates. We also com- lane iinker-(S)-bromoacetamidobenzyI-EDTA in 15 rabbits. Five groups pared the antibody response with the mirror image structures (enan- of three each were given 0.1, 1.0, or 10 mg of rain conjugate i.v., blood tiomers) (S)-BAD and (R)-BAD. samples were taken daily for 14 days and biweekly for 70 days, and the plasma Tw was calculated. A drop in plasma rain at 6-8 days coincided with the appearance of antibody on enzyme-linked immunosorbent assay. MATERIALS AND METHODS Specific anti-(S)-BAD, anti-(R)-BAD, anti-(S)-bromoacetamidobenzyl- All reagents and solvents were the purest as were commercially available EDTA, and anti-mouse IgG were measured. Rabbit IgG conjugates did not and were used without further purification, if not stated otherwise. Pure water elicit an immune response. Mouse IgG conjugates were immunogenic on (18Mfl/cm) was used throughout. Metal-free conditions were maintained dur- the first exposure, with both anti-l,4,7,10-tetraazacylododecane ing synthetic chemistry. All glass labware was washed with a mixed acid N,N',N",N'"-tetraacetic acid and anti-mouse responses. Anti-l,4,7,10- solution and rinsed thoroughly with pure water (3). All plastic labware was tetraazacylododecane N,N',N",N"'-tetraacetic acid was specific for the washed with 3 M HC1 and rinsed thoroughly with pure water. (S) or (R) enantiomer, but cross-reaction appeared with reboosting. A Thin Layer Chromatography. Thin layer chromatography was run on second injection of the opposite enantiomer gave a response to that enan- plastic-backed silica gel plates (0.2-mm-thick silica gel, 60 F254; Merck, tiomer. Lym-1 bromoacetamidobenzyl-EDTA produced anti-bromoacet- Darmstadt, Germany) using a 10% (w/v) ammonium acetate:methanol (1:1, amidobenzyl-EDTA and anti-mouse response. v/v) solution as the eluent. In this system, unchelated cobalt and conjugates remain at the origin while free chelates migrate to Rf 0.5-0.7. High Performance Liquid Chromatography. High performance liquid INTRODUCTION chromatography was carried out on a Rainin Rabbit HP system (Rainin In- The macrocyclic bifunctional chelating agent BAD, 3 incorporating struments, Woburn, MA). UV absorbance was measured at 214 nm using an DOTA, has been developed for use in radioimmunotherapy. The cur- ISCO Model UA-5 (ISCO, Inc., Lincoln, NE). The high performance liquid chromatography system was controlled and data were acquired using Rainin rent dose-limiting factor in cancer therapy with radioimmunoconju- Dynamax software on a Macintosh SE/30 computer. Size exclusion chroma- gates is bone marrow irradiation, which is increased by loss of radio- tography was done using a SynChropak GPC 300 (4.6 x 250 mm) column metals from unstable chelate conjugates to the bone marrow. Because operated at 0.3 ml/min using 0.1 M sodium phosphate, pH 7.2, with 1 mM DOTA forms highly stable, inert complexes with metals like yttrium, EDTA. negligible metal loss occurs in vivo. For these reasons [9~ Sephadex Gel Filtration (Centrifuged Column Method). A centrifuged conjugates of mAbs are ideal for radioimmunotherapy and human Sephadex G 50-80 column method (4-6) was used to separate proteins from trials are now under way. lower molecular weight compounds. In a Phase I/II trial of i.p. therapy in ovarian cancer, Kosmas et aL UV Spectrophotometry. Absorbance measurements at 280 nm were made (1) found the antibody-chelate conjugate [9~ on a Gilford Model 250 spectrophotometer using a 1-cm path length microcell. (HMFG1) highly immunogenic, with some patients developing serum Absorbances were measured on dilutions suitable to give absorbance readings sickness. To date, human anti-mouse antibody has been the only of 0.1-1.0. Radiation Counting. Gamma counting was done in a Beckman Model 310 counter with the appropriate energy windows set for 57Co. Thin layer chro- Received 7/21/93; accepted 12/14/93. matography plates containing radiolabeled materials were visualized with an The costs of publication of this article were defrayed in part by the payment of page AMBIS Radioanalytical Imaging System. charges. This article must therefore be hereby marked advertisement in accordance with Reagents. Lym-1, an anti-B-cell lymphoma IgG2a mAb (7), was obtained 18 U.S.C. Section 1734 solely to indicate this fact. t Supported by a Veterans Administration Merit Review grant (D. A. G.); National from Damon Biotech (Needham Heights, MA). Rabbit IgG and 2-IT [Traut's Cancer Institute Grants RO1-CA 48282, RO1-CA28343 (D. A. G.), and RO1-CA16861 reagent (8)] were purchased from Sigma Chemical Co. (St. Louis, Missouri). (C. F. M.); and United States Public Health Service Grants CA 28343, CA 48282 When IgG and Lym-1 were conjugated under identical experimental condi- (D. A. G.), and CA 16861 (C. F. M.). tions, no significant variation in the chelate/mAb ratio was observed when a 2 To whom requests for reprints should be addressed at Veterans Administration Medi- cal Center, Department of Nuclear Medicine (115), 3801 Miranda Ave., Palo Alto, CA new bottle of 2-IT was used. 94304. Conjugations. Conjugations were done as described by McCall et aL (9) in 3 The abbreviations used are: BAD, 2-(p-bromoacetamidobenzyl)-l,4,7,10-tetraazacy- a two-step procedure using Traut's reagent (8), which reacts with amino groups clododecanetetraacetic acid; DOTA, 1,4,7,10-tetra-azacylododecaneN,N',N",N"'-tetra- to produce mercaptobutyrimidyl groups, followed by alkylation of the mer- acetic acid; mAb, monoclonal antibody; BABE, bromoacetamidobenzyl-EDTA;2-IT, capto sulfur with the bifunctional chelating agent BAD or BABE. 2-imminothiolane linker; KLH, keyhole limpet hemocyanin; hTr, human transferrin; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; ACA, Macrocycles. (R)- and (S)-nitrobenzyl-DOTA were prepared according to anti-conjugate antibody. the method of Renn and Meares (10). Reduction of each to the respective 1049 Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1994 American Association for Cancer Research. IMMUNOGENI('ITY Ol: mAb ('ONJU(iATES p-amino compound was accomplished as described by McCall et al. (9). Three months after the first i.v. injection of Lyre- 1 BAD, the 0. I-rag-treated Conversion of the p-amino compounds to the p-bromoacetamido macrocycles rabbits were boosted with the same dosc of the same conjugate and reboosted BAD used a modification of the method of Mukkala et al. (11) and was done again 1 month later for a total of three doses. In the BABE boosting experiment according to the method of McCall et al. (9). the 10-rag-treated rabbit was given a 0.l-rag i.v. dose 4.7 months after the first Lym-I-2-IT-BAD Conjugations. The Lym-1 mAb solution (7.36 mg/ml) injection. was prepared for conjugation with a centrifuged gel filtration column (6) with In a "switch-boost" experiment 4.4 months after the first i.v. injection of 0.1 M tetramethylammonium phosphate, pH 8.9, as the column buffer. Excess Lyre-1 (S)- or (R)-BAD, the 1.0-mg rabbits were boosted with an i.v. injection BAD in aqueous solution and freshly prepared 100 mM 2-IT in triethanolamine of the opposite enantiomer. This was done to see if it was possible to circum- hydrochloride, pH 8.7 (final approximate concentrations: Lym-l, 6 mg/ml; vent the immune response that occurred after reinjection of the same enanti- BAD, 2.0 raM; 2-IT, 1.1 raM) were added to the collected effluent (6.93 mg/ml) omer. Blood samples were collected as before. in order. The pH of the solution was adjusted to 9.0 and the solution was Specificity of Antibody Response by ELISA. Flat-bottomed 96-well mi- incubated at 37~ for 30 rain. Excess BAD was removed and the conjugate crotiter plates were coated with 50/xl each (containing approximately 700 ng was placed in 0.1 M ammonium acetate solution, pH 7.0, with a centrifuged gel in PBS, pH 7.4) of hTr conjugate of 2-IT-(S)-BAD, 0.4 chelate/transferrin filtration column. The concentration of protein-bound chelators available for molecule, or 2-IT-(R)-BAD, 0.7 chelate/transferrin molecule, and incubated metal binding was estimated as described by Moi et al.