Immunogenicity in Rabbits and Mice of an Antibody-Chelate Conjugate: Comparison of (S) and (R) Macrocyclic Enantiomers and an Acyclic Chelating Agent

[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 humans, 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 monoclonal 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 clododecanetetraaceticacid; 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. (12). The purified for 24 h at 37~ in a humidified chamber. To block nonspecific binding, 5% conjugates were stored at -70~ until use. bovine serum albumin in PBS was added and the mixture was incubated for 1 IgG-2-IT-BAD/BABE Conjugations. IgG (22 mg) was dissolved in 0.9 ml h at 37~ After the plates were washed, they were incubated with 50/,1 of a 0.1 M tetramethylammonium phosphate, pH 8.9, and an excess of a BAD 1:10 dilution of rabbit test serum for 2 h at 37~ After three washes in PBS (18-24 raM) or BABE (40 mM) solution and a freshly prepared solution of 100 plus 0.05% Tween 80, the plates were incubated for 1 h with a 1:4000 dilution mM 2-IT in triethanolamine hydrochloride, pH 8.7, was added to give the final of mouse monoclonal anti-rabbit immunoglobulin-alkatine phosphatase con- approximate concentrations: IgG, 22 mg/ml; BAD, 2.0 mM; 2-IT, 1.1 raM). The jugate. The plates were then washed 3 times with PBS and incubated at 37~ pH of the solution was adjusted to 9.0 and the solution was incubated at 37~ with 50 /,1 of p-nitrophenyl phosphate, 1 mg/ml in 0.1 M glycine buffer for 30 min (for BABE: BABE, 4 mM; 2-IT, 2 mM, 22 h to obtain 9 chelates/ containing 1 mM MgCI2, and 1 mM ZnCIz (pH 10.4), and the absorbance was mAb). Excess BAD/BABE was removed and the conjugate was placed in determined after -30 min in a TiterTek Multiskan plate reader at 450 nm. A 0.1 M ammonium acetate solution, pH 7.0, with a centrifuged gel filtration >0.20 (background -< 0.20) was considered positive. Mouse mAb 2D12.5 column. The concentration of protein-bound chelators available for metal specific for Yttrium-DOTA (13) was used as a positive control. binding was estimated as described by Moi et al. (12). The purified conjugates For detection of specific anti-BABE antibody response, the ELISA was set were stored at -70~ until use. up as described above except 50 /,1 (300 ng)/well transferrin-2-IT-BABE The synthetic route and the stereochemistry of (S) and (R) nitrobenzyl- conjugate, 3.9 chelates/transferrin molecule was used on the plate as the DOTA and 2-IT-BABE are shown in Fig. 1. Conjugates of mouse mAb Lym-1 antigen and mAb WC3All specific for 2-IT-BABE, also obtained in our with (R)- and (S)-BAD, and also with (S)-BABE, were prepared by the laboratory by KLH immunization (14), was used as a positive control. For method of McCall et aL (9). detection of specific rabbit anti-mouse antibody response by ELISA, plates Radiolabeling Conjugates. The antibody conjugates were radiolabeled in were coated with 10 ng/well of mouse IgG. Polyclonal rabbit anti-mouse 0.1 M citrate (BABE) or 0.1 M ammonium acetate (DOTA) by adding the antibody was used as a positive control. solution to dried purified lnln and incubating at room temperature for 15 rain Serial Dilution and Competitive Inhibition. Sera with high absorbance (6). Any unbound ll]In was scavenged by adding EDTA and was removed by on ELISA, after boosting or reboosting with the same enantiomer, were diluted centrifugation in a Centricon filter followed by dilution in buffer. serially to 10 -t, 10 -2, or 10 -3, and absorbance was measured. For the inhibition Rabbit Blood Clearance Studies. Individual normal male rabbits weigh- studies, 50 ;,1 containing 0, 1, 10, or 100/xg/ml of hTr-2-IT-(S)- or (R)-BAD ing -3 kg were given i.v. injections of either 0.1, 1.0, or 10 mg (equivalent to conjugate were added to 50 /xl of sera, followed by incubation at 4~ over- 2.3-233.0 mg for a 70-kg human) of lnln radiolabeled conjugate (1.8-4.5 night. Fifty /,1 of this incubate was used in the same ELISA assay and chelates/molecule). In each experiment either the (S) or (R) enantiomer of absorbance was recorded. The sera with high anti-BABE ELISA was inhibited rabbit IgG-BAD, mAb Lym-I-BAD, or Lym-I-(S)-BABE was given (Table 1). in the same way with hTr-2-IT-BABE conjugate. Heparinized blood samples were collected from an ear vein daily for 14 days Preparation of Murine Monoclonal Anti-Chelate Antibodies. Since and approximately biweekly for 70 days. Whole blood and plasma samples specificity for the metal ion was desired, the chelate hapten was filled with the were counted and the percentage of the injected dose circulating in plasma desired stable element for immunization. The BABE conjugates contained calculated from the hematocrit and blood volume was estimated from body cobalt, indium, and gallium; the BAD conjugates contained yttrium and gal- weight. lium. BALB/c mice were immunized i.p. with 10 /~g of the KLH-coupled antigen in complete Freund's adjuvant. Two weeks later this was repeated in incomplete Freund's adjuvant followed in another 2 weeks by an i.v. booster dose of the conjugate alone. The mice were screened for antibody production after 1 week by measuring the 24-h retention of radiolabeled haptens by whole body counting with a Picker dual probe scintillation counter. The mice with 20--50% retention at 24 h (compared with ~1-3% in control, nonimmunized mice) were hyperimmunized with 30/~g i.v. conjugate; 3 days later their spleen HN cells were fused with PX63 AG8 myeloma cells. Hybridomas were selected in 9 NH~* "+ hypoxanthine-aminopterin-thymidine medium. Clones obtained were screened by ELISA using hTr-2-IT-hapten as the solid phase antigen coated on micro- titer plates.

RESULTS

eooH o 9 HOOr CZXm Rabbit Blood Clearance Studies. The data points and bars plot- ted in Figs. 2-7 represent measurements taken from individual bleedings. A semilog plot of the disappearance half-life of rabbit

o IgG-2-IT-BAD conjugates is shown in Fig. 2. The disappearance followed a single exponential curve with no break over 13 days. The ._,,,j,"-,,,,,,r ,, tot ~Amt,,,- T~ (S) = 6.72 + 1.24 (SD) days and (R) =- 4.43 + 0.21 days, Fig. 1. The stereochemistry of 2-IT-(S) and (R)-BAD and 2-1T-(S)-BABE mouse which indicates that the two preparations were similar but not iden- monoclonai antibody conjugates. tical (P < 0.05). Blood disappearance of Lym-1 2-IT-BAD conju- 1050

Table 1 Rabbit protocol: antibody half-life and specific immune response by ELISA Rabbit Dose (mg) Antibody Conjugate Chelate/antibody % of aggregates tu2 a (days) tl/2 [3 (days) ELISA absorbance 1 0.10 R-IgG a 2-IT-(S)-BAD 2.5/1 ND 1, 2, 3 Negative 2 1.00 R-lgG" 2-IT-(S)-BAD 2.5/1 ND (6.72 _+ 1.24)I' no break Negative 3 5.80 R-IgG" 2-IT-(S)-BAD 2.5/1 ND Negative 4 0.10 R-IgG 2-IT-(R)-BAD 4.5/1 ND 4, 5, 6 Negative 5 1.00 R-IgG 2-IT-(R)-BAD 4.5/1 ND (4.43 + 0.21) No break Negative 6 10.00 R-IgG 2-1T-(R)-BAD 4.5/1 ND Negative 7 0.10 Lym-1 2-IT-(S)-BAD 2.0/1 0 7, 8, 9 S+, R- 8 1.00 Lym-1 2-IT-(S)-BAD 2.0/1 0 (1.85 _+ 0.19) 0.83 -+ 0.09 S+, R- 9 10.00 Lym-1 2-IT-(S)-BAD 2.0/1 0 S+, R- 10 0.10 Lym-1 2-IT-(R)-BAD 2.0/1 0 I0, 11, 12 Negative 11 1.00 Lym-1 2-IT-(R)-BAD 2.0/1 0 1.98 + 0.15 0.6 R+, S- 12 10.00 Lyre-1 2-IT-(R)-BAD 2.0/1 0 (n = 1) R+, S- 13 0.10 Lym-I 2-IT-(S)-BABE 1.8/1 2 13, 14, 15 Negative 14 1.00 Lym-1 2-IT-(S)-BABE 1.8/1 2 1.96 + 0.25 0.98 + 0.04 Negative 15 10.00 Lym-1 2-IT-(S)-BABE 1.8/1 2 S+ (no R)

Reinjection 7 0.1 x 3 Lym-1 2-IT-(S)-BAD 2.0/1 0 ND ND S+, R+ 13 0.1 X 3 Lym-1 2-IT-(R)-BAD 2.0/1 0 ND ND R+, S+ 8 1.0 • 1 Lym-1 2-IT-(R)-BAD 2.0/1 0 ND ND R+, S+ 11 1.0 x 1 Lym-I 2-IT-(S)-BAD 2.0/1 0 ND ND R+, S+ 15 0.1 x 1 Lym-1 2-IT-(S)-BABE 1.8/1 2 ND ND S- o R-IgG, rabbit polycional immunoglobulin; ND, not done; Lym-l, mouse monoclonal anti-lymphoma mAb; x 3, 3 times; x 1, once.

RABBIT IgG 21T-(S)a(R)-BAD (R)-BAD) had anti-(R)-BAD antibody but none of the rabbits that were given injections of Lyre-1 2-IT-(S)-BAD had anti-(R)-BAD 100"00~176 I .... ~ L ! ~ , antibody after one exposure (Fig. 6). The target antigen on plate was hTr-2-IT-(R)-BAD. Of the three rabbits given injections of Lym-1 2-IT-BABE, the one that received 10 mg produced anti-BABE anti- - - -~-b&_--~-_~ body (Fig. 7). In this rabbit, a second injection of 0.1 mg Lym-1 2-IT-BABE, 4.7 months later, gave no detectable anti-2-IT-BABE on 10"00% f --~'-~=-_:~"` " i--:~: ELISA. Rabbits reboosted twice (a total of three injections) with the same enantiomer developed cross-reactivity to the opposite enanti-

I.I.I i] 9100ugm IS) omer (Table 1). After a second exposure to the same amount (1 mg) of the opposite enantiomer ("switch-boost" with Lym-1 2-IT-(S) or O * ling (S) ta 1.00% __ (R)-BAD) reactivity developed to both enantiomers. Nine rabbits o~ ~ " 10mg r were studied for anti-mouse immune response. Rabbits that were [] 100ugm (R) given injections of 1.0 and 10 mg of immunoconjugates showed ; o lmg (R) positive anti-mouse antibody. A 10mg (R) Concentration and Specificity by Dilution and Inhibition. The 0.10% i ...... Lym-1 (S)- and (R)-BAD ELISA became negative at a dilution of 0 2 4 6 8 10 12 14 TIME POST-DOSE DAYS 10 -3 in all strongly positive sera tested. In the 0.1-mg (R) and (S) Fig. 2. Semilog plot of blood disappearance half-life of JUln rabbit IgG-2-IT-(S)- and reboosting experiment, the ELISA was inhibited by a 100-/xg/ml hTr -(R)-BAD conjugates. The disappearance followed a single exponential curve with no break over 13 days. The T,~ (S) = 6.72 +_ 1.24 days and (R) = 4.43 --- 0.21 days, which indicates that the two preparations were similar but not identical (P < 0.05). LYM-1-21T-(S)&(R)-BAD gates was more rapid than rabbit IgG 2-IT-BAD conjugates 100.00% I i -- I (P < 0.001; Fig. 3). The T~ of the first phase of (S) = 1.85 _+ 0.19 days and (R) = 1.98 _+ 0.15 days (not significantly different). Three of the six Lym-1 2-IT-BAD [0.1 mg (S), 1.0 mg (S), and 1.0 mg (R)] showed biexponential curves with a break at 6-8 days, which 0 10.00% indicates the appearance of circulating antibody. The break was lo- cated by a sudden increase in the rate of disappearance of the second ~ i! z_ "~-."'4~ ~ 9 100 ugm (S) phase with T,,~ = 0.75 _+ 0.15 days (P < 0.01). Fig. 4 shows the m ~ ~ 1 mg (S) blood disappearance of Lym-1 conjugated to the acyclic chelator O a 2-IT-BABE. Two of the three curves (1.0 and 10 mg) were bi- 1.00% ...... -~-- 10 mg (S) exponential with a break at 6-8 days. The T,~ of the first phase = i_7 .... -___~ ...... ",, ~,, i o 100 ugm (R) ...... A'~ 9 1.96 + 0.25 days, and the T,~ of the second phase after the break = i " ,- i ~ 1 mg (R) 0.9 _+ 0.04 days (P < 0.05). These half-lives were not significantly 10 mg (R) different from the Lym-1 BAD data. 0.10% : Rabbit Immune Response to Conjugates. All three rabbits given 0 2 4 6 8 10 12 14 injections of Lym-1 2-IT-(S)-BAD produced anti-(S)-BAD antibody, TIME POST-DOSE DAYS but none of the rabbits that was given an injection of Lym-1 2-IT- Fig. 3. Blood disappearance of Lym-1 2-IT-(S)- and -(R)-BAD: more rapid than rabbit (R)-BAD produced anti-(S)-BAD antibody after one exposure (Fig. IgG-2-IT-(S)- and -(R)-BAD (P < 0.001). Three of the six: 0.1 mg (S), 1.0 mg (S) and 1.0 mg (R), showed biexponential curves with a break at 6-8 days. The Tv2 of the first phase 5). The target antigen on plate was hTr-2IT-(S)-BAD. Two of three of (S) = 1.85 -+ 0.19 days and that of (R) = 1.98 +_ 0.15 days (not significantly different); rabbits (those that were given injections of 1 or 10 mg Lym-1 2-IT- second phase Tv2 = 0.75 +_ 0.15 days (P < 0.01). 1051

LYM-1-21T-BABE cations of the results for radioimmunotherapy and diagnosis using these conjugates. Human anti-mouse antibody is a universal finding in 100.00% -~-~• ~ - --~-- I _ ~ ~ .... therapy with mAbs that severely limits their usefulness. However, the

4k. more serious reactions such as serum sickness have been rare and usually have occurred after large repeated doses in immunocompetent patients (15). While human anti-mouse antibody is well documented r~ O 10.00% in human mAb conjugate therapy trials, anti-conjugate antibody is O ._1 rarely measured. Such conjugates of small molecules or haptens with lid Z larger carrier molecules (mAb), are very efficient for generating a UJ (/) specific anti-hapten antibody response. This response is augmented O when the carrier is foreign (e.g., KLH or mouse protein), when the " 1.00% o-e conjugate/carrier ratio is high (>10/1), when the conjugate is injected ~: 9100 ugm i.p., and when the conjugate is coinjected with adjuvants or aggre- ,:- , ~ 1 mg gated protein. We have used this technique very efficiently in another "\ i, 10mg context to generate high affinity anti-hapten mAb WC3All specific 0.10% for cobalt-2-IT-BABE (13) and mAb 2D12.5 specific for yttrium- 0 2 4 6 8 10 12 14 DOTA (14). We are currently applying these mAbs in novel pretar- TIME POST-DOSE DAYS geted immunoscintigraphy and immunotherapy protocols in tumor Fig. 4. Blood disappearance of Lym-1 2-IT-(S)-BABE. Two of the three curves (1.0 mice (16). In this technique, injection of mAb, directly radiolabeled and 10 mg) were biexponentialwith a break at 6-8 days. The T,~ first phase = 1.96 --- 0.25 days; T,~ second phase --- 0.9 + 0.04 days (P < 0.05).

ANTIGEN ON ELISA PLATE: (SpBAD ANTIGEN ON ELISA PLATE: (R)-BAD

0.8 0.8

0.7 0.7

0.$ 0.6

0.5 0.5

d 0.4 0,4 d 0 0.3

0.2

0.1 0 ••dd• S-lOOug &ling &lOmO R-lOOt~l ~lmg ~lmg S,-lOOug S-ling S.lOmg R..lOOug R-ling R-lOmg Fig. 5. ELISAspecific for anti-(S)-BAD on all samples 0-70 days. Three rabbits that Fig. 6. ELISA specific for anti-(R)-BAD on all samples 0-70 days. Two of three were given injectionsof Lym-I 2-IT-(S)-BAD had anti-(S)-BAD at days 6-8; rabbits that rabbits that were given injectionsof Lym-1 2-IT-(R)-BAD had anti-(R)-BAD antibody at were given injections of Lym-1 2-1T-(R)-BAD produced no anti-(S)-BAD antibody after days 6-8; rabbits that were given injectionsof Lym-1 2-IT-(S)-BAD had no anti-(R)-BAD one exposure. O.D., Absorbance. antibody after one exposure. O.D., Absorbance.

conjugate of the same enantiomer [equivalent to --0.86/XM (S) or (R) ANTIGEN ON ELISA PLATE: 2IT-BABE BAD]. In the positive switch boost sera the ELISA was inhibited specifically by the opposite (switch) enantiomer, which indicates a 1 high concentration of specific antibody. The results were similar for 0, the positive (10-mg rabbit) Lym-1 BABE sera. 0.8 --,--- lOOug Mouse Immune Response to KLH Conjugates. For the 24-h retention measurements, the hapten-chelate was radiolabeled with the 0.7 ---o-- leg ~\ / 'L same metal as used for immunization. The 24-h percentage dose o. 10. retention of radiolabeled hapten is shown in Fig. 8. Control, nonim- q 0.5 munized mice retained only 1-3% at 24 h, the rest being rapidly 0 excreted by the kidneys. The mean 24-h retention of BABE hapten is 0.4 27.5 _+ 30% (n = 16) and BAD hapten retention is 86 +_ 18.4% (n = 0.3 15) (P < 0.0001). The immune response to the BABE conjugates was lower and more variable than BAD conjugates. 0.1 ~...~___-r------,_~,__.---.

DISCUSSION 0 I I I----I I I I I I I l 1 3 5 7 9 12 The goal of this study was to define the immune response to DAYS enantiomerically related hapten-chelate conjugates of mAbs in ani- Fig. 7. Of the three rabbits that were given injectionsof Lym-1 2-IT-(S)-BABE, the one mals, to devise strategies for avoiding it, and to consider the impli- receiving 10 mg produced anti-BABE antibody. O.D., Absorbance. 1052

one or other of the conditions that promote a response such as "hu- 1201 KLH IMMUNOGENS manizing" the carrier protein has the potential to reduce or abolish the / loo anti-hapten response. Partial success with chimeric or "humanized" mAbs in reducing antigenicity has been reported, although anti-idio- (3 type antibody still occurs (18). Work on human monoclonal antibodies z 8o while promising is at a very early stage (19). Z < Johnson et al. (20) recently have considered the question of an ACA IJJ~= 60 response in humans treated with genetically engineered "humanized"

u.I mAb drug conjugates. They chose to study the antigenic response to o 40 mouse mAb conjugates in rats as a suitable animal model for accu- rately simulating the effect of modifying a weakly immunogenic o~ humanized mAb on its antigenicity in humans. Rats that were given injections of unmodified mouse mAb 225 IgG1 directed against human epidermal growth factor receptor failed to mount anti-mouse BABE C n=15 BABE:n=16 BAD C n--a BAD n=15-- immune response, presumably due to their phylogenetic relatedness. Fig. 8. Twenty-four-h percentage of injected dose retention of radiolabeled hapten in In contrast, rats that were given injections of Vinca immunoconjugates immunized mice: control, nonimmunized mice; 1-3% at 24 h; BABE hapten; 27.5 _+ 30% of mAb 225 IgG1 mounted a strong anticonjugate antibody response (n = 16) and BAD hapten = 86 - 18.4% (n = 15, P < 0.0001). Bars, SD. when four repeat doses of mAb conjugate were given i.p. In vitro inhibition studies showed the response was directed primarily against the linker portion of the molecule. The presence of ACA had profound by chelate conjugation, is replaced with the sequential administration effects on the serum pharmacokinetics, with the peak serum concen- of anti-hapten mAb, followed by radiolabeled hapten. The physical tration inversely correlated with the level of reactivity of the ACA. and temporal separation of mAb and radioactive chelate-hapten in This finding agrees with our demonstration of a break in the plasma pretargeting has many advantages, particularly in reducing normal disappearance curve at approximately 6-8 days with a sudden increase tissue and marrow irradiation in radioimmunotherapy (13, 17), but it in the rate of disappearance of mAb (Figs. 3 and 4), coinciding with also greatly lowers the probability of an antibody response to the the appearance of antibody (Figs. 5 and 6). chelate hapten. Thus, one strategy used by these investigators to decrease the ACA In a Phase I/II trial of i.p. therapy in ovarian cancer, Kosmas et aL response was to change the linker used to attach the drug to the (1) found the [9~ (HMFG1) conjugate highly antibody. Higher serum values were observed when a different linker immunogenic with some patients developing serum sickness. Inter- was used with the same chelate. However, the peak serum value for estingly, in this study, a high titer of human anti-hapten antibody was the new linker was still slightly lower than that seen with unmodified present, with reactivity restricted to the DOTA epitope and very little mAb. This result corresponds to our finding of an incomplete escape antilinker antibody. This was shown by low reactivity of DOTA- from the anti-hapten chelate response in rabbits receiving subsequent positive sera against anti-2-IT-benzoic acid or anti-BABE when they injections of the opposite enantiomer. Nevertheless, the possibility of were used as the target antigen on the ELISA plate. developing linker and chelate chemistry with low immunogenic po- We have shown previously that the antibody response to a mAb tential was shown to be a feasible goal. Our finding in these experi- chelate conjugate can be restricted to the chelate moiety or may also ments of an absent response after one i.v. injection of homologous include the linker used to attach the chelate to the mAb. Our mouse mAb, even with immunogenic DOTA conjugates, also shows promise anti-hapten mAb 2D12.5 raised against yttrium-2-IT-BAD was re- for optimized conditions that include the use of humanized and even- stricted to yttrium-DOTA (13), whereas the 2-IT-BABE linker made tually human mAbs. up part of the epitope recognized by mAb WC3All, which we raised against KLH-2-IT-BABE (cobalt) (14). Heavily substituted proteins are known to be more immunogenic REFERENCES than lightly modified ones. 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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 1994 American Association for Cancer Research. 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, Claude F. Meares, et al.

Cancer Res 1994;54:1049-1054.

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