Bioconjugate Chem. 1004, 5,357-363
357
Monoclonal Antibodies to Thioguanine: Influence of Coupling Position on Fine Specificity Vibeke Mortensen Nerstram,’J Ulla Henriksen,t Peter E. Nielsen,! Ole Buchardt,* Kjeld Schmiegelow,l and Claus Kocht Research Center for Medical Biotechnology, Department of Immunology, Statens Seruminstitut, Artillerivej 5, DK-2300 Copenhagen S, Denmark, Research Center for Medical Biotechnology, Department of Organic Chemistry, The H.C. 0rsted Institute, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen 0, Denmark, Research Center for Medical Biotechnology, Department of Biochemistry B, The Panum Institute, University of Copenhagen, Blegdamsvej 3, DK-2200 Copenhagen N, Denmark, and Department of Pediatrics, Rigshospitalet, University Hospital, Blegdamsvej 9, DK-2100 Copenhagen 0, Denmark. Received November 17, 1993’
Thioguanine derivatives with reactive ester groups at positions 6,7, or 9 of the purine ring were synthesized and coupled to a protein carrier. The purified protein derivative of tuberculin was used as the carrier for immunizing bacillus Calmette-Guerin primed mice. This led to high antibody titers against the homologouslycoupled hapten, and spleen cells from the immunized mice were used to produce monoclonal antibodies against thioguanine. All monoclonal antibodies were selected for their ability to recognize free thioguanine and were analyzed for their fine specificity by inhibition experiments with a panel of thiopurine derivates. The specificity of the monoclonal antibodies showed a strong dependence on the coupling position of the thioguanine. Within each group of monoclonal antibodies, raised against one of the three different conjugates, there was a high degree of heterogeneity, with antibodies differing in their binding according to the substitution on the thioguanine analogues used in the inhibition experiments. This panel of antibodies may be used for quantitative assays of thiopurines and their metabolites in patients undergoing treatment with thioguanine, 6-mercaptopurine, and azathioprine.
INTRODUCTION
The thiopurines azathioprine (6-((l-methyl-4-nitroimidazol-5-yl)thio)purine),thioguanine (2-amino-6-purinethione), and 6-mercaptopurine (6-purinethione) are used for the treatment of systemic connective tissue diseases, leukemia, and lymphomas. Catalyzed by the hypoxanthine guanine phosphoribosyl transferase these antimetabolites are converted to thioguanine nucleotides which mediate the cytotoxic effect of the thiopurines through incorporation into DNA and RNA. In addition, the thiopurines inhibit de-novo purine synthesis, thus enhancing the incorporation of thioguanine nucleotides through the purine salvage pathway (Tidd et al., 1974). Treatment with thiopurines may, however, cause certain side effects, most of which are dose-related (McCormack and Johns, 1990). It is therefore of clinical interest to establish quantitative assays, not only for thioguanine itself, but also for thioguanine metabolites in blood samples from patients undergoing treatment with thioguanine. Current methods are mostly based on the extraction of thioguanine from plasma or blood, followed by HPLC analysis, and they are thus both cumbersome and time consuming (Bruunshuus and Schmiegelow,1989). An attractive alternative would be to set up assays based on the use of immunological reagents, provided antibodies can be prepared that recognize intact thioguanine. Also, antibodies that recognize relevant metabolites of thioguanine would be useful. From an immunological point of view, thioguanine is a hapten (MW 167 Da) and has to be coupled onto an + Statens Seruminstitut.
immunogenic carrier protein in order to be recognized as an antigen. A reactive linking group therefore must be introduced which can subsequently be employed for the couplingreaction. The couplingof the hapten to the carrier must be strong and stable, and the position of the reactive group may influence the fine specificity of the resulting antibodies. We have used three different coupling positions on thioguanine to obtain series of monoclonal antibodies against this hapten, and we have analyzed the reactivity of these antibodies to thioguanine and a series of 6-purinethiono and 6-purinethiolo derivatives. EXPERIMENTAL PROCEDURES
Materials and Methods. Chemicals for syntheseswere obtained from Aldrich, Steirheim, Germany. Chemicals for conjugations were obtained from Merck. The protein carrier PPD’ and rabbit antibodies against OA were from Statens Seruminstitut, Copenhagen, Denmark, and OA was from Sigma, St. Louis, MO. Compounds used for the screening of antibody specificity were obtained from Sigma or prepared as described. The derivatives are listed in Scheme 1 and Chart 1. Peroxidase-labeled rabbit antimouse immunoglobulin and antibodies for mouse immunoglobulin subclass determination were from DAKO, Ballerup, Denmark, and Zymed, San Francisco, CA, respectively. Microtiter plates (Maxisorp) were from NUNC, Roskilde, Denmark. Abbreviations used: BCG, bacillus Calmette-Guerin; DCC,
* The H.C. Orsted Institute.
N,N’-dicyclohexylcarbodiimide; DMF, dimethylformamide;
The Panum Institute. University Hospital. * To whom correspondence should be addressed. Tel: +45 32683595; Fax: +45 32683149. Abstractpublishedin Advance ACS Abstracts, June 15,1994. 1 The
@
1043-1802/94/2905-0357$04.50l0
DMSO, dimethyl sulfoxide; ELISA, enzyme-linked immunosorbent assay; EtOAc, ethyl acetate; EtOH, ethanol; MeCN, acetonitrile; MeOH, methanol; NHS,N-hydroxysuccinimide;OA, ovalbumin; OPD, 1,2-phenylenediamine hydrochloride; PBS, phosphate-buffered saline; PPD, purified protein derivative. 0 1994 American Chemical Society
958
Bloconlugate Chem., Vol. 5, No. 4, 1994
Nerstrom et al.
Scheme 1. Reaction Pathways for the Syntheses of Thioguanine Derivatives S- Substituted compounds S- and 7-substltuted compounds
S
CI
I
1
0
H 2
o o
1
BrCH2COOH
&Substituted compounds
HZNH*N%NHcoR AN NHz 10
1
POCIS
HZN 11
1
SC(NHz)z
S
Chart 1. Structures of the Compounds. Used in the Inhibition Assay to Characterize Reactivity of the mAbs s s CHzCOOH
f
S
-
13
Ribose
,CH3 N
Y
4,' 16
H
\
14
15
,CH3
? l
Riboie
N
HzN
k
N
AN 17
y N H
6-Purinethione (13), 6-purinethione riboside (14), 6-thioxanthine (15), 6-(methy1thio)purine riboside (16) and 2-amino6-(methylthio)purine (17).
UV spectra were recorded on a Hewlett-Packard 8452A diode array spectrophotometer, NMR spectra at 90 MHz on a JEOL FX-9OQ spectrometer, and mass spectra on a VG Masslab 20-250 quadropole spectrometer. Elemental analyses were performed on a Perkin-Elmer 240 elemental analyzer.
%-Amino-9-( carboxymethyl)-6-chloropurine(3) and 2-Amino-7-(carboxymethyl)-6-chloropurine (4). Bromoacetic acid (3.0 g, 21.7 mmol) and anhydrous K2C03 (8.3 mg, 60 mmol) were mixed in anhydrous DMF (30 mL) with stirring, and 2-amino-6-chloropurine (2)(3.1 g, 18.1 mmol) was added. After 3 h a t room temperature, water (ca. 100 mL) was added to give a clear solution. The g-(carboxymethyl) isomer 3 (2.4 g, 10.55 mmol, 58%)was precipitated by adjusting the pH to 3 with concentrated HC1. Further addition of water with cooling caused precipitation of the 7-(carboxymethyl)isomer 4 (0.5 g, 2.2 mmol, 12 %) contaminated with a small amount of 3.4 can be separated from 3 on a silica gel column. Elution with a gradient of EtOAc 0-50 7% in MeOH provided 3 followed by 4: MS (FAB+) mlz 228 (M + 1). Anal. Calcd for C7H&lN50~0.5H20: C, 35.53; H, 2.98; N, 29.59; C1,14.98. Found: C, 35.51; H, 2.69; N, 28.90; C1, 15.07. 2-Amino-9-(carboxymethyl)-6-purinethione (5). 2-Amino-9-(carboxymethyl)-6-chloropurine (3) (410 mg, 1.8 mmol) and thiourea (137 mg, 1.8 mmol) were mixed in EtOH (20 mL) and refluxed for 1 h. The resulting product 5 (350 mg, 1.56 mmol, 86%) was isolated after coolingand suspended in water (25mL), and solid NaHC03 was added. The solution was filtered and acidified to pH 3 with concentrated HC1, and the product was collected: MS (FAB+) mlz 226 (M + 1). Anal. Calcd for C7H7N502S-0.5H20: C, 35.89; H, 3.44; N, 30.03; S, 13.69. Found: C, 35.29; H, 3.15; N, 29.92; S, 13.82. Succinimidyl Thioguanin-9-ylacetate (7). 2-Amino9-(carboxymethyl)-6-purinethione(5) (225 mg, 1 mmol) and NHS (138 mg, 1.2 mmol) were dissolved in anhydrous DMF (5 mL). DCC (247 mg, 1.2 mmol) was added, and the mixture was left overnight at room temperature. After
Bioconjugate Chem., Vol. 5, No. 4, 1994
Thioguanlne Derivatives: Synthesis and Antibodies
filtration, the product was precipitated with ether, collected, and washed with MeOH and ether (155mg, 0.48 mmol, 48% ). The compound could not be purified further because of hydrolysis: MS (FAB+) m/z 323 (M + 1). 2-Amino-7-(carboxymethyl)-6-purinethione (6) was prepared from 2-amino-7-(carboxymethyl)-6-chloropurine (4)(595mg, 2.62 mmol) and thiourea (239mg, 3.14 mmol) as described for 5 (450mg, 2 mmol, 77%): MS (FAB+) m/z 226 (M + 1). Anal. Calcd for C7H7NsOzS.0.5H20: C, 35.89;H, 3.44;N, 30.03. Found: C, 35.94;H, 3.21;N, 29.78. Succinimidyl Thioguanin-7-ylacetate (8) was prepared from 2-amino-7-(carboxymethyl)-6-purinethione (6) (123mg, 0.55mmol), NHS (76mg, 0.66 mmol), and DCC (135mg, 0.66mmol) as described for 7 (45 mg, 0.14 mmol, 26%): MS (FAB+) m/z 323 (M 1). Succinimidyl 4- (Bromomethyl)benzoate. 4-(Bromomethy1)benzoicacid (1.18g, 5.5 mmol) and NHS (0.76 g, 6.6mmol) were dissolved in anhydrous dioxane (15mL). DCC (1.35 g, 6.6 mmol) was added, and the mixture was left overnight at room temperature, filtered, and evaporated to dryness. The product (1.21g, 3.9 mmol, 71%) was recrystallized from 2-propanol: lH NMR (CDC13) 6 8.11 (d, 2 H, J = 8 Hz), 7.53 (d, 2 H, J = 8 Hz), 4.50 (s, 2 H), 2.90 (s, 4 H). Succinimidyl 4 4 ((2-Amino-6-puriny1)thio)methy1)benzoate(9). Succinimidyl4-(bromomethyl)benzoate (343mg, 1.1 mmol), thioguanine (1) (167mg, 1 mmol), and excess Et3N were mixed in anhydrous DMF (5mL) with stirring. After 3 h at room temperature, the mixture was filtered, and the product was precipitated with ether, collected, and washed with CHCl3 (300mg, 0.75 mmol, 75%): MS (FAB+) m/z 399 (M + 1). Anal. Calcd for C17H14N604S.2.5H20: C, 46.05;H, 4.32;N, 18.95;S, 7.23. Found: C, 45.84;H, 4.34;N, 18.73;S, 7.19. 2,4-Diamino-5-acetamido-6-hydroxypyrimidine (loa) and 2,4-diamino-5-benzamido-6-hydroxypyrimidine (lob) were prepared as previously described (Wilson, 1948). 2-Amino-6-chloro-8-methylpurine (lla). 2,4-Diamino-5-acetamido-6-hydroxypyrimidine (loa) (986mg, 5.39 mmol) was refluxed in POC13 (25mL) for 5 min, benzyltriethylammonium chloride (2.46g, 10.4 mmol) in MeCN (25mL) was added, and refluxing was continued for 4 h. The mixture was evaporated to an oil, poured on icewater, neutralized with concentrated ammonium hydroxide, and boiled for 5min. The product (500mg, 2.75mmol, 51%) was precipitated by adjusting the pH to 3 with concentrated HC1, collected, and used without further purification. 2-Amino-6-chloro-8-phenylpurine (llb). 2,4-Diamino-5-benzamido-6-hydroxypyrimidine (lob, 826 mg, 3.37 mmol) was refluxed in Poc13 (15 mL) for 1 h, benzyltriethylammonium chloride (1.54g, 6.76 mmol) was added, and refluxing was continued for 1 h. The product was collected after cooling, suspended in water, neutralized with concentrated ammonium hydroxide, boiled for 5 min, chilled, collected, and used without further purification (480mg, 1.96 mmol, 58%) (modification of the method by Elion et al., 1951). 2-Amino-8-methyl-6-purinethione (12a) was pre(1 la) (260 pared from 2-amino-6-chloro-8-methylpurine mg, 1.42 mmol) and thiourea (129 mg, 1.7 mmol) as described for 5, except that Na2C03 was used in the purification instead of NaHC03 (193 mg, 1.07 mmol, 75%): MS (FAB+) m/z 182 (M + 1). ( l l a has been prepared previously by different methods (Daves, Jr., et al., 19601.)
+
350
2-Amino-8-phenyl-6-purinethione (12b) was pre(1lb) (150 pared from 2-amino-6-chloro-8-phenylpurine mg, 0.61 mmol) and thiourea (56mg, 0.73 mmol) analogously to 12a (100mg, 0.41 mmol, 68%): MS (FAB+)m/z 244 (M 1). Conjugation of ActivatedThioguanineDerivatives to Carrier Proteins. The activated thioguanine derivatives 7-9 were dissolved in DMSO at 40 mg/mL and were conjugated with PPD and OA. The protein solutions were adjusted to 1 mg/mL and dialyzed against 0.1M NaHC03, pH 8.3. Ten pL of thioguanine derivative was added per milliliter of protein solution and allowed to react for 2 h at room temperature under gentle mixing. The conjugate was finally dialyzed against PBS, pH 7.2,for 24 h with three buffer changes. Immunization Procedure. The thioguanine-PPD conjugates were adsorbed onto an Al(OH)3suspension to give a vaccine that contained 10 pg of PPD and 1 mg of A1 in 0.5 mL of vaccine. CFlxBALB/c mice that had been sensitized with two human doses of BCG 1 month previously were injected intraperitoneally with 0.5 mL of vaccine per immunization. Five mice were immunized with each conjugate. After 2 weeks, they were reimmunized with the same dose, and mouse sera were assayed for antibody levels 10days after the second immunizations. Four days prior to fusion the mice received a final intraperitoneal boost with the same vaccine. Fusion and Screening Procedure. Fusions were performed essentially as described by Kohler and Milstein with slight modifications (Kohler and Milstein, 1975; Reading, 1982). The spleen cells from immunized mice were fused with the myeloma cell line X63.Ag8.653. Selected clones were recloned by limiting dilution at least three times to ascertain monoclonality. Culture supernatants were screened for antibody activity against thioguanine by a capture ELISA, in which microtiterplates were coated with a rabbit antiserum to OA, at 10 pg/mL in 0.05M sodium carbonate buffer, pH 9.6, followed by the respective thioguanine derivative coupled to OA at 10-50 ng/mL in dilution buffer (PBS, pH 7.2 containing 1 % w/v bovine albumin and 0.05 % v/v Triton X-100). This was followed by incubation with dilutions of culture supernatant, horse radish peroxidase-labeled rabbit anti-mouse immunoglobulin (Code P260)diluted 1:1000,and finally with H202 and OPD substrate solution in phosphate-acetate buffer, pH 5.0. (Engvall and Perlmann, 1971;Tijssen, 1985). Culture supernatants which tested positive in the above system were immediately assayed for inhibition of the reaction with free thioguanine, and only those cultures whose reactions could be inhibited were further propagated. Assay for the Fine Specificity of the Monoclonal Antibodies. The fine specificities of the monoclonal antibodies were analyzed by inhibition experiments with a series of thiopurines (depicted in Chart 1). The density of the thioguanine-OA conjugates on rabbit anti-0Acoated plates was adjusted to give a maximal signal of about 3.5 OD at 490 nm in ELISA. Variations in titers of the monoclonal antibodies were adjusted by diluting the individual culture supernatants to give an OD signal of 2.0. The monoclonal antibodies were incubated overnight with varying concentrations from 3 X 10-5 to 6 X legM of the respective thiopurines before the mixture was transferred to the antigen-coated ELISA plate. The further procedure was as described above. The inhibitory potential was calculated as inhibition (7% = ((ODcontrol -
+
Nerstr~met at.
Bioconjugate Chem., Vol. 5, No. 4, 1994
300
Table 1. IH NMR and UV Data of 2-Aminopurines
compd
X
ba (X or N(l)Hb)
c1 c1
7
SH
Y CHzCOOH CHzCOOH CHzCONHS
11.98
8
SH
CHzCONHS
12.1
SCHzCsH4CONHS
H
3 4
CHz: 4.67s CeH4: 7.76 d; 8.03 d (J = 8 Hz) NHS, CzH4: 2.89 s a &valuesin DMSO-&. For 7 and 8. nm in MeOH. 9
OD,,,~)/OD,,tr,,~)lOO. Subclasses of individual monoclonal antibodies were determined by ELISA using a commercial kit.
6(YP (9)CHz: 4.88 s (7)CHz: 5.13 (9)CHz: 5.40 NHS, C2H4: 2.82 s (7)CHz: 5.97 8 NHS, C2H4: 2.80 s 12.5
H-P 8.32s 7.98s
(2)NHza 6.96 6.65 6.92
243; 310 243; 320 256; 344
8.28s
6.58
262; 348
7.90s
6.43
242; 314
8.10s
X-C
Arbitrary units 100
-
7s
-
RESULTS
Chemical Synthesis of ThioguanineDerivatives for Couplingto Proteins. The reaction sequences are shown in Scheme 1and described in the Materials and Methods. Alkylation of 2-amino-6-chloropurine with bromoacetic acid resulted in a mixture of 9- and 7-carboxymethyl isomers with 2-amino-9-(carboxymethyl)-6-chloropurine (3) as the major product. The assignment, 7- or 9-isomer, was done by lH NMR and UV spectroscopy (Table 1). The signals from H(8) and N-CHz are shifted upfield for the 9-isomers relative to the corresponding signals from the 7-isomers,whereas the (2)NHz signals appear at lower field for the 9-isomers (Kjellberg and Johansson, 1986; Green et al., 1990). The longest wavelength absorption maximum in the UV spectra exhibits a bathochromic shift for the 7-isomers compared to the 9-isomers (Green et al., 1990). The 6-chloro substituent of purines is exchangeablewith nucleophiles, and treatment of the 9- and 74carboxymethyl) isomers with thiourea gave the corresponding thiono derivatives 5 and 6. The reactive succinimidyl esters, 7 and 8, were then obtained by reacting 5 and 6 with NHS and DCC. Attempts to react 2-amino-6-chloropurine(2) or thioguanine (1) directly with succinimidyl bromoacetate failed, but reaction of thioguanine with the less reactive succinimidyl4-(bromomethyl)benzoateled to a usable thioguanine derivative reactive at position 6 (9 in Scheme 1). Immune Responsesto the ThioguanineConjugates. All three conjugates of the differently activated thioguanine derivatives induced a high antibody response (Figure 1). When defined as the reciprocal of the dilution giving half maximum signal value, the mean titers for five mice from each group were 150 000, 320 000, and 640 000 for the 7-, 9-, and S-substituted thioguanines, respectively. Monoclonal Antibodies against S-Substituted Thioguanine (the 138 Series). Sixty-eight positive clones were found, of which around 30% produced antibodies inhibitable with free thioguanine. Fourteen of these mAbs were selected, nine of which were analyzed in detail for their fine specificity. The pattern of cross-reactivities for this series of mAbs is displayed in Figure 2, showing the results for three representative antibodies, all of the IgG2a subclass. The antigen used for immunization was conjugated through the sulfur atom and could be regarded as a sustituted thiol (structures c and d in Scheme 2), but screening was
so -
25
0
4:
8 0
-
Serum dilution x 10-3 Figure 1. Immune response against thioguanine conjugated to a carrier protein through three different positions as measured in ELISA. Each curve represents mean responses in groups of five mice. Arbitrary units of 100 represent maximum response in the individual ELISA assays. A: 7-conjugated thioguanine. 0:%conjugated thioguanine. 0:S-conjugated thioguanine. performed with thioguanine, which is best represented by the thiono form (structure a or b in Scheme 2). Both 6-thiono- and 6-thio10 derivatives &e., (methy1thio)guanine (17)) are thus recognized by this series of antibodies. Changes at position 2 have varying effects on reactivity. Exchange of the amino group with hydrogen (Le., 6-purinethione (13)) or introduction of oxygen at position 2 (i.e., 6-thioxanthine (15)) only affected the reactivity of antibody 138-7 marginally, whereas it completely abolished the reactivity of the other two antibodies. Compounds with substituents at positions 7,8, or 9 were markedly restricted in their reactivity with these antibodies, but to varying degrees. Thus, substitution at position 7 (6) hardly affected the reactivity of 138-6,whereas 138-2 lost most and 138-7part of their reactivities. Substitution at position 8 (12aand 12b)diminished reactivity depending on the substituent. Substitution at position 9 further diminished reactivity of 138-7; compare 13 with 14. Monoclonal Antibodies against 9-Substituted Thioguanine (the 125 Series). One fusion gave a yield of 600 antigen-specific hybridomas, which were all inhib-
Thloguanine Derivatives: Synthesis and Antibodies
Bioconjugate Chem., Voi. 5, No. 4, 1994 % Inhibition
% Inhibition
1
361
17
13
15
6
1Za
1Zb
14
17
1
16
13
15
1Za
6
12b
14
16
Thiopurines
T h i op u r i n e s
Figure 2. Cross-reactivity pattern for three representative monoclonal antibodies raised against thioguanine conjugated to the carrier protein at position 6. The numbers are the different thioeuanine analogues 13 X 106 M) used in the inhibition assay andiefer to Chart 1.
Figure 3. Cross-reactivity pattern for three representative monoclonal antibodies raised against thioguanine conjugated to the carrier protein in position 9. The numbers are the different thioguanine analogues (3 x 10-8 M) used in the inhibition assay and refer to Chart 1.
Scheme 2
% Inhibition S
ion
.
80
rr-rrr
I....
I...rl..._.r
r... -r.1.-.1-11
60 SH 40
20
itable by free thioguanine. Six clones were subjected to repeated reclonings, and the resulting antibodies were analyzed for their fine specificity in the inhibition assay. The cross-reactivity patterns for this group of monoclonal antibodies is shown in Figure 3, where three antibodies, all of the IgG2a subclass, have been selected to represent the observed patterns of binding specificity. It appears that reactivity was primarily dependent on the structure of the pyrimidine ring. The S-substituted derivative (17) had markedly reduced reactivity. Changes at position 2 interfered with reactivity: exchange of the amino group with hydrogen (13) diminished the reactivities of the three antibodies, and introduction of oxygen at position 2 (15) practically abolished reactivity with antibodies 125-3and 125-5,whereas antibody 125-4,as an exception, maintained some reactivity. Substitution at position 7 (6) did not influence reactivity, whereas substitution at position 8 appeared to affect the reactivity differently (compare the reactivity of 12a and 12b for the three antibodies mentioned). Substitution with ribose at position 9 (14) seemed almost to restore the reactivity lost by removal of the amino group (13) but did not restore the reactivity lost by S-substitution (16). Monoclonal Antibodies against 7-Substituted Thioguanine (the 144 Series). Fusions gave avery large number of positive clones, of which almost all the antibodies could be inhibited by free thioguanine. Sixteen
0 1
17
13
15
6
12a
1Zb
14
TI1 i opu ri n es
Figure 4. Cross-reactivity pattern for two representative monoclonal antibodies raised against thioguanine conjugated to the carrier protein in position 7. The numbers are the different thioguanine analogues (3 X 106 M) used in the inhibition assay and refer to Chart 1.
mAbs were selected and further analyzed. This group of mAbs showed a higher degree of homogeneity than the two other. The general pattern of reactivity is represented by 144-4 (IgG1) and 144-5 (IgG2a) in Figure 4. The binding activity for the 144 series was mostly influenced by the structure of the pyrimidine ring. Thus, S-substitution (17) profoundly reduced reactivity, and changes at position 2 diminished reactivity, either slightly when the amino group was exchanged with hydrogen (13) or more profoundly with 6-thioxanthine (15). Substitution in the imidazole ring seemed to be less important. Compound 6 with substitution at position 7 inhibited just as well as thioguanine, as might be expected from the fact that this is the site of substitution in the immunizing conjugate. With substitution at position 8, the reactivity depended on the substituent (compare 12a
Nerstrom et al.
382 Bloconlugate Chem., Vol. 5, No. 4, 1994
and 12b), and substitution at position 9 did not seem to have much impact on reactivity (compare 13 and 14). Antibody 144-5 from this series showed a somewhat extraordinary reactivity pattern, in that almost all the thiopurines possessed some reactivity, though with different affinities. DISCUSSION
When the reactive thioguanine derivatives were allowed to react with free amino groups on a carrier protein (PPD), immunogenic hapten-carrier conjugates were obtained that led to the production of antibodies which recognized derivatives of thioguanine differentially depending on the structure of the exposed part of the molecule. Previous experience suggests that immunization of animals pretreated with live BCG vaccine with hapten conjugated to PPD gives rise to a remarkably potent carrier effect (Lachmann et al., 1986). This has been demonstrated when merthiolat was used as hapten2and also in systems involving peptides (Lussow et al., 1991) or lowimmunogenic protein^.^ The mechanism is presumed to involve the induction of a strong T-cell immunity to mycobacterial antigens by the BCG treatment, followed by the exploitation of these educated T cells as T helper cells in the subsequent proliferation of specific B cells presenting the hapten linked to fragments of mycobacterial protein (PPD). In the present experiments, BCG-prevaccinated mice responded after only two immunizations with antihapten antibody titers high enough to warrant cell fusion after a final boost. The initial screening of the mouse sera was performed with the homologous thioguanine derivative coupled onto an irrelevant carrier, OA. Thus, at this stage, we did not analyze whether the antibody response was directed against free or coupled forms of thioguanine. However, during the screening of hybridomas secreting antibodies against the homologous hapten-carrier conjugate, we immediately selected those for which antibody binding to the solid phase was completely inhibited by free thioguanine in solution. Several types of tautomerism have been proposed for thioguanine and related compounds (Pullman and Pullman, 1973;Elguero et al., 1976). The most important ones are shown in Scheme 2, Le., 9-7 tautomerism, e.g., a + b, and thiono-thiolo tautomerism, e.g., a +. c. Thioguanine has primarily the thiono structure (a or b), both in the solid state and in solution. A pronunced difference is observed in the UV spectra of thiono (Am= = 350 nm) and thiolo forms (A,,= = 320 nm) (cf. Table 1; Elion et al., 1959). Thioguanine is best represented by the canonical form b in the solid state (Bugg and Thewalt, 1970). Little is known about the 9-7 tautomerism in solution, but thioguanine is probably best represented by a mixture of the canonical forms a and b in solution, by analogy with guanine (Pfleiderer, 1961). The fine specificityof the selected monoclonal antibodies was analyzed through inhibition experiments with a series of thiopurine derivatives, substituted at various positions. The specificity was strongly dependent on the position used for coupling to the carrier molecule. When the S-position was chosen as the coupling site, the reactivity of the resulting antibodies was not influenced
* Klausen, J., Weiss Bentzon, M., and Koch, C. Immunization of BCG-pretreated mice with merthiolate conjugated to Purified Protein Derivative (PPD) of Tuberculin gives rise to high titre antibodies. (Manuscript in preparation). 3 Unpublished observation.
by substitution at the sulfur atom. The reactivities of these antibodies were strongly influenced by the substituents a t positions 2, 7, 8, and 9. When the 7- or 9-positions were used for conjugation, the reactivities of the resulting antibodies were mostly dependent on the structure of the pyrimidine ring and less influenced by substitutions at positions 7, 8, and 9. However, for all three series of mAbs we observed a high degree of heterogeneity, i.e., the influence of substitutions at various positions varied from no inhibition to complete inhibition of the homologous interaction with the antigen used for immunization. Among the structures used to define the fine specificity of the thioguanine-specific antibodies are some which are known to be metabolites of thioguanine in man and experimental animals after the injection or therapeutic use of thioguanine as an antimetabolite. The finding that such metabolites are recognized differently by the monoclonal antibodies that we have obtained provides the opportunity to measure thioguanine and certain of its metabolites in clinical samples. The present series of antibodies will therefore be employed in assays to monitor the treatment of patients with thioguanine. ACKNOWLEDGMENT
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