Clin 8iochem, Vol. 24, pp. 37--42, 1991
0009-9120/91 $3.00 + .00 Copyright © 1991 The Canadian Society of Clinical Chemists.
Printed in Canada. All right~ reserved.
Monoclonal Antibody Technology for Cyclosporine Monitoring VALERIE F.J. QUESNIAUX Sandoz Pharma AG, CH-4002 Basel, Switzerland Monitoring blood levels of Cyclosporine (CsA) has been the basis for adjusting individual dosage regimens in the clinic. Radioimmunoassays using polyclonal antisera reacted with CsA and some CsA metabolites, leading to overestimation when compared with highperformance liquid chromatographic measurements of CsA. Monoclonal antibodies (mAbs) have the potential to discriminate between closely related molecules. MAbs with high affinity for CsA have been prepared and their fine-specificity characterized by cross-reactivity studies using a large series of CsA-derivatives. According to the known sites of metabolism on the CsA molecule and to its threedimensional structure, it was possible to predict which mAb would be suitable for recognizing native Cs specifically.
KEY WORDS: monoclonal antibody; cyclosporine;therapeutic drug monitoring; immunoassay. Introduction
he original radioimmunoassays available for T Cyclosporine (CsA; Sandimmune ®) monitoring were poorly specific for the parent drug since they revealed many CsA metabolites as well (1,2). This was due to the cross-reactivity of the polyclonal antisera used in these assays for the CsA metabolites and underlined the need for a more specific immunoassay using monoclonal antibodies (mAbs). Because of their restricted specificity, mAbs potentially present more clear-cut cross-reactivities for closely related antigens with minor variations than polyclonal antisera do. As represented schematically in Figure 1, polyclonal antisera contain several populations of antibodies interacting with various epitopes on the antigen surface. When a polyclonal antiserum reacts with a slightly modified antigen analogue, the antibody populations specific for epitopes which have been modified in the analogue will be unable to recognize the modified antigen. However, all the remaining antibody populations which are specific for epitopes unmodified in the analogue will recognize the modified and the native antigen equally well. This will result in an intermediate cross-reactivity of the polyclonal antiserum
Correspondence:Valerie F.J. Quesniaux, SandozPharma AG, CH-4002 Basel, Switzerland. Manuscript received May 14, 1990; revised July 5, 1990; accepted July 16, 1990. CLINICAL BIOCHEMISTRY, VOLUME 24, FEBRUARY 1991
for the antigen analogue. In contrast, no or very low cross-reactivity for the antigen analogue will be obtained with mAbs specific for the region of the antigen which is modified in the analogue. These mAbs will not recognize their original epitope and therefore will not bind to the analogue. Conversely, complete cross-reactivity can occur with mAbs specific for regions of the antigen which are unmodified in the analogue. These mAbs will recognize equally well the original and the modified antigen. This was illustrated in the case of CsA and its metabolite 17 (AM1), since an intermediate crossreactivity was observed with polyclonal antisera (6-fold difference), whereas discrimination up to 150-fold could be observed with individual mAbs and, on the other hand, several mAbs recognized CsA and metabolite 17 (AM1) equally well (3). This example shows that this approach is valid when only one given analogue with a defined modification has to be discriminated from the original molecule. I m m u n o c h e m i c a l d e t e c t i o n of c y c l o s p o r i n e metabolites Since CsA is extensively metabolized, a monitoring immunoassay specific for the parent compound requires an antibody reacting with the native CsA but not with a dozen CsA metabolites presenting different modifications. Fortunately, all CsA metabolites identified so far are modified on residues 1, 4, 6 and 9, which are located on one half of the Cs molecule (4), delineating an area compatible with the size of a mAb epitope (see Figure 2). A mAb recognizing an epitope including these residues should therefore react specifically with CsA but not with the metabolites. As a first aim, it appeared important to develop a mAb able to discriminate between CsA and the most abundant metabolite 17 (AM1). Blood levels of AM1 can exceed those of Cs in certain clinical situations (5,6), whereas immunosuppressive activity seems to be significantly lower than that of CsA [consensus 10-20% of CsA activity (7)]. Metabolite 17 (AM1) is hydroxylated on the last carbon of the side chain of MeBmt CsA-residue 1 (4). A prerequisite for a discriminating mAb was therefore reac37
QUESZ~-L~UX a
Specificity for the homologous antigen:
polyclonala n t ~
=~=~> polyspecificity b
monoclonalantibodiesI and 2:
=:==~ monospecificity
Specificity for a modified antigen:
polyclonala n t i s e ~
monoclonalantibodies1and2:
===l> intermediatecross-reaction
===~> no cross-reaction
==:=~> completecross-reaction
Figure 1--Schematic representation of the specificity of polyclonal and monoclonal antibodies for native (a) and modified (b) antigen.
tion with an epitope including the last carbon of the residue 1 side chain. In order to obtain such antibodies and because free CsA is poorly immunogenic (8), it was necessary to design an immunogenic conjugate of Cs in which the last carbon of residue 1 would be accessible for antibody recognition.
Role of CsA conformation in immunoreactivity No information on the conformation of CsA in an aqueous environment is available, due to the poor water solubility of CsA. However, the three-dimen38
sional structure of CsA has been determined by X-ray diffraction analysis in crystals and by nuclear magnetic resonance in aprotic solvents, the main difference between these two conformations being the orientation of the 7 carbon side chain of residue 1 (9-11). With the assumption that the conformation in aqueous environment was similar to that observed in aprotic solvents, namely that the residue I side chain was protruding into the solvent, an immunogenic conjugate was prepared where CsA was coupled through residue 8, the residue opposite to the last carbon of residue 1. CLINICAL BIOCHEMISTRY, VOLUME 24, FEBRUARY 1991
M O N O C L O N A L ANTIBODIES TO CYCLOSPORINE TASLZ 1 Fine Specificityof Monoclonal Antibodies and PolyclonalAntisera to Thr2-CsA Amino Acid Residues Recognized Monoclonal Abs R14-85-1 V14-54-20 R14-7-20 V14-306 R45-200-7 R45-620-40 V14-343 R45-83-40 R14-212-6 R45-269-2 R45-180-7 V45-180-56 R45-109-22 R45-246-2 V45-187-15 R45-45-11 V45-271-10 V14-203
1
2
3
4
5
+ + +++ + + + +++ + + +++ + + + +++ + + + -
+++ + + + +++ + + + + + +++ +++ +++ +++ + +++ + -
+ + + + + + +++ + + + + + + + + + +++ + + + +++ +++ +++ +++ +++ +++ +++ +++ +++ +
+ + + + + + + + + + + + +++ + + + +++ +++ +++ +++ +++ +++ +++ +++ +++ -
+ +++ . + + + + + + + +++ +++ +++ +++ +++ +++ +++ +
+ +
+ +
+++ +++
+ +++
6
7
. + +++ .
8
.
. . .
. + + + + + + +++ +++ +++ +++ +++ +++ +++ -
9 .
. .
. . .
. . . . . . . . . + + + +
. . . . .
.
.
. . . . -
. . . .
. .
. . .
+ +
. . . . .
-
. . . .
11
.
. .
. . . . . .
10
+
+ +
+ -
+++ +++ + + +
. . . .
+ +++ + + +
. .
Polyclonal Abs 14 45
+ +
+ +
D
+ + + , + a n d - c o r r e s p o n d r e s p e c t i v e l y to r e s i d u e s s t r o n g l y , w e a k l y a n d n o t a t a l l r e c o g n i z e d b y t h e a n t i s e r a b a s e d o n t h e b i n d i n g to v a r i o u s C s A d e r i v a t i v e s : w h e n s e v e r a l C s A d e r i v a t i v e s modified a t a g i v e n r e s i d u e h a d to be added a t a > 3 0 - f o l d , ---3 to 3 0 - f o l d , o r < 3 - f o l d m o l a r e x c e s s c o m p a r e d t o u n m o d i f i e d C s A to r e a c h 5 0 % o f E L I S A i n h i b i t i o n .
Hyc (met. 10)
(met. 8, 17, 18, 25, 26)
bond (met. 18, 26)
(rnet. 9,'16)
(met. 1, 8, 9, 10, 16, 26)
Figure 2--Three-dimensional model of CsA showing the localization of the six main sites of metabolism of CsA (according t o Ref. 4): d e m e t h y l a t i o n a n d h y d r o x y l a t i o n o f N - M e - L e u 4 r e s i d u e , 8 ' - h y d r o x y l a t i o n a n d i n t r a m o l e c u l a r e t h e r b o n d o f N-methyl-(4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine ( N - M e - B m t ) r e s i d u e 1, a n d h y d r o x y l a t i o n o f N - M e - L e u 6 a n d N - M e - L e u 9 r e s i d u e s . T h e m e t a b o l i t e s m o d i f i e d a t t h e d i f f e r e n t p o s i t i o n s a r e m e n t i o n e d . A b u = a l p h a - A m i n o b u t y r i c acid; Sar = Sarcosine; met = metabolite.
C L I N I C A L B I O C H E M I S T R Y , V O L U M E 24, F E B R U A R Y 1991
39
QUESNIAUX TABLE 2 Fine Specificity of Monoclonal Antibodies and Polyclonal Antisera to D-LysS-CsA Amino Acid Residues Recognized
Monoclonal Abs 26-148 26-118 29-56 26-85 78-299 78-214 78-216 78-91 29-355 Polyclonal Abs 29 78 +++,
+,
-
1
2
3
4
5
6
7
8
9
i0
II
+ + +++ +++ +++ +++ + +++
-
+ + + + +++ + + +++
+ + + + + +++
+ + + + + + +++
+++ +++ +++ +++ +++ +++ +++ +++ +++
+ + + + .
+++ +++ +++ +++ +++ +++ +++ +++
+++ +++ +++ +++ +++ +++ +++ + .
+ + +
+ -
+
--
+
--
_
+÷+
--
÷
--
+
--
+
--
_
÷÷+
--
÷÷÷
÷
as in Table
.
.
1.
H o w e v e r , n o n e of s e v e r a l polyclonal a n t i s e r a a n d 110 m A b s r a i s e d a g a i n s t t h i s c o n j u g a t e could disc r i m i n a t e b e t w e e n CsA a n d m e t a b o l i t e 17 [AM1 (3)]. T h e fine-specificity of t h e s e m A b s w a s t h e n determined by cross-reactivity studies with more t h a n 60 C s A d e r i v a t i v e s modified a t t h e different r e s i d u e s a n d a l o n g t h e n i n e carbon-side c h a i n of r e s i d u e 1 (12). M o s t of t h e polyclonal a n t i b o d i e s a n d t h e m A b s o b t a i n e d recognized r e s i d u e s 1 to 6 (Table 1), a n d t h e y r e a c t e d only w i t h t h e first c a r b o n s of t h e r e s i d u e 1 side c h a i n (13). T h e s e r e s u l t s s u g g e s t e d t h a t w h e n CsA w a s dissolved in a q u e o u s buffer, t h e t e r m i n a l a t o m s of t h e r e s i d u e 1 side c h a i n w e r e not a v a i l a b l e for a n t i b o d y r e c o g n i t i o n a t t h e face of C s A opposite to r e s i d u e 8 (Figure 3). I t w a s t h e n h y p o t h e s i z e d t h a t t h e residue 1 side c h a i n could be folded b a c k a g a i n s t t h e r e s t of t h e molecule, as o b s e r v e d in crystals, r a t h e r than protruding. B a s e d on t h i s h y p o t h e s i s , a second series of a n t i bodies w a s r a i s e d u s i n g a c o n j u g a t e of CsC coupled t h r o u g h r e s i d u e T h r 2 , a r e s i d u e opposite to t h e l a s t c a r b o n of r e s i d u e 1 in t h e X - r a y c r y s t a l s t r u c t u r e . S e v e n out of 9 m A b s recognized m e t a b o l i t e 17 (AM1) 10 to 150-fold less well t h a n CsA (3). T h e finespecificity of two polyclonal a n t i b o d i e s a n d n i n e mAbs was determined by cross-reactivity studies w i t h C s A - d e r i v a t i v e s . M o s t a n t i b o d i e s recognized t h e l a s t c a r b o n s of t h e r e s i d u e 1 side c h a i n t o g e t h e r w i t h r e s i d u e s 6, 8 a n d 9 [Table 2 (12,13)]. T h e s e r e s u l t s i n d i c a t e d t h a t t h e l a s t c a r b o n of t h e r e s i d u e 1 side c h a i n w e r e close to r e s i d u e s 6, 8 a n d 9, as o b s e r v e d in t h e crystal, s u g g e s t i n g t h a t in a q u e o u s buffer, t h e side c h a i n of r e s i d u e 1 could be folded b a c k as o b s e r v e d in t h e c r y s t a l r a t h e r t h a n p r o t r u d i n g as s e e n in aprotic s o l v e n t s (13).
Criteria for antibody selection O n e m A b r e c o g n i z i n g m e t a b o l i t e 17 (AM1) 15040
.
fold less well t h a n CsA w a s selected a n d f u r t h e r t e s t e d for its c r o s s - r e a c t i v i t y w i t h o t h e r C s A m e t a b olites. T h i s m A b w a s s h o w n to d i s c r i m i n a t e 15 to 1000-fold b e t w e e n C s A a n d its m e t a b o l i t e s , h a v i n g less c r o s s - r e a c t i v i t y for t h o s e s e c o n d a r y m e t a b o l i t e s w i t h m u l t i p l e m o d i f i c a t i o n s (14). A second m A b specific for t h e r e g i o n of t h e C s A m o l e c u l e t h a t is not m e t a b o l i z e d w a s selected. T h i s m A b recognized a l m o s t e q u a l l y well CsA a n d m o s t of its m e t a b o lites, w i t h a s l i g h t d i s c r i m i n a t i o n for m e t a b o l i t e s modified on r e s i d u e 4. T h e s e two m A b s , r e f e r r e d to as t h e "specific" a n d t h e "nonspecific" m A b s for n a t i v e CsA, w e r e i n c o r p o r a t e d into r a d i o i m m u n o a s s a y s u s i n g t r i t i a t e d or i o d i n a t e d t r a c e r s . S e v e r a l studies, u n d e r t a k e n to a s s e s s t h e exclusive specificity of t h e "specific" m A b in clinical s a m p l e s , d e m o n s t r a t e d a h i g h correlation b e t w e e n the r e s u l t s o b t a i n e d u s i n g "specific" m A b - b a s e d r a d i o i m m u n o a s s a y (RIA) a n d h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h y (HPLC) (15,16). I n a m u l t i c e n t e r s t u d y w h e r e t h e perform a n c e s of t h e m A b s a n d of t h e o r i g i n a l polyclonal a n t i s e r u m w e r e c o m p a r e d in RIA, m u c h l o w e r res u l t s w e r e o b t a i n e d w i t h t h e "specific" m A b comp a r e d to t h e polyclonal a n t i s e r u m (17). C o n v e r s e l y , t h e r e s u l t s o b t a i n e d w i t h t h e "nonspecific" m A b w e r e h i g h e r t h a n t h o s e o b t a i n e d w i t h t h e polyclonal a n t i s e r u m , as e x p e c t e d f r o m t h e i r c r o s s - r e a c t i v i t y patterns.
Clinical relevance T h e clinical r e l e v a n c e of m o n i t o r i n g p a r e n t CsA a l o n e or t o g e t h e r w i t h t h e m e t a b o l i t e s is still n o t c l e a r l y e s t a b l i s h e d . T h e c o r r e l a t i o n of clinical situa t i o n s w i t h blood levels of p a r e n t C s A on t h e one h a n d , a n d of m e t a b o l i t e s on t h e o t h e r h a n d , h a s b e e n r e s t r i c t e d so f a r to t h e few l a b o r a t o r i e s e q u i p p e d to p e r f o r m s o p h i s t i c a t e d H P L C for i n d i v i d u a l m e tabolites. R o s a n o et al. (5) s h o w e d t h a t t h e concenCLINICAL
BIOCHEMISTRY,
VOLUME
24, FEBRUARY
1991
MONOCLONAL ANTIBODIES TO CYCLOSPORINE
a
~
val-5
N-Me-Leu-6 b
Val-5
D-Ala-8
Abu N-Me-VaI-11. ~
Figure 3ab (continued).
trations determined using the "nonspecific" mAb accounted for the total of parent compound plus the most abundant primary metabolites 17 (AM1) and 1 (AM9) in samples from transplanted patients with good renal function or undergoing rejection. Immunoassays based on the "specific" and "nonspecific" mAbs run in parallel could therefore provide a convenient way to follow the rate of metabolism and allow accumulation of data on the clinical significance of CsA metabolite blood levels. References 1. Donatsch P, Abisch E, Homberger M, Traber R, Trapp M, Voges R. A radiojmmunoassay to measure cyclosporine A in plasma and serum samples. J Immunoassay 1981; 2: 19-32. 2. Robinson WT, Schran HF, Barry EP. Methods to measure cyclosporine l e v e l s - high pressure liquid chromatography, radioimmunoassay and correlation. CLINICALBIOCHEMISTRY,VOLUME24, FEBRUARY 1991
Transplant Proc 1983; 15 (Suppl. 1/2): 2403-8. 3. Quesniaux VFJ, Schmitter D, Schreier MH, Van Regenmortel MHV. Monoclonal antibodies to cyclosporine are representative ofthe major antibodypopulations present in antisera of immunized mice. Mol Irnmunol 1990; 27: 227-36. 4. Maurer G, Loosli HR, Schreier E, Keller B. Disposition of cyclosporine in several animal species and man. I. Structural elucidation of its metabolites. Drug Metab Dispos 1984; 12: 120-6. 5. Rosano TG, Pell MA, Freed BM, Dybas MT, Lempert N. Cyclosporine and metabolites in blood from renal allograft recipients with nephrotoxicity, rejection or good renal function: comparative high-performance liquid chromatography and monoclonal radioimmunoassay studies. Transplant Proc 1988; 20 (Suppl. 2): 330-8. 6. Wang CP, Burckart GJ, Ptachcinski RJ, et al. Cyclosporine metabolite concentrations in the blood of liver, heart, kidney and bone marrow transplant patients. Transplant Proc 1988; 20 (Suppl. 2): 591-6. 7. Ryffel B, Foxwell BMJ, Mihatsch MJ, Donatsch P,
41
QUESNIAUX C
Abu-2
Abu-2
d
( Figure 3cd. Figure 3--Identification of the 11 amino acid residues of CsA on space filling models of CS conformation according to X-ray crystallographic analysis (a,c) and to two-dimensional nuclear magnetic resonance in aprotic solvent (b,d). In (a) and (b), the CsA molecule is oriented as in Figure 2, whereas in (c) and (d) the opposite side of the CsA molecule is shown. Amino acid abbreviations as in Figure 2.
8.
9.
10.
11.
12.
42
Maurer G. Biologic significance of cyclosporine metabolites. Transplant Proc 1988; 20 (Suppl. 2): 57584. Quesniaux VFJ, Himmelspach K, Van Regenmortel MHV. An enzyme immunoassay for the screening of monoclonal antibodies to cyclosporine. Immunol Lett 1985; 9: 99-104. Petcher TJ, Weber HP, Riiegger A. Crystal and molecular structure of an iodo-derivative of the cyclic undecapeptide cyclosporine. Helv Chim Acta 1976; 59: 1480--8. Loosli HR, Kessler H, Oschkinat H, Weber HP, Petcher TJ, Widmer A. The conformation of 'cyclosporine A' in the crystal and in solution. Helv Chim Acta 1985; 68: 682-704. Kessler H, Loosli HR, Oschkinat H. Assignment of the 1H, 13C, and 15N-NMR spectra of cyclosporine A in CDC13 and C6D6 by a combination of homo- and heteronuclear two-dimensional techniques. Helv Chim Acta 1985; 68: 661-81. Quesniaux VFJ, Tees R, Schreier MH, Wenger RM, Van Regenmortel MHV. Fine specificity and cross-
reactivity of monoclonal antibodies to cyclosporine. Mol Immunol 1987; 24: 1159-68.
13. Quesniaux VFJ, Wenger RM, Schmitter D, Van Regenmortel MVH. Study of the conformation of cyclosporine in aqueous medium by means of monoclonal antibodies. Int J Pept Protein Res 1988; 31: 173-85. 14. Quesniaux V, Tees R, Schreier MH, Maurer G, Van Regenmortel MHV. Potential of monoclonal antibodies to improve therapeutic monitoring of cyclosporine. Clin Chem 1987; 33: 32-7. 15. Schran HF, Rosario TG, Hassell AE, Pell MA. Determination of cyclosporine concentrations with monoclonal antibodies. Clin Chem 1987; 33: 2225-9. 16. Ball PE, Munzer H, Keller HP, Abisch E, Rosenthaler J. Specific 3H radioimmunoassay with a monoclonal antibody for monitoring cyclosporine in blood. Clin Chem 1988; 34: 257-60. 17. Holt DW, Johnston A, Marsden JT, et al. Monoclonal antibodies for radioimmunoassay of cyclosporine: a multicenter comparison of their performance with the Sandoz polyclonal radioimmunoassay kit. Clin Chem 1988; 34: 1091-6. CLINICAL BIOCHEMISTRY, VOLUME 24, FEBRUARY 1991
0009-9120/91 $3.00 + .00 Copyright © 1991 The Canadian Society of Clinical Chemists.
Printed in Canada. All right~ reserved.
Monoclonal Antibody Technology for Cyclosporine Monitoring VALERIE F.J. QUESNIAUX Sandoz Pharma AG, CH-4002 Basel, Switzerland Monitoring blood levels of Cyclosporine (CsA) has been the basis for adjusting individual dosage regimens in the clinic. Radioimmunoassays using polyclonal antisera reacted with CsA and some CsA metabolites, leading to overestimation when compared with highperformance liquid chromatographic measurements of CsA. Monoclonal antibodies (mAbs) have the potential to discriminate between closely related molecules. MAbs with high affinity for CsA have been prepared and their fine-specificity characterized by cross-reactivity studies using a large series of CsA-derivatives. According to the known sites of metabolism on the CsA molecule and to its threedimensional structure, it was possible to predict which mAb would be suitable for recognizing native Cs specifically.
KEY WORDS: monoclonal antibody; cyclosporine;therapeutic drug monitoring; immunoassay. Introduction
he original radioimmunoassays available for T Cyclosporine (CsA; Sandimmune ®) monitoring were poorly specific for the parent drug since they revealed many CsA metabolites as well (1,2). This was due to the cross-reactivity of the polyclonal antisera used in these assays for the CsA metabolites and underlined the need for a more specific immunoassay using monoclonal antibodies (mAbs). Because of their restricted specificity, mAbs potentially present more clear-cut cross-reactivities for closely related antigens with minor variations than polyclonal antisera do. As represented schematically in Figure 1, polyclonal antisera contain several populations of antibodies interacting with various epitopes on the antigen surface. When a polyclonal antiserum reacts with a slightly modified antigen analogue, the antibody populations specific for epitopes which have been modified in the analogue will be unable to recognize the modified antigen. However, all the remaining antibody populations which are specific for epitopes unmodified in the analogue will recognize the modified and the native antigen equally well. This will result in an intermediate cross-reactivity of the polyclonal antiserum
Correspondence:Valerie F.J. Quesniaux, SandozPharma AG, CH-4002 Basel, Switzerland. Manuscript received May 14, 1990; revised July 5, 1990; accepted July 16, 1990. CLINICAL BIOCHEMISTRY, VOLUME 24, FEBRUARY 1991
for the antigen analogue. In contrast, no or very low cross-reactivity for the antigen analogue will be obtained with mAbs specific for the region of the antigen which is modified in the analogue. These mAbs will not recognize their original epitope and therefore will not bind to the analogue. Conversely, complete cross-reactivity can occur with mAbs specific for regions of the antigen which are unmodified in the analogue. These mAbs will recognize equally well the original and the modified antigen. This was illustrated in the case of CsA and its metabolite 17 (AM1), since an intermediate crossreactivity was observed with polyclonal antisera (6-fold difference), whereas discrimination up to 150-fold could be observed with individual mAbs and, on the other hand, several mAbs recognized CsA and metabolite 17 (AM1) equally well (3). This example shows that this approach is valid when only one given analogue with a defined modification has to be discriminated from the original molecule. I m m u n o c h e m i c a l d e t e c t i o n of c y c l o s p o r i n e metabolites Since CsA is extensively metabolized, a monitoring immunoassay specific for the parent compound requires an antibody reacting with the native CsA but not with a dozen CsA metabolites presenting different modifications. Fortunately, all CsA metabolites identified so far are modified on residues 1, 4, 6 and 9, which are located on one half of the Cs molecule (4), delineating an area compatible with the size of a mAb epitope (see Figure 2). A mAb recognizing an epitope including these residues should therefore react specifically with CsA but not with the metabolites. As a first aim, it appeared important to develop a mAb able to discriminate between CsA and the most abundant metabolite 17 (AM1). Blood levels of AM1 can exceed those of Cs in certain clinical situations (5,6), whereas immunosuppressive activity seems to be significantly lower than that of CsA [consensus 10-20% of CsA activity (7)]. Metabolite 17 (AM1) is hydroxylated on the last carbon of the side chain of MeBmt CsA-residue 1 (4). A prerequisite for a discriminating mAb was therefore reac37
QUESZ~-L~UX a
Specificity for the homologous antigen:
polyclonala n t ~
=~=~> polyspecificity b
monoclonalantibodiesI and 2:
=:==~ monospecificity
Specificity for a modified antigen:
polyclonala n t i s e ~
monoclonalantibodies1and2:
===l> intermediatecross-reaction
===~> no cross-reaction
==:=~> completecross-reaction
Figure 1--Schematic representation of the specificity of polyclonal and monoclonal antibodies for native (a) and modified (b) antigen.
tion with an epitope including the last carbon of the residue 1 side chain. In order to obtain such antibodies and because free CsA is poorly immunogenic (8), it was necessary to design an immunogenic conjugate of Cs in which the last carbon of residue 1 would be accessible for antibody recognition.
Role of CsA conformation in immunoreactivity No information on the conformation of CsA in an aqueous environment is available, due to the poor water solubility of CsA. However, the three-dimen38
sional structure of CsA has been determined by X-ray diffraction analysis in crystals and by nuclear magnetic resonance in aprotic solvents, the main difference between these two conformations being the orientation of the 7 carbon side chain of residue 1 (9-11). With the assumption that the conformation in aqueous environment was similar to that observed in aprotic solvents, namely that the residue I side chain was protruding into the solvent, an immunogenic conjugate was prepared where CsA was coupled through residue 8, the residue opposite to the last carbon of residue 1. CLINICAL BIOCHEMISTRY, VOLUME 24, FEBRUARY 1991
M O N O C L O N A L ANTIBODIES TO CYCLOSPORINE TASLZ 1 Fine Specificityof Monoclonal Antibodies and PolyclonalAntisera to Thr2-CsA Amino Acid Residues Recognized Monoclonal Abs R14-85-1 V14-54-20 R14-7-20 V14-306 R45-200-7 R45-620-40 V14-343 R45-83-40 R14-212-6 R45-269-2 R45-180-7 V45-180-56 R45-109-22 R45-246-2 V45-187-15 R45-45-11 V45-271-10 V14-203
1
2
3
4
5
+ + +++ + + + +++ + + +++ + + + +++ + + + -
+++ + + + +++ + + + + + +++ +++ +++ +++ + +++ + -
+ + + + + + +++ + + + + + + + + + +++ + + + +++ +++ +++ +++ +++ +++ +++ +++ +++ +
+ + + + + + + + + + + + +++ + + + +++ +++ +++ +++ +++ +++ +++ +++ +++ -
+ +++ . + + + + + + + +++ +++ +++ +++ +++ +++ +++ +
+ +
+ +
+++ +++
+ +++
6
7
. + +++ .
8
.
. . .
. + + + + + + +++ +++ +++ +++ +++ +++ +++ -
9 .
. .
. . .
. . . . . . . . . + + + +
. . . . .
.
.
. . . . -
. . . .
. .
. . .
+ +
. . . . .
-
. . . .
11
.
. .
. . . . . .
10
+
+ +
+ -
+++ +++ + + +
. . . .
+ +++ + + +
. .
Polyclonal Abs 14 45
+ +
+ +
D
+ + + , + a n d - c o r r e s p o n d r e s p e c t i v e l y to r e s i d u e s s t r o n g l y , w e a k l y a n d n o t a t a l l r e c o g n i z e d b y t h e a n t i s e r a b a s e d o n t h e b i n d i n g to v a r i o u s C s A d e r i v a t i v e s : w h e n s e v e r a l C s A d e r i v a t i v e s modified a t a g i v e n r e s i d u e h a d to be added a t a > 3 0 - f o l d , ---3 to 3 0 - f o l d , o r < 3 - f o l d m o l a r e x c e s s c o m p a r e d t o u n m o d i f i e d C s A to r e a c h 5 0 % o f E L I S A i n h i b i t i o n .
Hyc (met. 10)
(met. 8, 17, 18, 25, 26)
bond (met. 18, 26)
(rnet. 9,'16)
(met. 1, 8, 9, 10, 16, 26)
Figure 2--Three-dimensional model of CsA showing the localization of the six main sites of metabolism of CsA (according t o Ref. 4): d e m e t h y l a t i o n a n d h y d r o x y l a t i o n o f N - M e - L e u 4 r e s i d u e , 8 ' - h y d r o x y l a t i o n a n d i n t r a m o l e c u l a r e t h e r b o n d o f N-methyl-(4R)-4-[(E)-2-butenyl]-4-methyl-L-threonine ( N - M e - B m t ) r e s i d u e 1, a n d h y d r o x y l a t i o n o f N - M e - L e u 6 a n d N - M e - L e u 9 r e s i d u e s . T h e m e t a b o l i t e s m o d i f i e d a t t h e d i f f e r e n t p o s i t i o n s a r e m e n t i o n e d . A b u = a l p h a - A m i n o b u t y r i c acid; Sar = Sarcosine; met = metabolite.
C L I N I C A L B I O C H E M I S T R Y , V O L U M E 24, F E B R U A R Y 1991
39
QUESNIAUX TABLE 2 Fine Specificity of Monoclonal Antibodies and Polyclonal Antisera to D-LysS-CsA Amino Acid Residues Recognized
Monoclonal Abs 26-148 26-118 29-56 26-85 78-299 78-214 78-216 78-91 29-355 Polyclonal Abs 29 78 +++,
+,
-
1
2
3
4
5
6
7
8
9
i0
II
+ + +++ +++ +++ +++ + +++
-
+ + + + +++ + + +++
+ + + + + +++
+ + + + + + +++
+++ +++ +++ +++ +++ +++ +++ +++ +++
+ + + + .
+++ +++ +++ +++ +++ +++ +++ +++
+++ +++ +++ +++ +++ +++ +++ + .
+ + +
+ -
+
--
+
--
_
+÷+
--
÷
--
+
--
+
--
_
÷÷+
--
÷÷÷
÷
as in Table
.
.
1.
H o w e v e r , n o n e of s e v e r a l polyclonal a n t i s e r a a n d 110 m A b s r a i s e d a g a i n s t t h i s c o n j u g a t e could disc r i m i n a t e b e t w e e n CsA a n d m e t a b o l i t e 17 [AM1 (3)]. T h e fine-specificity of t h e s e m A b s w a s t h e n determined by cross-reactivity studies with more t h a n 60 C s A d e r i v a t i v e s modified a t t h e different r e s i d u e s a n d a l o n g t h e n i n e carbon-side c h a i n of r e s i d u e 1 (12). M o s t of t h e polyclonal a n t i b o d i e s a n d t h e m A b s o b t a i n e d recognized r e s i d u e s 1 to 6 (Table 1), a n d t h e y r e a c t e d only w i t h t h e first c a r b o n s of t h e r e s i d u e 1 side c h a i n (13). T h e s e r e s u l t s s u g g e s t e d t h a t w h e n CsA w a s dissolved in a q u e o u s buffer, t h e t e r m i n a l a t o m s of t h e r e s i d u e 1 side c h a i n w e r e not a v a i l a b l e for a n t i b o d y r e c o g n i t i o n a t t h e face of C s A opposite to r e s i d u e 8 (Figure 3). I t w a s t h e n h y p o t h e s i z e d t h a t t h e residue 1 side c h a i n could be folded b a c k a g a i n s t t h e r e s t of t h e molecule, as o b s e r v e d in crystals, r a t h e r than protruding. B a s e d on t h i s h y p o t h e s i s , a second series of a n t i bodies w a s r a i s e d u s i n g a c o n j u g a t e of CsC coupled t h r o u g h r e s i d u e T h r 2 , a r e s i d u e opposite to t h e l a s t c a r b o n of r e s i d u e 1 in t h e X - r a y c r y s t a l s t r u c t u r e . S e v e n out of 9 m A b s recognized m e t a b o l i t e 17 (AM1) 10 to 150-fold less well t h a n CsA (3). T h e finespecificity of two polyclonal a n t i b o d i e s a n d n i n e mAbs was determined by cross-reactivity studies w i t h C s A - d e r i v a t i v e s . M o s t a n t i b o d i e s recognized t h e l a s t c a r b o n s of t h e r e s i d u e 1 side c h a i n t o g e t h e r w i t h r e s i d u e s 6, 8 a n d 9 [Table 2 (12,13)]. T h e s e r e s u l t s i n d i c a t e d t h a t t h e l a s t c a r b o n of t h e r e s i d u e 1 side c h a i n w e r e close to r e s i d u e s 6, 8 a n d 9, as o b s e r v e d in t h e crystal, s u g g e s t i n g t h a t in a q u e o u s buffer, t h e side c h a i n of r e s i d u e 1 could be folded b a c k as o b s e r v e d in t h e c r y s t a l r a t h e r t h a n p r o t r u d i n g as s e e n in aprotic s o l v e n t s (13).
Criteria for antibody selection O n e m A b r e c o g n i z i n g m e t a b o l i t e 17 (AM1) 15040
.
fold less well t h a n CsA w a s selected a n d f u r t h e r t e s t e d for its c r o s s - r e a c t i v i t y w i t h o t h e r C s A m e t a b olites. T h i s m A b w a s s h o w n to d i s c r i m i n a t e 15 to 1000-fold b e t w e e n C s A a n d its m e t a b o l i t e s , h a v i n g less c r o s s - r e a c t i v i t y for t h o s e s e c o n d a r y m e t a b o l i t e s w i t h m u l t i p l e m o d i f i c a t i o n s (14). A second m A b specific for t h e r e g i o n of t h e C s A m o l e c u l e t h a t is not m e t a b o l i z e d w a s selected. T h i s m A b recognized a l m o s t e q u a l l y well CsA a n d m o s t of its m e t a b o lites, w i t h a s l i g h t d i s c r i m i n a t i o n for m e t a b o l i t e s modified on r e s i d u e 4. T h e s e two m A b s , r e f e r r e d to as t h e "specific" a n d t h e "nonspecific" m A b s for n a t i v e CsA, w e r e i n c o r p o r a t e d into r a d i o i m m u n o a s s a y s u s i n g t r i t i a t e d or i o d i n a t e d t r a c e r s . S e v e r a l studies, u n d e r t a k e n to a s s e s s t h e exclusive specificity of t h e "specific" m A b in clinical s a m p l e s , d e m o n s t r a t e d a h i g h correlation b e t w e e n the r e s u l t s o b t a i n e d u s i n g "specific" m A b - b a s e d r a d i o i m m u n o a s s a y (RIA) a n d h i g h - p e r f o r m a n c e liquid c h r o m a t o g r a p h y (HPLC) (15,16). I n a m u l t i c e n t e r s t u d y w h e r e t h e perform a n c e s of t h e m A b s a n d of t h e o r i g i n a l polyclonal a n t i s e r u m w e r e c o m p a r e d in RIA, m u c h l o w e r res u l t s w e r e o b t a i n e d w i t h t h e "specific" m A b comp a r e d to t h e polyclonal a n t i s e r u m (17). C o n v e r s e l y , t h e r e s u l t s o b t a i n e d w i t h t h e "nonspecific" m A b w e r e h i g h e r t h a n t h o s e o b t a i n e d w i t h t h e polyclonal a n t i s e r u m , as e x p e c t e d f r o m t h e i r c r o s s - r e a c t i v i t y patterns.
Clinical relevance T h e clinical r e l e v a n c e of m o n i t o r i n g p a r e n t CsA a l o n e or t o g e t h e r w i t h t h e m e t a b o l i t e s is still n o t c l e a r l y e s t a b l i s h e d . T h e c o r r e l a t i o n of clinical situa t i o n s w i t h blood levels of p a r e n t C s A on t h e one h a n d , a n d of m e t a b o l i t e s on t h e o t h e r h a n d , h a s b e e n r e s t r i c t e d so f a r to t h e few l a b o r a t o r i e s e q u i p p e d to p e r f o r m s o p h i s t i c a t e d H P L C for i n d i v i d u a l m e tabolites. R o s a n o et al. (5) s h o w e d t h a t t h e concenCLINICAL
BIOCHEMISTRY,
VOLUME
24, FEBRUARY
1991
MONOCLONAL ANTIBODIES TO CYCLOSPORINE
a
~
val-5
N-Me-Leu-6 b
Val-5
D-Ala-8
Abu N-Me-VaI-11. ~
Figure 3ab (continued).
trations determined using the "nonspecific" mAb accounted for the total of parent compound plus the most abundant primary metabolites 17 (AM1) and 1 (AM9) in samples from transplanted patients with good renal function or undergoing rejection. Immunoassays based on the "specific" and "nonspecific" mAbs run in parallel could therefore provide a convenient way to follow the rate of metabolism and allow accumulation of data on the clinical significance of CsA metabolite blood levels. References 1. Donatsch P, Abisch E, Homberger M, Traber R, Trapp M, Voges R. A radiojmmunoassay to measure cyclosporine A in plasma and serum samples. J Immunoassay 1981; 2: 19-32. 2. Robinson WT, Schran HF, Barry EP. Methods to measure cyclosporine l e v e l s - high pressure liquid chromatography, radioimmunoassay and correlation. CLINICALBIOCHEMISTRY,VOLUME24, FEBRUARY 1991
Transplant Proc 1983; 15 (Suppl. 1/2): 2403-8. 3. Quesniaux VFJ, Schmitter D, Schreier MH, Van Regenmortel MHV. Monoclonal antibodies to cyclosporine are representative ofthe major antibodypopulations present in antisera of immunized mice. Mol Irnmunol 1990; 27: 227-36. 4. Maurer G, Loosli HR, Schreier E, Keller B. Disposition of cyclosporine in several animal species and man. I. Structural elucidation of its metabolites. Drug Metab Dispos 1984; 12: 120-6. 5. Rosano TG, Pell MA, Freed BM, Dybas MT, Lempert N. Cyclosporine and metabolites in blood from renal allograft recipients with nephrotoxicity, rejection or good renal function: comparative high-performance liquid chromatography and monoclonal radioimmunoassay studies. Transplant Proc 1988; 20 (Suppl. 2): 330-8. 6. Wang CP, Burckart GJ, Ptachcinski RJ, et al. Cyclosporine metabolite concentrations in the blood of liver, heart, kidney and bone marrow transplant patients. Transplant Proc 1988; 20 (Suppl. 2): 591-6. 7. Ryffel B, Foxwell BMJ, Mihatsch MJ, Donatsch P,
41
QUESNIAUX C
Abu-2
Abu-2
d
( Figure 3cd. Figure 3--Identification of the 11 amino acid residues of CsA on space filling models of CS conformation according to X-ray crystallographic analysis (a,c) and to two-dimensional nuclear magnetic resonance in aprotic solvent (b,d). In (a) and (b), the CsA molecule is oriented as in Figure 2, whereas in (c) and (d) the opposite side of the CsA molecule is shown. Amino acid abbreviations as in Figure 2.
8.
9.
10.
11.
12.
42
Maurer G. Biologic significance of cyclosporine metabolites. Transplant Proc 1988; 20 (Suppl. 2): 57584. Quesniaux VFJ, Himmelspach K, Van Regenmortel MHV. An enzyme immunoassay for the screening of monoclonal antibodies to cyclosporine. Immunol Lett 1985; 9: 99-104. Petcher TJ, Weber HP, Riiegger A. Crystal and molecular structure of an iodo-derivative of the cyclic undecapeptide cyclosporine. Helv Chim Acta 1976; 59: 1480--8. Loosli HR, Kessler H, Oschkinat H, Weber HP, Petcher TJ, Widmer A. The conformation of 'cyclosporine A' in the crystal and in solution. Helv Chim Acta 1985; 68: 682-704. Kessler H, Loosli HR, Oschkinat H. Assignment of the 1H, 13C, and 15N-NMR spectra of cyclosporine A in CDC13 and C6D6 by a combination of homo- and heteronuclear two-dimensional techniques. Helv Chim Acta 1985; 68: 661-81. Quesniaux VFJ, Tees R, Schreier MH, Wenger RM, Van Regenmortel MHV. Fine specificity and cross-
reactivity of monoclonal antibodies to cyclosporine. Mol Immunol 1987; 24: 1159-68.
13. Quesniaux VFJ, Wenger RM, Schmitter D, Van Regenmortel MVH. Study of the conformation of cyclosporine in aqueous medium by means of monoclonal antibodies. Int J Pept Protein Res 1988; 31: 173-85. 14. Quesniaux V, Tees R, Schreier MH, Maurer G, Van Regenmortel MHV. Potential of monoclonal antibodies to improve therapeutic monitoring of cyclosporine. Clin Chem 1987; 33: 32-7. 15. Schran HF, Rosario TG, Hassell AE, Pell MA. Determination of cyclosporine concentrations with monoclonal antibodies. Clin Chem 1987; 33: 2225-9. 16. Ball PE, Munzer H, Keller HP, Abisch E, Rosenthaler J. Specific 3H radioimmunoassay with a monoclonal antibody for monitoring cyclosporine in blood. Clin Chem 1988; 34: 257-60. 17. Holt DW, Johnston A, Marsden JT, et al. Monoclonal antibodies for radioimmunoassay of cyclosporine: a multicenter comparison of their performance with the Sandoz polyclonal radioimmunoassay kit. Clin Chem 1988; 34: 1091-6. CLINICAL BIOCHEMISTRY, VOLUME 24, FEBRUARY 1991
