ANALYTICAL
202,%-39
BIOCHEMISTRY
(19%)
Detection of Catalytic Dan S. Tawfik,*vt
Monoclonal
Antibodies’
Bernard S. Green,? and Zelig Eshhar*
*Department of Chemical Immunology, Weizmunn Institute of Science, Rehovot 76100, Israel; and TDepartment of Pharmaceutical Chemistry, Faculty of Medicine, Hebrew University, P.O. Box 12065, Jerusalem 91120, Israel
Received
September
19,199l
Several laboratories have now shown that monoclonal antibodies having enzyme-like properties can be generated. The generation of catalytic antibodies makes use of the same basic procedures that have been used for the generation of binding monoclonal antibodies, yet the process involves an additional crucial step: screening for catalytic activity. In this paper we address the unique problems involved in the detection of inefficient catalytic activity that is accompanied by uncatalyzed background reaction. An analysis that allows optimization of assay conditions and estimation of the minimal antibody concentration required to observe catalysis is presented. The results indicate that the structure of the substrate should be optimized to increase its affinity (i.e., decrease its K,) and reduce its concentration to pseudo-first-order conditions (S, -6 Km) so that the signal observed in the presence of a catalytic antibody (AP,,) is significantly higher than that of the background (A Pwt ). Other factors involved in the screening procedures, e.g., sensitivity of the assay, solubility and reactivity of the substrate, and purity of the antibody preparation, are also discussed. The effect of these assay parameters on the ability to detect catalytic activity is demonstrated with pnitrophenyl ester-hydrolyzing antibodies. o 1992 Academic P~MS, IDC.
During the past few years monoclonal antibodies (MABs)~ having enzyme-like properties have been successfully raised and a variety of reactions have been cat1 This research was supported in part by AID-CDR Grant DPE5544-G-SS-6020-00 and by U.S. Army Medical Research Grant DAMD 17-99-Z-0010. 2 Abbreviations used: MAB, monoclonal antibody; catMAB, catalytic monoclonal antibody; KLH, keyhole limpet hemocyanin; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; CIEIA, competitive inhibition immunoassay; TSA, transition state analog; TBS, tris(hydroxymethyl)aminoethane-buffered saline; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Ab, antibody; S, substrate; P, product. ooo3-2697/92
Copyright All rights
35
$3.00
0 1992 by Academic of reproduction
alyzed with considerable rate enhancement, turnover, and substrate specificity (1). Today, the basic procedures for generating catMABs do not differ from those used for conventional binding antibodies, although differences may develop in the future. Two unique points are crucial for the successful production of catMABs: (i) the design of the hapten and (ii) the process of selecting those few MABs that are catalytic from among all the antibodies that bind the hapten but do not catalyze the reaction. Catalytic monoclonal antibodies are currently raised by immunization with a hapten-protein carrier conjugate and subsequent fusion; the resulting hybridoma cells are then screened in order to select those antibodies exhibiting binding affinity for the hapten. (An alternative method for selecting potential catMAB-secreting clones on the basis of their affinity to a “short transition state analog” has also been used (2)). These clones are then propagated in large quantities (generally in ascites fluid) and purified to allow their screening for catalytic activity. Relative to enzymes, antibody-mediated catalysis is still very modest; the largest turnover number, keat, reported for a catMAB is 0.3 min-’ (3), while hczz,, values as high as lo6 min-’ are not uncommon for enzymes (4). In addition, the nature of most of the reported antibodycatalyzed reactions is such that they proceed with measurable rates even in the absence of any catalyst. The catalytic power of antibodies is therefore generally described in terms of k,,,lku,,, (n), i.e., the rate enhancement of the reaction, rather than the absolute rate, k,, commonly used to describe enzymatic activity. Rate enhancements (n) by catMABs are generally in the range of 102-105, which is still lower than those by enzymes by orders of magnitude. The detection of catalysis by antibodies therefore presents unique difficulties that are addressed in this paper. The most important factors involved in being able to detect antibody-mediated catalytic activity are: (i) the concentration and purity of the MAB in the preparation used for the assay and (ii) the choice (structure) and
in any
Press, Inc. form
reserved.
TAWFIK,
51P ! P
0-y-(W&-C
- NH(CH&
GREEN,
SSAKLH
OH
0
0-;-(CH&C-NH(CH&COOH
‘W
OH
0
0
0-!-(C”,&N”(CH,)&OOH---+
OH
B tl HO-C-(CH2)3C-NH(CHz)5-COOH
+
O-i-C,, 5
P
0-C-(CH,)&OH
B
6
FIG. 1. Structure of hapten (l), inhibitors (4-6) used and the catalyzed reaction.
(2, 3), and substrates
concentration of the substrate. In this study we present data showing that detection of antibody-mediated catalysis requires not only relatively high concentrations of purified MABs but also the use of an appropriately designed substrate. Unlike enzymes, catalytic antibodies are tailormade catalysts. The structure of the substrate must obviously be related to the structure of the hapten used and the reaction that is to be catalyzed by the resulting antibodies; yet, various possibilities for the design of the substrate always exist. Other important points, e.g., sensitivity of the assay to be used and solubility and reactivity of the substrate, pH, and buffer, are also briefly discussed. This paper thus addresses most of the aspects involved in detecting catalytic antibodies. MATERIALS
AND
METHODS
Mice were immunized with the KLH conjugate of a p-nitrophenyl phosphonate hapten (1) (Fig. 1). After fusion, the resulting hybridomas were first screened by ELISA for binding to the hapten BSA conjugate 1. Potential catalytic clones were then selected using a competitive inhibition assay (CIEIA) on the basis of their affinity for the “short” TSA 3. Selected clones were propagated as ascites; the MABs were then purified by protein A affinity chromatography and dialyzed against 30 mM TBS, pH 8. The purified antibodies were screened for catalytic activity by measuring the rate of hydrolysis of ester 4 (Fig. 1). All experimental procedures used were as previously described (2). Protein concentration of the purified antibodies was determined by measuring optical density at 280 nm.
AND ESHHAR
Two clones, CNJ 157 and CNJ 206, having, respectively, the highest and the lowest catalytic activity within a set of anti- 1 catMABs, were used in this study. MAB homogeneity was judged by SDS-PAGE, which yielded only heavy and light chains under reducing conditions, using Coomassie blue staining. As previously described (2), the following were used to ensure that catalysis occurs at the antibody binding site (and is not due to minor enzyme impurities in the antibody preparation): substrate specificity, stoichiometric inhibition of catalytic activity by the hapten inhibitor (2), and correlation of the binding specificity of the MABs (as determined by CIEIA) with inhibition of catalytic activity, using the same series of inhibitors. Reaction rates were determined by measuring the release of p-nitrophenol (absorption at 405 nm) using a Dynatech MR-5000 microplate reader. Substrates, pnitrophenyl esters 4 and 6, as acetonitrile 40-100 mM stock solutions, were diluted with TBS and added to antibody solutions (the final organic solvent concentrations were ~1%). Product formation, AP,, and APuncat, was measured as AOD,,,, for the first 3 min of the reaction in the presence of the catalytic antibody and a hapten-binding, noncatalytic antibody (CNJ 251). The signal ratio A P,,IA P,,,,, was calculated from these data. The kct,, and K,,, values were determined using a Lineweaver-Burk analysis. The observed rates (v,, ,t) were corrected for the uncatalyzed rate of hydrolysis in buffer in the presence of a hapten-binding, noncatalytic antibody hoObserved). uncat The uncatalyzed rate (kcat) was determined in the absence of any antibody using initial rates analysis and extrapolated to zero buffer concentration. RESULTS
A theoretical analysis was performed in order to develop a model for estimating the minimal antibody concentrations required to detect catalytic activity and for analyzing the factors, other than rate enhancement (kc,,/&,,) by the antibody, that determine the ability to detect even inefficient catalysts. The analysis is made for a first-order reaction: substrate, S + product, P. Catalytic antibodies, like enzymes, catalyze reactions via the formation of an enzyme-substrate complex and therefore obey the classical Michaelis-Menten kinetics (Eq. [l]). The initial velocity of the catalyzed reaction (v,, cat) is therefore Ah “ocat
=
- kc,
- So
So + Km
(where Abe is the initial MAB concentration, kat is the catalyzed first-order rate constant, So is the initial substrate concentration, and Km is the Michaelis constant). The initial velocitv of the uncatalyzed reaction, i.e.,
DETECTION
OF
CATALYTIC
37
ANTIBODIES
the initial velocity observed under the same conditions in the absence of a catalytic antibody,3 is simply
PI
vouncat= klcat * so.
The ratio of the catalyzed (Eq. [l]) and uncatalyzed (Eq. [2]) initial velocities is equal (if measured in the same time period, At) to the ratio of the products released by these reactions (AP): -=uocat Abo(L/kd vouncat So + km
= Abe - n =- APat So + km APuneat
L31
APcat 6 O.l.S,. A Pcatl A Puncat is the signal ratio pa, i.e., the ratio of products released under the same conditions, by the reaction catalyzed by a catMAB, and in the presence of a noncatalytic antibody. Choosing a low signal ratio, PR, will result in decreasing selectivity; that is, the number of “false positives” will be largely increased. Rearranging Eq. [3] and substituting the assay parameters pL,and pRgives -n a
Ab,.n
(AP,,tIAP,,,,t)Wo
3 &&,
+ 10.~~).
+ K3
5
10 WI
(where n represents the catalytic power and is equal to k&mcat) The values of both AP,, and So are dictated by various parameters of the assay used to detect the catalytic activity. APcat is limited by the minimal detectable amount of product, i.e., by the assay sensitivity ps (ps < AP,,). The initial substrate concentration to be used, So, must exceed, by at least 10 times, the concentration of the product’:
Abe
0
Wal Ml
Ab, * n (which is equal to Ab,(lz,,l&,,)) represents the relative enzyme units, i.e., the overall measured activity relative to uncatalyzed or background reaction (enzyme units, or v,, is the overall activity in absolute 3 k,,-* is the first-order rate constant, or pseudo-first-order rate constant in cases where other species having a constant concentration are also involved in the reaction (for example, hydroxyl anion or water). In many cases the reaction rate is enhanced by the buffer or by the presence of adventitious protein; the observed rate (v:~-?) in these cases is higher. For the purpose of detection of catalytic activity, V= and therefore n“b”“d and k”b”“’ are more relevant. The theoretical antibody catalytic power, R, is usually calculated by using &a*, which is determined in the absence of any antibody and extrapolated to zero buffer concentration (see Materials and Methods) and is therefore higher than nobti. For the hydrolysis of PNP ester 4, is two- to threefold larger than Lt. L*oh& ’ For the analysis of this model, this limitation is required to maintain initial velocity conditions (5). Practically, as demonstrated later, it also results from obvious need that S,, > Ab, .
15
( PM)
FIG. 2.
AP,,IAP,,,-t determined for esters 4 (open symbols) and 5 (solid symbols) in the presence of various concentrations of purified CNJ157 (Cl, W) and 206 (0,O) and under the same conditions in the presence of a noncatalytic MAB (AP--*). Final substrate concentration (S,,) was 0.1 mM.
terms and is equal to E,. kcator Ab, . kat for a catMAB). For a certain value of Ab, * n, varying combinations of antibody concentrations (Ab,) and catalytic power (n) can be ascribed. Ab, . n enable us to estimate the minimal MAB concentration required for the detection of antibodies exhibiting a given catalytic activity. If even weak catalytic activity (i.e., low n) is to be observed, higher MAB concentration must be used, and vice versa; using a lower MAB concentration would enable the detection of only relatively highly active antibodies (i.e., having high n). The value of Ab, n as a function of the assay parameters is determined by Eq. [4b]. Using a more sensitive assay (i.e., a smaller value ps), choosing a lower signal ratio (,.&a),and the availability of a substrate with higher affinity (i.e., lower K,,,) would all result in lowering Abe * n and thereby enable the detection of less powerful antibodies while using the same MAB concentration. The effects of antibody concentration, substrate affinity (Fig. 2), and concentration (Fig. 3) are demonstrated with the p-nitrophenyl ester-hydrolyzing antibodies CNJ 157 and CNJ 206 and substrates 4 and 5. The kinetic parameters for these MABs are given in Table 1. The experimental results, as well as the model, indicate that the minimal antibody concentration required for detection of catalytic activity is primarily affected by the catalytic power n. Using ester 4 as a substrate and a minimal signal ratio (PR = AP&AP,,,) of 10, a minimal concentration of 2.3 pM (ca. 0.35 mg/ml) is needed for CNJ 206 (n = 1600) whereas only ca. 0.35 PM (0.05 mg/ml) is needed for the more powerful CNJ 157 (n = 9700) (Fig. 2). Ab, . n calculated for this system by Eq. [4] (using the values PR = 10; Km = 0.1 mM, and cc, = 5 X lo-’ M) is 1.5 X 10V3 M. Thus, the minimal antibody concentration that can be used to detect moderately active antibodies (e.g., CNJ157 with n = 104) should be 0.15 pM. Yet, under assay conditions, nobserved, l
38
TAWFIK,
o! 0.0
GREEN,
1 0.2
0.4
So
0.8
0.8
1.0
(mM)
FIG. 3.
AP,,fAP,,, determined for CNJ157 (5.5 PM) in the presence of various concentrations of ester 4; APwt was measured in the presence of a noncatalytic MAB under the same conditions.
which is smaller than the theoretical n3, is more relevant, yielding Ab, of ca. 0.4 pM. For the less active CNJ 206 (n = 1600), the calculated minimal Ab, is 2.5 pM. The experimental results (Fig. 2) indicate that the observed Ab,, for these antibodies are in accordance with the calculated values for this system. As derived from Eq. [4a], Ab, . n is dependent upon substrate affinity. A substrate with higher affinity, i.e., ester 4 (K, = 0.11 mM), significantly increases detection ability. When a “shorter” substrate having lower affinity is used, i.e., ester 5 (K, values of 1.07 and 0.90 mM for CNJ 157 and CNJ 206, respectively), the activity of the less efficient antibody (CNJ 206, n = 1600) is noticeable only at high MAB concentration (AP,,I = 10 at 15 PM antibody concentration; Fig. 2). @mat As predicted (Eq. [4a]), increasing the substrate concentration results in a lowering of the measured signal (A P,,IA P,,n,t) (Fig. 3). At relatively high substrate concentrations (>0.5 InM) the observed decrease in signal ratio is higher than that predicted (Eq. [3]) due to the inactivation of these particular catMABs by the substrate that becomes significant at S, 9 K,. Inactivation is due to acylation of the antibody active site by the substrate as the result of nonproductive binding. The lability of p-nitrophenyl esters is the main cause; yet, substrate-induced inactivation or nonproductive binding are not unexpected in many other antibody-catalyzed reactions.
AND
ESHHAR
concentrations required to detect any authentic catalytic activity. As expected, rather high concentrations of purified antibody (0.5-15 PM; ca.O.l-2.0 mg/ml) are required (Fig. 2). Unlike the catalytic power of the detected antibodies (n), other parameters of the assay can be predetermined. The effects of substrate affinity (K,) and concentration (S,), which are described in Eq. [4], are clearly observed (Figs. 2 and 3). These data emphasize that it is practically impossible to detect MABs exhibiting modest catalytic activity under nonoptimal conditions. In order to increase the ability to detect less powerful catalytic antibodies, one should design a substrate with the maximal affinity, i.e., having the lowest K,. It is well established that the binding affinity of antibodies to a substance is generally increased when its structure not only mimics the hapten structure, but also the linker or a even a portion of the protein carrier structure (6). In this case, the c-amino caproate moiety in ester 4, which mimics the carrier protein lysine residue portion of the hapten conjugate structure, was necessary to provide a substrate with sufficient affinity. In some cases of nonhydrolytic reactions, for example, bimolecular reactions, cyclization reactions, or Diels-Alder reactions, where the product resembles the hapten, product inhibition should be considered. Yet, it appears that sufficient affinity of the substrate is also required in these cases (7). When a substrate is designed to be used to detect antibody-mediated catalytic activity other factors should be considered as well: (i) sol&i&-hydrophilic or ionic groups, such as carboxyl in this case, contribute to substrate solubility (ester 4 is soluble in buffer up to 20 mM); insufficient solubility of the substrate requires the addition of relatively high concentrations of miscible organic solvents, which causes a decrease in reactivity (data not shown). (ii) Stability-some structures are unfavorable due to intrinsic features of the molecule that result in high uncatalyzed rates of reaction. When ester 6, having a L-t value ca. loo-fold higher than that of ester 4, was used as a substrate, even the more active antibody (CNJ 157) could not be detected (A P,,lA P,,., = 1.0 at 10.5 pM antibody concentration). In this case,
TABLE DISCUSSION
The model presented provides a simple tool for the optimization of any assay designed for the detection of catalytic activity that is accompanied by a measurable uncatalyzed, background reaction. This analysis is valid not only for the detection of catalytic antibodies but also for any enzyme or other catalyzed reaction that has the same character. The relative enzyme units Ab,, * n (or E, * n) allow us to evaluate the antibody, or enzyme,
Kinetic
Parameters
MAB
K’, (-0
K”, bM)
157 206
0.11 0.11
1.07 0.90
D The If, values for esters (see Materials and Methods). ’ kat was determined using
1
and
of CNJ157
4 and ester
k (min”‘l)
b
2.39 0.41 5 were
determined
4 as substrate.
206”
n 0.97 x 10’ 0.16 X 10’ as indicated
DETECTION
OF
CATALYTIC
increased reactivity of 6 is due to intramolecular catalysis by the carboxyl group (8). As noted, the lowest possible substrate concentration should be used in the detection assay. The effect of substrate concentration (S,) on the signal ratio (AP,,I Eq. [4a], Fig. 3) is the result of the different A puncat ; kinetics of the catalyzed and uncatalyzed reactions. While the uncatalyzed reaction rate is proportional to the substrate concentration [2], the catalyzed reaction [l] exhibits saturation kinetics; i.e., at higher substrate concentrations (S, % K,) the rate becomes independent of substrate concentration ( v0 = v,&. The minimal substrate concentration (S,) is primarily dictated by the sensitivity of the assay (cc, < AP,,, AF,, < 0.1. S,), but also by the antibody concentrationP Other parameters such as buffer and pH should be considered as well. In hydrolytic reactions the pH may greatly affect both kc, and k,,ncat and thereby the ability to detect catalytic activity. Optimal pH for detection should be selected so that the resulting rate of reaction For the system prek, = kncat - n) is measurable. sented, pH 7.5-8.3 was found to be suitable. The ability to detect catalytic activity, i.e., the value of Ab, * n, is also dependent on the sensitivity of the assay [4b]. Various detection methods that are routinely used to assay enzymatic activity were employed for catalytic antibodies, e.g., HPLC, optical absorbance, and fluorescence. The sensitivity, i.e., the minimal detectable amount of product, differs for each method. Optical absorbance measurements, direct or after HPLC separation, are generally in the micromolar range. Yet, other methods, e.g., fluorescence, are more sensitive and allow the detection of nanomolar or even lower concentrations. Is the use of highly sensitivity assay always advantageous? The K,‘s of the more efficient MABs, as is the case for enzymes, are in the millimolar range (1~). The model predicts that, under typical assay conditions, i.e., signal ratio pLR= 10 and K, = 0.1 mM, Ab, . n is 1.1 X 10T3 and 1.0 X lop3 for assay sensitivity of 1 FM and 1 IIM, respectively [4b]. Thus, increasing the assay sensitivity by three orders of magnitude results in an insignificant change in the ability to detect catalytic activity. On the other hand, if the same catMABs are screened using a substrate with higher affinity (e.g., Km = 0.01 mM), then the gain in sensitivity (i.e., pus = 1 nM) is more effective (Ab, * n = 0.1 X 10p3). An interesting conclusion derived from this model is, therefore, that using highly sensitive assay techniques does not directly or linearly increase the ability to detect catalytic antibodies that are present in low concentrations. Finally, it is critical to ensure that the detected catalytic activity occurs at the antibody binding site and is not due to minor quantities of highly active enzyme impurities (Id, 2). The purity of the antibody preparation is equally important when screening is performed using a high concentration of antibodies produced from
39
ANTIBODIES
ascites or growth media. The use of protein A or G affinity columns, as was done with CNJ157 and CNJ206, is effective, yet solid evidence that catalysis occurs at the antibody binding site must be provided; see Materials and Methods and (2). Ideally, it would be desirable to directly screen hybridoma supernatants for catalytic activity rather than select those clones that bind the hapten, produce large quantities of purified antibodies, and then assay for catalytic activity. Direct screening procedures are unavoidable if the new, recombinant DNA-based, nonhybridoma methodologies for the production of antibodies are used (9). It is clear from the data presented that, regardless of the sensitivity of the assay used, the antibody concentrations needed to detect moderate or even highly active catalytic antibodies (n > 104) are higher than those generally present in hybridoma supernatants under normal growth conditions (1-5 pg/ml). For many reactions the observed kuncat in hybridoma supernatants is much higher than that in buffer due to the presence of various enzymes that catalyze the same reaction. The detection of catalytic antibodies present in low concentrations and in the presence of enzyme contaminants requires the development of novel techniques that are not only highly sensitive but very selective as well; i.e., the product must be detected in the presence of a large excess of unreacted substrate. The analysis presented in this paper may also be helpful for evaluating techniques that will allow direct screening of antibodymediated catalytic activity. ACKNOWLEDGMENTS We thank Ms. Romy Zemel and Ms. Rachel Chap for their assistance in the production of the MABs. We are also grateful to Professor Jacob Bar-Tanah for reviewing the manuscript.
REFERENCES 1. (a) Lerner, R. A., Benkovic, S. J., and Schultz, P. G. (1991) Science 262, 659-667; (b) (1991) Catalytic Antibodies, Ciba Foundation Symposium No. 159 Wiley, New York; (c) Green, B. S., and Tawfik, D. S. (1989) Trends Bioteehnol. 7, 304-310; (d) Schultz, P. G. (1989) Angew. Chem. Znt. Ed. Engl. 28,1285-1295. 2. Tawfik, D. S., Zemel, R. R., Arad-Yellin, R., Green, B. S., and Eshhar, Z. (1990) Biochemistry 29,9916-9921. 3. Tramontano, A., Ammann, A. A., and Lerner, R. A. (1988) J. Am. Chem. Sot. 110,2282-2286. 4. Fersht, A. (1985) Enzyme Structure and Mechanism, 2nd ed., Freeman, New York. 5. I. H. Segel 6. Pressman, Antibody
(1975)
Enzyme
Kinetics,
D., and Grossberg, Specificity, Benjamin,
Wiley,
A. (1968) The New York.
New
York.
Structural
7. Tramantano, A., and Schloeder, D. (1989) in Methods mology (Langone, J. J., Ed.), Vol. 178, pp. 531-550, Press, San Diego.
Basis
of
in EnzyAcademic
8. Kirby, A. J. (1980) Adv. Phys. Org. Chem. 17,183-277. 9. Winter, G., and Milstein, C. (1991) Nature 349, 293-299; Huse, W. D., Sastry, L., Iverson, S. A., Kang, A. S., Alting-Mees, M., Burton, D. R., Benkovic, S. J., and Lerner, R. A. (1989) Science, 246,1275-1281.
202,%-39
BIOCHEMISTRY
(19%)
Detection of Catalytic Dan S. Tawfik,*vt
Monoclonal
Antibodies’
Bernard S. Green,? and Zelig Eshhar*
*Department of Chemical Immunology, Weizmunn Institute of Science, Rehovot 76100, Israel; and TDepartment of Pharmaceutical Chemistry, Faculty of Medicine, Hebrew University, P.O. Box 12065, Jerusalem 91120, Israel
Received
September
19,199l
Several laboratories have now shown that monoclonal antibodies having enzyme-like properties can be generated. The generation of catalytic antibodies makes use of the same basic procedures that have been used for the generation of binding monoclonal antibodies, yet the process involves an additional crucial step: screening for catalytic activity. In this paper we address the unique problems involved in the detection of inefficient catalytic activity that is accompanied by uncatalyzed background reaction. An analysis that allows optimization of assay conditions and estimation of the minimal antibody concentration required to observe catalysis is presented. The results indicate that the structure of the substrate should be optimized to increase its affinity (i.e., decrease its K,) and reduce its concentration to pseudo-first-order conditions (S, -6 Km) so that the signal observed in the presence of a catalytic antibody (AP,,) is significantly higher than that of the background (A Pwt ). Other factors involved in the screening procedures, e.g., sensitivity of the assay, solubility and reactivity of the substrate, and purity of the antibody preparation, are also discussed. The effect of these assay parameters on the ability to detect catalytic activity is demonstrated with pnitrophenyl ester-hydrolyzing antibodies. o 1992 Academic P~MS, IDC.
During the past few years monoclonal antibodies (MABs)~ having enzyme-like properties have been successfully raised and a variety of reactions have been cat1 This research was supported in part by AID-CDR Grant DPE5544-G-SS-6020-00 and by U.S. Army Medical Research Grant DAMD 17-99-Z-0010. 2 Abbreviations used: MAB, monoclonal antibody; catMAB, catalytic monoclonal antibody; KLH, keyhole limpet hemocyanin; BSA, bovine serum albumin; ELISA, enzyme-linked immunosorbent assay; CIEIA, competitive inhibition immunoassay; TSA, transition state analog; TBS, tris(hydroxymethyl)aminoethane-buffered saline; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Ab, antibody; S, substrate; P, product. ooo3-2697/92
Copyright All rights
35
$3.00
0 1992 by Academic of reproduction
alyzed with considerable rate enhancement, turnover, and substrate specificity (1). Today, the basic procedures for generating catMABs do not differ from those used for conventional binding antibodies, although differences may develop in the future. Two unique points are crucial for the successful production of catMABs: (i) the design of the hapten and (ii) the process of selecting those few MABs that are catalytic from among all the antibodies that bind the hapten but do not catalyze the reaction. Catalytic monoclonal antibodies are currently raised by immunization with a hapten-protein carrier conjugate and subsequent fusion; the resulting hybridoma cells are then screened in order to select those antibodies exhibiting binding affinity for the hapten. (An alternative method for selecting potential catMAB-secreting clones on the basis of their affinity to a “short transition state analog” has also been used (2)). These clones are then propagated in large quantities (generally in ascites fluid) and purified to allow their screening for catalytic activity. Relative to enzymes, antibody-mediated catalysis is still very modest; the largest turnover number, keat, reported for a catMAB is 0.3 min-’ (3), while hczz,, values as high as lo6 min-’ are not uncommon for enzymes (4). In addition, the nature of most of the reported antibodycatalyzed reactions is such that they proceed with measurable rates even in the absence of any catalyst. The catalytic power of antibodies is therefore generally described in terms of k,,,lku,,, (n), i.e., the rate enhancement of the reaction, rather than the absolute rate, k,, commonly used to describe enzymatic activity. Rate enhancements (n) by catMABs are generally in the range of 102-105, which is still lower than those by enzymes by orders of magnitude. The detection of catalysis by antibodies therefore presents unique difficulties that are addressed in this paper. The most important factors involved in being able to detect antibody-mediated catalytic activity are: (i) the concentration and purity of the MAB in the preparation used for the assay and (ii) the choice (structure) and
in any
Press, Inc. form
reserved.
TAWFIK,
51P ! P
0-y-(W&-C
- NH(CH&
GREEN,
SSAKLH
OH
0
0-;-(CH&C-NH(CH&COOH
‘W
OH
0
0
0-!-(C”,&N”(CH,)&OOH---+
OH
B tl HO-C-(CH2)3C-NH(CHz)5-COOH
+
O-i-C,, 5
P
0-C-(CH,)&OH
B
6
FIG. 1. Structure of hapten (l), inhibitors (4-6) used and the catalyzed reaction.
(2, 3), and substrates
concentration of the substrate. In this study we present data showing that detection of antibody-mediated catalysis requires not only relatively high concentrations of purified MABs but also the use of an appropriately designed substrate. Unlike enzymes, catalytic antibodies are tailormade catalysts. The structure of the substrate must obviously be related to the structure of the hapten used and the reaction that is to be catalyzed by the resulting antibodies; yet, various possibilities for the design of the substrate always exist. Other important points, e.g., sensitivity of the assay to be used and solubility and reactivity of the substrate, pH, and buffer, are also briefly discussed. This paper thus addresses most of the aspects involved in detecting catalytic antibodies. MATERIALS
AND
METHODS
Mice were immunized with the KLH conjugate of a p-nitrophenyl phosphonate hapten (1) (Fig. 1). After fusion, the resulting hybridomas were first screened by ELISA for binding to the hapten BSA conjugate 1. Potential catalytic clones were then selected using a competitive inhibition assay (CIEIA) on the basis of their affinity for the “short” TSA 3. Selected clones were propagated as ascites; the MABs were then purified by protein A affinity chromatography and dialyzed against 30 mM TBS, pH 8. The purified antibodies were screened for catalytic activity by measuring the rate of hydrolysis of ester 4 (Fig. 1). All experimental procedures used were as previously described (2). Protein concentration of the purified antibodies was determined by measuring optical density at 280 nm.
AND ESHHAR
Two clones, CNJ 157 and CNJ 206, having, respectively, the highest and the lowest catalytic activity within a set of anti- 1 catMABs, were used in this study. MAB homogeneity was judged by SDS-PAGE, which yielded only heavy and light chains under reducing conditions, using Coomassie blue staining. As previously described (2), the following were used to ensure that catalysis occurs at the antibody binding site (and is not due to minor enzyme impurities in the antibody preparation): substrate specificity, stoichiometric inhibition of catalytic activity by the hapten inhibitor (2), and correlation of the binding specificity of the MABs (as determined by CIEIA) with inhibition of catalytic activity, using the same series of inhibitors. Reaction rates were determined by measuring the release of p-nitrophenol (absorption at 405 nm) using a Dynatech MR-5000 microplate reader. Substrates, pnitrophenyl esters 4 and 6, as acetonitrile 40-100 mM stock solutions, were diluted with TBS and added to antibody solutions (the final organic solvent concentrations were ~1%). Product formation, AP,, and APuncat, was measured as AOD,,,, for the first 3 min of the reaction in the presence of the catalytic antibody and a hapten-binding, noncatalytic antibody (CNJ 251). The signal ratio A P,,IA P,,,,, was calculated from these data. The kct,, and K,,, values were determined using a Lineweaver-Burk analysis. The observed rates (v,, ,t) were corrected for the uncatalyzed rate of hydrolysis in buffer in the presence of a hapten-binding, noncatalytic antibody hoObserved). uncat The uncatalyzed rate (kcat) was determined in the absence of any antibody using initial rates analysis and extrapolated to zero buffer concentration. RESULTS
A theoretical analysis was performed in order to develop a model for estimating the minimal antibody concentrations required to detect catalytic activity and for analyzing the factors, other than rate enhancement (kc,,/&,,) by the antibody, that determine the ability to detect even inefficient catalysts. The analysis is made for a first-order reaction: substrate, S + product, P. Catalytic antibodies, like enzymes, catalyze reactions via the formation of an enzyme-substrate complex and therefore obey the classical Michaelis-Menten kinetics (Eq. [l]). The initial velocity of the catalyzed reaction (v,, cat) is therefore Ah “ocat
=
- kc,
- So
So + Km
(where Abe is the initial MAB concentration, kat is the catalyzed first-order rate constant, So is the initial substrate concentration, and Km is the Michaelis constant). The initial velocitv of the uncatalyzed reaction, i.e.,
DETECTION
OF
CATALYTIC
37
ANTIBODIES
the initial velocity observed under the same conditions in the absence of a catalytic antibody,3 is simply
PI
vouncat= klcat * so.
The ratio of the catalyzed (Eq. [l]) and uncatalyzed (Eq. [2]) initial velocities is equal (if measured in the same time period, At) to the ratio of the products released by these reactions (AP): -=uocat Abo(L/kd vouncat So + km
= Abe - n =- APat So + km APuneat
L31
APcat 6 O.l.S,. A Pcatl A Puncat is the signal ratio pa, i.e., the ratio of products released under the same conditions, by the reaction catalyzed by a catMAB, and in the presence of a noncatalytic antibody. Choosing a low signal ratio, PR, will result in decreasing selectivity; that is, the number of “false positives” will be largely increased. Rearranging Eq. [3] and substituting the assay parameters pL,and pRgives -n a
Ab,.n
(AP,,tIAP,,,,t)Wo
3 &&,
+ 10.~~).
+ K3
5
10 WI
(where n represents the catalytic power and is equal to k&mcat) The values of both AP,, and So are dictated by various parameters of the assay used to detect the catalytic activity. APcat is limited by the minimal detectable amount of product, i.e., by the assay sensitivity ps (ps < AP,,). The initial substrate concentration to be used, So, must exceed, by at least 10 times, the concentration of the product’:
Abe
0
Wal Ml
Ab, * n (which is equal to Ab,(lz,,l&,,)) represents the relative enzyme units, i.e., the overall measured activity relative to uncatalyzed or background reaction (enzyme units, or v,, is the overall activity in absolute 3 k,,-* is the first-order rate constant, or pseudo-first-order rate constant in cases where other species having a constant concentration are also involved in the reaction (for example, hydroxyl anion or water). In many cases the reaction rate is enhanced by the buffer or by the presence of adventitious protein; the observed rate (v:~-?) in these cases is higher. For the purpose of detection of catalytic activity, V= and therefore n“b”“d and k”b”“’ are more relevant. The theoretical antibody catalytic power, R, is usually calculated by using &a*, which is determined in the absence of any antibody and extrapolated to zero buffer concentration (see Materials and Methods) and is therefore higher than nobti. For the hydrolysis of PNP ester 4, is two- to threefold larger than Lt. L*oh& ’ For the analysis of this model, this limitation is required to maintain initial velocity conditions (5). Practically, as demonstrated later, it also results from obvious need that S,, > Ab, .
15
( PM)
FIG. 2.
AP,,IAP,,,-t determined for esters 4 (open symbols) and 5 (solid symbols) in the presence of various concentrations of purified CNJ157 (Cl, W) and 206 (0,O) and under the same conditions in the presence of a noncatalytic MAB (AP--*). Final substrate concentration (S,,) was 0.1 mM.
terms and is equal to E,. kcator Ab, . kat for a catMAB). For a certain value of Ab, * n, varying combinations of antibody concentrations (Ab,) and catalytic power (n) can be ascribed. Ab, . n enable us to estimate the minimal MAB concentration required for the detection of antibodies exhibiting a given catalytic activity. If even weak catalytic activity (i.e., low n) is to be observed, higher MAB concentration must be used, and vice versa; using a lower MAB concentration would enable the detection of only relatively highly active antibodies (i.e., having high n). The value of Ab, n as a function of the assay parameters is determined by Eq. [4b]. Using a more sensitive assay (i.e., a smaller value ps), choosing a lower signal ratio (,.&a),and the availability of a substrate with higher affinity (i.e., lower K,,,) would all result in lowering Abe * n and thereby enable the detection of less powerful antibodies while using the same MAB concentration. The effects of antibody concentration, substrate affinity (Fig. 2), and concentration (Fig. 3) are demonstrated with the p-nitrophenyl ester-hydrolyzing antibodies CNJ 157 and CNJ 206 and substrates 4 and 5. The kinetic parameters for these MABs are given in Table 1. The experimental results, as well as the model, indicate that the minimal antibody concentration required for detection of catalytic activity is primarily affected by the catalytic power n. Using ester 4 as a substrate and a minimal signal ratio (PR = AP&AP,,,) of 10, a minimal concentration of 2.3 pM (ca. 0.35 mg/ml) is needed for CNJ 206 (n = 1600) whereas only ca. 0.35 PM (0.05 mg/ml) is needed for the more powerful CNJ 157 (n = 9700) (Fig. 2). Ab, . n calculated for this system by Eq. [4] (using the values PR = 10; Km = 0.1 mM, and cc, = 5 X lo-’ M) is 1.5 X 10V3 M. Thus, the minimal antibody concentration that can be used to detect moderately active antibodies (e.g., CNJ157 with n = 104) should be 0.15 pM. Yet, under assay conditions, nobserved, l
38
TAWFIK,
o! 0.0
GREEN,
1 0.2
0.4
So
0.8
0.8
1.0
(mM)
FIG. 3.
AP,,fAP,,, determined for CNJ157 (5.5 PM) in the presence of various concentrations of ester 4; APwt was measured in the presence of a noncatalytic MAB under the same conditions.
which is smaller than the theoretical n3, is more relevant, yielding Ab, of ca. 0.4 pM. For the less active CNJ 206 (n = 1600), the calculated minimal Ab, is 2.5 pM. The experimental results (Fig. 2) indicate that the observed Ab,, for these antibodies are in accordance with the calculated values for this system. As derived from Eq. [4a], Ab, . n is dependent upon substrate affinity. A substrate with higher affinity, i.e., ester 4 (K, = 0.11 mM), significantly increases detection ability. When a “shorter” substrate having lower affinity is used, i.e., ester 5 (K, values of 1.07 and 0.90 mM for CNJ 157 and CNJ 206, respectively), the activity of the less efficient antibody (CNJ 206, n = 1600) is noticeable only at high MAB concentration (AP,,I = 10 at 15 PM antibody concentration; Fig. 2). @mat As predicted (Eq. [4a]), increasing the substrate concentration results in a lowering of the measured signal (A P,,IA P,,n,t) (Fig. 3). At relatively high substrate concentrations (>0.5 InM) the observed decrease in signal ratio is higher than that predicted (Eq. [3]) due to the inactivation of these particular catMABs by the substrate that becomes significant at S, 9 K,. Inactivation is due to acylation of the antibody active site by the substrate as the result of nonproductive binding. The lability of p-nitrophenyl esters is the main cause; yet, substrate-induced inactivation or nonproductive binding are not unexpected in many other antibody-catalyzed reactions.
AND
ESHHAR
concentrations required to detect any authentic catalytic activity. As expected, rather high concentrations of purified antibody (0.5-15 PM; ca.O.l-2.0 mg/ml) are required (Fig. 2). Unlike the catalytic power of the detected antibodies (n), other parameters of the assay can be predetermined. The effects of substrate affinity (K,) and concentration (S,), which are described in Eq. [4], are clearly observed (Figs. 2 and 3). These data emphasize that it is practically impossible to detect MABs exhibiting modest catalytic activity under nonoptimal conditions. In order to increase the ability to detect less powerful catalytic antibodies, one should design a substrate with the maximal affinity, i.e., having the lowest K,. It is well established that the binding affinity of antibodies to a substance is generally increased when its structure not only mimics the hapten structure, but also the linker or a even a portion of the protein carrier structure (6). In this case, the c-amino caproate moiety in ester 4, which mimics the carrier protein lysine residue portion of the hapten conjugate structure, was necessary to provide a substrate with sufficient affinity. In some cases of nonhydrolytic reactions, for example, bimolecular reactions, cyclization reactions, or Diels-Alder reactions, where the product resembles the hapten, product inhibition should be considered. Yet, it appears that sufficient affinity of the substrate is also required in these cases (7). When a substrate is designed to be used to detect antibody-mediated catalytic activity other factors should be considered as well: (i) sol&i&-hydrophilic or ionic groups, such as carboxyl in this case, contribute to substrate solubility (ester 4 is soluble in buffer up to 20 mM); insufficient solubility of the substrate requires the addition of relatively high concentrations of miscible organic solvents, which causes a decrease in reactivity (data not shown). (ii) Stability-some structures are unfavorable due to intrinsic features of the molecule that result in high uncatalyzed rates of reaction. When ester 6, having a L-t value ca. loo-fold higher than that of ester 4, was used as a substrate, even the more active antibody (CNJ 157) could not be detected (A P,,lA P,,., = 1.0 at 10.5 pM antibody concentration). In this case,
TABLE DISCUSSION
The model presented provides a simple tool for the optimization of any assay designed for the detection of catalytic activity that is accompanied by a measurable uncatalyzed, background reaction. This analysis is valid not only for the detection of catalytic antibodies but also for any enzyme or other catalyzed reaction that has the same character. The relative enzyme units Ab,, * n (or E, * n) allow us to evaluate the antibody, or enzyme,
Kinetic
Parameters
MAB
K’, (-0
K”, bM)
157 206
0.11 0.11
1.07 0.90
D The If, values for esters (see Materials and Methods). ’ kat was determined using
1
and
of CNJ157
4 and ester
k (min”‘l)
b
2.39 0.41 5 were
determined
4 as substrate.
206”
n 0.97 x 10’ 0.16 X 10’ as indicated
DETECTION
OF
CATALYTIC
increased reactivity of 6 is due to intramolecular catalysis by the carboxyl group (8). As noted, the lowest possible substrate concentration should be used in the detection assay. The effect of substrate concentration (S,) on the signal ratio (AP,,I Eq. [4a], Fig. 3) is the result of the different A puncat ; kinetics of the catalyzed and uncatalyzed reactions. While the uncatalyzed reaction rate is proportional to the substrate concentration [2], the catalyzed reaction [l] exhibits saturation kinetics; i.e., at higher substrate concentrations (S, % K,) the rate becomes independent of substrate concentration ( v0 = v,&. The minimal substrate concentration (S,) is primarily dictated by the sensitivity of the assay (cc, < AP,,, AF,, < 0.1. S,), but also by the antibody concentrationP Other parameters such as buffer and pH should be considered as well. In hydrolytic reactions the pH may greatly affect both kc, and k,,ncat and thereby the ability to detect catalytic activity. Optimal pH for detection should be selected so that the resulting rate of reaction For the system prek, = kncat - n) is measurable. sented, pH 7.5-8.3 was found to be suitable. The ability to detect catalytic activity, i.e., the value of Ab, * n, is also dependent on the sensitivity of the assay [4b]. Various detection methods that are routinely used to assay enzymatic activity were employed for catalytic antibodies, e.g., HPLC, optical absorbance, and fluorescence. The sensitivity, i.e., the minimal detectable amount of product, differs for each method. Optical absorbance measurements, direct or after HPLC separation, are generally in the micromolar range. Yet, other methods, e.g., fluorescence, are more sensitive and allow the detection of nanomolar or even lower concentrations. Is the use of highly sensitivity assay always advantageous? The K,‘s of the more efficient MABs, as is the case for enzymes, are in the millimolar range (1~). The model predicts that, under typical assay conditions, i.e., signal ratio pLR= 10 and K, = 0.1 mM, Ab, . n is 1.1 X 10T3 and 1.0 X lop3 for assay sensitivity of 1 FM and 1 IIM, respectively [4b]. Thus, increasing the assay sensitivity by three orders of magnitude results in an insignificant change in the ability to detect catalytic activity. On the other hand, if the same catMABs are screened using a substrate with higher affinity (e.g., Km = 0.01 mM), then the gain in sensitivity (i.e., pus = 1 nM) is more effective (Ab, * n = 0.1 X 10p3). An interesting conclusion derived from this model is, therefore, that using highly sensitive assay techniques does not directly or linearly increase the ability to detect catalytic antibodies that are present in low concentrations. Finally, it is critical to ensure that the detected catalytic activity occurs at the antibody binding site and is not due to minor quantities of highly active enzyme impurities (Id, 2). The purity of the antibody preparation is equally important when screening is performed using a high concentration of antibodies produced from
39
ANTIBODIES
ascites or growth media. The use of protein A or G affinity columns, as was done with CNJ157 and CNJ206, is effective, yet solid evidence that catalysis occurs at the antibody binding site must be provided; see Materials and Methods and (2). Ideally, it would be desirable to directly screen hybridoma supernatants for catalytic activity rather than select those clones that bind the hapten, produce large quantities of purified antibodies, and then assay for catalytic activity. Direct screening procedures are unavoidable if the new, recombinant DNA-based, nonhybridoma methodologies for the production of antibodies are used (9). It is clear from the data presented that, regardless of the sensitivity of the assay used, the antibody concentrations needed to detect moderate or even highly active catalytic antibodies (n > 104) are higher than those generally present in hybridoma supernatants under normal growth conditions (1-5 pg/ml). For many reactions the observed kuncat in hybridoma supernatants is much higher than that in buffer due to the presence of various enzymes that catalyze the same reaction. The detection of catalytic antibodies present in low concentrations and in the presence of enzyme contaminants requires the development of novel techniques that are not only highly sensitive but very selective as well; i.e., the product must be detected in the presence of a large excess of unreacted substrate. The analysis presented in this paper may also be helpful for evaluating techniques that will allow direct screening of antibodymediated catalytic activity. ACKNOWLEDGMENTS We thank Ms. Romy Zemel and Ms. Rachel Chap for their assistance in the production of the MABs. We are also grateful to Professor Jacob Bar-Tanah for reviewing the manuscript.
REFERENCES 1. (a) Lerner, R. A., Benkovic, S. J., and Schultz, P. G. (1991) Science 262, 659-667; (b) (1991) Catalytic Antibodies, Ciba Foundation Symposium No. 159 Wiley, New York; (c) Green, B. S., and Tawfik, D. S. (1989) Trends Bioteehnol. 7, 304-310; (d) Schultz, P. G. (1989) Angew. Chem. Znt. Ed. Engl. 28,1285-1295. 2. Tawfik, D. S., Zemel, R. R., Arad-Yellin, R., Green, B. S., and Eshhar, Z. (1990) Biochemistry 29,9916-9921. 3. Tramontano, A., Ammann, A. A., and Lerner, R. A. (1988) J. Am. Chem. Sot. 110,2282-2286. 4. Fersht, A. (1985) Enzyme Structure and Mechanism, 2nd ed., Freeman, New York. 5. I. H. Segel 6. Pressman, Antibody
(1975)
Enzyme
Kinetics,
D., and Grossberg, Specificity, Benjamin,
Wiley,
A. (1968) The New York.
New
York.
Structural
7. Tramantano, A., and Schloeder, D. (1989) in Methods mology (Langone, J. J., Ed.), Vol. 178, pp. 531-550, Press, San Diego.
Basis
of
in EnzyAcademic
8. Kirby, A. J. (1980) Adv. Phys. Org. Chem. 17,183-277. 9. Winter, G., and Milstein, C. (1991) Nature 349, 293-299; Huse, W. D., Sastry, L., Iverson, S. A., Kang, A. S., Alting-Mees, M., Burton, D. R., Benkovic, S. J., and Lerner, R. A. (1989) Science, 246,1275-1281.
