Download Monoclonal Antibodies Binding Renal Renin

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Monoclonal Antibodies Binding Renal Renin
VICTOR DZAU, M.D., MEREDITH MUDGETT-HUNTER, P H . D . ,
GEOFFREY KAPLER, B.A., AND EDGAR HABER,
M.D.
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SUMMARY Somatic-cell fusion of normal antibody-producing spleen cells with cells from a plasmacytoma culture results in a culture of hybrid cells from which a monoclonal line may be selected. These lines are
immortal and may be amplified as tumors in syngeneic animals to produce large quantities of antibodies
characterized by molecular homogeneity. We report the application of this technique to the production of antibodies binding canine renin. Balb/c mice were immunized with pure canine renal renin and their spleen cells
fused with the NS-1 myeloma line. In two separate fusions, nine clones of cells were isolated that bound canine
renal renin but did not cross-react with a number of protein antigens tested. One of these antibodies crossreacted with renins of several different species, including human renin. Binding inhibition studies carried out
with one of these monoclonal antibodies demonstrated a dissociation constant for renin of 10"' M. These monoclonal antibodies hare great potential in answering significant questions concerning the structure, biosynthesis, tissue localization, and physiologic actions of renin. (Hypertension 3 (suppl II): II-4-II-8, 1981)
KEY WORDS
• renin antibody
• monoclonal antibody
A
NTIBODIES specific for renin have recently
become available with the purification of the
enzyme from tissues of several different species.^ These antibodies have been used in the direct
measurement of renin by immunoassay*"7 as inhibitors of renin in vivo,* in the identification of extrarenal renin-like enzymes,*"11 and in the examination of
renin biosynthesis.1*"1* Although these studies have
yielded information of interest, antibodies produced
by the process of conventional immunization are
necessarily limited with respect to quantity, reproducibility, and homogeneity. The recent development of
methods for the production of monoclonal antibodies
has overcome these problems;" antibodies can now be
obtained as homogeneous proteins in unlimited quantity. We have applied these methods to the selection
and propagation of hybrid clones from mice immunized with canine rcnin and report here the
preliminary characterization of nine monoclonal antibodies binding this antigen.
Methods
Immunization
Balb/c mice were injected intraperitoneally with 50
jig of purified canine renal renin1 in complete Freund's
adjuvant 4 weeks prior to fusion. Three days before fusion the animals were intravenously boosted with 5 ^g
of the same antigen.
Cell Fusion
The procedure for fusion is a modification of one
reported by Marshak-Rothstein et al.1* We fused 10s
spleen cells from an immunized mouse with 107 mouse
myeloma cells from the NS-1 line (kind gift of Dr.
Malcolm Gefter) in the presence of 30% polyethylene
glycol for 5 minutes. After 48 hours of growth in
Delbecco's modified Eagle medium (DMEM) supplemented with 20% fetal calf serum, 50 Mg/ m l gentamicin, and 580 Mg/ m l glutamine, the cells were resuspended in hypoxanthine aminopterin thymidine
(HAT) medium (1 X 10"4 M hypoxanthine, 4 X 10"7 M
aminopterin, and 1.6 X 10"* M thymidine)19 and distributed into four Costar 96-well microtiter plates.
After 14 days of growth in the HAT medium, supernatants from wells exhibiting cell growth were assayed
utilizing an immunoradiometric assay for reninspecific antibody.
From the Hypertension Unit, Bngham and Women's Hospital,
Cardiac Unit, Massachusetts General Hospital, and Department of
Medicine, Harvard Medical School, Boston, Massachusetts.
Supported by a grant from Reynolds Industries, AHA Established Investigatorship No. 79-189 (Dr. Mudgett-Hunter), Clinical
Investigatorship 5K08-OO75O (Dr. Dzau) from the National Heart,
Lung, and Blood Institute, and Grant RO8 HL-OO75O-O1 from the
NIH.
Address for reprints. Edgar Haber, M.D., Cardiac Unit, Massachusetts General Hospital, Boston, Massachusetts 02114.
II-4
MONOCLONAL ANTIBODIES TO RENIN/Dzau et al.
Results
Immunoradiometric Assay
20
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The general approach described by Klinman et a/.
was utilized. Flexible polyvinyl chloride microtiter
plates (Cooke Laboratories) were first coated with a
solution of canine renin or one of the following control
antigens: pepsin, trypsin, hemoglobin (or bovine
serum albumin). The plates were then washed with
20% horse serum followed by incubation with varying
dilutions of culture supernatants, ascites fluid, or fractionated monoclonal antibody. After a water wash,
1Jt
I goat antimouse Fab (the antibody is directed at
Fab so that all hybridoma protein isotypes may be
detected by the reagent) was added to each well; and,
after a final wash, the wells were separated and
counted in a gamma scintillation counter. To determine the capacity of soluble renin to inhibit the binding of monoclonal antibodies to immobilized renin, increasing concentrations of renin were added to the
antibody prior to application to the microtiter plate.
Subcloning and Expansion of Hybridomas
Utilizing mouse 3T3 cells or non-immune spleen
cells as a feeder layer, cells from wells exhibiting
renin-specific antibody were subcloned by limiting
dilution. Positive clones were injected into pristaneprimed Balb/c mice (0.5 ml pristane i.p. 1-2 weeks
prior to cell injection) at a dose of 10* cells per mouse.
After visible evidence of ascites production, the abdomens of the mice were tapped every other day, and
fluid from several animals pooled.
SOUD PHASE IMMUNORADIOMETRIC ASSAY
OF AB 191-2E7
Two separate fusions, F5 and F9, yielded renin antibody-secreting clones (table 1). Antibodies of the
isotypes IgGx and IgM were identified. Canine renin
appears to be an effective antigen in the mouse, and
the yield of positive clones is abundant, exceeding the
laboratory's capacity for isolation. The antibodies
bound renin at a considerable dilution. In this assay
the half-maximal point of antibody binding to the immobilized antigen is both a measure of concentration
and avidity. Representative dilution plots are shown in
figure 1 for antibodies from fusions 5 and 9. The antibodies representative of each of the two fusions may
be diluted considerably with continued antigen-bind-
TABLE 1. Yields of Antibody-Producing Clones in Fusions
F5andF9
Renin
Wells
Cell antibody
assayed growth producClones
Fusion
(no.)
(%)
tion
isolated
Isotype
F5
400
95
9
1G11-2E7
IGM
F9
400
80
78
2B3-2H10
IgG,
2B3-1H4
IgG,
IgG,
2B3-IA6
2B3-IA5
IgG,
SOUD PHASE IMMUNORADIOMETRIC ASSAY
OF AB 2B3-2H10
1600-
1200-
800-
400-
100
11-5
1000
IGM DILUTION
10,000
id
MOLAR ANTIBODY CONCENTRATION
FIGURE 1. Left: Solid-phase immunoradiometric assay of antibody 2E7 Renin is bound to the plastic
microtiter plate and then incubated with the monoclonal antibody solution. The ordinate represents binding
ofiul goat antimouse Fab to IgM monoclonal antibody 1G11-2E7. The abscissa represents dilution of the
antibody solution that had an initial concentration of 1.2 X 10^ M. Right: Immunoradiometric assay of
antibody 2B3-2H10, using same method. Abscissa is molar concentration of antibody.
II-6
PROCEEDINGS/INTERAMERICAN SOCIETY
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ing activity. Figure 1 right, which examines the I g d
antibody, 2B3-2H10, from fusion 9, is particularly instructive. The antibody concentration was determined in this experiment, and it is remarkable that
half-maximal binding to insolubilized antigen occurs
at 8 X 10~u M antibody concentration, indicating considerable avidity for immobilized renin.
The antibodies appear to be specific for purified
renin. Table 2 indicates that no significant binding occurred to pepsin, trypsin, hemoglobin, digoxin conjugated to hemocyanin, or to the mixture of proteins
contained in either 10% horse serum or 20% fetal calf
serum. Cross reactivity can be demonstrated with
renins of other species, as can be seen in table 3. It is
of considerable interest that antibody 2E7, while being
fully specific for canine renin, recognized the renins of
several other species equally well, including human
renin. Apparently this is an antibody to an epitope
common among mammalian renins. This epitope is
not prominently recognized in conventional renin antisera since there is usually little antigenic cross reactivity between human and animal renins. As expected,
another mouse monoclonal antibody specific for
digoxin (our laboratory code: 26-10) did not bind
renin in this assay.
Measurement of the binding of antibody to immobilized renin affords only a measurement of the illdefined quantity, avidity. Since there is opportunity
for both monovalent and bivalent binding to antigen,
the half-maximal concentration for antibody binding
is dependent in part on the density of antigen substitution on the solid support. Affinity is best determined
utilizing soluble antigen. Since monoclonal antibodies recognize but a single determinant on the surface of a non-repeating antigen such as renin, affinity
may be measured utilizing the same approach applied
to antibody-hapete binding. In figure 2 the binding of
IgGi antibody to immobilized renin is examined in the
presence of varying concentrations of soluble renin.
Half-maximal binding occurs at a concentration of
10"7 M renin, which corresponds to the dissociation
constant of the antibody for the single epitope on renin
that is bound.
SUPP II, HYPERTENSION, VOL 3, N o 6, N O V / D E C , 1981
TABLE 3. Cross Reactivity of Monoclonal Antibody 2E7
with Renins of Several Species
Bound I25I goat
antimouse Fab
(CPM)
Species
Dog
2700
2600
2600
2000
2300
80
Human
Hog
Bovine
Mouse
Control
INHIBITION OF ANTIBODY BINDING BY CANINE RENIN
0.0001
0001
001
01
RENIN CONCENTRATION
1
FIGURE 2. Competitive radioimmunometric assay of antibody 2B3-1A6. The assay b carried out as indicated in figure
1 except that varying concentrations of soluble renin
(abscissa) are added to the antibody solution prior to its
application to immobilized renin.
TABLE 2. Binding of Monoclonal Antibodies to a Variety of Antigens
Antigens
CPM-bound 12eI goat antimouse Fab
, Monoclonal
, . antibody
1A5
- 2H10
1A6
1H4
Medium
10
XIOE6
Renin
5019
3638
3911
4600
Trypsin
Pepsin
Hemoglobin
274
150
539
257
319
88
283
213
398
236
203
257
136
238
225
121
Horse
serum
(10%)
475
182
1402
265
82
Fetal
calf
serum
(20%)
1740
310
300
1145
101
Digoxinhemocyanin
281
199
264
250
100
MONOCLONAL ANTIBODIES TO REWN/Dzau et al.
Discussion
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There are a number of significant research problems
that could be attacked effectively if monoclonal antibodies specific for renin were available. Most obvious
is the value of renin antibody or Fab fragments as
specific physiological reagents to delineate the role of
renin in the control of the normal or diseased circulation. While these studies can be carried out with conventionally derived antibodies, the difficulty in isolating renin limits the amount of antigen available, and
thus, supplies of antibody are also severely limited.
Since each antiserum is comprised of a different mixture of antibodies varying in both specificity and
affinity," comparative experiments among different
laboratories is difficult. We have noted an additional
problem. The effectiveness of a mixed immunoglobulin population in inhibiting the enzymatic activity of renin is markedly reduced when papain
cleavage to produce Fab is carried out.M This may be
explained by demonstrating that the IgG fraction of a
renin-specific antiserum is comprised of antibodies
that recognize not only the catalytic site of renin but
also other epitopes that are remote from this site. Intact antibodies, being bivalent, precipitate renin and
thus take it out of solution, regardless of whether the
catalytic site or another part of the molecule is bound.
On the other hand, Fab, which is monovalent, will
only inactivate the enzyme if the catalytic site is
bound. Since a minority of antibodies are catalyticsite specific, the effectiveness of Fab from a mixture of
antibodies is far less as has been observed.
Monoclonal antibodies solve all these problems. A
small amount of antigen can yield an infinite amount
of antibody because of the immortality of the cell
culture that produces the antibody. The availability of
large quantities of antibody will allow for comparative studies among different laboratories, but more
importantly, the selection of catalytic site-specific
monoclonal antibodies will solve the difficulty of
producing ample quantities of Fab, a useful physiologic reagent.
A major question in renin research relates to the
identity of inactive renins found in various physiologic fluids. Are these prorenins in a biosynthetic
sense, or do they represent a post-synthetic modification? The mapping of epitopes on the surface of renin
and prorenin with a variety of monoclonal antibodies
should resolve this problem.
There has been considerable interest in the identification of renin-like proteins in tissues other than
the kidney. Conflicting results have been reported with
different antisera." 1 " Are these related to cross-reacting antibodies recognizing epitopes common to several
proteins, or is renin really present in a number of
tissues? Mapping the profile of renal renin with a set of
monoclonal antibodies should allow identification of
the cross-reacting species of other tissues.
In this preliminary communication, we have described a set of monoclonal antibodies that appear to
be specific for renin. They bind immobilized renin with
seemingly different avidities, but do not cross react
II-7
with the other antigens tested. A monoclonal antibody specific for digoxin does not bind renin in the
assay used. One of the monoclonal antibodies recognizes an unusual epitope that is common to the renins
of a number of species, including man. None of the antibodies inhibits the enzymatic activity of renin. This
may be a product of the unique specificity characteristic of monoclonal antibodies in that they are
directed against a single epitope on the surface of a
molecule. The monoclonal antibodies examined thus
far may not recognize renin's catalytic site or a region
sufficiently near it to hinder access of substrate. An alternative possibility is that the antibodies are not specific for native renin but recognize a denatured form
of the enzyme. An epitope on denatured renin may not
be shared by the native enzyme. A remote explanation, though unlikely in view of the number of
monoclonal antibodies examined, is that the antibody
is directed to a contaminating protein present in a
quantity too small to be detected by conventional analytic procedures and not to renin itself. The interest
and excitement of this line of investigation will be
found in future studies that further characterize these
as well as other renin-specific hybridoma proteins.
References
1 Dzau VJ, Slater EE, Haber E. Complete purification of dog
renal renin Biochemistry 18: 5224, 1979
2. Slater EE, Cohn RC, Dzau VJ, Haber E Purification of human
renal renin. Clin Sci Mol Med 55: 117, 1978
3 Corvol P, Devaux C, Ito T, Sicard P, Ducloux J, Menard J.
Large scale purification of hog renin' Physicochemical
characterization Circ Res 41: 616, 1977
4. Inagami T, Murakami K. Pure renin- Isolation from hog
kidney and characterization J Biol Chem 252: 2978, 1977
5 Guyene TT, Galen FX, Devaux C, Corvol P, Menard J. Direct
immunoassay of human renin' comparison with rerun activity in
plasma and amniotic fluid. Hypertension 2: 465, 1980
6. Michelakis AM, Yoshida H, Menzie J, Murakami K, Inagami
T A radioimmunoassay for the direct measurement of renin in
mice and its application to submaxillary gland and kidney studies Endocnnol 94: 1101, 1974
7. Mailing C, Poulsen K. A direct radioimmunoassay for plasma
renin in mice and its application to the direct radioimmunoassay of renin in various organs Biochim Biophys Acta
491: 532, 1977
8 Dzau VJ, Kopelman RI, Barger AC, Slater EE, Haber E.
Renin-specific antibody for study of cardiovascular homeostasis. Science 207: 1091, 1980
9 Dzau VJ, Brenner A, Emmett N, Haber E. Identification of
renin and renin-like enzymes in rat brain by a renin-spccific
antibody. Clin Sci 59: 45s, 1980
10 Dzau VJ, Emmett N, Kapler G, Brenner A, Churchill S, Haber
E. Characterization of renin-like enzyme(s) in the rabbit uterus
by renin specific antibody Circulation 62 (suppl III): III-238,
1980
11 Slater EE, Defendini R, Zimmerman EA Wide distribution of
immunoreactive renin in nerve cells of human brain Proc Natl
Acad Sci USA 77: 5458, 1980
12. Hirose A, Yokosawa H, Inagami T Immunochemical identification of renin in rat brain and distinction from acid
proteases Nature (London) 274: 392, 1978
13. Pratt R, Dzau VJ, Ouellette A Abundant androgen regulated
mRNA in mouse submandibular gland cell retranslation of
renin precursor mRNA Nuclei Acid Res 9: 3433, 1981
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PROCEEDINGS/INTERAMERICAN SOCIETY
14. Carlson W, Dzau V, Quay S, Kreisberg J, Haber E.
Biosynthesis of rerun in the dog kidney: evidence for the
presence of prorenin. Circulation 62 (suppl III)- III-187, 1980
15 Re R, Fallon JT, Dzau V, Quay S, Haber E. Remn synthesis by
arterial smooth muscle cells. Circulation 59 and 60 (luppl II):
11-10, 1979
16. Poulsen K, Vuust J, Lykkegaard S, Hoj Nielsen A, Lund T.
Renin is synthesized as a 50,000 dalton single-chain polypeptide
in cell-free translation systems. FEBS Lett 98: 135, 1979
17 Kohler G, Howe CS, Milstein C. Fusion between
immunoglobulin-secreting and nonsecreting myeloma cell lines.
Eur J Immunol 6: 292, 1976
18. Marshak-Rothstein A, Fink P, Gridley T, Raulet D, Bevan M,
Gefter ML, Properties and applications of monoclonal antibodies directed against determinants of the Thy-1 locus. J Im-
SUPP
II,
HYPERTENSION, VOL
3, No 6,
NOV/DEC,
1981
munol 122: 2491, 1979
19 Littlefield JW. Selection of hybrids from matings of fibroblasts
in vitro and their presumed recombinants. Science 145: 709,
1964
20. Klinman NR, Pickard AR, Sigal NH, Gearhart PJ, Metcalf
ES, Pierce SK. Assessing B cell diversification by antigen receptor and precursor cell analysis. Ann Immunol (Paris) 127C:
489, 1976
21 Haber E, Margohes MN, Cannon LE. Origins of antibody
diversity: Insights gained from amino acid sequence studies of
elicited antibodies. Cold Spring Harbor Symposium on Quantitative Biology. Vol XLI: 647, 1977
22. Dzau VJ, Kopelman RI, Barger AC, Haber E. In vivo and in
vitro studies with antirenin Fab fragments. Circulation 62
(suppl III). 111-89, 1980
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Monoclonal antibodies binding renal renin.
V Dzau, M Mudgett-Hunter, G Kapler and E Haber
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Hypertension. 1981;3:II-4
doi: 10.1161/01.HYP.3.6_Pt_2.II-4
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