0013-7227/90/1272-0658$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society

Vol. 127, No. 2 Printed in U.S.A.

Mapping of an Assembled Epitope of Human FollicleStimulating Hormone-/? Utilizing Monoclonal Antibodies, Synthetic Peptides, and Hormone-Receptor Inhibition* DILIP D. VAKHARIA, JAMES A. DIAS, ARVIND N. THAKUR, THOMAS T. ANDERSEN, AND ALAN O'SHEA Wadsworth Center for Laboratories and Research (D.D. V., J.A.D.), New York State Department of Health, Albany, New York 12201-0509; School of Public Health, State University of New York at Albany, Albany, New York 12201; Department of Molecular Endocrinology (A.O., A.N.T.), Middlesex Hospital Medical School, University of London, London, W1N8AA, United Kingdom; and Department of Biochemistry (T.T.A.), Albany Medical College, Albany, New York 12208

product of the lowest mass of both peptide and antibody which gave a positive result was used to rank the peptides for their binding with mab 3G3. Peptides were ranked in the following descending order of potency: 33-53, 49-67, 66-85>»16-36,120, 95-103, 52-65, 81-100, and 103-110. Ability of the mabs to inhibit binding of [125I]hFSH to bovine testis membrane receptor (Rec) was also studied. When [125I]hFSH was preincubated with increments of each mab for 2 h at 25 C before adding Rec with further incubation for 16 h, all mabs inhibited [l26I]hFSH binding to Rec. The data suggest that most of the hFSH/3 molecule has a conformation enabling all antibody recognizable regions to be in close proximity to each other. The present study provides evidence for an assembled epitope comprising in part, amino acids 33-53, which has been previously shown to be involved in receptor binding. Peptide sequences 49-67 and 66-85 are neighboring sequences in this assembled epitope which contains the determinants for receptor binding. (Endocrinology 127: 658666,1990)

ABSTRACT. Monoclonal antibodies (mabs) to human (h) FSH were utilized to probe epitopes of the /3-subunit of hFSH (hFSH/3). These mabs had an average approximate affinity constant (KJ of ICM" 1 for hFSH/3 and IO'M" 1 for heterodimeric hFSH. Hormone specificity of mabs for hFSH/3 was demonstrated by a lack of cross-reactivity with hCGa, FSHa, or LHa. Epitope specificity of each mab was initially assessed by determining whether solid phase mab could bind to [125I]hFSH already bound to mabs in liquid phase. In addition, it was determined whether [126I]mab could bind to hFSH already bound to solid-phase mabs. Both epitope cross-matching protocols indicated that all mabs bound to the same epitopes on hFSH/3. Next, synthetic peptides corresponding to the sequence of hFSH/3 were used in an enzyme-linked immunosorbent assay to map this epitope. All mabs bound to peptides 7-19, 1-20, 33-53, and 6685 but did not bind or bound weakly to peptides 81-100, 95-103, and 103-110. Titration experiments were performed using different concentrations of peptide (0.3-41 nmol) and one mab 3G3 (500 ng-25 ng) in the enzyme-linked immunosorbent assay. The

T

Monoclonal antibodies (mabs) to hFSH which appear to recognize overlapping epitopes of FSH have been described (9,10). Other studies have utilized immunological approaches and chemical cross-linking (11-17) in an attempt to elucidate the spatial arrangements of gonadotropins in hormone-receptor complexes. Our own immunochemical studies of the interaction of FSH with its receptor using polyclonal antisera suggested that some antigenic sites on each subunit of FSH were masked when the hormone was bound to its receptor (11). However, assignment of specific sequences of hFSH/3 which are antigenic determinants has not been made, and whether any of these are near the receptor binding site is not known. In the present study, we have analyzed a panel of hFSH/3-specific mabs for their antigen specificity and receptor binding neutralizing property, and identified peptide sequences that comprise the epitope recognized by these mabs.

HE amino acid sequences of human (h) FSH/3 and FSHa have been determined by protein sequencing (1, 2) and translation of the coding sequence of each gene (3-6). A crystal structure has not been reported and therefore, little is known about specific tertiary structural features of individual subunits and heterodimeric hFSH. A synthetic peptide corresponding to hFSH/3 3353 inhibits FSH binding to its receptor and is a partial agonist of FSH action (7). Synthetic peptides corresponding to hFSH/3 76-118 and 22-33 had immunological activity in a RIA for hFSH suggesting that these sequences represented unique epitopes on hFSH/3 (8). Received March 16, 1990. * Supported by USPHS Grant HD-18407 (to J.A.D.) and Research Career Development Award HD-00682 (to J.A.D.). Address requests for reprints to: James A. Dias, Ph.D., Wadsworth Center for Laboratories and Research, New York State Department of Health, Empire State Plaza, P.O. Box 509, Albany, New York 122010509.

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MAPPING OF HUMAN FSH/3 EPITOPE Materials and Methods Hormone Preparations Highly purified hFSHa (N-594-A), hFSH0 (N-594-B; AFP1194-B), hFSH (LER-1781-2,4,000 IU/mg; AFP-4822-B, 3,100 IU/mg), hLHa (1793-A), and hCGa were obtained from the National Hormone and Pituitary Program (Baltimore, MD) and Dr. Leo E. Reichert, Jr (Albany Medical College, Albany, NY). hFSH utilized as immunogen was 2794 IU/mg supplied by Professor W. Butt (Birmingham, UK). mabs Male BALB/c mice were immunized (ip) with hFSH (20 /xg) in Freund's complete adjuvant. Subsequent injections were made in Freund's incomplete adjuvant (sc) 2 weeks (20 ng) and 4 weeks (10 ^g) after the first immunization. Two weeks after the last immunization, 10 ng hFSH in saline were injected (ip). The fusion of JK Ag 8.6.5.3 myeloma cells (5.5 x 107) with splenocytes of the immunized males (7.0 X 107 cells) was performed as previously described (18) using 35% 4,000 mol wt polyethylene glycol. The hybridomas were cloned by serial dilution from single colonies, resultant from the primary fusion plating. The cell lines were cloned once and maintained in culture for 2-3 weeks. Data obtained from dose-displacement curves generated with increments of unlabeled hFSH/? were analyzed using the computer program LIGAND. The analysis demonstrated that each mab exhibits a homogeneous antibody population with single affinity for ligand. Thus, by two lines of evidence, these mabs were judged to be derived from a single clone. Ascites fluid containing high concentrations of mab was obtained by injecting BALB/c mice ip with 0.5 ml 2,6,10,14tetramethylpentadecane (Aldrich Chemical Co., Dorset, UK) followed 10 days later by 5 X 106 hybridoma cells, mabs were tested for receptor-binding inhibition (11) after dialysis of the ascites fluid overnight at 4 C. Control nonspecific ascites was also utilized, and in some cases mabs were purified by proteinA affinity chromatography according to manufacturer's instructions (Bio-Rad, Richmond, CA) or ion exchange chromatography utilizing a Pharmacia (Piscataway, NJ) FPLC system, a mono-Q column (5 cm X 5 mm), and a gradient of 0-1 M KC1 developed in a 0.01 M Tris, pH 8.3, buffer. Radioligand assays Iodination of hFSH was performed as previously described (11). Immature bovine testes were utilized as a source of FSH receptor (19, 20), and the receptor assay was performed as previously described (21). A double antibody procedure was utilized to separate antibody-bound hormone from free hormone as follows. Mab (100 Ml), [125I]FSH (50,000 cpm, 100 MD, test samples (200 M1)> and second antibody (rabbit anti-mouse immunoglobulin G, 1:40, 200 n\), were combined and brought to a final volume of 1 ml. All reagents, with the exception of mabs, were diluted in 0.01 M phosphate buffer, pH 7.4, 0.14 M NaCl (PBS) containing 0.1% gelatin, mabs were diluted in 0.05 M EDTA-PBS containing 1:400 normal mouse serum. After overnight incubation at 4 C, 1 ml ice-cold PBS was added to each tube followed by centrifugation (1250 x g) for 50 min at 4 C. Data from displacement curves were analyzed by a com-

659

puter program to determine slope and ED60 (22). Analysis of ligand binding data was performed to determine the affinity and concentration of mabs (23). The molarity of mab present in each fraction was determined by analysis of dose-displacement curves generated with increments of unlabeled hFSH. A one-site model and a stoichiometry of 2 molecules of hormone bound per antibody molecule with no interaction between the two binding sites was assumed. Preparation of solid-phase antibodies Solid-phase mabs were prepared as follows (24). To 5 g of microparticulate cellulose (Sigmacell, type 20) were added 20 ml 0.15 M l,l'-carbonyldiimidazole (Sigma, St. Louis, MO; 0.61 g/25 ml acetone), and the mixture was allowed to react at 20 C for 60 min with vigorous stirring. The activated, imidazolylcarbamate cellulose was recovered by filtration over a sinteredglass funnel and washed with three 100-ml aliquots of acetone. The activated cellulose was then allowed to air-dry and stored in a tightly sealed container at —20 C until required for protein coupling. The activated cellulose was equilibrated to ambient temperature, and 200 mg were weighed into a polystyrene tube. One hundred microliters of mab-containing ascites fluid, in 0.9 ml 0.05 M barbital buffer, pH 8.0, were added, briefly vortexed to form a slurry, stoppered, and rotated end-over-end for 16-18 h at ambient temperature. The immunoadsorbent was washed repeatedly with 10 ml aliquots of various buffers in the order listed below and recovered by centrifugation (10 min at 1,200 X g) after each wash cycle. 1) 0.5 M bicarbonate buffer, pH 8.0, rotate 20 min; 2) repeat; 3) 0.1 M acetate buffer, pH 4.0, rotate 60 min; 4) 0.1 M acetate buffer, pH 4.0, sonicate for 30 sec, rotate 16-20 h, 5) assay buffer {i.e. 0.1 M phosphate, 0.1% BSA, pH 7.4) rotate 20 min; and 6) repeat. The immunoadsorbent was then stored in 0.1% gelatin-PBS, at 4 C. Synthesis of peptides Solid phase synthesis of peptides representing overlapping sequences of hFSH0 peptides 1-20, 16-36, 33-53, 49-67, 5265, 66-85, and 95-103 was performed on an automated peptide synthesizer (Biosearch model 9500, San Rafael, CA) using tBoc amino acids and diisopropylcarbodiimide activation of the carboxyl group. Methylbenzhydrylamine resin was utilized for all the peptides except that corresponding to the C-terminal of hFSH/?, for which the standard Merrifield resin was utilized. Peptides were cleaved from the resin using anhydrous hydrofluoric acid, followed by acetic acid extraction and rotoevaporation. Other peptides, 7-19 and 103-110, were synthesized utilizing Fmoc chemistry in a rapid amide multiple peptide synthesis system (Dupont, Boston, MA) according to manufacturer's instructions. Peptide 81-100 was synthesized on an Applied Biosystems (Foster City, CA) 431A peptide synthesizer using Fmoc chemistry. Cleavage from the resin was done with 95% trifluoroacetic acid. The peptides obtained were further extracted by first dissolving in a minimal volume of trifluoroacetic acid followed by precipitation with absolute ether in dry ice-acetone bath. After four washes of the precipitate with ethylacetate-ethyl ether (1.5:1), the precipitate was dissolved in a minimal volume of 1 N acetic acid and diluted 50 times its volume in Milli-Q water and lyophilized. Deprotection of cys-

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660

MAPPING OF HUMAN FSH/3 EPITOPE

teines with mercuric acetate was performed followed by gel filtration in acetic acid and lyophilization. The lyophilized material was used after Sephadex G-10 filtration developed in 0.1 M pyridine acetate buffer. Synthetic peptides were analyzed for their amino acid composition by the Wadsworth Center, protein sequencing and amino acid analysis facility, using an automatic Beckman System Gold Analyzer (Beckman Instruments, San Ramon, CA). The determined amino acid composition of all the peptides was comparable to the theoretical value. The mass (nanomoles) of each peptide was derived from the calculations of the amino acid analysis. Epitope mapping using cross-matching protocols Inhibition of radiolabeled P25IJhFSH binding to immobilized mabs. Epitope cross-matching studies were performed in which epitope disparity can be detected by a failure of soluble antibody to prevent [125I]hFSH from binding to a nonidentical immobilized mab. Bidirectional data evaluation (cross-validation) was used to confirm epitope identity or steric hindrance. One hundred microliters (1:500 dilution) of mab were preincubated with 100 MI [125I]hFSH (80,000 cpm) overnight with shaking; then 100 fA solid-phase antibody (1:50) were added and again incubated overnight. The next day the reaction mixture was diluted to 2 ml with PBS, and the solid phase antibody was recovered by centrifugation, effecting the separation of bound from free [125I]hFSH. Two site immunoradiometric assay, mabs were radiolabeled by standard chloramine T-methodology. Briefly, antibody 10-25 Mg in 20-40 /il phosphate buffer (0.5 M, pH 7.2), 1 mCi 125I-Na, and 20 MI chloramine T (2 mg/ml; 0.05 M phosphate buffer, pH 7.2) were reacted for 30 sec at room temperature. The reaction was stopped by addition of 50 MI sodium metabisulfite (3.5 mg/ ml, 0.05 M phosphate buffer, pH 7.2) and the separation of radiolabeled mab from free iodine was effected by adsorption to immobilized protein A according to manufacturer's instructions (Bio-Rad). Soluble unlabeled hFSH (7 IU, 100 MD was incubated with 100 jul solid phase mabs (1:50) for 4 h at 37 C. Solid phase mab not incubated with hFSH served as controls for nonspecific binding of [125I]mab. Unbound FSH was removed by washing the solid phase mabs in 0.1% gelatin PBS. Then [125I]mab (detection antibody) was added and incubated for 2 h at 37 C. If the labeled mab recognizes the same epitope on hFSH as the antibody on the solid phase, then it cannot bind to hFSH. If the epitopes are different, then radiolabeled mab will bind to hFSH bound to immobilized mab. Utilizing this approach, the possibility of false positives is very small. A false negative may occur due to very low affinity or iodination damage of the radiolabeled mab. To rule out this possibility, solid phase antihFSHa was included as a control to determine the amount of [125I]mab that could bind to hFSH. Epitope mapping using synthetic peptides in an enzyme-linked immunosorbent assay (ELISA). Epitope mapping was performed in 96-well Immulon I plates (Dynatech Lab., Chantilly, VA). All the assays were carried out in duplicates. Alkaline phosphatase conjugated goat-antimouse immunoglobulin

Endo • 1990 Vol 127 • No 2

(Tago, Inc., Burlingame, CA) and substrate (P-nitrophenyl phosphate) kit (Bio-Rad) were used for detecting immunoglobulin bound to peptide and for color development, respectively. Stock solutions of peptides (5-10 mg/ml) were prepared in 0.003 M potassium phosphate buffer, pH 6.3. Peptides 1-20 and 16-36 were dissolved completely by adding 50 n\ 1 M KOH to 1 ml 0.003 M potassium-phosphate buffer. Peptides, hFSH, hFSH/3, hFSHa, or hTSHa, were coated on plates by incubating them at 37 C for 2 h in 0.05 M Tris, pH 9.5, buffer. Peptides in concentrations ranging from 0.3-41 nmol/50 n\ • well were used for coating. Heterodimeric FSH or subunits were used in concentrations ranging from 5 ng-50 ng/50 /ttlwell. One percent BSA (7-globulin-free) in 0.05 M Tris buffer, pH 9.5, or 5% nonfat milk solution (Carnation milk powder) in bicarbonate buffer (15 mM sodium-carbonate, 35 DIM sodiumbicarbonate, 3 mM sodium-azide), pH 9.6 was added for 1 h at room temperature to block the free sites in the wells. For peptides 1-20 and 16-36 better results were obtained with 1% BSA block. After washing, purified mabs were added to wells for 1 h at 37 C. mabs were diluted in binding buffer (0.1 mM EDTA, 0.05% Tween, 0.25% BSA in PBS, pH 7.4). After washing, alkaline phosphatase labeled goat-anti-mouse immunoglobulin, diluted 1:1000 in binding buffer, was added and incubated for 1 h at room temperature. The wells were washed again and substrate was added. The color which developed after the addition of substrate was quantitated at 0.5-4 h and 16-18 h utilizing a Dynatech plate reader, with a 410-nm filter setting. The color development quantitated in wells containing antibody alone and peptides or hormone subunits alone was considered as nonspecific and was subtracted from the values obtained from wells containing corresponding peptides or hormone subunits and mab. a) Specificity of mabs: All mabs (3G3, 3B7, 3B8, 4E9, and 1B10) were tested against hFSH, hFSHa, hTSHa, and hFSH/3 in the ELISA test for their specificity, mab 46.3H6.B7, which is specific for HFSH and hFSH/3, and mab 10.5.F1, which is specific for hFSH and hFSHa or hTSHa, were used in the assay as positive controls. b) Specificity of mabs for hFSHf3 peptides: Peptides were titrated against an excess (500 ng/well) amount of one of the mabs, 3G3. The least concentration or concentration 2-fold higher of peptide resulting in a positive signal was selected to titrate the minimum amount of mab concentration needed for a positive signal. In order to determine nonspecific binding, all the peptides were screened again with the mabs at minimum equimolar concentration along with mabs which were preincubated with hFSH/3. To determine the concentration of hFSH/3 needed to saturate the binding sites of mab, a saturation standard curve for 3G3 was derived using RIA. It was determined that 125 ng hFSH/3 were needed to inhibit binding of [125I]hFSH/3 to 20 ng 3G3 by 90%. Thus for ELISA, 2 Mg/ml mab 3G3 were incubated overnight at 4 C with 3.5 Mg/ml hFSH/3 to saturate binding sites of antibody. All other mabs at concentrations equimolar to 3G3 were adsorbed with hFSH/3 in a similar manner. Statistical analysis The data obtained from two epitope cross-matching experiments were subjected to one-way analysis of variance using

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MAPPING OF HUMAN FSH/3 EPITOPE Duncan's multiple range test. The procedure was performed utilizing SPSS release 3.1 (SPSS, Inc., Chicago, IL) at the computing facility of the State University of New York at Albany. The data obtained from ELISA were analyzed by Student's paired t test.

Results FSH binding characteristics of mab

Five mabs that were positive for human FSH binding activity were utilized throughout this study. The FSH« and FSH/? binding characteristics of these antibodies were studied utilizing a double-antibody RIA (Fig. 1). As can be seen from the data in Fig. 1, hFSHa subunit at very high dose level (10 fig) exhibited no cross-reactivity with the mab IB 10. This fidelity of recognition was also true for all the other four mabs (3G3, 3B7, 3B8, 4E9) utilized in this study. In Table 1, a summary of the hFSH and hFSH/? binding characteristics of the five mabs is presented. Without exception, mabs reacted with /3-subunit with greater affinity than they reacted with the heterodimer. The mean mass of hFSH required to displace 50% of radioligand was 508 ± 54 ng/tube, whereas only 53 ± 8 ng of hFSH/? was required to elicit the same degree of displacement. Thus, hFSH had only 10% of the. activity of hFSH/3 subunit despite the fact that hFSH was utilized as the immunogen. There appeared to be no difference in the affinity constants (Ka) (Table 1, column 6) of these mabs when they were tested in the assay, where

displacement curves were generated using [125I]hFSH and hFSH or hFSH/?. However, the Ka values appeared tfhFSH A

•

o

100

-

80

--

^

h•z. LLJ

hFSH >v

\

CD

60 --

0hFSH

O 40 LU

^

\

v \

°- 20 U CLONE 1B10 I 1

100

10

1,000

ng/TUBE FIG. 1. Double-antibody RIA for hFSH (LER-1781-2) and hFSH/3 (N594-B) using monoclonal antibody 1B10. Monoclonal antibody 1B10 (1:5,000, 100 MI), [126I]hFSH (50,000 cpm, 100 MD and unlabeled hormone were combined with rabbit anti-mouse antiserum (1:40, 200 yX) and normal mouse serum (1:400, 100 MU in a final volume of 1 ml. The assay was incubated at 4 C overnight. Separation of bound from free ligand was achieved by centrifugation at 1250 x g for 50 min. Subunit preparations hFSHa (N-594-A), hCGa and LHa (1793-a) when tested at 1 and 10 /ug/tube had no activity in the RIA.

661

different when mabs were tested in the displacement assay where [125I]hFSH/? and hFSH,8 were used (Table 1, column 7). mab 3G3 had highest affinity for hFSH/? and mab 3B8 the least. The Ka values derived from displacement curves utilizing labeled and unlabeled hFSH/? were taken into consideration in the subsequent discussion. This was because these mabs were specific for hFSH/? and had higher affinity for hFSH/? than hFSH. Epitope cross-matching Two methods were initially used to assess whether the epitope specificity of each antibody was shared or different. The results are presented in Tables 2 and 3. The values in each column are compared with each other. The results in Table 2 indicate that the epitope on hFSH blocked by soluble mab 3G3 or IB 10 is identical to the one recognized by all the solid phase mabs. The epitope blocked by soluble mab 4E9 is identical to the one recognized by solid phase mab IB 10, 3B8, and 3G3. However, it differed from solid phase 3B7. Soluble mab 3B7 was not able to block the epitope site on hFSH that was identified by other mabs. The epitope blocked by soluble mab 3B8 was identical to the one recognized by solid phase mabs 4E9, 1B10, and 3G3. However, mab 3B8 was not able to block the epitope recognized by solid phase mab 3B7 and an additional epitope recognized by mab 4E9. The data suggest that all mabs recognized identical or near identical epitope on hFSH molecule, and that mab 3B7 did not identify an identical epitope seen by other mabs. The results in Table 3 indicate that solid phase mabs 3G3, 1B10, 4E9, and 3B8 blocked the epitope on hFSH molecule recognized by mab 3G3, thereby preventing the binding of radiolabeled 3G3. Radiolabeled 3G3 recognized an epitope that was not identified by mab 3B7. Binding of radiolabeled 3G3 to hFSH bound to solid phase mab 10.4.B6 (antibody specific to a-subunit of hFSH) was significant suggesting that radiolabeled 3G3 could identify epitopes on /3-subunit portion of hFSH molecule. The counts bound to solid phase mab 8A11 (which did not react with hFSH) represented nonspecific counts. Binding of radiolabeled mab IB 10 to hFSH was blocked by solid phase 3G3, 1B10, 4E9, 3B7, and 3B8. Except for the positive control (10.4.B6), the counts obtained due to binding of radiolabeled 4E9 to solid phase mabs were not different from nonspecific counts due to binding to 8A11. This suggested that the epitope recognized by 4E9 is identical to those recognized by 3G3, 3B7, 3B8, and 1B10. The same was true of mab 3B7 and 3B8. Binding of radiolabeled 10.4.B6 to solid phase mabs 3G3, IB 10, 4E9, 3B7, and 3B8 confirm the presence of hFSH on solid phase mab. The binding to solid phase

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MAPPING OF HUMAN FSH/3 EPITOPE

662

Endo • 1990 Vol 127 • No 2

TABLE 1. hFSH and hFSH/3 binding characteristics of mabs

hFSH 686 ± 404 ± 580 ± 453 ± 418 ± 508 ±

3G3 1B10 4E9 3B7 3B8

Means

Ka (hFSHj8)° Ka(hFSH/3)6

ED 50

ED50

Mab

Slope 1.8 1.22 0.92 1.12 1.02 1.12

171 90 352 62 127 54

± ± ± ± ± ±

hFSH/8 61 56 76 34 38 53

0.08 0.34 0.15 0.03 0.213 0.06

±24 ±18 ±34 ±6 ±12 ±8

X10 8 M" 1

Slope 1.72 1.63 1.78 1.41 1.42 1.59

± ± ± ± ± ±

0.03 0.18 0.16 0.01 .17 0.08

3.0 3.4 2.5 4.2 3.2

± ± ± ± ±

0.6 1.1 0.29 0.75 1.0

X10 8 M" 1

6.6 3.5 1.8 1.6 0.6

± ± ± ± ±

1.16 0.50 0.41 0.18 0.11

The ED60 values are presented as nanograms of hFSH or hFSH/3 subunit per tube. Final assay volume was 1 ml; [126I]hFSH was the radioligand. Slope and ED60 values were determined with the computer program NIHRIA (22). No displacement was observed with 1 ng pure hFSHa, hCGa, or hLHa subunit. The affinity constants of hFSH/J (footnotes a or b) for each mab were calculated utilizing the computer program LIGAND (23). "Displacement curve was generated using [125I]hFSH. 6 Displacement curve was generated using [126I] hFSH/3. TABLE 2. Inhibition of [126I]hFSH binding to solid-phase mabs by soluble mabs

The data in both epitope-cross-matching studies suggest that all five mabs identified nearly identical epitopes on hFSH/3 molecule, but that mab 3B7 may not identify the epitope recognized by other mabs.

Soluble antibody

Solid phase antibody

3G3

1B10

97 ± 2° 100 ± 5° 97 ± 1° 97 ± 4 ° 98 ± 1° 104 ± 2°

3G3 1B10 4E9 3B7

66 50 64 120 ± 6° 83 ± 28° 91 100 ± 4° 103 ± 3° 71

3B8

3B8

3B7

4E9 ± 14°''' ±10 fc ± 15°>c ±12° ±6"

C

20 ±6 35 ±16° c 49 ±10 63 ± 8 ° 61 ± 17"

50 51 73 83 74

± 106>c

±9" ±8°'° ±16° ±9°

The values are the percent of inhibition of [126I]hFSH binding to solid-phase mab by soluble mab. Inhibition is observed when solid phase and soluble mab recognize identical epitopes. Data from four experiments were pooled and an analysis of variance performed, partitioning the variance for block effects. Comparisons (Duncans multiple range test, P < 0.01) were made within rows. Values with the same superscript in each column are not different from each other.

3B7 was not statistically different from binding to 8A11 or 10.4B6. This suggested that the amount of hFSH bound to solid phase 3B7 was significantly lower than that bound to other hFSH/3 specific mabs. Alternatively, 3B7 may sterically hinder 10.4B6 binding to hFSH.

Epitope mapping by ELISA. All five mabs were specific for hFSH and hFSH/3 and did not react with hFSHa or hTSHa (data not shown) confirming the RIA studies. The results of the determination of the least mass of peptide and mab 3G3 required for a positive signal are presented in Table 4. The product of smallest amount of peptide and mab 3G3 was used to rank peptide binding to mab 3G3. Peptides 33-53, 49-67, and 66-85 were considered to bind mab 3G3 strongly, mab 3G3 did not bind to peptide 81-100 and 103-110 at the highest concentration of peptide and mab tested. To determine the specificity of the binding of mabs to peptides, all mabs, used at equimolar concentration, were adsorbed with excess of hFSH/3 and then tested for their binding to peptides in ELISA. Table 5 gives the comparison of binding of hFSH/3 adsorbed or unadsorbed mabs

TABLE 3. Immunoradiometric epitope cross-matching analysis

Radiolabeled mab Solid phase mab 1B10

3G3 3G3 1B10

4E9 3B7 3B8 10.4B6 8A11

562 349 653 2,077 762 57,553 1,044

± ± ± ± ± ± ±

16° 156° 84° 356 124° 249 284°

2,322 ± 372 ± 2,672 ± 2,773 ± 2,035 ± 13,830 ± 1,361 ±

162°-* 124 336* 1736 24°-6 359 364°

4E9 4,028 ± 1,650 ± 6,045 ± 5,167 ± 5,300 ± 57,488 ± 2,572 ±

531 b-c 266° 476c>d 582" 677Cid 131 12°'6

3B7 307 1,417 1,726 612 1,266 12,052 930

± ± ± ± ± ± ±

40° 127° 46° 8° 120° 1,216 70"

3B8 1,398 589 1,048 263 513 11,856 1,151

± ± ± ± ± ± ±

366 7°' 6 76°-° 117° 127°'° 522 7°

10.4B6 55,943 ± 264 36,703 ± 1,106 24,816 ± 2,753 3,399 ± 417° 16,634 ± 167 499 ± 392° 1,407 ± 325°

Solid-phase mabs were saturated with unlabeled human FSH and washed to remove unbound FSH followed by addition of radiolabeled mab. After incubation, the solid-phase antibodies were washed again. The actual counts per minute in the table represent the data after the nonspecific binding (counts present in absence of FSH) are subtracted. Any solid-phase mab that had radiolabeled mab bound would not be significantly different from 10.4B6 and would indicate that the labeled mab recognized a different epitope from the solid-phase mab. Antibody 10.4B6 is an hFSHa-specific mab utilized as a positive control. Antibody 8A11 is a nonspecific negative control. For statistical analysis, each column was subjected to a one-way analysis of variance after the data were uniformly transformed to remove any negative numbers. Duncan's multiple range test was utilized to test for differences between means within columns. Means within the same column that share the same superscript are not different from each other at P 0.01 level.

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MAPPING OF HUMAN FSH/3 EPITOPE TABLE 4. Minimum amount of peptide (nmol) and mab 3G3 (ng)

receptor-binding inhibition before dialysis (data not shown). Therefore, it is essential to dialyze ascites before testing for receptor-binding inhibition. As can be seen in column 4 of Table 6, 3G3 and IB 10 were more potent inhibitors of hFSH binding to receptor than antibodies 3B7, 3B8, and 4E9. mabs 3G3 and 1B10 required 1.0 and 1.5 X 10~12 mol antibody to inhibit FSH binding to receptor by 50% whereas other mabs tested (3B7, 3B8, and 4E9) required 3 to 6 times more antibody to cause the same inhibition. This could be due to differences in the affinity for hFSH/? between the two groups of mabs (Table 1, column 7). The molarity of mab in each ascites preparation differed, not unexpectedly (Table 6, column 2).

required in the ELISA test Peptide

Concentration of Concentration of Product of peptide0 mab 3G36 peptide x mab Rank (nmol) (ng) 0.3 0.3

33-53 49-67 66-85 16-36 1-20 95-103 52-65

25 50 25 25 25 500 250

5.0 17.0 41.0 5.0

37.0

7.5 15 125 425

1025 2500 9250

663

1 2 3 4 5 6 7

Different concentrations (0.3-41 nmol) of each peptide were coated on the plate and tested against excess mab 3G3 (500 ng) in ELISA as described in Materials and Methods. The smallest concentration of each peptide which resulted in a positive signal (absorbance 0.1 or more, 1.5 h after adding the substrate) was determined. This concentration of each peptide was then used to titer out mab 3G3 (500-25 ng) in the ELISA. The table gives the values of smallest amounts of peptide and mab required for positive signal. The product of these two values for each peptide was determined. The smallest value was ranked first.

Discussion The structure-function relationships of the gonadotropin hormones has been an intensive area of research for many years (25, 26). In the absence of a crystal structure of hFSH, indirect methods must be used to determine antigenic sites and the receptor binding domain(s) on hFSH. The mabs used in these studies were raised after immunization of mice with heterodimeric hFSH. mabs were specific for /3-subunit of hFSH and bound with higher affinity to hFSH/? as the free subunit than to hFSH/? complexed with hFSHa in heterodimeric hFSH. The data suggest that free hFSH/? subunit has a larger epitope which is accessible to mabs than hFSH/? in heterodimeric hFSH because some of this epitope may be blocked by the a-subunit. The animals were immunized with heterodimeric hFSH and not free hFSH/? subunit. It is not known whether subunits dissociate when heterodimeric FSH is vigorously emulsified in adjuvants and injected. After immunization, these protein molecules may be processed and released into the circulation, where B cells can bind the processed species. It is likely that during this processing, the subunits in heter-

to different peptides. The data suggest that none of the mabs bound to peptide 103-110. 3G3 bound to all the remaining peptides. None of the other mabs showed binding to peptide 52-65. In addition mab 3B7 did not bind peptides 16-36 and 95-103. mab 3B8 did not bind peptide 49-67. Of all the peptides giving a positive signal, 95-103 was weakest. Peptide 81-100 was not tested. Effect of mab on FSH binding to receptor It was of interest to assess directly whether mabs could neutralize receptor-binding activity of hFSH. Each of the five mabs, as well as two control ascites fluids, were dialyzed, and then tested for their effect on the binding of hFSH to receptor (Figs. 2 and 3 and Table 6). A doserelated inhibition of hFSH binding to receptor was observed for all five mabs, but as expected, no inhibition was observed when either of the control ascites fluids were used. Control ascites 8A11 and 1H12 demonstrated

TABLE 5. Specificity of mab 3G3, 3B7, 3B8, 4E9, IB 10 for different hFSHjS peptides in the ELISA. Comparison of binding of hFSHjS adsorbed (Ad) and unadsorbed (U) mabs 3G3 Peptide hFSH/3 7-19 1-20 16-36 33-58 49-67 52-65 66-85 95-103 103-110

4E9

1B10

3B8

3B7

IVlttoo

(nmol) (5ng) (6) (41) (17) (.3) (.3) (37) (5) (5) (40)

Ad

U 1.42 2.0 1.78 1.44 0.58 0.93 0.83 1.63 0.20 0.14

± ± ± ± ± ± ± ± ± ±

0 0 0.05 0.04 0.01 0.05 0.07 0 0.01 0.00

0.12 ± 1.7 ± 0.40 ± 0.21 ± 0.24 ± 0.17 ± 0.24 ± 0.32 ± 0.07 ± 0.15 ±

0.02 0.06 0.07 0.06 0.03 0.01 0.02 0.02 0.00 0.016

U 1.83 1.14 1.22 0.44 0.34 0.75 0.44 0.89 0.30 0.12

± ± ± ± ± ± ± ± ± ±

Ad

U

Ad

0.30 2±0 0.09 -0.01 ± 0.02 0.06 0.81 ± 0.00 1.39 ± 0.05 0.99 0.07 0.93 ± 0.03 0.69 ± 0.00 0.37 0.05 0.12 ± 0.02 0.58 ± 0.00 0.37 c 0.02 0.24 ± 0.01 0.26 ± 0.01 0.19 d 0.25 ± 0.00 0.02 0.02 0.46 ± 0.06 0.02 0.36 ± 0.05* 0.17 ± 0.00 0.32 0.05 0.42 ± 0.01 1.59 ± 0.00 0.72 0.19 ± 0.00 0.07 0.01 -0.05 ± 0.00 6 0.02 0.08 ± 0.01 -0.01 ± 0.01 -0.02

± 0.06 ± 0.05 ± 0.00 ± 0.00 ± 0.01 ± 0.00 ±0.1" ± 0.07 ± 0.02 ± 0.006

U 1.54 1.07 0.35 0.66 0.27 0.54 0.02 0.43 0.19 0.07

± ± ± ± ± ± ± ± ± ±

U

Ad 0.26 0.21 ± 0.01 0.06 0.77 ± 0.01 0.03 0.13 ± 0.02 0.09 0.54 ± 0.016 0.00 0.17 ± 0.00 0.04 0.17 ± 0.01 0.00 0.01 ± 0.00* 0.03 0.14 ± 0.01 0.00 0.189 ± 0.01" 0.02 0.03 ± 0.003"

1.37 0.83 1.65 1.17 0.35 0.47 0.47 1.51 0.22 0.12

± ± ± ± ± ± ± ± ± ±

Ad 0.15 0.01 0.24 0.09 0.01 0.02 0.05 0.05 0.08 0.02

0.93 0.64 0.79 0.64 0.13 0.39 0.41 0.88 -0.004 0.11

± ± ± ± ± ± ± ± ± ±

0.06 0.00 0.00° 0.03 0.01 0.05" 0.016 0.08 0.03 0.0086

mab 3G3 (2 jig/ml) and equimolar concentrations of other mabs were incubated with 3.5 fig/m\ hFSHjS overnight at 4 C. hFSH/3-adsorbed and unadsorbed mab were then tested for their binding to peptides in the ELISA as described in Materials and Methods. The values (mean ± SE) represent absorbance at 410 nm. Student's paired t test was applied to determine the differences between adsorbed and unadsorbed value for each peptide. Values without superscripts were significantly different at P < 0.05. Values with superscript a, c, and d were significantly different at P values 0.1, 0.09, and 0.07, respectively. Values with superscript b were not different P

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MAPPING OF HUMAN FSH/3 EPITOPE

664

Endo • 1990 Vol 127 • No 2

TABLE 6. Determination of FSH receptor binding inhibitory activity of mabs

3G3

1B10 4E9 3B7 3B8

100

1,000

10,000

100,000

RECIPROCAL ANTIBODY DILUTION FIG. 2. Inhibition of hFSH receptor binding by monoclonal antibodies, 3B7, 3B8, and 1B10. Each tube contained [125I]hFSH (100,000 cpm) and various dilutions of antiserum, preincubated at 25 C for 2 h. Then testis membranes containing FSH receptor were added (350 nl, 10 ng) followed by further incubation at 25 C overnight. The reaction was terminated by addition of 1 ml ice-cooled Tris buffer (0.05 M pH 7.5) and centrifuged at 1250 x g for 30 min. All ascites including the control (1H12) were dialyzed before use in the assay. 100% indicates 9,500 cpm FSH bound to receptor.

100

1,000

10,000

100,000

RECIPROCAL ANTIBODY DILUTION FIG. 3. Inhibition of hFSH receptor binding by mabs 3G3 and 4E9. The experimental protocol is identical to the legend description in Fig. 2. Control ascites utilized was 8A11.

odimeric hFSH are dissociated. It is known that subunits in heterodimeric hFSH easily dissociate at acidic pH. The processing of antigens for example by macrophages takes place at acidic pH (27). Although immunization was with heterodimeric hFSH, the immune system appears to have identified and responded to epitopes on individual hFSH/3 molecule, mabs so obtained bound with higher affinity to hFSH/3 as the free subunit than to hFSH/3 complexed with hFSHa in heterodimeric hFSH. On the basis of conventional epitope mapping studies, all mabs except for mab 3B7 appeared to recognize a similar epitope. In the case of the synthetic peptide ELISA also, mab 3B7 was different from other mabs. It

ID 6 0

Molarity" X 10"6

X 10" (ml)

X 10"12 (moles)'

2.32 2.14 1.88 0.19 1.09

0.047 0.068 0.36 1.67 0.42

1.03 1.46 6.78 3.17 4.57

3

6

° Moles of antibody per liter assuming two sites per molecule estimated by computerized curve fitting (23). "Volume of ascites fluid required to inhibit 50% of [125I]hFSH binding to FSH receptor. c Moles of antibody required to inhibit 50% of [126I]hFSH binding to FSH receptor.

did not bind peptides 16-36 or 95-103, whereas other mabs did albeit weakly. All mabs bound peptides 7-19, 1-20, 33-53, and 66-85. However, none of the mabs bound the C-terminal peptide 81-100 or 103-110. No mabs except 3G3 showed binding to peptide 52-65. Positive control mab 46.3H6.B7, which is specific for hFSH/? and obtained from a different fusion, showed peptide specificity similar to that of 3G3 (data not included). From the epitope mapping study, it appears that except for the C-terminal end (region of hFSH,S, 81-100, 103110, 95-103), the remaining segment of hFSHjS is highly antigenic. The binding of mab or receptor to more than one region of a hormone molecule has been reported by others (25, 26) using synthetic peptides in RIA or receptor inhibition assays. These studies have shown that 57 peptides, of different regions of hCG, bind to the same mab or inhibit binding of hormone to receptor with different potencies (ED50). In our study we have tried to rank the peptides in terms of their interaction with the mabs by determining the least amount of peptide and mab required for a positive signal in the ELISA. From the titration data using different concentrations of peptides or mab 3G3, peptides 33-53, 49-67, and 66-85 were considered to be more antigenic than others. This was because these peptides, together with mab 3G3, were required in lesser amounts than other peptides for positive signal in the ELISA test. The data also suggest that most of the hFSH/? molecule has a conformation enabling all the antibody recognizable regions to be in close proximity to each other. It seems reasonable to suggest that because there are 6 disulfide bridges in hFSH/?, amino acids far apart in sequence may be brought closer after folding. One mab, specific for hCG/3 molecule, has been reported by Bidart et al. (28) to bind hCG/? peptide sequences 1-7 and 82105. As suggested by them, these regions of hCG/3 could have been brought in close proximity by disulfide bridges between cystine residues at positions 9 and 90. Fujiki et al. (29) have assigned disulfide bridges between cystine

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MAPPING OF HUMAN FSH/3 EPITOPE SECONDARY STRUCTURE OF hFSHP 1 TO 111 50

75

100

5.0 HW Hydrophilicily •5.0 200.0 Log Surface Prob. 0.1 1.2 Flexibility 0.8 1.7 Antigenic Index •1.7

CF Turns CF Alpha Helices CF Beta Sheets GOR Turns GOR Alpha Helices GOB Beta Sheets Glycosyl. Sites

100 AMINO ACID NUMBER FIG. 4. Antigenicity index (AI) profile of hFSH/J derived from the program PEPTIDESTRUCTURE and PLOTSTRUCTURE of the sequence analysis software package of the Genetics Computer Group (version 5). Regions 1-4, 12-19, 37-49, 69-73, 83-96, and 107-111 of hFSH/8 are predicted to be antigenic by this method. These regions contain AI values greater than zero and 4 or more values with AI greater than 0.3. Region 51-68 contains 8 values greater than 0.4 and 10 values less than zero. The present epitope mapping study confirms that regions 1-4, 12-19, and 69-73 are antigenic. These antibodies do not appear to recognize predicted regions 83-96 and 107-111. Our studies show that region 49-67 is also antigenic.

residues at positions 3-28, 17-51, and 32-104 of the hFSHjS molecule. This would suggest that amino acid residues 50-100 can be brought in close proximity to the N-terminal residues 1-32. The positions of other three disulfide bridges between cystine residues at positions 20, 66, 82, 84, 87, and 94 are as yet not assigned. Thus it is likely that mabs used in the present study are recognizing an assembled epitope comprising amino acids present predominantly in peptide sequences 33-53, 49-

665

67, and 66-85. A number of mutually overlapping continuous and discontinuous epitopes can exist on a protein surface and the whole surface can be antigenic (30, 31). Synthetic peptides of longer length were used in this study to provide an opportunity to generate some of the secondary and perhaps even tertiary structural specificity of the antigen (30). Therefore, it may be that the mabs in the present study are reacting against conformationally defined determinants of the assembled epitope. It is as yet not clear whether these determinants are present as a continuous or discontinuous stretch of the peptide sequence. Jameson and Wolf (32), have designed a computer program that integrates flexibility parameters (from Chou-Fasman and Robson-Garnier secondary structure predictions) with hydropathy (Hopp and Wodd predictions) or surface accessibility (predictions by Janin et al., and Emini et al.,) values in a weighted fashion, to produce a plot of surface contour, referred to as the antigenic index (AI). According to that analysis, regions 1-4, 1219, 37-49, 69-73, 83-96, and 107-111 of hFSH/3 are predicted to be antigenic (Fig. 4). These regions contain all amino acids with AI greater than zero and 4 or more amino acids with AI greater than 0.3. Region 51-68 contains 8 amino acids whose AI is 0.45 or more and 10 amino acids whose AI is less than zero. The present epitope mapping study partly confirms this prediction. The epitope mapping study has identified peptides 1-20, 33-53, and 66-85 of hFSH/3 to which all the mabs bind. These peptides include the regions 1-4, 12-19, 37-49, and 69-73 predicted to be antigenic by the method of Jameson and Wolf. Our data do not confirm the prediction that C-terminal end region (85-95 and 107-111) is antigenic. Region 51-68 of hFSHjS is interesting in that it contains 44% of the amino acids with AI 0.45 or more and remaining 56% with AI less than zero. Furthermore, amino acids in this region follow a pattern in which two amino acids with AI equal to 0.45 or more are followed by three amino acids with AI less than zero. Peptides 49-67 and 52-65 fall in between this region. Our studies have shown that four out of five mabs react with peptide 49-67 and do not react with peptide 52-65. All mabs were able to inhibit binding of [125I]hFSH to the receptor. Four times less concentrations of 3G3 and 1B10 were required than 3B7,3B8, or 4E9 in the receptor inhibition study. The difference in the receptor inhibition property of these mabs could be attributed to their different affinities for hFSH/3. It is notable that peptide sequence 33-53 of hFSH/? has been shown to inhibit binding of [125I]hFSH to its receptor (7, 33). Our preliminary studies using synthetic peptides to inhibit binding [125I]hFSH to receptor have also identified peptide 3353 and additionally peptides 49-67 and 66-85 to be strong

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MAPPING OF HUMAN FSH/? EPITOPE

666

inhibitors of [125I]hFSH binding to receptor. The ED50 (mean ± SE), values for peptides 33-53, 66-85, and 4967 are 68 ± 1.34, 76.5 ± 2.1, and 96.1 ± 7.2 nmol, respectively. The present study has determined that except for C-terminal end (region 81-100, 95-103, 103110), the remaining segment of hFSH/3 is highly antigenic. The data also suggest that most of the hFSH/3 molecule has a conformation enabling all antibody recognizable regions to be in close proximity to each other, mabs used in the present study recognize an assembled epitope comprising amino acids present predominantly in peptide sequences 33-53, 49-67, and 66-85. This assembled epitope also contains the determinant for receptor binding.

Acknowledgments We thank Ms. Elizabeth Larkins for typing this manuscript. The gift of hormone and hormone subunits from the National Pituitary Agency and the NIADDK (Baltimore, MD) is highly appreciated.

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Endo • 1990 Vol 127 • No 2

13. Pierce JG, Bloomfield GA, Parson TF 1979 Purification and receptor binding properties of complexes between lutropin and monovalent antibodies against its alpha subunit. Int J Pept Protein Res 13:54 14. Moyle WR, Ehrlich PH, Canfield RE 1982 Use of monoclonal antibodies to subunits of human chorionic gonadotropin to examine the orientation of the hormone in its complex with receptor. Proc Natl Acad Sci USA 79:2245 15. Gighi RR, Moudgal NR 1983 Use of alpha and beta subunit specific antibodies in studying interaction in hCG with Leydig cell receptors. Arch Biochem Biophys 225:490 16. Hwang J, Menon KMJ 1984 Spatial relationships of the human chorionic gonadotropin subunits in the assembly of the hCGreceptor complex in the luteinized rat ovary. Proc Natl Acad Sci USA 81:4667 17. Berger P, Kofler R, Wick G 1984 Monoclonal antibodies against human chorionic gonadotropin: affinity and ability to neutralize the biological activity of hCG. Am J Reprod Immunol 5:157 18. Claflin L, Williams K 1978 Mouse myeloma-spleen cell hybrids: enhanced hybridization frequencies and rapid screening procedures. Curr Top Microbiol Immunol 81:107 19. Dias JA, Huston JS, Reichert Jr LE 1981 Effect of the structure-

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