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Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India
¹ Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560012, India

(Requests for offprints should be addressed to R R Dighe; Email: rdighe{at}mrdg.iisc.ernet.in)

	Abstract

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Human chorionic gonadotrophin (hCG) is secreted during early pregnancy and is required for implantation and maintenance of the pregnancy. Active or passive immunoneutralization of hCG results in termination of pregnancy and this forms the basis of the hCG-based female contraceptive vaccine. However, the ß subunit of hCG possesses 85% sequence homology with the first 114 amino acids of the ß subunit of pituitary human LH (hLH), which is required for ovulation and maintenance of the corpus luteum function during the menstrual cycle. Immunization against hCG or its ß subunit leads to generation of antibodies that can neutralize hLH due to many shared epitopes and hence may cause abnormal menstrual cycles. Therefore, it is essential to identify epitopes that are different in the two hormones. In the present study, we report a monoclonal antibody (MAb) specific for hCG that shows no binding to the isolated subunits. Interestingly, the MAb also does not bind hLH at all. The epitope mapping analysis revealed that this antibody recognizes a unique discontinuous epitope present only in the heterodimeric hCG and is distinct from the unique C-terminal extension of hCGß that is absent in hLHß. The MAb, either as IgG or its recombinant single-chain variable region fragment, inhibited the response to hCG, but not to hLH. Thus, the epitope recognized by this MAb is an ideal candidate antigen for immunocontraception.

	Introduction

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Human chorionic gonadotrophin (hCG) is secreted by the trophoblastic cells of the placenta during early pregnancy (Fox 1970). hCG plays an important role in the rescue of the corpus luteum and maintenance of pregnancy (Strauss et al. 1995). Active or passive immunoneutralization of hCG prevents implantation of the embryo, which is the basis of the female contraceptive vaccine (Talwar et al. 1994, Dirnhofer & Berger 1995, Stevens 1996, Delves 2004). One such vaccine has completed phase II efficacy trials in women (Talwar et al. 1994). The immunogen for this vaccine consists of a heterospecies dimer (HSD) of the ß subunit of hCG (hCGß) associated non-covalently with the subunit of ovine luteinizing hormone (oLH) and conjugated to either tetanus or diphtheria toxoid as carrier (Talwar et al. 1994). The results of the phase II clinical trials with this HSD vaccine revealed that sexually active women with normal ovulatory cycles did not become pregnant as long as the circulating antibodies were above 50 ng/ml, with no effect on the luteal phase length, and exhibited menstrual regularity, demonstrating the contraceptive efficacy of the vaccine.

However, hCGß possesses 85% sequence homology with the first 114 amino acids of the ß subunit of human LH (hLH), which is not a pregnancy-specific hormone but plays a critical role in ovulation and maintenance of the corpus luteum function in the luteal phase of the female reproductive cycle. Immunization against hCG can thus lead to immunological cross-reaction with hLH due to many shared epitopes between the two hormones, and although the phase II trial of the HSD vaccine did not show any effect on hLH function, prolonged immunization may eventually lead to defects in ovulation and luteal phase defects (Dirnhofer & Berger 1994). In order to circumvent possible cross-reactivity with hLH, attempts have been made to use as the immunogen the 30 amino acid C-terminal peptide (CTP) that is unique to hCGß but absent in hLHß. Antibodies against this peptide are highly specific for hCG, but cannot neutralize the biological activity of the hormone (Louvet et al. 1974). In addition, the CTP is a poor immunogen and generates antibodies with low affinity and lower bioneutralization capacity (Mitchison 1990). Hence, it is essential to identify the epitopes in hCG molecule, other than the CTP region, that are absent in hLH so that these epitopes can serve as potential targets for eliciting highly specific hCG bioneutralizing antibodies in humans.

In the present study, we report a monoclonal antibody (MAb) designated as E12 that displays very-high-affinity binding to hCG specifically and does not cross-react with hLH, as well as the subunits of hCG. Using different strategies, a conformation-specific epitope within the hCG molecule distinct from CTP has been identified.

	Materials and methods

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Chemicals and biological materials

Hormones
The hormones and their subunits – hCG (CR127), hLH, human follicle-stimulating hormone (hFSH), human thyrotrophin (hTSH), hCG, hCGß (CR123) – used in this study were obtained from The National Hormone and Pituitary Program, UCLA, USA.

Biochemicals and radiochemicals
¹²⁵I NaI and [1,2,6,7,16,17-³H]-testosterone were purchased from Perkin-Elmer Life Sciences, Boston, MA, USA. All the restriction enzymes and modifying enzymes required were purchased from Boehringer Mannheim, or from MBI Fermentas (Hanover, MD, USA). Phenyl Sepharose, HiTrap Protein G-Sepharose columns, HiTrap chelating columns used for purification of IgG and recombinant single-chain variable region fragment (ScFv) were purchased from Amersham Pharmacia Biotech. All other reagents were obtained from Sigma Chemical Company.

Production, characterization and purification of MAbs

MAbs against hCG and Pichia-expressed recombinant hCG used in the present study were raised and characterized as described previously (Dighe et al. 1990a). The ability of these antibodies to bind all human glycoprotein hormones or their subunits was demonstrated using ¹²⁵I-labelled probes or ELISAs. Ascites were produced in Balb/c mice and were used for purification of IgG by Protein G-Sepharose chromatography. All animal experiments were carried out under expert supervision after obtaining appropriate clearances from the Institutional Ethical Committee that oversees the usage of animals in the institute.

RIA
To test binding of the antibody to various antigens, ¹²⁵I-hCG/hCG/hCGß/hFSH/hTSH/hLH, were incubated overnight with the desired amount of antibody at room temperature. The antigen–antibody complexes were precipitated by the addition of anti-rabbit IgG raised in goat and polyethylene glycol 6000 as described earlier (Dighe et al. 1990a, Sen Gupta & Dighe 1999), centrifuged at 5000 g, the supernatant discarded and the radioactivity in the pellet counted using a Perkin-Elmer autogamma counter.

ELISA
hCG, hLH and single-chain derivative of hCG (hCGß) and its mutants (50 ng/well) were coated on ELISA plates followed by 1% BSA solution in PBS as blocking buffer. The MAbs (purified IgG or ScFv) were then added to the wells and incubated at 37 °C for at least 1 h and binding was determined as described earlier (Gadkari et al. 2003).

Cloning of single-chain antibody (ScFv)
The single-chain antibody (ScFv) (Huston et al. 1988) was generated from the hybridoma cells producing MAb E12 using the kit purchased from Pharmacia Biotech employing the protocol provided by the manufacturer. The antigen binding ability of the ScFv was determined by ELISA and its ability to inhibit hCG–receptor interaction was determined by a radioreceptor assay (RRA) as described in the case of the parent antibody. The cDNA encoding the ScFv was cloned into Pichia expression vector, pGAPZA (Invitrogen) and fermentation with the clone secreting the ScFv was carried out as described for hCG (Gadkari et al. 2003). ScFv secreted into the medium was purified using a combination of hydrophobic interaction chromatography and Ni²⁺-affinity chromatography as described in the Current Protocols in Protein Science, Volume I Supplement 1 CPPS Unit 9.4.

RRA
The effect of antibodies on hCG–receptor interaction was determined by incubating the antibodies with ¹²⁵I-hCG for 1 h at 37 °C followed by addition of particulate rat LH receptor and continuing the incubation for additional 1 h. ¹²⁵I-hCG bound to the receptor was pelleted by centrifugation at 5000 g and counted in a gamma counter (Sen Gupta & Dighe 1999). The total binding was determined by incubating the receptor with either buffer or normal rabbit serum, while the non-specific binding was determined by incubating ¹²⁵I-hCG with the receptor in the presence of 400 ng/ml unlabelled hCG and was subtracted from the total binding to obtain specific binding.

In vitro bioassay
The in vitro bioassay was performed using mouse testis cell preparation as the source of the Leydig cells as described earlier (Dighe & Moudgal 1983). Briefly, the Leydig cells were obtained by mechanical dispersion of adult mouse testes and suspended in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 50 mM Hepes and 0.1% BSA. hCG was preincubated with the desired antibody diluted in DMEM, Hepes and BSA for 1 h, after which the cell preparation was added to it, and the incubation was continued for another 4 h. At the end of this incubation period, the medium was separated from the cells by centrifugation and testosterone secreted into the medium was estimated by RIA as described earlier (Dighe & Moudgal 1983).

	Results

Top Abstract Introduction Materials and methods Results Discussion References

 
Generation and characterization of MAbs

MAb E12 was raised against the recombinant hCG expressed using the Pichia pastoris expression system. The fusion and selection of the clones secreting the antibodies were carried out as described earlier. The antibody was selected based on its ability to bind to urinary hCG by ELISA and confirmed by demonstrating binding to ¹²⁵I-hCG. Other MAbs used in this study have been described earlier (Dighe & Moudgal 1983, Dighe et al. 1990a).

Characterization of MAb E12

The antigen specificity of MAb E12 was determined using ELISA and confirmed by binding assays using radioiodinated hCG, hLH, hFSH, hTSH, hCG and hCGß. As shown in Fig. 1a and b, MAb E12 could bind hCG, both in the ELISA and in the radioiodinated hCG binding assay, but in the same experiments failed to bind hLH, clearly demonstrating that the antibody recognizes an epitope present in hCG that is completely absent in hLH. MAb E12 also failed to bind ¹²⁵I-hCG, ¹²⁵I-hFSH and ¹²⁵I-hTSH, indicating that the epitope is not present in the subunit (Fig. 1c). Similarly, it also failed to bind hCGß in both assays, demonstrating that the MAb specifically recognizes the unique epitope present in the heterodimeric hCG (Fig. 1c). Lack of binding to the isolated ß subunit also suggested that the antibody does not recognize the C-terminal portion of hCG ß subunit that distinguishes it from the hLH ß subunit.

View larger version (24K): [in this window] [in a new window]   Figure 1 Characterization of E12 antibody. The antigen specificity of the antibody was determined by ELISA, as well as by RIA. The values presented are the means of duplicates and each experiment was carried out at least twice. (a) Binding of MAb E12 to hCG and hLH was carried out using ELISA with a large excess of the MAb (1 µg/well). In the same experiment, binding of both antigens to the polyclonal hCG antiserum was also determined to demonstrate the integrity of the antigens. (b) Binding of 125I-hCG/hLH to increasing concentrations of MAb E12 IgG was tested by RIA. The non-specific binding was determined by incubating the radioiodinated probes in the absence of MAb E12 and was subtracted from the total binding obtained in each case. (c) Cross-reactivity of the antibody to other members of the glycoprotein hormone family, as well as the subunits of hCG was determined by RIA using 125I-hCG/hCGß/hCG/hFSH/hTSH as probes. The data shown here represent the percentage of the total input radioactivity that was bound to the antibody in each case. (d) Binding of 125I-hCG to the antibody was determined in the presence of increasing concentrations of unlabelled hCG and the binding data obtained were converted into a Scatchard plot. n=3–4.

Effect of MAb on binding of ¹²⁵I-hCG to the receptor

MAb E12 was preincubated with ¹²⁵I-hCG for 1 h at room temperature followed by addition of the receptor and determination of the receptor-bound hormone. As shown in Fig. 2, MAb E12 inhibited binding of ¹²⁵I-hCG to the receptor, indicating that the epitope recognized by the antibody is important for hormone–receptor interaction.

View larger version (11K): [in this window] [in a new window]   Figure 2 RRA. The effect of antibody on binding of hCG to its receptor was tested by RRA using 125I-hCG as the tracer and rat testis membrane preparation as the source of LH receptors. 125I-hCG (approximately 200 000 c.p.m.) was preincubated with either 1 µg MAb E12 IgG, buffer or normal mouse IgG for 1 h at room temperature. The receptor preparation was added and incubation continued for 1 h more. At the end of incubation, the receptor-bound radioactivity was determined. The non-specific binding was determined by incubating the labelled hormone with the receptor preparation in the presence of a large excess of unlabelled hCG. n=3–4.

The ability of MAb E12 to inhibit responses to hCG and hLH was next investigated using the mouse Leydig cell bioassay. As shown in the Fig. 3, MAb E12 or its ScFv could inhibit the response to hCG, but not to hLH. These data confirmed that MAb E12 recognizes the unique epitope present in hCG that is not present in hLH.

View larger version (18K): [in this window] [in a new window]   Figure 3 In vitro Leydig cell bioassay. Effect of antibody on response to hCG and hLH was determined by preincubating the hormones with either MAb E12 IgG or E12 ScFv expressed using the Pichia pastoris expression system for 1 h at room temperature followed by addition of the mouse Leydig cell preparation and continuing the incubation at 32 °C for another 4 h. Testosterone secreted into medium was determined by RIA. Values presented are means ± S.D. of quadruplicates. n=3–4.

Having demonstrated the ability of MAb E12 to distinguish between hCG and hLH, three different approaches were employed to locate the epitope.

The first approach employed the strategy of carrying out RIA displacement analysis with several chemically modified derivatives of hCG, as described by Venkatesh & Murthy (1997) and Venkatesh et al.(1999). Different chemical modifications or enzymatic digestions of hCG were carried out under non-reducing conditions and the ability of each derivative to inhibit binding of ¹²⁵I-hCG to the MAb was determined. Depending on the retention of the activity as estimated by displacement analysis following a specific modification, the contribution of specific residues towards the formation of epitopes can be determined. The contribution of the specific residues is likely to be minimal if the modification results in the retention of more than 70% of the RIA displacement activity. Residues are likely to be proximal to the epitope if 20–70% of the activity is retained, while less than 20% activity means the residues are at the core of the epitope. The exact location of the epitope can be predicted taking into account the sequence and the three dimensional structure of the protein. Table 1 summarizes the modifications carried out, residues modified and the retention of the hCG-like activity by RIA with MAb E12 with ¹²⁵I-hCG as the probe. Binding of hCG to the antibody was completely abolished when the residues R, Y, M, H and acidic residues either D or E were chemically modified. In addition, the epitopic region seemed to be very sensitive to digestion with trypsin and chymotrypsin, confirming the contributions of the basic and aromatic residues to the epitope. Lack of effect of carboxypeptidase on the activity also confirms that the C-termini of both subunits do not contribute to the epitope. Finally, treatment with formic acid resulted in more than 80% loss in activity, confirming that the epitope is generated only when both subunits are annealed, generating the heterodimer.

View larger version (18K): [in this window] [in a new window]   Figure 4 ELISA with mutants of hCG. Equal quantities of urinary and recombinant hCG (phCG), single-chain derivatives of hCG (hCGß) and its mutants were coated onto the ELISA plate and binding of MAb E12 to each one of them was determined by ELISA. The single-chain mutant hCGP38Aß has been designated as hCG*ß, hCGT54Aß as hCGNß and hCGQ13K, E14K, P16K, Q20Kßas hCG +ß. n=3–4.

View larger version (30K): [in this window] [in a new window]   Figure 5 Comparison of hCG and hLH. Shown in dark grey is the subunit, while the ß subunit is shown in light grey. The horizontal line marks the boundary of the upper and the lower parts of hCG. The regions in hCGß that are different from those in hLHß have been encircled. The diagram was constructed using the ‘Setor’ program (Evans 1993).

View larger version (42K): [in this window] [in a new window]   Figure 6 Sequence alignment. The sequence alignment between hCGß and hLHß was obtained using the ‘malign’ program. The residues that show variations between the two hormone subunits are highlighted in grey. The residues that are at the interface are marked with ‘!’ below. The structural environment of all the residues is represented using ‘JOY’ (Mizuguchi et al. 1998).

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

MAb E12 was generated by immunizing a mouse with Pichia-expressed hCG (Gadkari et al. 2003), but was selected on the basis of binding to urinary hCG. Interestingly, the antibody also does not bind either subunit of hCG and therefore recognizes a unique epitope that is generated when the two subunits combine to give rise to a heterodimeric hormone capable of binding to the receptor and eliciting a biological response. The antibody is quite intriguing as it does not recognize hLH at all, signifying that this epitope is present in hCG, but not in hLH. Further, the ability of this antibody to inhibit binding of the hormone and a response suggests that the epitope is important for the bioactivity of the hormone and therefore an ideal epitope for eliciting an immunological response that neutralizes hCG, but not hLH. The antibody failed to decrease binding of the hormone to the receptor if the latter was preincubated with the hormone, suggesting that the epitope is not accessible to the antibody when the hormone is complexed with the receptor (data not shown). These results were similar to those obtained with the polyclonal antisera (Dighe & Moudgal 1983).

It is evident that MAb E12 recognizes a discontinuous epitope. Exact location of such epitopes in complex molecules such as hCG is usually a very difficult task (Charrel-Dennis et al. 2004). We have used three different approaches to identify the MAb E12 specific epitope. The most striking difference between hCG and hLH is the C-terminal 35 amino acids present in hCGß that are absent in hLHß. However, the antibody is not likely to recognize this region of hCGß as it does not bind the isolated ß subunit of hCG. The sequence alignment of hCGß and hLHß reveals several differences between the two subunits at positions 8, 10, 15, 42, 47, 51, 58, 77, 82, 83, 89, 91, 92, 98, 112 and 114–145, the last being the CTP present in hCGß. Based on the RIA analysis of chemically modified derivatives of hCG, it can be concluded that the residues that are likely to contribute to the epitope are methionine, arginine, aspartic acid, tyrosine, phenylalanine and histidine. In view of these data and also the differences between hCGß and hLH, the residues that are most likely to contribute are ßM41, ßR43, ßG47, ßA51, ßN58 and ßD61, and E56, Y65, F74, E77 and H79. These residues are present in the L2 loop of the ß subunit and the L3 loop of the subunit respectively (Lapthorn et al. 1994, Wu et al. 1994). Some contribution from the L1 loop of the subunit to the epitope is also possible as the antibody failed to bind single-chain hCG^{Q13K, E14K, P16K, Q20K}ß (Sunita 2003, S Setlur and R R Dighe, unpublished observation).

Based on these data, we conclude that the epitope recognized by MAb E12 is present in the L2 loop of the ß subunit and L3 loop of the subunit (Fig. 7). In the past, one such antibody recognizing hCGß L2 loop that binds hCG heterodimer with higher affinity as compared with the free hCGß subunit (epitope designated as c3) has been reported by Berger et al.(2002). However, the exact location of the epitopic region has not been reported. Studies carried out by Schwarz and co-workers revealed that the antibodies belonging to the groups c1 and c2 are closely related to c3 group of antibodies recognizing the epitopes generated only on heterodimerization of hCG subunits and thus are similar to MAb E12 reported in the present study. These MAbs show inhibitory effects on hCG–receptor interaction similar to those shown by MAb E12 (Schwarz et al. 1988). We have located the epitopes of two other ß subunit-specific MAbs capable of recognizing both hCG and hLH in the determinant loop of the ß subunit. An subunit-specific MAb was shown to bind hCG and hLH in the L3 loop of the subunit (R A Gadkari and R R Dighe, unpublished observations). In contrast, MAb E12, which has unique specificity, binds hCG in the L2 of the ß subunit and the L3 of the subunit. Additional evidence in support of this argument is that the surface potential around the proposed epitopic region in hCGß is significantly different from that in hLHß (Fig. 8). The charge on the hCG molecule in this region is more negative than that in hLH.

View larger version (43K): [in this window] [in a new window]   Figure 7 Epitopic region. The figure depicts the epitopic region of MAb E12. The subunit has been shown in dark grey, while the ß subunit is in light grey. The residues of both subunits identified as important for antibody binding have been shown as a ‘ball and stick’ model. The diagram was constructed using the ‘Setor’ program (Evans 1993).

View larger version (98K): [in this window] [in a new window]   Figure 8 Surface models of hCG and hLH. The surface models of hCG and hLH were generated using the ‘Grasp’ program (Nicholls et al. 1991). The proposed epitopic region of MAb E12 has been encircled in both hCG and hLH models to highlight the differences in the potentials of the two hormones in that region.

MAb E12 can also be used for passive immunization in cases of emergency contraception. The ScFv that has been produced in this study is functional in terms of binding the hormone and blocking its response. We have expressed this antibody using Pichia expression vector, established fermentation protocols with the clone producing the antibody, purified it, and are in the process of characterizing it further.

	Acknowledgements

 
The authors wish to thank Dr G Satyanarayan Murthy for providing the modified derivatives of hCG. The work presented here was supported by grants from the Department of Biotechnology, Government of India, the Indian Council of Medical Research and the University Grants Commission, New Delhi, the Wellcome Trust, London (senior fellowship in Biomedical Science to N S) and the Council of Scientific and Industrial Research, New Delhi (fellowships to S R and N). The authors wish to thank Cadila Pharmaceuticals Limited, Ahmedabad, India for their generous support. The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work.

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Received 8 February 2005
Accepted 21 March 2005

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