AMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY 23:22-25 (C) 1990 MUNKSGAARD

Toward Regulation of Gonadal Function by a Synthetic Hybrid Molecule Composed of Gonadotropin and Fc Fragment of Immunoglobulin G G.A. JOHNSON, C. WILKEN, E.A. VAN KIRK, E.L. BELDEN, Department of Animal Science, University of Wyoming, Laramie

AND

A conjugate of human chorionic gonadotropin (RCG) and Fc fragment of immunoglobulin G was prepared by covalent cross-linking using the heterobifunctional reagent, N-succinimidyl 3-(2-pyridyldithio) propionate. Mouse Leydig tumor cells expressing receptors for luteinizing hormone were specifically lysed in vitro as a consequence of complement fixation via the Fc component of the hybrid molecule. Furthermore, administration of RCG-Fc to rams caused an acute depression in circulatory testosterone. This novel concept of targeted inhibition of gonadal function could prove to have future applications in control of reproductive processes. (Am J Reprod Immunol. 1990; 23:22--25.) Key words: Testis, human chorionic gonadotropin, immunoregulation, complement, cytolysis. ABSTRACT:

INTRODUCTION

Reproductive behavior and fertility of domesticated animals have traditionally been manipulated by castration. This surgical procedure undoubtedly involves stress and can be complicated by hemorrhage and/or microbial infection. We have begun to consider an alternative, possibly more humane strategy, toward regulation of animal reproduction. The basic notion is to instigate the immune system into site-directed cytolysis, the specific target being steroidogenic gonadal cells. Human chorionic gonadotropin (HCG) was chemically linked to the Fe component of immunoglobulin (Ig) G. The hormone and fragment of antibody theoretically constituted gonadal cellular recognition and lethal (i.e., complement-fixing) moieties of the conjugate, respectively (Fig. 1). MATERIALS AND METHODS

Preparation of HCG-Fc Pyridyldithio propionate (PDP) was introduced at a 1:1 ratio into an epsilon amino group of a residue of lysine of HCG and isolated Fc by reaction with N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP).4 Thiol-disulfide exchange (conjugation) was carried out by mixing PDP-HCG with thiolated Fc (Fig. 2). Conjugate was purified by gel filtration (Fig. 3).

Submitted for publication January 18, 1990; accepted April 5, 1990. Address reprint requests to W.J. Murdoch, Department of Animal Science, University of Wyoming, Laramie, WY 82071-3684.

W.J. MURDOCH

Binding of HCG-Fc to Leydig Cells Competition of HCG and HCG-Fc (ng HCG equivalent) with radioiodinated (lactoperoxidase method) ovine LH (3 ng; NIADDK-I-3) for cellular binding sites was assessed utilizing mouse Leydig tumor cells (MLTC).5 Cells were maintained in RPMI-1640 culture media (Gibco, Grand Island, NY) containing 25 mM HEPES, 25 mM sodium bicarbonate, 100 units/ml penicillin, 0.1 mg/ml streptomycin and 5% fetal calf serum (pH 7.4) in an atmosphere of 5% CO 2 at 37 C. Aliquots of cells (50,000) were dispensed into polypropylene culture tubes (coated with bovine serum albumin) and hormonal preparations were added (final volume of 0.11 ml of 0.05 M Tris HCI buffer containing 0.1% gelatin, 1 mM CaCI 2 , and 1 mM NaN 3 ; pH 7.4). Incubations were carried out for 16 h at 25°C. Cells were pelleted by centrifugation (15 min; 17,000g). Supernatants were discarded and cells counted in a gamma spectrometer. Nonspecific binding of radiolabelled LH was determined in the presence of excess HCG (10 u.g). Bioactivity of the Fc Component of the Conjugate Conjugate or free Fc were aggregated with Protein A (1:640 dilution in veronal-buffered saline [VBS]; Enzyme Center Inc., Boston, MA). Guinea pig complement was added to the reaction mixture (1:15 dilution in VBS; Gibco; 30 min at 25°C). Supernatant was removed and serially titrated. Consumption of complement was measured by incubating dilutions of supernatant in a 2% solution of hemolysin-sensitized sheep erythrocytes (30 min at 37°C). Hemolysis was considered a positive test for undepleted complement. In Vitro Killing A lytic effect of conjugate and HCG + Fc (unconjugated mixture), as monitored by cellular release of chromium (51Cr),6 was investigated using dispersed Leydig cells incubated either in the absence or presence of complement. Chromium-labelled cells (50,000/ 0.15 ml Tris-gel buffer) were allotted to wells of microtiter plates, with or without complement (0.1 ml of 1:10 dilution in VBS), and incubated for 90 min at 37°C. Radioactivity was quantified in the supernatant recovered from cells following centrifugation (5 min; 1000g). Data were expressed as a percentage of specific maximal release of 51Cr ([experimental counts per min background releasel/ltotal releasable counts per min background release] x 100). Maximal release was estimated by exposing cells to 3% Triton-X. Background release was determined by incubating cells (± complement) without hormonal preparations. Conjugate and HCG + Fc (high dose) were also tested against a line of cells completely void of receptors for gonadotropin (Sp2 mouse hybridoma), The experiment was repeated eight

23

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In Vivo Action of RCG-F c on Testicular Function Yearlin g rams were injecte d intrave nously with RCG-F c (0.04 mg RCG equiva lent), an unconj ugated mixtur e of RCG and Fc (amoun ts equal to each component of conjug ate), or 5 ml vehicle (PBS). Six animal s were random ly assigne d to each group. Blood sample s were collected by jugula r venipu ncture at hourly intervals for 8 h beginn ing just before (0 h) admini stratio n of treatm ents. Individ ual sample s were then collected on days 12 and 21 post-tr eatmen t. Serum was analyze d for concen tration s of testost erone by radioim munoa ssay." Data within time of sample collecti on were subjected to one-wa y analys is of varianc e. Specific means were compar ed by least signifi cant differen ce only when a signifi cant F-test was indicat ed.

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entFig. 1. Proposed mechani sm by which RCG-Fc incites complem mediated lysis (classica l pathway ) of the plasma membran e of Leydigs cells. Antigens expresse d on the surface of microbes, by cancerou cells, or by cells impacted in autoimm une disorders , can be uniquely latrecognized by Fab compone nts of antibodie s. Antibodi es migrate erally and aggregat e along the surface of the target cell upon bindsites ing-rem iniscent of protein-l ike hormone s.' This brings receptor for complem ent located within the Fe tail of the antibody close enough together to allow for fixation of complem ent, formatio n of the "attack" complex, and cellular perforati on.v" Chorioni c gonadotr opin binds The with high affinity to the receptor for luteinizi ng hormone (LR).' reFab compone nts of a complem ent-fixin g antibody were simply of placed with RCG in order to alter the cellular recogniti on capacity the lytic molecule.

RESULT S

A high degree of compet itive displac ement of radioiodinat ed LR by the conjug ate (versus RCG) was exhibited (Fig. 4). It was also ascerta ined that the Fc compo nent of the conjug ate was as capable of fixing comple ment as unconj ugated Fc when bound to Protein A (i.e., supern atant recove red from reactio n mixtur es did not cause lysis of sensitiz ed sheep red blood cells at dilutio ns at which incuba tions withou t conjug ate or Fc exhibit ed definit ive hemoly sis).

times (each determ ination within experim ent was performed in triplica te). The averag e effect agains t MLTC s of each dose of conjug ate was contras ted to the unconj ugated mixtur e of RCG and Fc by Dunne tt's test (percen tages were transfo rmed [arc sin V%] for the purpos e of statisti cal analysi s).

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of Fig. 2. Method of conjugat ion of RCG and Fe. (A) Preparat ion was PDP-RC G. One mg RCG (Sigma Chemica l Co., St. Louis, MO) to dissolved in 2 ml phosphat e-buffere d saline (PBS) and subjected ultrafiltr ation (20 min; 2,000g) over a 30,000 molecula r weight cutoff was membra ne (Amicon, Danvers , MAl. Retentat e containi ng RCG was recovere d (2 min; 1000gl and diluted to 1 ml in PBS. This solution mixed for 30 min with 0.04 mg SPDP (Sigma) in 0.05 ml absolute ethanol (25°C). Derivati zed RCG was separate d from excess reagent and low molecula r weight reaction product by centrifug ation, recovOne ered, and reconstit uted in 1 ml PBS. (B) Preparat ion of PDP-Fc. mg chromop ure sheep Fe prepared from IgG by cleavage with papain in (Jackson Immuno reasearc h, West Grove, PAl was diluted to 1 ml was PBS. Reaction of Fe with SPDP (0.16 mg in 0.05 ml ethanol) carried out for 30 min at 25°C and PDP-Fc was isolated as indicated

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Fc above. An average of one PDP was introduc ed per mole of RCG and as determin ed by spectrop hotometr ic measure ment of release of pyri-l dine-2-th ione from reduced products . (C) Reductio n of the 2-pyridy disulfide group in PDP-Fc. Retentat e containi ng PDP-Fc was diluted in 2 ml 0.1 M sodium acetate buffer containin g 0.15 M sodium chlofor ride and 50 mM dithiothr etiol (DTT; Sigma) (pl-l 4.5) and reacted n, 30 min at 25°C. Thiolate d Fe was purified by ultracent rifugatio recovere d, and reconstit uted in 1 ml PBS. (0) Conjuga tion of PDPRCG with thiolated Fe (25°C). The reaction was terminat ed at 1 hand pyridine -2-thione was removed from the reaction mixture by ultrafilbetration. Romocon jugates are not formed to an apprecia ble extent of cause thiol-dis ulfide exchang e occurs much faster than oxidation thiol groups.

24

JOHNSON ET AL.

MILLILITERS

Fig. 3. Chromatographic separation of HCG-Fc from unreacted HCG and Fc by filtration over Sephadex G-75 (l x 95 cm column). Proteins were eluted with PBS. Milliliters 5 to 8 (containing conjugate) were pooled. A mixture of unconjugated HCG and Fe was voided from the column between fractions 12 and 16. Conjugate was stored at 4°C in a sealed ampule purged with nitrogen gas. Subsequent chromatographic analysis indicated that the hybrid molecule was stable for several weeks under these conditions.

Specific killing of Leydig cells in suspension was induced by RCG-Fc under conditions in which incubation medium was supplemented with complement. Cells not expressing receptors for LR were not lysed by RCG-Fc (Fig. 5). Within 1 h after initiation of treatments to rams it was apparent that both RCG-Fc and RCG + Fc caused a rise in circulatory concentrations of testosterone versus vehicle controls. Injection with conjugate was followed by a precipitous fall in serum concentrations of testosterone (Fig. 6). A depression in level of testosterone apparently persisted until 12 days following administration of conjugate (Fig. 7). DISCUSSION

Initial testing of cellular lysis by RCG-Fc in vitro indicated a statistically significant effect. Nevertheless, the percentage of specific killing seemed relatively low. We became more encouraged by these data when we determined retrospectively (unpublished observation) by autoradiography that only about 10% of MLTCs expressed detectable receptors for LR within a period of several hours after removal from the culture

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flask (i.e., if data were expressed as a percentage of sensitive cells, then the kill would have been near 100% in the case of the medium dose of conjugate). It was evident that administration of conjugate caused an increase, then a rather prompt depression, in serum concentrations of testosterone within the systemic circulation of rams. An analogous pattern in ovarian secretion of progesterone is observed in ewes during functional luteolysis induced by prostaglandin Fz

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by C3b, and become activated.P'"? An antibody-dependent, cell-mediated killing mechanism has been implicated in the treatment of RIV-1 disease. The rationale is that cells infected by RIV-1 or virus itself would be targeted for elimination by a hybrid molecule constructed of a CD4 receptor domain of T cells/monocytes and the constant regions of the heavy chains ofIgG. l l Additionally, melanoma cells were lysed by cytotoxic T lymphocytes in the presence of a monoclonal antibody linked to an analog of melanocyte-stimulating hormone. 1Z The nature of the described compound has potentially distinct advantages over other related tactics of targeted cellular killing, such as with immunotoxins or hormonal-toxin conjugates. 13-16 The approach employs a potent system of natural foundation. The hybrid need not be internalized and dissociated within the cell to be cytotoxic. And, the conjugate has the potential of eradicating unresponsive cells in the immediate vicinity of direct attack (i.e., the attack complex of complement can attach to any lipid bilayer within an effective diffusion radiusl.l? It appears that functional Leydi9' cells can arise from undifferentiated (precursor) cells. 7 The submitted method of regulation of gonadal function using a hybrid molecule consisting of gonadotropin and Fc has significant conceivable implications within the pet and livestock industries. It could also be ofpos-

IMMUNOREGULATION OF GONADAL FUNCTION

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sible benefit in control of populations of animals within the wild. In human beings, it might be implemented into the treatment regimen of hormonal-dependent cancers (e.g., gonad, prostate gland, breast, uterus/cer-

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cellular binding sites and/or Fc might be liberated from BCG on the cellular surface before initiation of CI binding, thereby diminishing a lytic response to BCGFc. Perhaps stable genetically-engineered fusion proteins made up' of the receptor-specific subunit of gonadotropins and a lethal element ofFc could be efficiently manufactured. ACKNOWLEDGMENTS

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vix). Finally, compounds of this design could prove to be powerful tools to probe basic mechanisms regulating reproductive phenomenon. It is rather expensive to produce small quantities of BCG-Fc by chemical procedures and the disulfide bridge linking BCG and Fc is suspectedly fragile. Such instability in vivo would be less than ideal. Free BCG could presumably compete with intact conjugate for

This research was generously supported by a grant from the Animal Assistance Foundation, Denver, CO. REFERENCES 1. Dufau ML. Endocrine regulation and communicating functions of

the Leydig cell. Ann Rev Physiol. 1988; 50:483-508. 2. Muller-Eberhard HJ. The membrane attack complex of complement. Ann Rev Immunol. 1986; 4:503-528. 3. Morgan EL, Weigle WOo Biological activities residing in the Fe region of immunoglobulin. Adv Immunol. 1987; 40:61-134. 4. Carlsson J, Drevin H, Axen R. Protein thiolation and reversible protein-protein conjugation. Biochem J. 1978; 173:723-737. 5. Rebois RV. Establishment of gonadotropin-responsive murine Leydig tumor cell line. J Cell BioI. 1982; 94:70-76. 6. Belden EL, Peng H-M. Bovine antibody dependent cell-mediated cytotoxicity (ADCC) effector cells. Vet Immunol Immunopathol. 1987; 16:157-171. 7. Murdoch WJ, Dunn TG. Alterations in follicular steroid hormones during the preovulatory period in the ewe. Bioi Reprod. 1982; 27:300-307 . 8. Murdoch WJ. Treatment of sheep with prostaglandin F 2'" enhances production of a luteal chemoattractant for eosinophils. Am J Reprod Immunol Microbiol. 1987; 15:52-56. 9. Becker EL. Chemotactic factors of inflammation. Trends Pharmacol Sci. 1983; 4:223-225. 10. Ross GD. Immunobiology of the Complement System. Orlando: Academic Press, 1986 . 11. Capon DJ, Chamow SM, Mordenti J, Marsters SA, Gregory T, Mitsuya H, Byrn RA, Lucas C, Wurm FM, Groopman JE, Broder S, Smith DH. Designing CD4 immunoadhesions for AIDS therapy. Nature 1989; 337:525-531. 12. Liu MA, Nussbaum SR, Eisen HN. Hormone conjugated with antibody to CD3 mediates cytotoxic T cell lysis of human melanoma cells. Science 1988; 239:395-398. 13. Vitetta ES, Krolick KA, Miyarna-Inaba M, Cushley W, Uhr JW. Immunotoxins: A new approach to cancer therapy. Science 1983; 219:644-650. 14. Myers DA, Murdoch WJ, Villemez CL. Protein-peptide conjugation by a two-phase reaction. Biochem J. 1985; 227:343. 15. Oeltmann TN. Synthesis and in vitro activity of a hormone-diphtheria toxin fragment A hybrid. Biochem Biophys Res Commun. 1985; 133:430-435. 16. Singh V, Sairam MR, Bhargavi GN, Akhras RG. Hormonotoxins. J Bioi Chern. 1989; 264:3089-3095. 17. Teerds KJ, de Rooij DG, Rommerts FFG, van den Hurk R, Wensing CJG. Proliferation and differentiation of possible Leydig cell precursors after destruction of the existing Leydig cells with ethane dimethyl sulphonate: The role ofLH/human chorionic gonadotrophin. J Endocrinol. 1989; 122:689-696.

Toward regulation of gonadal function by a synthetic hybrid molecule composed of gonadotropin and Fc fragment of immunoglobulin G.

A conjugate of human chorionic gonadotropin (HCG) and Fc fragment of immunoglobulin G was prepared by covalent cross-linking using the heterobifunctio...
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