Phase I clinical trial of monoclonal anti-human chorionic gonadotropin antibody in women with an ectopic pregnancy*

Transcription

1 FERTILITY AND STERILITY Copyright 1989 The Anleri8n Fertility Soiety VoL 52, No.5, November 1989 Printed on aid-free paper in U.S.A. Phase I linial trial of monolonal anti-human horioni gonadotropin antibody in women with an etopi pregnany* Rene Frydinan, M.D.t:j: Herve Fernandez, M.D.t Frederi Troalen, Ph.D. Pasal Ghillani, Ph.D. Jean Daniel Rainhorh, M.D.t Dominique Bellet, M.D;, Ph.D. Hopital A. Belere, Unite Institut National de Ia Sante et de Ia Reherhe Mediate (INSERM) 187, Clamart, and Unite'd'Inimunohimie, Institut Gustave Roussy, Villejuif, Frane A phase-1 linial trial of a mouse monolonal anti-human horioni gonadotropin (hcg) antibody designated as HT13 was onduted in three patients treated for etopi pregnany. Doses of 5 and 25 mg of purified antibody were injeted into patients. Monolonal antibody serum levels dropped to 0 within 1 to 5 days. Human antibodies direted against mouse immunoglobulins were not deteted at up to 41 days after injetion. In one. patient, the tubal pregnany resolved, as onfirmed by hcg levels of <10 muljml and by tubal pateny at hysterosalpingography. The time of resolution was 30 days. In two patients, salpingetomy was performed beause of persistene of elevated hcg levels, whereas HT13 had a striking effet on progesterone (P) and estradiol (E 2) serum levels. The injetion of anti-hcg antibody did not appear to interfere with the subsequent fertility of patients, and two out of three patients later developed a suessful pregnany. While the preise role of antibody injetion in the interruption of etopi pregnany remains speulative, the injetion of monolonal anti-hcg antibody appears to indue a dramati derease in the prodution of both P and E 2 Fertil Steril 52:734, Hi89 The inidene of etopi pregnany is inreasing in most populations. 1 Early diagnosis by both serum human horioni gonadotropin (hcg) level determination and uitrasonography has led to the development of onservative tehniques. Several authors have proposed medial treatments 2-5 inluding therapeuti abstention, 6-8 while other groups have evoked the possibiiity of interrupting early pregnany by an immunotherapeuti approah, using either polylonal or monolonal antibodies direted to different antigeni sites present ort plaental hormones, trophoblasti tissues, Reeived August 22, 1988; revised and aepted July 13, * Supported in part by a grant from Lafon Corporation, Paris, Frane. t Hopital A. Belere, Servie de Gyneologie Obstetrique. :j: Reprint requests: Rene Frydman, Servie de Gyneologie Obstetrique, Hopi tal Antoine Belere, Unite INSERM 187, 157 rue de Ia Porte de Trivaux, Clamart, Frane. Unite d'immunohimie, Institut Gustave Roussy. sperm, or the zona pelluida. 9 In this study, we present a phase I linial trial of a monolonal antibody direted against hcg for the treatment of etopi pregnany. Patients MATERIALS AND METHODS Between January and Deember, 1986, three patients were hospitalized in our Department of Gyneology with suspiion of etopi pregnany, as suggested by linial examination, serum hcg levels> 0 muljml and ultrasonography showing both an enipty uterus and the absene of fluid in the pouh of Douglas. The diagnosis of ampullary pregnanies was onfirmed by laparosopy performed either on the same day or within 24 hours. No patient had tubal rupture or hemoperitoneum of more than 100 ml. 734 Frydman et al. Anti-hCG antibody in etopi pregnanies

2 The three patients were fully informed of the therapeuti protool aording to the priniples defined by our University Ethial Committee. They agreed to omply with the requirements for linial and biologial monitoring after having been informed of the possibility of a seond look laparosopy in ase of either sudden development of pain or seondary rise in serum hcg onentration. Preparation of Monolonal Antibody The seletion proess and harateristis of mouse monolonal anti-hcg antibody designated as HT13 have been previously desribed. 10 Antibody HT13 was raised against hcg and is of a ')'-1 isotype immunoglobulin (IgG1). As HT13 is direted to an antigeni site present on the a subunit, this antibody binds not only to hcg but also to luteinizing hormone (LH), follile-stimulating hormone (FSH), or thyroid-stimulating hormone (TSH). HT13 has an affinity onstant of 3 X M- 1 for hcg. Asiti fluids ontaining HT13 were olleted aseptially. After ammonium sulfate preipitation and entrifugation, the pellet was dissolved in sterile water and applied to a sterile protein-a Sepharose (Pharmaia Pisataway, NJ) olumn (10 ml) for isolation oflgg1 immunoglobulins. The olumn was then washed thoroughly with 0.1 M sterile phosphate buffer, ph 8.5; the adsorbed lgg 1 was eluted with 0.05 M sterile itrate solution, ph 5.5. The ph of the eluate was adjusted to neutral and onentrated by an ultrafiltration Amion system (Danvers, MA). The onentrated solution (5 mg/ml) was filtered, under sterile onditions, through 0.22 JLM Millex filters (Millipore Bedford, MA) and gave negative results in the Limulus amoeboyte lysate assay (Mallinkrodt Saint Louis, MO) at a onentration of 0 g/ml. The immunoglobulin was quantified by protein quantifiation assay (Bio Rad, Rihmond, CA). Treatment with Monolonal Antibody On the day of treatment (DO) pyrogen free monolonal antibody HT13 mixed with 125 ml of isotoni gluose 5% was injeted intravenously in 1 hour. The dose injeted into patient 1 was 5-mg of HT13, whereas the other two patients reeived a dose of 25 mg, eah. Follow-up of patients was based on linial examination and serum hcg, estradiol (E 2) and progesterone (P) determination performed daily. During the first week of monitoring, the three patients were hospitalized; then, monitoring was arried out biweekly in an out pa- tient basis until hcg levels fell below 10 mul/ml. After one week, the three patients left the hospital. The funtional result of this protool was determined several months later by hysterosalpingography. The follow-up period of these patients is urrently between 12 and 24 months. Detetion of Cirulating Mouse Immunoglobulin A two-step immunoradiometri assay was arried out to detet mouse immunoglobulins. Briefly, goat anti-mouse lg antibody (Nordi, Immunologial laboratories, Tilburg, The Netherlands) was adsorbed on a solid phase support by inubating polystyrene beads with a 1/1000-fold dilution of the antiserum in M biarbonate buffer ph 9.0 overnight at room temperature. After washing steps, antibody-oated beads were inubated overnight at 4 o with 0 ul of either serum sample or purified antibody HT13 at different dilutions in fetal alf serum as standard. After three washing steps, sheep anti-mouse lg (Fab') 2 (IM 1310 Amersham, UK) was added. The reation lasted for 1 hour at C followed by a washing step, and radioativity bound was determined. Detetion of Human Antimouse Antibody Serum samples olleted from patients at various time were tested for the presene of human anti-mouse lg antibody by a two-step immunoradiometri assay. Briefly, monolonal antibody HT13 used as apture antibody was bound to a solid phase support as previously desribed. 10 Antibody-oated beads were washed three times with deionized water and then inubated with 0 ul of serum sample or goat anti-mouse lg antibody (Nordi, Tilburg, The Netherlands) used as standards and diluted in fetal alf serum. After a 2 hour inubation at C, the beads were extensively washed and labeled monolonal antibody HT13 was added as radioative traer. The reation was arried out for 1 hour at oc, followed by a washing step, and radioativity bound was determined. Biohemial, Hormonal, and Immunologial Assays Several biologial parameters were tested in serum samples using ommerial assays aording to the manufaturer's instrutions: in brief, samples were tested for reatinin, aspartate aminotransferase (AAT), TSH, E 2, P, tri-iodothyronin (T 3), tetra-iodothyronine (T 4), and irulating immune Vol. 52, No.5, November 1989 Frydman et al. Anti-hCG antibody in etopi pregnanies 735

3 8,000 2,000 0!e_n_t_ 60 II) L'!!! (.) <... - ".... Cl. 1- :z: 2,000, "(.) 60. 8,000 L!!'J _I "' (.) ". man horioni gonadotropin levels remained onsistently high in patients 1 and 2 until surgery and subsequently dropped below 10 mul/ml 1 month after treatment. In patient 3, hcg levels remained stable until day 13 and then dereased to normal level at day 30. E 2 and P serum levels dereased rapidly within 1 hour to 2 days after injetion in the three patients (Fig 2). While a subsequent elevation of serum E 2 and P levels ourred in patient 1, hormone levels remained stable in the sera of the other two patients. One month after treatment, E 2 and P levels were within the normal ranges in the sera ofthe three patients. A derease in serum TSH was learly assoiated with the presene of mouse immunoglobulins. Furthermore, the disappearane of mouse immunoglobulins was aompanied by an inrease in serum TSH to normal range. A transient derease in T3 was observed in patients 1 and 3 between 2 and 5 days after injetion, whereas T4 serum values were not modified by the treatment. Finally, biohemial parameters, and partiularly, serum reatinin and serum asparatate aminotrans- Days after antibody Injetion Figure 1 Levels of monolonal antibody HT13 and of hcg and/or BhCG determined in serie of the three patients as desribed under "Patients" in "Materials and Methods". omplexes. Serum hcg and Beta hcg levels were measured by a radioimmunoassay using antiserum AC RESULTS Levels of antibody HT13 found in the serum of the three patients are presented. Two hours after injetion, a low-serum level was deteted in patient 1, who had been injeted with 5-mg of HT13, whereas higher levels were found in the other two patients injeted with 25 mg. Subsequent evolution shows in patient 3, the HT13 serum level dropped dramatially within 1 day after injetion. In ontrast, in patient 1 and 2, HT13levels remained elevated for 4 days after the injetion, and subsequently dereased between 4 and 6 days after the injetion (Fig. 1). Human antibodies against mouse immunoglobulins were not deteted in samples olleted from the three patients studied up to 41 days after treatment. Moreover, irulating immune omplexes were not deteted in these patients. Hu '/'JEE./>/!_Ij 1,0 1, CJO H f'_t_ifj.nt.!i"' - 8 e " "' 4 2 i.. i w 1 0 ; ; I 0 CJO H f'_t!n_t_ IIJ/ CJO H injetion Days after antibody injetion Figure 2 Serial study of progesterone and estradiol serum levels in three patients: H + 1 means one hour after antibody injetion Frydman et al. Anti-hCG antibody in etopi pregnanies

4 ferase, were unmodified, as observed from various serum samples olleted after injetion of HT13. For patients 1 and 2, salpingetomies were performed on days 11 and 19, respetively, after injetion, due of asymptomati persistene ofhigh-hcg levels. A radial surgial treatment was proposed beause the two patients had a history of infertility with tubal fators for whih surgial orretion had been attempted many years earlier. In these two ases, the volume of hematosalpinx was unhanged. In patient 3, the tubal pregnany resolved, as onfirmed by hcg levels whih remained at <1 ng/ml 30 days after injetion. The use of monolonal anti-hcg antibody did not appear to interfere with subsequent fertility. Two out of the three patients (no. 1 and 3) beame pregnant, 6 and 8 months after treatment and delivered a healthy baby. DISCUSSION Surgial treatment is not the only therapeuti approah for managing etopi pregnany, and various other medial treatments have been proposed by different groups. 2-5 Methotrexate has been suessfully used to treat an interstitial pregnany. 2 However, the use of this drug is assoiated with systemi toxiity and may inrease the inidene of ongenital abnormalities in patients previously treated, even though there is no evidene that methotrexate is responsible for ongenital abnormalities in subsequent pregnanies At the same time, early diagnosis of etopi pregnany has enabled the development of onservative surgial tehniques using laparotomy or laparosopy. Sine 1975, hybridoma tehnology has permitted the prodution of monospeifi antibodies. Suh antibodies direted to aner-assoiated antigens have been used in vivo for either the loalization or the treatment of malignant tumors Treatment of etopi pregnany using monolonal antibodies might offer an alternative to surgery or the potential toxiity of methotrexate. A phase I linial trial was aimed at studying the effets of a monolonal antibody direted against hcg and injeted in vivo into patients with an etopi pregnany. Monolonafantibody HT13 was seleted for injetion on the basis of its high-affinity for hcg (Kasn = 3.0 X M- 1 ). The dose of injeted HT13 was alulated as the dose of antibody apable of binding, at least, the amount of hcg irulating in patient serum. In the three patients studied, no linial or biologial side effets were observed. The derease in the TSH anti-t 3 serum levels at the time of the injetion was transient. It is likely that the derease in TSH serum levels was due to the ross-reativity of HT13 with TSH. The utilization of antibodies with a unique speifiity for hcg might prevent suh a derease. Moreover, it is partiularly noteworthy that neither human anti-mouse immunoglobulin antibodies nor irulating immune omplexes were deteted in the serum of our patients. This is not surprising, sine a human immune response to mouse immunoglobulins is more likely to our after the seond injetion of mouse monolonal antibodies, as already desribed. 15 Suh an immune response might limit the utilization of mouse anti-hcg antibodies. In fat, the risk of sensitization of the woman would limit the utility of suh treatment to one time in an individual's lifetime and thus would severely limit the linial utility of this approah as an effetive therapeuti option. However, the preparation of himeri antibodies onstruted with mouse variable and human onstant immunoglobulin domains offers a new opportunity for overoming this limitation. 16 It is lear from these data that the injetion of anti-hcg antibodies has a diret effet on both E 2 and P serum levels, sine a dramati derease in their levels was onsistently observed in the patients after HT13 injetion. It is noteworthy that the drop of both E 2 and P serum levels might not be explained by the derease of hcg levels whih remained onsistently hight in patient 1 and 2 but might be related to the binding of HT13 to hcg. Suh development of hcg-antibody omplexes would inhibit the in vivo formation of hormone reeptor omplexes as that has been demonstrated by in vitro experiments. 17 Thus it is likely that the injetion of monolonal anti-hcg antibody has a diret effet on the prodution of hormones neessary to the development of pregnany. The influene of antibody injetion on the interruption of pregnany remains speulative, sine two out of three patients had salpingetomy, whereas a hemial interruption was observed in the third patient. Finally, it is likely that the use of mouse monolonal anti-hcg antibody for the treatment of etopi pregnany may have severe limitation. However, the injetion of anti-hcg antibody does not appear to ompromise the subsequent fertility of patients: two out of three patients have developed a suessful pregnany. These preliminary data justify further studies to disset the potential effet of anti-hcg antibody injetion on steroid hormone Vol. 52, No.5, November 1989 Frydman et al. Anti-hCG antibody in etopi pregnanies 737

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