Proc. Natl. Acad. Sci. USAVol. 89, pp. 4304-4308, May 1992Medical Sciences

Design of a long-acting follitropin agonist by fusing the C-terminalsequence of the chorionic gonadotropin f3 subunit to the follitropin(8 subunit

(biologic half-life/O-linked carbohydrate/glycoprotein hormone)

FUAD A. FARES*, NOBUHIKO SUGANUMA*, KEuI NISHIMORIt, PHILIP S. LAPOLTt, AARON J. W. HSUEHt,AND IRVING BOIME**Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, MO 63110; and tDivision of ReproductiveBiology, Department of Gynecology and Obstetrics, Stanford University Medical Center, Stanford, CA 94305-5317

Communicated by Oliver H. Lowry, February 5, 1992

ABSTRACT Follitropin (FSH) is a pituitary glycoproteinhormone that is essential for the development of ovarianfollicles and testicular seminiferous tubules. FSH is used clin-ically to stimulate follicular maturation for in vitro fertilizationand treatment of anovulatory women. One issue regarding theclinical use of FSH is its short half-life in the circulation. Toaddress this point, we constructed chimeric genes containingthe sequence encoding the C-terminal peptide of the chorionicgonadotropin 13 subunit (CG1) fused to the translated sequenceof the human FSH P subunit (FSH(8). This region of CGI3 isimportant for maintaining the prolonged plasma half-life ofhuman CG dimer. The presence of the C-terminal peptidesequence did not significantly affect assembly ofFSH(3 with thea subunit or secretion of the dimer. In vitro receptor bindingand steroidogenic activity of dimer bearing the FSHf-C-terminal peptide chimera were the same as wild-type FSH.However, both the in vivo potency and half-life in circulation ofthe dimer bearing either one or two C-terminal peptide unitswere enhanced. Dimers containing FSH(-CGI3 chimeras couldserve as potent FSH agonists for clinical use, and the presentstrategy may have wide applications for enhancing the in vivohalf-life of diverse proteins.

Follitropin (follicle-stimulating hormone; FSH) is a pituitaryglycoprotein hormone essential for maintenance of ovarianfollicle and testicular tubule development. FSH, together withluteinizing hormone (LH), thyroid-stimulating hormone, andhuman chorionic gonadotropin (hCG), constitute a family ofglycoprotein hormones that are heterodimers containing twononidentical subunits, a and (3(1, 2). Within an animal species,the amino acid sequences of the a subunits are identical, and,although the ( subunits determine the biologic specificity ofthe hormones, there is significant amino acid sequence simi-larity among them (1, 2). This is readily apparent for the LH,6 and CG ( subunits (LH,8 and CG,() as they share >80%osequence identity, which is presumably responsible for thesimilar biologic activity of their dimers. CG.B is distinguishedamong the ( subunits because of the presence of a C-terminalextension with four O-linked oligosaccharides. Sequence anal-ysis showed that a 1-base-pair (bp) deletion and 2-bp insertionin CGP3 relative to the LH(3 coding sequence accounts for theextended open reading frame of CGP (3). This extension isbelieved to play a role in maintaining the prolonged half-life ofhCG compared to the other hormones (4-7). Recent studiesusing site-directed mutagenesis and gene transfer techniquesindicated that the dimer containing CG(8 devoid of the C-ter-minal peptide (CTP) was 3-fold less active than native hCG instimulating ovulation in rats (7).

Gene transfer techniques together with site-directed muta-genesis have been valuable tools for elucidating structure/function determinants of the glycoprotein hormone family (7-12). Similar methodology provides a powerful approach fordesigning therapeutic analogs of these hormones. FSH is usedclinically to stimulate development of ovarian follicles for invitro fertilization (13-16) and to initiate follicular maturation inanovulatory women with chronic anovulatory syndrome (17) orluteal phase deficiency (18). One major issue regarding theclinical use ofFSH is its relatively short half-life in vivo (19, 20).To address this issue, one or two copies of the CTP of hCG/3were fused to the human FSH (3-subunit (FSH,8) coding se-quence. We reasoned that FSH dimers containing these exten-sions would have a prolonged half-life and enhanced bioactivityin vivo, as suggested from the experiments withhCG lacking theCTP sequence. These constructs were transfected into Chinesehamster ovary (CHO) cells together with the wild-type asubunit and stable clones were selected. Compared to wild-typeFSH, the addition ofCTP sequences did not significantly affectassembly, secretion, or stimulation of steroidogenesis in vitro.However, the in vivo potency of the chimera containing one ortwo CTP units was substantially increased. The data show thepotential for these FSH chimeras as therapeutic agents. Inaddition, the presence of the CTP sequence may represent ageneral method for enhancing the in vivo longevity of differentproteins.

EXPERIMENTAL PROCEDURESMaterials. [35S]Cysteine (>1000 Ci/mmol; 1 Ci = 37 GBq)

and [32P]dATP were obtained from Amersham. The DNAvector M13mp19 and restriction enzymes were purchasedfrom New England Biolabs or Bethesda Research Labora-tories. T4DNA and Klenow polymerases were obtained fromUnited States Biochemical. Oligonucleotides used for muta-genesis were prepared by the Washington University ProteinChemistry Laboratory (St. Louis).

Cell culture media and reagents were prepared by theWashington University Center for Basic Cancer Research.Dialyzed calf serum and G418 were purchased from GIBCO.Fetal calf serum was obtained from Washington UniversityCenter for Basic Cancer Research, and Immunoprecipitinwas obtained from GIBCO. Polyclonal antisera directedagainst either FSH dimer or FSH(3 were obtained from theNational Hormone and Pituitary Distribution Program (Na-tional Institute of Diabetes and Digestive and Kidney Dis-eases, National Institutes of Health).

Construction of Expression Vectors. To create hFSH3chimera bearing a single CTP unit, a Hind1II site was created

Abbreviations: FSH, follitropin; FSH,3, FSH ,( subunit; hCG, humanchorionic gonadotropin; CG,3, CG subunit; LH, luteinizing hor-mone; LH(3, LH subunit; CTP, C-terminal peptide.

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Proc.- Natl. Acad. Sci. USA 89 (1992) 4305

in codon 111 (the stop codon) of the hFSH.8 gene and in thehCG/3 gene at codon 118 (Fig. 1A). The HindIII/HindIIIfragment from the FSH,8 gene was ligated in-frame to theCG/3 HindIII/BamHI fragment. As a result of the ligation,Ser-118 was changed to Ala-118; oligonucleotide-directedmutagenesis was used to convert the alanine back to serine,which resulted in loss of the HindIII site in exon III.We also engineered a FSH analog to contain two tandemly

arranged CTP units. We reasoned that the in vivo potency ofthis molecule would be greater than a FSH dimer containingthe 8 subunit with only one CTP unit. A chimera containingtwo tandem CTP repeats was constructed by creating anHindIII site in the stop codon of the FSH3--CTP chimera(Fig. 1B). The HindIII/HindIII fragment was ligated to theHindIII/BamHI fragment from hCG3. Finally, the HindIIIsite at the 5' end of each chimeric gene was changed to aBamHI site by using Klenow enzyme and a BamHI linker(Fig. 1B). The BamHI/BamHI fragments containing theFSHf3-CTP or FSHp-(CTP)2 genes were inserted at theBamHI site of vector pM2 (21). The correct orientation wasconfirmed by restriction enzyme analysis and the entireregion ofexon III was sequenced to confirm the specificity ofthe mutagenesis. The chimera bearing the second CTP unitcontained a serine to alanine change as a consequence ofgenerating an additional HindIII site.The chimeras were transfected alone or with pM2 contain-

ing the a-subunit gene into CHO cells and were screened asdescribed (21). Selected clones were maintained in F-12medium containing 0.125 mg of G418 per ml in a humidified5% C02/95% air incubator (21).

Metabolic Labeling and Protein Analysis. Cells were platedinto six-well dishes (720,000 cells per well) and labeled for 6h in F-12 cysteine-free medium containing 5% dialyzed calfserum and 25 ,Ci of [35S]cysteine per ml. Media and celllysates were immunoprecipitated and proteins were identi-fied on SDS gels as described (21).In Vitro Bioassay and Radioligand Receptor Assay. FSH and

chimeric proteins in conditioned medium obtained from cellsincubated without serum were quantitated with a FSH im-munoradiometric assay and a double-antibody RIA (Diag-nostic Products, Los Angeles). The in vitro bioactivity ofFSH and FSH-CTP chimeras was determined by the gran-ulosa cell aromatase bioassay as described (9, 22). Stimula-tion of estrogen production by wild-type FSH and the chi-meras was compared after a 3-day culture period. For theradioligand receptor assay, human FSH was iodinated by thelactoperoxidase method (23). Decapsulated testes from 15-day-old rats were homogenized in ice-cold Dulbecco's phos-

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phate-buffered saline (PBS) containing 0.1% bovine serumalbumin, using a Kontes ground glass tissue grinder. Thehomogenate was centrifuged at 20,000 x g for 30 min at 4°C,and the crude membrane preparation was resuspended inPBS. Aliquots of the membrane preparation were incubatedat room temperature with 125I-labeled hFSH (Q30,000 cpm)in the presence or absence of the FSH derivatives for 18-20h. Binding was quantitated by a y counter and the displace-ment of 125I-hFSH binding to receptors by the chimeras wasplotted semilogarithmically.In Vivo Half-Life and Biopotency Determinations. The half-

life of wild-type FSH and the chimeras was determined afteri.v. injection of 10 international units of dimers in immature(21-22 days old) female rats (Johnson Laboratories, Bridge-ville, IL). At selected intervals after injection, blood sampleswere withdrawn by cardiac puncture under halothane anes-thesia and FSH immunoreactivity was determined by RIA.To measure bioactivity, immature female Sprague-Dawley

rats were implanted with a 10-mm Silastic capsule containingdiethylstilbestrol. Rats were maintained in standard vivariumfacilities, with food and drinking water available ad libitum.Beginning the 4th day after estrogen treatment, rats receivedtwo equivalent intraperitoneal (i.p.) injections of wild-typeFSH or the chimeras at 24-h intervals. These hormones werequantitated by the radioreceptor assay and by radioimmu-noassay with monoclonal antibody, and the determinationswere in agreement within 15%. In the receptor assay, theamount of hormone required to achieve 50o binding wasused to quantitate the derivatives. Forty-eight hours after theinitial injection, animals were sacrificed by cervical disloca-tion, and ovaries were removed. The ovaries were decapsu-lated and weighed, and granulosa cells were isolated todetermine levels of aromatase, a specific indicator of FSHbioactivity. Induction of aromatase activity was determinedby the ability of isolated granulosa cells to convert an-drostenedione to estrogens in vitro. Granulosa cells isolatedfrom ovaries described above were cultured (300,000 viablecells per well) in McCoy's SA medium (GIBCO) containing0.3 AM androstenedione (Sigma) for 6 h at 37°C in a humid-ified atmosphere of5% C02/95% air. After culture, the mediawere analyzed for estrogen content by RIA as described (24,25). The viability of the cells was unaffected by the wild-typeFSH or the chimeras.

RESULTSWe examined whether addition of the CTP sequences to thecoding region of FSH,8 interferes with secretion or assembly

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FIG. 1. Construction of gonadotropin chimeras. (A) FSHP-CTP chimera. HindIII sites in the stop codon of FSHB gene at position 111 andin the hCGB gene at position 118 were created. The HindIII/HindIII fragment from FSH, gene was ligated in-frame with the HindIII/BamHIfragment of the CGB gene. This chimera contains the translated sequence of hFSHB gene (stippled box) and the CG3-CTP gene (solid box).(B) FSHA-CTP)2 chimera. An additional HindIII site was introduced in the stop codon ofthe FSH-CTP chimera. The resulting HindIII/HindIIIfragment was ligated in-frame to the HindIII/BamHI fragment from hCGB.

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4306 Medical Sciences: Fares et al.

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FIG. 2. Expression of uncombined ,8 subunits (A) and hormonedimers (B) in CHO cells. Transfection ofCHO cells with pM2 vectorbearing gene constructs encoding the CG,8, FSH(3, and FSHP-CTPchimeras, and clone selection were performed as described (7).Selection of clones expressing the corresponding dimers is describedin Experimental Procedures. (A) CHO cells were labeled for 6 h with25 1ACi of [35S]cysteine per ml and equivalent aliquots of the medium(lanes M) and lysate (lanes L) were immunoprecipitated with hCG.8(lanes 1 and 2) or hFSH(3 (lanes 3-6) antiserum and prepared forSDS/PAGE as described. Molecular mass markers (kDa) (Amer-sham) are indicated. (B) Synthesis of hFSH dimers in CHO cellsunder conditions described in A. WT, wild type.

of the chimeras. Secretion of the chimeras and wild-typeFSHP from stable cell lines was compared to hCG/3 (Fig. 2).Cells were labeled for 6 h with [35S]cysteine and the intra-cellular (lysate; lanes L) and extracellular (medium; lanes M)fractions were immunoprecipitated with subunit-specificpolyclonal antisera. As previously reported (8, 21), two majorhCGP3 forms appeared in the media (Fig. 2A, lane 2), whichreflect the presence of one or two N-linked oligosaccharides.The large shift in mobility between the lysate (lane 1) andmedium (lane 2) forms is due to addition of the 0-linkedoligosaccharides, a late event in the secretion of hCGP (21).hCG/3 is rapidly and quantitatively secreted from transfectedcells (8). By contrast, as reported previously (9), FSHPaccumulates intracellularly (lane 3) and little, if any, immu-noprecipitated material appears in the medium (lane 4).However, when either one or two CTP units are fused to the

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FSH coding sequence, both chimeras are secreted efficiently;the presence of the hCGf C-terminal region enhances secre-tion of FSH/3 (lanes 5-8). This is consistent with previousdata implicating the C-terminal sequence of the pituitary (3subunits as a determinant for secretion (8) (see Discussion).It is also evident that there is a direct relationship between themobility of the lysate/medium forms and the presence ofoneor two CTP units. The chimeras also undergo a mobility shiftcharacteristic of O-linked oligosaccharide addition seen withhCG(3. These data show that the O-linked glycosylationrecognition site of the C-terminal region is preserved eventhough the sequence is fused to a different protein.Assembly of the FSH3-CTP chimeras with the a subunit

was examined by transfecting stable clones expressing the asubunit with either hCGf3, FSHB, or the FSH3 chimeras. Thedimers and the corresponding free subunits were precipitatedwith FSH/3 subunit-specific antiserum (Fig. 2B). Immuno-precipitation of the a subunit with this 3-subunit antiserumproves that dimerization has occurred. As shown previously(9), the a subunit and FSH/3 migrate at similar rates andcannot be resolved by polyacrylamide gel electrophoresis(Fig. 2B, lanes 1 and 2). However, because very little of theuncombined P subunit is secreted (Fig. 2A, lane 4), theappearance of protein precipitated by the FSH/3 antiserum inthe medium indicates the presence of FSH dimer. FSH israpidly secreted and little accumulates intracellularly (9). Theincrease in molecular size caused by the fusion of one or twoofthe CTP units and the associated O-linked oligosaccharidespermits resolution of the FSH.8 chimera and the a subunit(Fig. 2B, lane 4). Precipitation with FSHP antisera revealsthe presence of both subunits (lane 6). The band migrating atthe 46-kDa marker is apparently nonspecific. Both single anddouble FSHP-CTP chimeras combined quantitatively sincethe amount ofa subunit precipitated by antiserum against thea subunit was comparable to that precipitated by the /3-sub-unit specific antiserum (data not shown). Thus, the presenceof either one or two CTP units on FSH/8 did not significantlyinterfere with assembly.

Receptor Binding and in Vitro Biologic Activity ofChimeras.Although the CTP in the CG/3 subunit is not critical forbioactivity of hCG in vitro (7, 26, 27), the additional 29 (oneCTP) or 58 (two CTP) amino acids on the heterologous

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FIG. 3. In vitro bioactivity of FSH and chimeras: Receptor binding (A) and steroidogenesis (B). (A) Displacement of '25I-FSH binding tothe FSH receptor in rat testes by wild-type FSH and chimeras. Testes membranes from 15-day-old rats were incubated at 230C with 125I-FSHand various concentrations of wild-type (WT) FSH or FSH chimeras as described. Total binding (100%1) was determined as the binding of125I-FSH in the absence of competitors. Mean (±SEM) of three separate experiments was determined from triplicate experiments. (B)Dose-dependent stimulation ofestrogen production from granulosa cells by wild-type FSH and chimeras. Granulosa cells were treated for 3 daysin medium containing the aromatase substrate androstenedione with increasing concentrations of wild-type FSH or chimeras produced by CHOcells. Estrogen concentration in the medium was determined by RIA (mean ± SEM). Similar results were obtained in three separate experiments.

Proc. Natl. Acad. Sci. USA 89 (1992)

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FIG. 4. In vivo biologic activity of wild-type FSH and the FSH-CTP chimeras. (A) Ovarian weight. Immature estrogen-treated female ratswere injected once daily with 2.5 IU of wild-type FSH or the FSH-CTP chimeras. Two days after the initial treatment, ovarian weight wasdetermined. Eight to 10 ovaries were used for each group. (B) Aromatase induction. Granulosa cells were isolated from the ovaries and incubatedex vivo for 6 h with 0.3 AM androstenedione for determination of aromatase activity as indicated by estrogen secretion, which was measuredby RIA. Mean ± SE of four or five determinations. WT, wild-type; FC1, FSH-CTP; FC2, FSH-(CTP).

subunit may affect signal transduction of the modified FSHproteins. Chimeras were quantitated in conditioned mediumby using a monoclonal antibody-based RIA and receptorbinding was assessed in a radioligand receptor assay byquantitating the displacement of 1"I-hFSH binding to rattestes membranes. Addition ofone or two CTP units to FSHPdid not affect receptor binding of the dimer (Fig. 3A).Because FSH specifically induces aromatase enzyme ingranulosa cells (28), in vitro signal transduction of the mod-ified FSH dimers was assessed in the granulosa cell aro-matase bioassay by measuring hormone-stimulated estrogenproduction (Fig. 3B). The steroidogenic activity ofFSH-CTPchimeras was comparable to that of wild-type FSH. Thus,modification at the carboxyl end of FSH.3 by adding the CTPsequence did not significantly affect in vitro receptor bindingor signal transduction. In addition, the chimeras have no LHbioactivity as tested in a LH radioligand receptor assay (datanot shown).In Vivo Biopotency/Half-Life of Chimeras. FSH treatment

stimulates ovarian granulosa cell differentiation and in-creases follicle growth and antrum formation (28). The in vivobiopotencies of wild-type FSH and the chimeras were ex-amined by determining ovarian weight augmentation andgranulosa cell aromatase induction. The hormones wereinjected i.p. into immature, estrogen-primed rats at 24-hintervals. Twenty-four hours after the second injection, ratswere killed and the ovaries were removed. Induction ofgranulosa cell aromatase activity was studied ex vivo byincubating granulosa cells with a saturating dose of thearomatase substrate androstenedione for 6 h. The ovarianweight increased significantly between animals treated withwild-type FSH and the FSH chimeras (Fig. 4A). In addition,estrogen production by granulosa cells from chimera-treatedrats increased 3- to 5-fold over that seen in rats treated withwild-type FSH (Fig. 4B). The difference between the FSH-CTP and FSH-(CTP)2 is not significant.Because the increased biopotency of the chimeras may

reflect a change in their in vivo longevity, we determined thecirculatory half-life of the hormones (Fig. 5). To obtain themaximal effect we examined the derivative containing twoCTP units. Wild-type FSH or the FSH-(CTP)2 chimera wasinjected i.v. into immature female rats and the plasma half-lifewas monitored by RIA. The clearance patterns of the wild-type and chimera reveal multiple phases. Within the first 4 hthere is already an =64fold difference in the level of chimeraand wild-type hormones. The slower phase, extending be-yond 4 h, also shows that the clearance ofthe chimera is muchslower than for the wild-type FSH; presumably, this phasereflects renal excretion. RIA determinations show that a high

level of the chimera is still detectable in serum after 24 h andyet injected wild-type hFSH reaches basal level between 8and 24 h. Thus, the data suggest that more than one mech-anism of clearance is affected by the presence of the CTP.

DISCUSSIONHere we describe the construction of chimeric FSH genesthat contain the CTP coding region of hCGB fused to FSHB.Compared to wild-type FSH, the presence of either one ortwo CTP units did not interfere with combination with the asubunit or secretion of dimers. Similarly, in vitro receptorbinding and steroidogenic activity ofthese chimeras were notsignificantly affected. The data are consistent with earlierstudies demonstrating that deletion of the CTP from hCG,3does not affect secretion or in vitro receptor binding/signaltransduction ofhCG dimer (7, 26). However, fusion of one ortwo CTP units to FSHB clearly increases the in vivo serumhalf-life and biologic activity of the chimeras.Due to the relatively rapid clearance of native FSH in vivo,

the commonly used therapeutic protocol requires frequentinjection of the hormone. The chimeras described here couldbe effective long-acting agonists for therapeutic use. More-over, CTP chimeras could be constructed for diverse proteinsto enhance their in vivo biologic half-life. Presumably, thisapproach requires that the active site is not at the carboxyl

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FIG. 5. In vivo half-life of wild-type FSH and FSH-(CTP)2chimera. Immature female rats were injected i.v. with 10 IU ofwild-type (WT) FSH or FSH4CTP)2 chimera. Blood samples weredrawn at the indicated times. Serum FSH immunoactivity wasdetermined by RIA. Mean ± SE of 3-5 determinations. Basal FSHlevels before treatment were <0.04 IU/ml.

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end of the molecule. The addition of the CTP could elicitimmune reactions. However, several studies have demon-strated that the CTP region of CG,3 is weakly immunogenic(29, 30). In addition, because the native hCG,3, which con-tains the CTP, is normally secreted in both men and women,the immune system may not recognize the chimera as aforeign protein.

Although the kidney is the main site of clearance for FSH(31), much less hCG is cleared by this route (32, 33). It hasbeen suggested that more negatively charged forms ofhumanFSH (i.e., forms with increased sialic acid content) havelonger half-lives, which may be related to a decreased glo-merular filtration (34). Thus, the presence of the CTP with itssialylated O-linked oligosaccharides may prolong the circu-lating half-life of the hormone secondary to a decrease inrenal clearance. Alternatively, as proposed previously (7) theCTP sequences may affect the processing of asparagine-linked oligosaccharides, which in turn could influence extra-cellular levels.

It is somewhat surprising that the presence of 29-58 aminoacids did not interfere with folding ofFSHI3. This is likely dueto the presence of the O-linked oligosaccharides, which havebeen postulated to give a rigid and extended conformation ofthe peptide core to which they are attached (35). This finding,together with the observation that the CTP sequence does notcontribute greatly to the folding of hCG(3 (1), may explainwhy the presence of CTP sequences did not greatly perturbthe overall function of the dimer.

Significantly, the CTP sequence can be shuttled to a differ-ent protein and still be an acceptor for 0-linked oligosaccha-rides. This suggests a potential model system for elucidatingthe recognition sequences that determine why particular ser-ine residues can accept 0-linked oligosaccharide chains. Sincethe CTP sequence terminates the carboxyl end of the subunit,and since this sequence apparently does not affect folding ofhCG/3, the CTP is a convenient sequence for site-directedmutagenesis. This is critical because if the O-linked glycosy-lation sites are in the interior of the subunit, amino acidreplacements could lead to pronounced position effects.

Previously, we showed that very little uncombined thy-roid-stimulating hormone ( subunit, LH,8, and FSHI3 aresecreted from transfected CHO cells (8, 36). In contrast,hCG(3 is secreted efficiently and quantitatively. This wasattributed in part to the presence of a retention signal at theC-terminal region of LH(8 (8) and FSH(3. The current datasupport this hypothesis since, although only a small amountof FSH,3 appeared in the medium, secretion of FSH3-CTPwas comparable to that of hCGf3. Presumably, the CTPincreases the exit rate of chimeras from the endoplasmicreticulum. The data support previous observations that theC-terminal region contains determinants associated with se-cretion and sorting for the pituitary hormones (8, 36).

In summary, fusion of the CTP sequence to a heterologousprotein did not interfere with folding of the subunit sincesecretion, assembly, receptor binding, and the in vitro ste-roidogenic activity of the dimers containing FSHf3-CTPchimera were not substantially affected. In contrast, thepresence of the CTP sequence significantly increased the invivo half-life and biologic potency of the engineered chime-ras. The data establish a rationale for using these chimeras aslong-acting FSH analogs that can be used clinically whereFSH replacement therapy is indicated. Treatment of anovu-latory women or of men with impaired testicular functionswould be candidates for such therapy.

The authors thank Dr. Merlie for comments regarding the manu-script. This work was supported in part by National Institutes ofHealth Grant HD-9-2922 and by a grant from Monsanto. F.A.F. wassupported by a postdoctoral fellowship from the Rockefeller Foun-dation.

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