METHODS FOR PRODUCING GONADOTROPIN AND TSH SUPER-AGONISTS

Abstract: Methods are provided for producing super-agonist hormones, which hormones are those having the common alpha subunit of the gonadotropins, generally comprising the transformation of host cells with recombinant DNA comprising a first DNA sequence coding for an alpha subunit from an equine animal species and a second DNA sequence coding for a beta subunit from a non-equine species. The preffered hormone comprises an equine alpha subunit and a bovine, porcine or human beta subunit of LH, FSH, CG or TSH and exhibits a greatly increased activity.

Description: 90/02812 - -- METHODS FOR PRODUCING GONADOTROPIN AND TSH SUPER-AGONISTS Field of Invention This invention relates to the expression, by recombinan DNA technology, of unique gonadotropin hybrid molecules. Gonadc- trσpin molecules are di eric in nature, containing a common alpha subunit and a hormone-specific beta subunit. This application describes the technology to produce large amounts of a gonadotro¬ pin that is composed of an alpha subunit ordinarily found in one animal and the beta subunit from another species. Background of Invention It has been known for almost 20 years that the glyco- protein hormone-.:, luteinizing hormone (LH) , follicle stimulating hormone (FSH) and thyroid stimulating hormone (TSH) are dimeric molecules consisting of a common alpha subunit and a hormone specific beta subunit (G. Bousfield et al., Mol. Cell. Endoc. fi:67-77 [1985]; T-H Liao et al., J. Biol. Che . 2__i_ -3275-3281 [1970]; J.G. Pierce et al., Ann. Rev. Biochem. 50:465-495 [1981}) While the genes that encode each of the three beta subunits are unique and have different chromosomal assignments, a single gene is responsible for the production of the same alpha subunit for each hormone (J.C. Fiddes et al. f In: Bioregulators of Reproduc¬ tion, Acad. Press. pp279-304 [1981]). Recombination of the beta subunit of LH, FSH or TSH with the alpha subunit of another results in the production of a hybrid molecule with biologic activity of the beta subunit (G. Bousfiβld £_ς ___ . , Mol. Cell. Endoc. 4_2.:67-77 [1985]; L.E. Reichert <_____, s_λ. , Endocrin. $7:531- 534 [1970]; J.G. Pierce si al-, » Biσl. Chem. 246:2312-2324 [1971]). Thus, dissociation of bovine LH into the alpha and beta εubunits and mixing of the bovine LH alpha with free bovine beta TSH will produce a hybrid molecule with bovine TSH bioactivity. It waε also noted that free εubunits obtained from dissociated hormones exhibit little or no biologic action when added to target cells (K.J. Catt e£ __ \ ∑. , J. Clin. Endocrin. Metabol. 2£:73-79 [1973], J.F. Williams ≤£ al. , Endocrin. 2<2£:1353-1359 (1980]). Only di ers of the alpha and beta εubuni are biologically active. Studies have demonstrated that both th alpha and beta εubunits must interact with the receptor to elici a biological response (W.R. Moyle gt fil., Proc. Natl. Acad. Sci. USA 2≤.:2245-2250 [1982]). Further, it has been demonstrated tha the carbohydrate moieties present upon the subunitε (in particu¬ lar, the alpha subunit) are essential for transduction of this extracellular hormonal signal to an intracellular biological action, usually through the action of the adenylate cyclase syst (H.R. Saira __rt al. , Science 229.65-67 [1985]). Scientists have examined the effects of recombination εubunitε obtained from hormones of different animals. When the amino acid sequence is compared between the ovine, bovine, por- cine, and human alpha subunits, a striking sequence homology is observed (J.G. Pierce ej; ___]_.. , Ann. Rev. Biσche . ££:465-495 [1981)). It has been demonstrated that human alpha subunit and _ - ovine LH beta εubunit, whan combined, exhibited LH bioactivity (L.E. Reichert ≤£ al. , Endocrin. 7:531-534 [1970]). Aε a result of the gene conservation of gonadotropin subunits between species, almost all inter-species hybrid molecules have been shown to be biologically active. Many other studies have confirmed that recombining subunits of LH obtained from different species re¬ sulted in the production of hormones with approximately equivalent biologic activities (T-H Liao e£ _%2_, , J, Biol. Che . 245.3275- • 3281 [1970]; L.E. Reic ert £ al. , Endocrin. £7:531-534 [1970] ? J.G. Pierce _£ &]_. , Ann. Rev. Biochem. Jϊ_2:465-495 [1981]). Much less is known about structure-function relation¬ ships of equine gonadotropins. Equine LH and FSH have recently been purified frc horse pituitary glands and the biochemical structure and biologic activity of the molecules studied. These molecules exhibit some unique features. First, cborionic gonadotropin (the hormone of pregnancy analogous to human chori- onic gonadotropin) exhibits both LH and FSH bioactivity (Y. Combarnous ≤i al., Endocrin. X_U_ :1821-1827 [1984]). Secondly, equine FSH exhibits much greater potency in a number of bioasεay systems, when compared on a molar basis, to rat or porcine FSH (F. Guillou et aj.. , Biochem. Biophys. Acta. ££7:196-203 (19B6])). Thirdly, the equine LH molecule exhibits at least 100 times great¬ er bioactivity on a molar basis than LH molecules purified from sheep or porcine pituitaries (G. Bousfield et a_l. , Mol . Cell. Endocrin. A& 69-77 [1985]). Finally, the equine LH beta subunit exhibits a c-teπuinal extension that is rich in εerine-linked (O- linked) oligoεaccharides, unlike other LH subunits, but quite similar to the C-terminal peptide of hCG (G.R. Bousfield e a -, J. Biol. Chem. 252:1911-1921 [1984]). When hybrid gonadotropin molecules were formed with purified equine εubunits combined with the corresponding subunit of other species, yet another interesting phenomenon was observe A hybrid LH molecule, that contains the alpha subunit of equine and the bovine or porcine β subunit of LH, results in an LH mole cule with 10-40 times the biopotency of the corresponding homolo gous LH molecule. No teaching, however, was provided in order t produce εuch molecules in useful quantities or purities. In the original paper that describes the unusually amplified bioactivity of hybrid LH molecules with equine alpha ( to 49 times as active aε the homologous LH species) the authors suggest that this phenomenon may be the result of the unique tyrosine-hiεtidine transposition observed at the C-terminus of this equine subunit (G. Bousfield et al., Mol. Cell. Endocrin. _UL ;69 -77 [1985]). Superovulation of cows and other animals is performed routinely to expand herd size with genetically superior animals. Induction of εuperovulation with a hormone of greater bioactivit would be highly desired since such require less hormone, thus reducing costs, while it appears that a hybrid gonadotropin mig accomplish this, only conventional biochemical methods are avail able to produce these unique hybrid molecules. Such methods wou rely upon pituitary glands obtained from horses and cows which — D - , could not be supplied in adequate quantities for continuous com¬ mercialization. Additionally, the cost of purification, dissocia¬ tion, rehybridization and purification would be much too great to be of conventional value. 5 It iε one aspect of the present invention to provide methods and technology to produce large amounts of a superactive hybrid agonist of bovine FSH. As the activity of the beta subunit can be greatly enhanced by the addition of an equine alpha subunit, it should be 0 understood that any known utility of proteins having the common alpha subunit of the gonadotropinε, such as human cho ionic gonad¬ otropin, luteinizing hormone, follicle stimulating hormone and thyroid stimulating hormone, in any species ether than equine, can be greatly enhanced by creating a hybrid molecule with an equine 5 alpha subunit and the beta subunit of the hormone of interest. Thus, the treatment of sterility in humans can also be greatly enhanced by using a chimeric hybrid of human FSH, for example, and an equine alpha unit. Thus, it is another aspect of the present invention to 0 provide methods and technology to produce large amounts of a super-active hybrid agonist of any hormone having the common alpha subunit of the gonadotrσpins, including human hormones. Summary of Invention 2.5 In accordance with these aspects of the instant inven¬ tion, there are provided new methods for producing large quanti¬ ties of super-active hormones having the common alpha subunit of O 90/02812 _ £___ PCT/EP89/01017 the gonadotropinε, such as LH, FSH, CG, and TSH, and preferably bovine gonadotropins, utilizing recombinant DNA methods. The preferred molecules advantageously would be used in the εuperovul¬ ation of bovine animals for the breeding of preferred traits. The new methods of the instant invention produce the hybrid molecule (preferably equine α/bovine LH β or, more preferably, bovine FSH β) super-agonist in gram amountε. The agoniεt of the instant invention may be advantageously produced at generally reduced cost, aε compared with conventional techniques. Further aspects of the present invention include host cells and germ cells transformed according to the present inven¬ tion aε well as the essentially pure hybrid chimeric proteins produced according to the present invention. Brief Description of the Figures Further undertaking may be had by study of the accompa¬ nying figures wherein: Figure 1 shows the nucleotide and a ino acid sequence o equine α cDN ; Figure 2 shows the nucleotide and amino acid εeguence o bovine β FSH; Figure 3 shows bovine Papillo a virus based expression vector with BamHl and Bglll cloning site. Detailed Description of Invention and Best Mode In order to produce the essentially pure hybrid hormones in accordance with the present invention, a eukaryotic host cell iε transformed with a vector comprising a promoter region, a first DNA sequence coding for an equine α subunit of a hormone which has the common a. subunit of the gonadotropins, and a second DNA sequence coding for a β subunit of a hormone which has the common subunit of the gonadotropins, from a .non-equine animal species. The first and second DNA sequences are in transcriptional reading frame with the promoter. The transformed eukaryotic host cells are then cultured under conditions which allow production of the ■ hybrid hormone. The eukaryotic cell is preferably a mammalian cell and the vector preferably further includes a DNA sequence providing for self-replication. In the most preferred embodiment, mammalian cell expres¬ sions that employ mouse C127 cells (epithelioid) , capable of co- and post-translational modification of the molecules encoded by the cDNAs inserted within the expression vector, will be used. Equivalent amounts of a vector containing the equine alpha cDNA and a vector containing the bovine FSH (or LH) cDNA will be ideal¬ ly transfected into C127 or other suitable host cells. Trans¬ formed cells will be identified by their characteristic piled-up appearance. The cell lines are then ideally expanded, culture producing within the culture medium, and large amounts of the recombinant DNA derived hybrid equine/bovine FSK which can then be purified. Example 1: Ecruinβ Aloha Subunit The cDNA clone for the equine common alpha subunit was isolated from an equine cDNA library constructed in the plasmid pBR322 at its Pstl site by G-C tailing. Fresh horse pituitary glands were obtained within 10 minutes of slaughter by dissection and quick-frozen in liquid nitrogen. RNA was prepared from the -.tissue by pulverization and ho ogenization in a 1:1 mixture of phenol:lOOmM Na-acetate (pH=5.2) that contained 0.5% SDS at 65 * C. After quick cooling on ice, the phases were separated by centrif gation at 5000 X 9 for ten minutes. The hot phenol extraction wa repeated twice more followed by two extractions with chloroform : isoa yl alcohol (24:1). RNA waε precipitated from the final pooled aqueous phase by the addition of 2.5 volumes of ethanol at -20'C. Poly A+ messenger RNA was separated from total pituitar RNA by chromatography across an oligo (dT) - cellulose column. The mixture was passed over the column in lO M Tris-HCl (pH=7.5) containing 0.5H NaCl and washed several times with this buffer. Poly A+ RNA was eluted with lOmM Tris-HCl (pH=7.5) containing l_r EDTA and 0.05% SDS. The cDNA library waε constructed from the poly A÷ RNA by standard procedures using i) avian yeloblaεtoεiε virus (AMV) reverse transcriptase to prepare firεt strand cDNA, and ii) both AMV reverse transcriptase and the Klenow fragment of the E.coli DNA poly erase to prepare the second strand. Following treatmen with SI nuclease, the double stranded cDNA was tailed by the addition of dc residues to the 3 ' hydroxyl group by the use cf t calf-thymus deoxynucleotidyl transferase. Tailed cDNA was annealed to dG-tailed pBR322 and the mixture was used to transfor E.σoli MC1061 (available from the American Type culture Collec¬ tion, Rockville Maryland 20852) to tetracycline resistance. The library was screened with a 32 P labeled bovine alph cDNA probe purchased from R. Maurer at the University of Iowa. Positively hybridizing clones were isolated and grown up for characterization by restriction enzyme digest and nucleotide sequencing. The nucleotide sequence and predicted alpha subunit protein are provided in Figure 1. Example 2 . Bovine FSH Beta The bovine FSH beta subunit cDNA was obtained from a bovine cDNA library that was established by methods substantially the same as those described for equine in Example 1. The library was screened with a partial FSK beta clone purchased from Dr. R. Maurer (from the University of Iowa) . Full length clones were identified, sequenced and amino acid predictions were made as shown in Figure 2. Example 3: Expression Vector Each of the isolated and purified cDNAs (for equine alpha and bovine FSH beta) was inserted into the expression cas¬ sette as detailed in Figure 3. This mammalian cell expresεion vector advantageously contains the bovine papillo a virus (BPV) genome, the rat metallothionein gene promoter region, a unique — 1o - cloning " site for insertion of the cDNA of interest and etallo- thionein poly A sequenceε. The conεtruction of this vector is n critical; those skilled will recognize that a plethora of altern tives abound which can be used in substitution. It will be reco nized that conventional techniques such aε those described in Maniatas, "Molecular Cloning, A Laboratory Manual", will readily suffice. Example 4: Transfection of Cells To produce cell lines that synthesize and secrets the hybrid FSH molecule, C127 mouse epithelioid cells are transfacte with a mixture of two plas ids, the equine alpha cDNA in the metallothioneir.-BPV plasmid and the bovine FSH cDNA in this same vector. This is ideally accomplished as follows. Ten microgram of each plasmid are added to 0.5ml of a 250mM CaCl 2 solution containing lOμg of salmon sperm DNA as a carrier. The mixture i bubbled into 0.5ml of 280 mM NaCl, 50mM HEPΞS and 1.5 ml*. Ka phos phate. The calcium phosphate precipitate is allowed to form for 30 minutes at 23*C. Twenty-four hours prior to transfection, 5X10 5 mouse C127 cells are transferred to a 100mm culture dish. Immediately before adding exogenous DNA, the cells are fed with fresh cultur medium containing 10% fetal bovine serum. One ml of the calcium phosphate precipitate is added to each dish (10ml) and the cells are incubated at 37*C for 8h. The medium is aspirated and re¬ placed with 5ml of 20% glycerol in phosphate-bu ered saline (PB (pH=7.0) for two minutes at room temperature. The cells are washed once with PBS, fed with 10ml of medium and incubated at 37 - c for 24h. Within 3 weeks, foci of transformed cells, charac- terized by their piled-up appearance, are identified, transferred to T-25 flasks and line expansion begun. The equine alpha/bovine FSH di er is ideally purified by a three step process including trisacryl Blue chro atography, ion exchange and gel filtration. Following purification and analysis of the product by a bovine beta FSH-specific radioim unoassay, a series of jji vitro FSH bioasεays can optionally be performed to determine the relative potency of this hybrid molecule compared with recombinant equine, bovine and human FSH. In addition, in yivo bioaεsayε can optimally be performed to determine' the potency of each of the preparations in a rodent model. The hybrid mole¬ cule will ideally exhibit enhanced biopotency. While the preferred examples of the present invention use equine alpha subunit and bovine FSH beta subunit, those of ordinary skill in the art will understand that the present inven¬ tion can be practiced in a similar manner using the beta subunit from any protein which has the common alpha subunit of the gonado- tropins. This includes not only the gonadotropins themselves, but also thyroid stimulating hormone (TSH) which also shares the common alpha subunit of the gonadotropinε. Furthermore, the beta subunit may be from any desired species. In all cases the pres¬ ence •_.£ thώ βyuiiie tal liei subunit will cause a great increase in the bio-activity of the hormone of the beta subunit in the species from which that beta subunit was taken. Thus, for example, if it - 11 - is desired to produce a human FSH with greatly enhanced bio¬ activity, a recombinant chimeric molecule can be produced in the manner discuεsed above to provide analpha equine subunit bound to the human FSH beta subunit. Accordingly, in its broadest sense, the present invention relates to the production of recombinant hybrid chimeric gonadotropin hormones (or hormones having the common alpha subunit of the gonadotropin hormones) having an ■&c_u.i-'_ Slpha Sub ΛiC aiid the beta subunit of the hormone whose activity it is desired to increase. It should further be understood that the hybrid mole¬ cules of the present invention produced by recombinant DNA tech¬ nology can be produced in essentially pure form and, in particular,- they are produced without contamination by other hormones and hormone subunits. Thus, the essentially pure hybri molecules of the present invention are novel as prior art hybrid gonadotropins produced with classical protein chemistry techniqu cannot be obtained in esεentially pure form and, in particular, cannot be obtained without contamination with other hormones and hormone εubunitε. Thus, the essentially pure hybrid hormones of the present invention are novel and obtaining such novel hormone would not have been obvious to one of ordinary skill in the art having knowledge only of the classical protein chemistry tech- niqueε for the production of such hormones. Those skilled in the art will readily recognize that numerous departures may be made from the procedures taught in th example without departing from either the spirit or scope of the present invention.

Claims: VT f- . is Claimed is : 1. A method for producing a hormone super-agonist, comprising: a) transforming eukaryotic host cells with a vector 5 comprising a promoter region and a first DNA εequence coding for an equine a subunit of a hormone which has the common εubunit of the gonadotropins and a second DNA sequence coding for a β subunit of a hormone which has the common 10 subunit of the gonadotropins, from a non-equine animal species, said first and second DNA sequence being in transcriptional reading frame with said promoter? and b) culturing said transformed eukaryotic host cells 15 under conditions which allow production of the hormone super-agonist. 2. The method of Claim 1 wherein said vector further comprises a DNA sequence providing for self-replication. 20 3. The method of Claim 1 wherein said hormone having the common subunit of the gonadotropins iε chorionic gonadotropin, follicle stimulating hormone, luteinizing hormone or thyroid stimulating hormone. -25 4. The method of Claim 1 wherein said non-equine animal species iε bovine. 5. The method of Claim 3 wherein εaid second DNA sequence codes for the β subunit of a bovine gonadotropin selected from the gro consisting of FSH and LH. 6. The method of Claim 5 wherein said vector furthering compris at leaεt a portion of the bovine papillo a virus genome. 7. The method of Claim 6 wherein εaid promoter region comprises metallothionein gene promoter region. 8. The host cell transformed according to the method of Claim 1 9. The host cell of Claim 8 being a mammalian host cell. 10. The host cell of Claim 9 being a C127 cell. 11. The hormone super-agonist produced by the host cell of any claims 8 to 10. 12. A hormone super-agonist in essentially pure form, free of contamination from other hormones or hormone subunitε, comprisin a hybrid chimeric molecule with an equine a subunit of a hormone having the common a subunit of the gonadotropins and a non-equin β subunit of a hormone having the common α subunit of the gonadotropins. - - 13. A hormone super-agonist in accordance with claim 12, wherei the hormone of εaid equine subunit is not the same aε the hormone of said non-equine β subunit. 14. A hormone super-agonist in accordance with claim 12, wherei εaid non-equine β subunit is from a bovine, ovine, porcine or human gonadotropin or thyroid stimulating hormone.

Inventors: CHAPPEL SCOTT C, NUGENT NOREEN P, CHAPPEL, SCOTT, C., NUGENT, NOREEN, P.
Applicants: APPLIED RESEARCH SYSTEMS
Application year: 1989
Main classification: C12P21
IPC codes: C07K1, C07K14, C07K19, C12N5, C12N15, C12P21, C12R1