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Europaisches Patentamt 
European Patent Office 
Office europeen des brevets 



© 



EUROPEAN PATENT SPECIFICATION 



© Date of publication of patent specification: 15.03.95 © Int. CI. 6 : C12N 15/16, C07K 14/575, 

C12P 21/02, A61K 38/22, 
© Application number: 86307586.7 ^g-j ^ 39/395 

© Date of filing: 02.10.86 



@ Nucleic acid encoding the alpha or beta chains of inhibin and method for synthesizing 
polypeptides using such nucleic acid. 



5 

CD 

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m 

o 
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© 


Priority: 03.10.85 US 783910 


© 


Proprietor: GENENTECH, INC. 




07.02.86 US 827710 




460 Point San Bruno Boulevard 




12.09.86 US 906729 




South San Francisco 


© 






California 94080 (US) 


Date of publication of application: 








20.05.87 Bulletin 87/21 


© 


Inventor: Mason, Anthony John 








1206 6th Avenue 


© 


Publication of the grant of the patent: 




San Francisco 




15.03.95 Bulletin 95/11 




California 94122 (US) 


© 






Inventor: Seeburg,Peter Horst 


Designated Contracting States: 




Erzazkerweg 5 




AT BE CH DE ES FR GB GR IT LI LU NL SE 




69 Heidelberg (DE) 


© 


References cited: 








WO-A-86/00078 


© 


Representative: Stuart, Ian Alexander et al 




WO-A-86/06076 




MEWBURN ELLIS 








York House 




Biochem. Biophys. Res. Commun., vol. 129 




23 Kingsway 




(1985), pages 396-403, K. Miyamoto 




London WC2B 6HP (GB) 




PROCEEDINGS OF THE NATIONAL ACADEMY 








OF SCIENCES OF THE UNITED STATES OF 








AMERICA, vol. 83, no.16 f August 1986, Bal- 








timore, USA; K.E. MAYO et a I., pages 








5849-5853 







CD 

CM 
CM 
CM 



Q. 

UJ 



Note: Within nine months from the publication of the mention of the grant of the European patent, any person 
may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition 
shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee 
has been paid (Art. 99(1) European patent convention). 



Rank Xerox (UK) Business Services 

I3.10/3.0S/3.3.3I 



EP0 222 491 B1 



NATURE, vol. 318, 19/26 Dec. 1985, New York, 




London; A.J. MASON et al, pages 659-663 




NATURE, vol. 321, 19 June 1986, New York, 




London; W. VALE et al., pages 776-779 




NATURE, vol. 321, 19 June 1986, New York, 




London, N.LING et al., pages 779-782 




PROCEEDINGS OF THE NATIONAL ACADEMY 




OF SCIENCES OF THE UNITED STATES OF 




AMERICA, vol. 82, no. 12, June 1985, Bal- 




timore USA* C H LI pt al nanp<; 4041-4044 




FEBS LETTERS, vol. 175, no. 2, October 1984, 




Amsterdam; N.G. SEIDAH et al., pages 




349-355 





2 



EP 0 222 491 B1 



Description 

BACKGROUND 

5 This invention relates to methods for making proteins in recombinant cell culture which contain the a or 
£ chains of inhibin. In particular, it relates to methods for obtaining and using DNA which encodes inhibin, 
and for making inhibin variants that depart from the amino acid sequence of natural animal or human 
inhibins and the naturally-occurring alleles thereof. 

Inhibin is a protein produced in the gonad which acts specifically at the pituitary level to inhibit the 

/o secretion of follicle-stimulating hormone (FSH). The existence of inhibin was first postulated by McCullagh 
in 1932 ("Science" 76: 19-20). Such preferential regulation of the gonadotropin secretion has generated a 
great deal of interest and has prompted many laboratories in the past fifty years to attempt to isolate and 
characterize this substance from extracts of testis, spermatozoa, rete testis fluid, seminal plasma and 
ovarian follicular fluid, using various bioassays. Although many reports have appeared in the literature 

75 claiming the purification of inhibin-like material with molecular weights ranging from 5,000 to 100,000 
daltons, subsequent studies have shown that these substances were not homogenous, did not have the high 
specific activity expected of true inhibin and/or failed to exhibit the molecular characteristics of inhibin as 
described herein (de Jong, Inhibin-Factor Artifact, "Molecular & Cellular Endocrin." 13: 1-10 (1979); Sheth 
et al ., 1984, "F.E.B.S." 165(1) 11-15; Seidah e\a\. t 1984, "F.E.B.S." 175(2):349-355; Lilja e^aj., March 1985, 

20 n F.E.B.S. M 182(1 ):1 81 -184: Li et al ., June 1985, "Proc. Nat Acad. Sci. USA" 82:4041-4044; Seidah et al ., 
H F.E.B.S. W 167(1 ):98-1 02; and Beksac eTal., 1984, "Intern. J. Andrology" 7:389-397). 

A polypeptide having inhibin activity was purified from bovine or ovine follicular fluid (PCT 86/00078, 
published January 3, 1986). This protein was reported to have a molecular weight of 56,000±1,000 on SDS- 
PAGE and was dissociable into two subunits having apparent molecular weights of 44,000±3,000 and 

25 14,00012,000. Amino terminal sequences for each subunit were described. 

Miyamoto, K. et aj., Biochem. Biophys: Res. Comm. , 129(2), 1985, 396-403 disclose isolation of at least 
four inhibin-like proteins from porcine follicular fluid. One of Mr 32K was further separated into bands I, II 
and III. Band II was reduced and dissociated into two bands of Mr 20K and 13K having N-terminal 
sequences Ser-Thr-Ala-Pro and Gly-Leu-Glu-Cys respectively. 

30 EP-A-210 461 (Ajinomoto) which is part of the state of the art by virtue of Art 54(3)EPC discloses 
isolation of a polypeptide from malignant human monocytic cells. The polypeptide had a molecular weight 
of 25HKD under non-reducing, and 16±1KD under reducing SDS electrophoresis. The amino terminal 
sequence and that of a cyanogen bromide cleavage fragment of the 16KD polypeptide were determined. 
WO86/06076 (Biotechnology Australia) which is part of the state of the art by virtue of Art. 54(3)EPC 

35 discloses DNA sequences encoding inhibin. 

Two proteins both having a molecular weight of about 32,000 daltons and having inhibin activity have 
been successfully isolated from porcine follicular fluid. Purification of porcine inhibin to substantial ho- 
mogeneity, ie. about 90% by weight of total protein in the fraction, was achieved through a combination of 
protein separation procedures including heparin-Sepharose affinity chromatography, gel filtration and 

40 reverse-phase, high-performance liquid chromatography (RP-HPLC). 

These proteins were isolated to substantial homogeneity from material obtained from swine and are 
referred to as Protein A and Protein B. Each protein has a molecular weight of about 32,000 daltons (32K) 
and is composed of . two polypeptide chains having molecular weights of 18,000 and 14,000 daltons, 
respectively, the chains being linked together in the hormonally-active protein by disulfide bonding. The 

45 amino-terminal amino acid residue sequence of the 18,000 daltons (18K) or alpha chain of both proteins 
was determined to be 

Ser-Thr-Ala-Pro-Leu-Pro-Trp- 
50 Pro-Trp-Ser-Pro-Ala-Ala-Leu-Arg-Leu-Leu-Gln-Arg-Pro-Pro-Glu-Glu- 
Pro-Ala-Val. 

The amino-terminal amino acid residue sequence of the 14,000 dalton (14K) or beta chain of Protein A was 
55 determined to be 



3 



EP 0 222 491 B1 



Gly-Lcu-Glu-X-Asp-Gly-Lys-Val-Asn-Ile-X-X-Lys-Lys-Gln-Phe- 
Phe-Val-Ser-Phe-Lys-Asp-Ile-Gly-Trp-Asn-Asp-Trp-Ile-Ile-Ala 

5 

and of Protein B was determined to be 

Gly-Lcu-Glu-X-Asp-Gly-Arg-Thr-Asn- 
10 Lcu-X-X-Arg-Gln-Gln-Phe-Phe-Ile-Asp-Phe-Arg-Lcu. 

Proteins A and B have been completely characterized. Each 32K protein exhibits inhibin activity in that it 
specifically inhibits the basal secretion of FSH but does not inhibit secretion of luteinizing hormone (LH). 
15 The individual chains were not hormonally-active. 

After the filing of the parent application hereto, inhibin B-chain dimers were shown to exist in follicular 
fluid as naturally-occurring substances, termed activin, which are capable of stimulating FSH release by rat 
anterior pituitary cells (Vale et al., 1986, "Nature" 321 :776-779 and Ling et al., 1986, "Nature" 321 :779-782). 

The amino acid sequence of the a and 0 chains of inhibin from humans remained unknown until the 
20 invention herein. The large quantities of human follicular fluid required to parallel the studies conducted with 
animal inhibins are not readily available, nor is there any assurance that human and animal inhibins would 
be sufficiently similar that purification using a parallel procedure would be effective. Accordingly, methods 
are needed for determining the characteristics and amino acid sequence for human inhibin. 

Also needed are economical methods for making the a and 0 chains of inhibin in large quantities, 
25 preferably entirely and completely free of proteins from the species homologous to the inhibin in question, 
which inhibin preferably also is biologically active. 

These and other objects will be apparent from consideration of the invention as a whole. 

SUMMARY 

30 

Nucleic acid now has been isolated and cloned in replicable vectors which encodes the mature porcine 
and human a and & chains of inhibin and their precursor prepro and pro forms. Sequencing of inhibin- 
encoding cDNA has led to the identification of prodomain regions located N-terminal to the mature inhibin 
chains that represent coordinately expressed biologically active polypeptides. The prodomain regions or 

35 prodomain immunogens are useful in monitoring preproinhibin processing in transformant cell culture or in 
experiments directed at modulating the clinical condition or reproductive physiology of animals. Thus a or 0 
chain nucleic acid is used to prepare prodomain sequences from the precursor forms of the inhibin chains, 
to transform host cells for the recombinant expression of mature inhibin o and/or & chains, and in diagnostic 
assays. In particular, regions from inhibins o and/or 0 chains are expressed in recombinant cell culture by a 

40 method comprising ligating the nucleic acid encoding the region into a replicable vector under the control of 
a promoter, transforming a host cell with the vector, culturing the host cell and recovering the prodomain, 
activin or inhibin from the cultured cell. Inhibin, activin and prodomains produced by the method of this 
invention are entirely free of homologous source proteins and can be produced in biologically active form. 
The nucleic acids identified herein encode the o, £ A and /3 B chains of porcine or human inhibin. 

45 Recombinant cells are transformed to express a0 A or a/3 B inhibins, or to express 0-chain heterodimers or 
homodimers (which are collectively referred to in the literature as activin). /5-chain dimers as products of 
recombinant cells expression are free of homologous proteins with which they ordinarily are associated in 
nature. 

Inhibin or activin and their nontoxic salts, combined with a pharmaceutical^ acceptable carrier to form a 
so pharmaceutical composition, are administered to mammals, including humans, for control of fertility. 
Administration of inhibin decreases fertility in female mammals and decreases spermatogenesis in male 
mammals, and administration of a sufficient amount induces infertility. Inhibin is also useful in tests to 
diagnose infertility. Activin has been shown in the literature to be capable of stimulating FSH release from 
pituitary cells and accordingly is useful as a fertility inducing therapeutic. 
55 The method of this invention also facilitates the convenient preparation of inhibin, activin and prodomain 
variants having primary amino acid sequences and/or glycosylation differing from the native analogues, in 
particular fusions of immunogenic peptides with inhibin, activin or prodomain sequences. 



4 



EP 0 222 491 B1 



Brief Description of the Drawings 

Fig. 1A is a schematic representation of the porcine o-chain mRNA. Overlapping cDNA clones used in 
the sequence determination are shown above the diagram of the mRNA structure. Black boxes on the 3' 

5 ends of X clones indicate that these clones were obtained by specific priming. Untranslated sequences are 
represented by a line, coding sequences are boxed. The unfilled portion represents the coding region for 
the signal peptide and pro-sequences, and the cross-hatched areas indicates the 134 amino acid a-chain. 
The scale is in nucleotides from the 5* end of the longest cDNA clone. 

Fig. 1B shows the nucleotide and predicted amino acid sequence of the porcine a-chain precursor. 

70 Nucleotides are numbered at the left and amino acids are numbered throughout. The amino acid sequence 
underlined was used to design a long synthetic DNA probe. The 364 amino acid precursor includes a 
hydrophobic signal sequence, a pro-region, and the mature a-chain (amino acids 231-364). The proteolytic 
processing site Arg-Arg (black bar) immediately precedes the NH2-terminus of the mature alpha chain. 
Several other putative dibasic processing sites present in the pro-region are indicated by open bars. The 

75 two potential N-linked glycosylation sites are shown by the cross-hatched bars. The AATAAA box close to 
the 3' end of the mRNA is underlined. 

Fig.2A is a schematic representation of the porcine /S A and 0 B subunit mRNAs with coding sequences 
boxed. The /9 A and /S B subunits (dashed) are encoded towards the 3* end of the coding sequences. The 3' 
and 5' untranslated regions are shown as a line. The length of the 5' and 3' untranslated region of the &B 

20 subunit mRNA is inferred from the size of the mRNA (Fig. 3) and its obvious similarity to the 0 A mRNA. 
Tentative regions of the cDNAs are shown as dashed in the diagram. The relative positions of the 
overlapping oligo-dT primed cDNA clones and the randomly primed clones (XPINj8 A 5s, XPIN0 B 1s, and 
XPIN0 B 2s) are indicated. The scale is in nucleotides from the 5' end of the 4.5 kb mRNA. 

Fig. 2B is the nucleotide sequence and deduced amino acid sequence of the porcine inhibin 0-subunit 

25 precursors. The £ B sequence is aligned with the j8 A sequence for maximum homology. The NH2 -terminal of 
the jS-subunit precursors are indicated by bracket and arrows. Cysteine residues are shaded, possible 
processing sites are indicated by open bars, and a potential glycosylation site is shown by the cross- 
hatched box. A very GC-rich region present 3' to the termination codon intron sequences is underlined and 
overlined in both sequences. Amino acid sequences used to design oligonucleotide probes are underlined, 

30 as is the AATAAA polyadenylation signal. There was one nucleotide difference between XPIN-/8 A 8 and other 
clones covering this area. A G-to-A change causes a change of amino acid 278 from a glycine to a serine. 
The proteolytic processing site Arg Arg Arg Arg Arg (black bar) immediately precedes the NFfe terminus of 
the mature j8 A subunit, with the prosequences located upstream. The amino acids for the 0 A subunit only 
are numbered. 

35 Fig. 3 is a Northern blot analysis of porcine ovarian mRNA with a, £ A and 0 B subunit cDNA 
hybridization probes. Lanes a, b, c, d, and f are polyA* mRNA and e and g are total RNA. The position of 
the 28S and 1 8S ribosomal RNAs are shown. Lanes a, d, and e were hybridized with an a-subunit cDNA 
probe; lanes d, e and g with a 0 A subunit specific probe, and lane c with a 0 B subunitspecific probe. The a- 
subunit mRNA is approximately 1.5 kb, the 0 A subunit mRNAs are approximately 4.5 kb. The hybridizations 
40 shown in lanes a, b, and c were performed with probes of approximately equal length and specific activity 
in order to judge relative mRNA levels. 

Fig. 4A is a comparison of the human 0-TGF amino acid sequence and porcine inhibin 0 A and 0 B 
amino acid sequences. The sequences were aligned around the cysteine residues. Identical residues are 
boxed, while conservative changes are designated by an asterisk. 
45 Fig. 4B compares the a-subunit sequence with the 0 A -inhibin sequence. 

Fig. 5 depicts the construction of a representative recombinant expression plasmid for porcine inhibin. 
Fig. 6 shows the nucleotide sequence and deduced amino acid sequence of the human a-inhibin cDNA. 
The 335 amino acid pro-or inhibin sequence is numbered from the hypothesized signal cleavage site. 
Sixteen amino acids of the signal sequence are numbered -1 through -16. Homology with the porcine 
so sequence predicts a further 12 amino acid residues in the signal sequence. In this and other figures, 
putative dibasic processing sites are shown by the open bars, glycosylation sites indicated by cross- 
hatched bars, and amino terminal mature chain processing sites are depicted as black -bars. The poly (A) 
additional signal sequence is underlined. Cysteine residues are shaded. 

Fig. 7 is a comparison of the human and porcine a-inhibin protein sequences. Spaces are introduced to 
55 maximize the homology; positions of non-identity are indicated by stars. Numbering is as for the porcine 
sequence, which is one amino acid shorter than the human. 

Fig. 8 shows that the nucleotide and deduced amino acid sequence of the human 0 A inhibin signal 
sequence (residues -28 through -1) is 28 amino acids with the precursor being 378 amino acids in length. 



5 



EP 0 222 491 B1 



The basic processing site is indicated by a black bar, and a potential glycosylation site in the precursor is 
indicated by a cross-hatched bar above the sequence. Cysteine residues are shaded. 

Fig. 9 illustrates the nucleotide and deduced amino acid sequence of human 0 B inhibin cDNA. The 
sequence commences at a cysteine residue (position 7), which lines up with the cysteine present at residue 
s 7 in the j8 A sequence (see Fig. 8). The processing site for the mature 0 B inhibin is shown as a black bar and 
a potential glycosylation site as a cross-hatched bar. Cysteine residues are shaded. 

DETAILED DESCRIPTION 

10 The polypeptides of this invention are the a and £ chains of inhibin, as well as their multimer forms 
(activin and inhibin), their prepro forms and their prodomains, together with glycosylation and/or amino acid 
sequence variants of each chain or form thereof. Inhibin (including alleles) from human or animal sources 
inhibits the basal release of FSH but not of LH from anterior pituitary cells while activin does the opposite 
(hereinafter referred to as "hormonally active" activin or inhibin). 

76 Generally, amino acid sequence variants will be substantially homologous with the relevant portion of 
the porcine or human a or j3 chain sequences set forth in Figs. 1B ( 2B, 6, 8 and 9. Substantially 
homologous means that greater than about 70% of the primary amino acid sequence of the candidate 
polypeptide corresponds to the sequence of the porcine or human chain when aligned in order to maximize 
the number of amino acid residue matches between the two proteins. Alignment to maximize matches of 

20 residue includes shifting the amino and/or carboxyl terminus, introducing gaps as required and/or deleting 
residues present as inserts in the candidate. For example, see Figs. 2B and 7 where the /9 A and 0b subunits 
or human and porcine a-inhibin sequences are aligned for maximum homology. Typically, amino acid 
sequences variants will be greater than about 90% homologous with the corresponding native sequences 
shown in Figs. 1B, 2B, 6, 8 and 9. 

25 Variants that are not hormonally-active fall within the scope of this invention, and include polypeptides 
that may or may not be substantially homologous with either a mature inhibin chain or prodomain 
sequence, but which are 1) immunologically cross-reactive with antibodies raised against the native 
counterpart or 2) capable of competing with such native counterpart polypeptides for cell surface receptor 
binding. Hormonally inactive variants are produced by the recombinant or organic synthetic preparation of 

30 fragments, in particular the isolated 0 chains of inhibin, or by introducing amino acid sequence variations so 
that the molecules no longer demonstrate hormonal activity as defined above. 

Immunological or receptor cross-reactivity means that the candidate polypeptide is capable of competi- 
tively inhibiting the binding of the hormonally-active analogue to polyclonal antisera raised against the 
hormonally-active analogue. Such antisera are prepared in conventional fashion by injecting goats or rabbits 

35 S.C. with the hormonally-active analogue or derivative in complete Freunds adjuvant, followed by booster 
intraperitoneal or S.C. injections in incomplete Freunds. 

Variants that are not hormonally active but which are capable of cross-reacting with antisera to 
hormonally-active inhibin, activin, or prodomains are useful (a) as reagents in diagnostic assays for the 
native analogues or their antibodies, (b) when insolubilized in accord with known methods, as an agent for 

40 purifying anti-native analogue antibodies from antisera, and (c) as an immunogen for raising antibodies to 
hormonally-active analogues. 

This invention includes the pro and/or prepro sequences of the inhibin a or /? chain precursors, or their 
immunologically or biologically active, fragments, substantially free of the corresponding mature inhibin 
. chains. These sequences for porcine and human inhibin are shown in Figs. 1 B, 2B, 6, 8 and 9. The prepro 

45 sequence for the porcine a subunit precursor is the polypeptide comprised by residues 1 to about 230, 
while the £ A subunit pro sequence is comprised by residues t to about 308. These sequences shall be 
referred to herein as encompassing prodomain sequences. 

The a and 0 subunit prodomain sequences are comprised of several domains bounded by proteolysis 
sites, any one of which is synthesized herein separately or in combination with other domains. The principal 

so porcine j9 A domains fall within residues 1 to about 70 (domain I), about 70 to about 110 (domain II), about 
110 to about 180 (domain III), about 180 to about 260 (domain IV), and about 270 to about 309 (domain V). 
In particular, the porcine £ A domains are 



55 



6 



EP 0 222 491 B1 



GHSAAPDCPSCA1ATLPKDVPNSQPEMVEAV , 
HIliQflilLKXRPDVTQFVFKAALLNAI , LHVCKVGENGYVELEDDIG, 
AEMNELMEQTSEIITFAEAGRAJUCTI^ 
5 SIRLFQQQ, PQGSADAGEEAEDVGFPEEKSEVLI SEXWDA , 

STWHimSSSIQRllI>C^AlJ>IRTACE^ and 
CHSAAPDCPSCAIATU^VPNSQPEMVI^^ . 

70 

The porcine 0 B domains comprise RAAHILLHAVRVSGWLNL as well as homologous 0 domains having the 
same sequences. The porcine a domains comprise 

GPElI>RELVLAK\niAlJlJDALGPPAVTGEGGDPGV and 
75 GSEPEEEDVSQAI1JTATGARCGAEPAAGE1>R£^ 
DRQGMAAANSSGPUJDIXA^ 
SCSARPEATPFXVAHTRARPPSGGERA . 

20 

A typical combination domain polypeptide would be 0 A domain II linked at its C-terminus to the Nhfe- 
terminus of £ A domain III. In addition, these domains are fused together by the proteolysis sites found in the 
sequences shown in Figs. 1B or 2B, by 1 to 4 residue polypeptides that are resistant to hydrolysis (for 
example, glutaminyl or histidyl residues), or are directly fused, whereby, in all three instances, combination 

25 domain polypeptides are produced. 

Principal human a chain prodomains are approximately residues 30-199 and 1 to 29, human 0A 
prodomains are approximately residues 1-30, 32-40, 43-59, 62-80, 83-185 and 186-230 while human 0 B 
prodomains are approximately residues 1-13, 15-30, 32-59, 62-145, 148-195 and 198-241 (referring to the 
numbering system adopted in Figs. 6, 8 and 9, respectively). Combination prodomain polypeptides are 

ao within the scope hereof, for example, the jS A prodomain at about 43-80, and 0 B prodomains at about 1-30 
and about 32-145. The preferred human a, 0 A and /3 B chain prodomains are about residues 1-29, about 43- 
80 and about 1 -30, respectively. 

The intact isolated prepro or prodomain & A , fi B or a sequences are best synthesized in recombinant cell 
culture. The individual subcomponent domains are synthesized by routine methods of organic chemistry or 

35 by recombinant cell culture. They then are labelled with a radioisotope or other detectable group such as an 
enzyme or fluorophore in accord with known methods and used in standard competitive immunoassays to 
detect the levels of prepro or pro forms of inhibin, including individual domains, in transformants with DNA 
encoding such forms or their precursors. This assay is useful in determining whether proteolytic hydrolysis 
of proinhibin is occurring in the host transformants or their culture media. The assay also is useful in 

40 determining whether a rate limiting step in recombinant synthesis is translation of mRNA into the prepro 
forms or processing of the prepro forms into mature inhibin. For example, high levels of prepro or pro 
inhibin in cell lysates, but relatively low levels of secreted mature inhibin, would suggest that the host cell is 
adequately transcribing and translating the inhibin ONA, but is not processing the precursors at an adequate 
rate. Thus, in this case one would select an alternate host cell rather than concentrating on improving the 

45 transcription or translation efficiency of the transforming plasmid, e.g., by selecting an alternate promoter. 
The prodomain sequences also are believed to be involved in coordinate modulation of animal physiology 
in reproductive cycles and fertility. 

Amino acid sequence variants are any one of 1) hormonal ly-active, 2) cross reactive with antibodies 
raised against mature inhibin or prodomain a or j9 chain sequences, or 3) cross-reactive with inhibin/activin 

so cell surface receptors, but are characterized by a primary amino acid sequence that departs from the 
sequence of natural inhibins or prodomain sequences. These derivatives ordinarily are prepared by 
introducing insertions, deletions or substitutions of nucleotides into the DNA encoding the target DNA to be 
modified in order to encode the variant, and thereafter expressing the DNA in recombinant cell culture. 
Polypeptides having up to about 100-150 residues also are conveniently prepared by in vitro synthesis. 

55 Such variants are characterized by the predetermined nature of the variation, a feature that sets them apart 
from naturally occurring allelic or interspecies variation. The variants may exhibit the same qualitative 
biological activity as the naturally-occurring analogue or may act antagonistically towards such analogues. 



7 



EP 0 222 491 B1 



While the site for introducing a sequence variation is predetermined, it is unnecessary that the mutation 
per se be predetermined. For example, in order to optimize the performance of mutation at a given site, 
random mutagenesis may be conducted at the target codon or region and the expressed inhibin mutants 
screened for the optimal combination of desired activity. Techniques for making substitution mutations at 

5 predetermined sites in DNA having a known sequence is well known, for example M13 primer mutagenesis. 
Mutagenesis is conducted by making amino acid insertions, usually on the order of about from 1 to 10 
amino acid residues, or deletions of about from 1 to 30 residues. Deletions or insertions preferably are 
made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, 
insertions or any subcombination may be combined to arrive at a final construct. Insertions include amino or 

10 carboxyl-terminal fusions, e.g. a hydrophobic extension added to the carboxyl terminus. Preferably, 
however, only substitution mutagenesis is conducted. Obviously, the mutations in the encoding DNA must 
not place the sequence out of reading frame and preferably will not create complementary regions that 
could produce secondary mRNA structure. 

Not all mutations in the DNA which encode the polypeptides herein will be expressed in the final 

75 secreted product. For example, a major class of DNA substitution mutations are those in which a different 
secretory leader or signal has been substituted for the native porcine or human a or & chain secretory 
leader, either by deletions within the leader sequence or by substitutions, wherein most or all of the native 
leader is exchanged for a leader more likely to be recognized by the intended host. For example, in 
constructing a procaryotic expression vector the porcine or human a or & chain secretory leader is deleted 

20 in favor of the bacterial alkaline phosphatase or heat stable enterotoxin II leaders, and for yeast the leader is 
substituted in favor of the yeast invertase, alpha factor or acid phosphatase leaders. However, the porcine 
and human secretory leaders are recognized by many heterologous higher eukaryotic cells. When the 
secretory leader is "recognized" by the host, the host signal peptidase is capable of cleaving a fusion of the 
leader polypeptide fused at its C-terminus to the mature inhibin or prodomain such that mature inhibin or 

25 prodomain polypeptide is secreted. 

Another major class of DNA mutants that are not expressed in final form as amino acid sequence 
variations are nucleotide substitutions made in the DNA to enhance expression, primarily to avoid 5' stem 
and loop structures in the transcribed mRNA (see de Boer et al., EP 75.444A) or to provide codons that are 
more readily transcribed by the selected host, e.g. the well-known preference codons for E. coli or yeast 

30 expression. These substitutions may or may not encode substituted amino acid residues, but preferably do 
not. 

Insertional and deletional amino acid sequence variants are proteins in which one or more amino acid 
residues are introduced into or removed from a predetermined site in the target inhibin, activin, prodomain 
or proform of inhibin or activin. Most commonly, insertional variants are fusions of heterologous proteins or 

35 polypeptides to the amino or carboxyl terminus of the a or j8 chains, the prodomains or other inhibin 
derivatives. Immunogenic derivatives are made by fusing an immunogenic polypeptide to the target 
sequence, e.g. a prodomain polypeptide, by synthesis in vitro or by recombinant cell culture transformed 
with DNA encoding the fusion. Such immunogenic polypeptides preferably are bacterial polypeptides such 
as trpLE, beta-galactosidase and the like, together with their immunogenic fragments. Other insertions entail 

40 inserting heterologous eukaryotic (e.g. the herpes virus gD signal) or microbial secretion signal or protease 
processing sequences upstream from the NH2 -terminus of the protein to be secreted. Deletions of cysteine 
or other labile residues also may be desirable, for example in increasing the oxidative stability of the a or & 
chain. Deletion derivatives will produce a or & chain fragments. Such fragments, when biologically or 
immunologically active, are within the scope herein. For instance, a fragment comprising £b or & A residues 

45 about from 11 to 45 (numbered from mature Glyi ) is to be included within the scope herein. 

Immunogenic conjugates of prodomain polypeptides, inhibin and activin are readily synthesized in 
recombinant cell culture as fusions with immunogenic polypeptides, e.g. beta-lactamase or viral antigens 
such as herpes gD protein, or by preparation of the polypeptides in unfused form (by recombinant or in 
vitro synthetic methods) followed by covalent cross-linking to an immunogenic polypeptide such as keyhole 

so limpet hemocyanin or STI using a divalent cross-linking agent. The immunogenic polypeptides are 
formulated with a vaccine adjuvant, e.g. alum or Freunds. Methods for preparing proteins in adjuvants and 
for cross-linking are well-known per se and would be employed by one skilled in the art, as are methods for 
vaccinating animals. The immunogenic conjugates are useful in preparing antibodies to the prodomain 
region for use in monitoring inhibin manufacture or for in vivo vaccination with the objective of raising 

55 antibodies capable of modulating animal physiology in reproductive cycles and fertility. Typically, the 
prodomain or its immunogen is administered in varied doses to fertile laboratory animals or swine and the 
reproductive cycles and fertility of the animals monitored, together with assays of serum levels of anti- 
immunogen or prodomain by routine competitive or sandwich immunoassay. 



8 



EP 0 222 491 B1 



Substitution derivatives are produced by mutating the DNA in a target codon, so that thereafter a 
different amino acid is encoded by the codon, with no concomitant change in the number of residues 
present in the molecule expressed from the mutated DNA. Substitutions or deletions are useful for example 
in increasing the stability of the proteins herein by eliminating proteolysis sites, wherein residues are 

5 substituted within or adjacent to the sites or are deleted from the sites, or by introducing additional disulfide 
bonds through the substitution of cysteine for other residues. Substitutions are useful for facilitating the 
synthesis or recovery of mature or prodomain a or 0 chains. For example, methionine residues within the 
mature inhibin sequences are substituted or deleted, prepro sequences deleted, methionine inserted at the 
-1 site immediately NH2 terminal to the mature NH2 terminal residue and another sequence inserted N- 

70 terminal to the exogenous methionine. The inhibin derivative in this case is expressed as a fusion having an 
intermediate methionyl residue, which in turn is cleaved at this residue by cyanogen bromide in accordance 
with known practice. The mature inhibin derivative released from the fusion is recovered. 

Exemplary porcine inhibin derivatives are [Asn26 6->Gln]lnha (to remove the putative glycosylation site), 
[QyS32s o r Cys324->A]!nha, [CyS36i or Cys 3 6 3->A]lnha, [LVS321 or Lys322->A]lnh£ A or [Lys322->His or 

75 Ser]lnh/9 A (to inactivate a potential proteolysis site), [Lys3is->Arg; Val3u->Thr] lnh£ A (to create a £ A /jS B 
hybrid), [Cys388 or Cys39o->A]lnhj8 A , [Lys4i i->Gln]lnh/S A , [Arg 3 i5->Lys, Val 3 u->Thr]lnh0 B (to create a 
jSb/0 a hybrid), [Cys 3 i9 or Cys32<r>A]lnhjS B [Prossi Gly 38 2-> Pro Phe Gly]lnh0 B , and [Arg 395 ->Gln]lnh0 B , 
wherein inh is an abbreviation for inhibin and the residue members for lnh/J B are those used for the 
corresponding lnh£ A residue (see Fig. 2B). 

20 The lnh/8 A amino acid positions which are principal candidates for mutational substitution or deletion (or 
adjacent to which residues may be inserted) include residues 293-297, 364-376 and 387-398 (Fig. 8). 
Preferably, the proline, cysteine and glycine residues within these sequences are not modified. Candidates 
having greater potency than inhibin or activin, or which serve as inhibin or activin antagonists, are identified 
by a screening assay wherein the candidate is diluted into solutions containing constant amounts of inhibin 

25 or activin and the compositions assayed in the rat pituitary cell assay. Candidates which neither antagonize 
or agonize inhibin or activin are screened for utility in immunoassays for inhibin or activin by measuring 
competitive immunodisplacement of labelled inhibin or activin of the native hormones from polyclonal 
antibody directed against the native hormones. Exemplary contemplated sequence variants of lnh£ A include 
Phe 3 o2->He or Leu; Gln 29 7->Asp or Lys; Trp 30 7->Tyr or Phe; Trp 3 io->Tyr or Phe; llesti->Phe or Val; 

30 Tyr 3 i7->Trp or Thr; HiS3is->Lys; AIa 3 ig->Ser; Asn 3 2o->Gln, Tyr or His; Tyr 3 2i->Thr or Asp, Phe34o->Tyr (a 
TGF-j8/j8 A intrachain hybrid); His 3 53->Asp; His 353 ->Lys (a 0 A /j8 B hybrid); Phe 35 6->Tyr; Vals64->Phe; Val 36 4- 
>Leu; Tyr 37 5->Thr; Tyr 3 76->Trp; Asn 38 s->Gln, His or Lys; lle 3 gi->Leu or Thr; Metsgo ->Leu or Ser; Val392- 
>Phe, Glu, Thr or lie. Comparable modifications are made in the human 0 B chain. For example, lnhj8 A 
contains a phenylalanyl residue at position 302, and Inhfo also contains a phenylalanyl residue at a 

35 homologous position (264, Fig. 9) when aligned in the same fashion as is shown for porcine 0 B in Fig. 4A. 
Thus, since the Phe 30 2residue of 0 A is described above as substituted by isoleucinyl or leucinyl, the Phe264 
of 0b is substituted with the same residues. 

A factor in establishing the identity of a polypeptide as inhibin, activin or an inhibin variant is the ability 
of antisera which are capable of substantially neutralizing the hormonal activity of mature inhibin or activin 

40 to also substantially neutralize the hormonal activity of the polypeptide in question. However it will be 
recognized that immunological identity and hormonal activity are not necessarily coextensive. For example, 
a neutralizing antibody for inhibin may not bind a candidate protein "because the neutralizing antibody 
happens to not be directed to specifically bind a site on inhibin that is critical to its activity. Instead, the 
antibody may bind an innocuous region and exert its neutralizing effect by steric hinderance. Therefore a 

45 candidate protein mutated in this innocuous region might no longer bind the neutralizing antibody, but it 
would nonetheless be inhibin in terms of substantial homology and biological activity. 

It is important to observe that characteristics such as molecular weight, isoelectric point and the like for 
a native or wild type mature inhibin or activin obtained from follicular fluid or other tissue sources are 
descriptive only for the native form. Variants contemplated by the foregoing definition will include other 

50 polypeptides which will not exhibit all of the characteristics of native analogue. For example, inhibin 
derivatives like the insertion mutants, deletion mutants, or fusion proteins described above will bring inhibin 
outside of the molecular weight established for the corresponding native inhibin because fusion proteins 
with mature inhibin or proinhibin itself as well as insertion mutants will have a greater molecular weight than 
native, mature inhibin. On the other hand deletion mutants of native, mature inhibin will have a lower 

55 molecular weight. Finally, post-trahslational processing of preproinhibin chains in heterologous cell lines 
may not be accomplished with the fidelity exercised by the homologous host cell, thereby resulting in some 
variation in the amino termini of the a and/or 0 chains. This variation may be encountered as residual 
prosequence remaining with the mature protein, or the loss of several mature residues that are cleaved off 

9 



EP 0 222 491 B1 



with the prosequence. The same is true with processing of the preprotein in heterologous recombinant cells. 

Covalent modifications of inhibin, activin or prodomains are included within the scope hereof and 
include covalent or aggregative conjugates with other chemical moieties. Covalent derivatives are prepared 
by linkage of functionalities to groups which are found in the inhibin amino acid side chains or at the N-or 

s C-termini, by means known in the art. For example, these derivatives will include: aliphatic esters or amides 
of the carboxyl terminus or residues containing carboxyl side chains, e.g., aspartyl residues; O-acyl 
derivatives of hydroxyl group-containing residues such as seryl or alanyl; and N-acyl derivatives of the 
amino terminal amino acid or amino-group containing residues, e.g. lysine or arginine. The acyl group is 
selected from the group of alkyl-moieties (including C3 to C10 normal alky I), thereby forming alkanoyl 

io species, and carbocyclic or heterocyclic compounds, thereby forming aroyl species. The reactive groups 
preferably are difunctional compounds known p_er se for use in cross-linking proteins to insoluble matrices 
through reactive side groups, e.g. m-Maleimidobenzoyl-N-hydroxy succinimide ester. Preferred derivatiza- 
tion sites are at histidine residues. 

Covalent or aggregative derivatives of mature inhibin, activin or prodomain sequences are useful as 

15 reagents in immunoassay or for affinity purification procedures. For example, inhibin or prodomain is 
insolubilized by covalent bonding to cyanogen bromide-activated Sepharose® by methods known per se or 
adsorbed to polyolefin surfaces (with or without glutaraldehyde cross-linking) for use in the assay or 
purification of anti-inhibin or anti-prodomain antibodies or cell surface receptors. Inhibin or a prodomain 
sequence also is labelled with a detectable group, e.g., radioiodinated by the chloramine T procedure, 

20 covalently bound to rare earth chelates or conjugated to another fluorescent moiety for use in diagnostic 
assays, especially for diagnosis of inhibin or prodomain levels in biological samples by competitive-type 
immunoassays. 

DNA which encodes the complete a and £ chains of inhibin/activin is obtained by chemical synthesis, 
by screening reverse transcripts of mRNA from ovary, or by screening genomic libraries from any cell. It 

25 may be more efficient to simply synthesize portions of the DNA desired since screening is required to 
identify DNA to cDNA or genomic libraries that encode the a and 0 chains. Synthesis also is advantageous 
because unique restriction sites can be introduced at the time of preparing the DNA, thereby facilitating the 
use of the gene in vectors containing restriction sites otherwise not present in the native sequence, and. 
steps can be taken to enhance translational efficiency as discussed above, without the need to further 

30 modify the DNA as by mutagenesis or the like. cDNA encoding the a or 0 chains is free of untranslated 
intervening sequences (introns) as well as free of flanking DNA encoding other proteins homologous to their 
source. 

DNA encoding the q and 0 chains is obtained from other sources than porcine and human by (a) 
obtaining a cDNA library from the ovary of the target animal, (b) conducting Southern analysis with labelled 

35 DNA encoding porcine or human a and £ chains or fragments thereof (generally, greater than 100 bp) in 
order to detect clones in the cDNA library that contain homologous sequences, (c) analyzing the clones by 
restricting enzyme analysis and nucleic acid sequencing so as to identify full-length clones and, if full length 
clones are not present in the library, recovering appropriate fragments from the various clones and ligating 
them at restriction sites common to the clones to assemble a clone encoding the full-length molecule. As 

40 shown infra , any sequences missing from the library can be obtained by the 3' extension on ovarian mRNA 
of synthetic oligodeoxynucleotides complementary to cDNA identified by screening the library, or homolo- 
gous sequences are supplied from known animal cDNAs. This is particularly useful in constructing pre or 
prepro inhibin sequences to facilitate processing of preproinhibin to mature inhibin from the desired 
species. 

45 Porcine and human ovarian cDNA libraries initially were probed for DNA encoding inhibin sequences 
using labelled oligonucleotides whose sequence was based on the partial amino acid sequence determined 
from analysis of purified porcine inhibin or, in the case of human cDNA, porcine cDNA probes. However, 
once having described cDNA encoding human and porcine inhibin and prodomains, one skilled in the art 
would realize that precisely hybridizing probes can be prepared from the described sequences in order to 

so readily obtain the remainder of the desired human or porcine gene. 

Nucleotide sequence analyses of identified porcine and human cDNA clones revealed the structures of 
the biosynthetic precursors of both forms of inhibin. Interestingly, the two inhibin chains are not derived 
from a single processed precursor. Instead, the two chains are translated from separate mRNAs and then 
assembled into the disulfide crosslinked two-chain molecule. 

55 Figs. 1 B and 23 and 6, 8 and 9 depict the DNA encoding the polypeptide chains constituting porcine 
and human preproinhibin and preproactivin. Obviously, degenerate codons may be substituted for those 
disclosed in these figures where the same amino acid is encoded. The DNA of Figs. 1 B, 2B, 6, 8 and 9 is 
mutated in order to encode the amino acid variants of the a and jSchains described above. In particular, the 



10 



EP 0 222 491 B1 



prepro sequences are deleted and a start codon inserted immediately 5' to the mature chain in question so 
that the chain is expressed directly in recombinant culture. The DNA also is labelled, e.g. with radioactive 
phosphorous, and used to screen ovarian cDNA libraries from other species to identify o or )3 chain 
encoding DNA from such other species as is generally described above. 

5 Covalent labelling of this DNA is accomplished with a detectable substance such as fluorescent group, 
a radioactive atom or a chemiluminescent group by methods known per se. The labelled DNA is then used 
in conventional hybridization assays. Such assays are employed in identifying vectors and transformants as 
described in examples infra , or for in vitro diagnosis such as detection of mRNA in tissues. 

Lengthy sequences desirably are synthesized in host cells transformed with vectors containing DNA 

70 encoding them, e.g. inhibin or prodomain sequence. Vectors are used to amplify the DNA which encodes 
, the chains, either in order to prepare quantities of DNA for further processing (cloning vectors) or for 
expression of the chains (expression vectors). An expression vector is a replicable DNA construct in which a 
DNA sequence encoding an a or $ chain is operably linked to suitable control sequences capable of 
effecting their expression in a suitable host. Cloning vectors need not contain expression control sequences. 

is Such control sequences include a transcriptional promoter, an optional operator sequence to control 
transcription, a sequence encoding suitable mRNA ribosomal binding sites (for prokaryotic expression), and 
sequences which control termination of transcription and translation. The vector should include a selection 
gene to facilitate the stable expression of the desired polypeptide and/or to identify transformants. However, 
the selection gene for maintaining a and/or £ chain expression can be supplied by a separate vector in 

20 cotransformation systems using eukaryotic host cells. 

Vectors comprise plasmids, viruses (including phage), and integratable DNA fragments i.e., fragments 
that are integratable into the host genome by recombination. The vectors described herein for use in 
eukaryotic cell expression of inhibin a and/or 0 chains contain plasmid sequences for cloning in microbes, 
where the plasmid replicates autonomously from the host genome, but the DNA is believed to integrate into 

25 the eukaryotic host cell genome upon transformation. Similarly, bacillus vectors that genomically integrate 
by homologous recombination in bacillus also are useful. However, all other forms of vectors which serve an 
equivalent function and which are, or become, known in the art are suitable for use herein. 

Suitable vectors generally will contain replicon (origins of replication, for use in non-integrative vectors) 
and control sequences which are derived from species compatible with the intended expression host. 

30 Transformed host cells are cells which have been transformed or transfected with vectors containing inhibin 
a and/or j8 chain encoding DNA. Transformed host cells contain cloned DNA and, when transformed with an 
expression vector, also express the o and/or £ chains. The expressed polypeptides will be deposited 
intracellular^ or secreted into either the periplasmic space or the culture supernatant, depending upon the 
-host cell selected and the presence of suitable processing signals in the expressed protein, e.g. homolo- 

35 gous or heterologous signal sequences. 

DNA regions are operably linked when they are functionally related to each other. For example, DNA for 
a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a 
preprotein which participates in the secretion of the polypeptide; a promoter is operably linked to a coding 
sequence if it controls the transcription of the sequence; or a ribosome binding site is operably linked to a 

40 coding sequence if it is positioned so as to permit translation. Generally, operably linked means that the 
DNA sequences being linked are contiguous and, in the case of secretory leaders, contiguous and in 
reading phase. 

Suitable host cells are prokaryotes, yeast or higher eukaryotic cells. Prokaryotes include gram negative 
or gram positive organisms, for example E. coli or Bacilli. Higher eukaryotic cells include established cell 

45 lines of mammalian origin as described below. A preferred host cell is E. coli 294 (ATCC 31,446) although 
other prokaryotes such as E. coli B, E. coli X1776 (ATCC 31 537), E. coli W3110 (ATCC 27 325), 
pseudomonas species, or Serratia Marcesans are suitable. 

Expression vectors for host cells ordinarily include an origin of replication (where extrachromosomal 
amplification is desired, as in cloning, the origin will be a bacterial origin), a promoter located upstream from 

so the inhibin coding sequences, together with a ribosome binding side (the ribosome binding or Shine- 
Dalgarno sequence is only needed for prokaryotic expression), RNA splice site (if the inhibin DNA contains 
genomic DNA containing one or more introns), a polyadenylation site, and a transcriptional termination 
sequence. As noted, the skilled artisan will appreciate that certain of these sequences are not required for 
expression in certain hosts. An expression vector for use with microbes need only contain an origin of 

55 replication recognized by the intended host, a promoter which will function in the host and a phenotypic 
selection gene, for example, a gene encoding proteins conferring antibiotic resistance or supplying an 
auxotrophic requirement. Inhibin DNA is typically cloned in E. coli using pBR322, a plasmid derived from an 
E. coli species (Bolivar, et aj. ( 1977 "Gene" 2: 95). pBR322 contains genes for ampicillin and tetracycline 



11 



EP 0 222 491 B1 



resistance and thus provides easy means for identifying transformed cells. 

Expression vectors, unlike cloning vectors, must contain a promoter which is recognized by the host 
organism. This is generally a promotor homologous to the intended host. Promoters most commonly used 
in recombinant DNA construction include the ^-lactamase (penicillinase) and lactose promoter systems 

5 (Chang et aU 1978, "Nature", 275: 615; and Goeddel et al., 1979, "Nature" 281: 544), a tryptophan (trp) 
promoter system (Goeddel et al.. 1980, "Nucleic Acids Res." 8: 4057 and EPO Appl. Publ. No. 36,776) and 
the tac promoter [H. De Boer et al., 1983, "Proc. Natl Acad. Sci. U.S.A." 80: 21-25]. While these are the 
most commonly used, other known microbial promoters are suitable. Details concerning their nucleotide 
sequences have been published, enabling a skilled worker operably to ligate them to DNA encoding inhibin 

w in plasmid vectors (Siebenlist et al., 1980, "Cell" 20: 269) and the DNA encoding inhibin or its derivative. 
Promoters for use in prokaryotic expression systems also will contain a Shine-Dalgarno (S.D.) sequence 
operably linked to the DNA encoding the inhibin, i.e., the S.D. sequence is positioned so as to facilitate 
translation. Generally, this means that the promoter and S.D. sequences located upstream from the second 
codon of a bacterial structural gene are substituted for the sequences of prepro inhibin located 5' to the 

75 mature a and/or £ chains. 

In addition to prokaryotes, eukaryotic microbes such as yeast cultures are transformed with inhibin- 
encoding vectors. Saccharomyces cerevisiae , or common baker's yeast, is the most commonly used 
among lower eukaryotic host microorganisms. However, a number of other strains are commonly available 
and useful herein. Yeast vectors generally will contain an origin of replication from the 2 micron yeast 

20 plasmid or an autonomously replicating sequence (ARS), a promoter, DNA encoding the a and/or 0 chain, 
sequences for polyadenylation and transcription termination, and a selection gene. A suitable plasmid for 
expression in yeast is YRp7, (Stinchcomb et al., 1979, "Nature", 282: 39; Kingsman et al., 1979, "Gene", 7: 
141; Tschemper et al., 1980, "Gene", 10:157). This plasmid already contains the trp1 gene which provides 
a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example ATCC 

25 No. 44076 or PEP4-1 (Jones, 1977, "Genetics", 85: 12). The presence of the trpl lesion in the yeast host 
cell genome then provides an effective environment for detecting transformation by growth in the absence 
of tryptophan. 

Suitable promoting sequences in yeast vectors include the promoters for metallothionein, 3- 
phosphoglycerate kinase (Hitzeman et al., 1980, "J. Biol. Chem.", 255: 2073) or other glycolytic enzymes 

30 (Hess et al., 1968, "J. Adv. Enzyme Reg.", 7: 149; and Holland et a|., 1978, "Biochemistry", 17, 4900), such 
as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylate, phosphofruc- 
tokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate 
isomerase, phosphoglucose isomerase, and glucokinase. Suitable vectors and promoters for use in yeast 
expression ar further described in R. Hitzeman et al., EP 73.657A. 

35 Other yeast promoters, which have the additional advantage of transcription controlled by growth 
conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, 
degradative enzymes associated with nitrogen metabolism, and the aforementioned metallothionein and 
glyceraldehyde-3- phosphate dehydrogenase, as well as enzymes responsible for maltose and galactose 
utilization. In constructing suitable expression plasm ids, the termination sequences associated with these 

40 genes are also ligated into the expression vector 3' of the inhibin or derivative coding sequences to provide 
termination and polyadenylation of the mRNA. 

Cultures of cells derived from multicellular organisms are the preferred host cells herein because it is 
believed that expression of hormonally. active inhibin or activin will only occur in such cells, with microbial 
expression resulting at most only in immunological cross-reactivity. In principle, any higher eukaryotic cell 

45 culture is workable, whether from vertebrate or invertebrate culture. Propagation of vertebrate cells in 
culture per se has become a routine procedure in recent years [ Tissue Culture , Academic Press, Kruse and 
Patterson, editors (1973)]. 

Suitable host cells for expressing a or 0 chains in higher eukaryotes include: monkey kidney CVI line 
transformed by SV40 (COS-7, ATCC CRL 1651); baby hamster kidney cells (BHK, ATCC CRL 10); Chinese 

so hamster ovary-cells-DHFR (described by Urlaub and Chasin, PNAS (USA) 77: 4216, [1980]); mouse Sertoli 
cells (TM4, Mather, J.P., Biol. Reprod. 23: 243-251 [1980]); monkey kidney cells (CVI ATCC CCL 70); 
african green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, 
ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 
1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary 

55 tumor (MMT 060652, ATCC CCL 51); rat hepatoma cells (HTC, M1, 54, Baumann, M., et al., J. Cell Biol. 85: 
1-8 [1980]) and TRI cells (Mather, J.P. et al., Annals N.Y. Acad. Sci. 383: 44-^68 [1982]). 

The transcriptional and translation control sequences in vertebrate cell expression vectors preferably are 
provided from viral sources. For example, commonly used promoters are derived from polyoma, Adenovirus 



12 



EP 0 222 491 B1 



2, and most preferably Simian Virus 40 (SV40). The early and late promoters of SV40 are particularly useful 
because both are obtained easily from the virus as a fragment which also contains the SV40 viral origin of 
replication (Fiers et al., 1978, "Nature", 273: 113). Smaller or larger SV40 fragments may also be used, 
provided the approximately 250 bp sequence extending from the Hind II! site toward the Bgl I site located in 

6 the viral origin of the replication is included. Further, it is also possible to utilize the genomic promoters, 
control and/or signal sequences normally associated with the a or /S-chains, provided each control 
sequences are compatible with and recognized by the host cell. 

An origin of replication may be provided either by construction of the vector to include an exogenous 
origin, such as may be obtained from SV40 or other viral (e.g. Polyoma, Adenovirus, VSV, or BFV) source, 

70 or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the 
host cell chromosome, the latter is often sufficient. 

Rather than using vectors which contain viral origins of replication, mammalian cells are cotransformed 
with DNA encoding a selectable marker and DNA encoding the a and/or $ chains. An example of a suitable 
selectable marker is dihydrofolate reductase (DHFR) or thymidine kinase. Such markers are proteins, 

75 generally enzymes that enable the identification of transformant cells, i.e., cells which had been competent 
to take up exogenous DNA. Generally, identification is by survival of transformants in culture medium that is 
toxic to untransformed cells or from which the cells cannot obtain a critical nutrient without having taken up 
the marker protein. 

In selecting a preferred host, mammalian cell for transfection by vectors which comprise DNA 

20 sequences encoding both inhibin and DHFR, it is appropriate to select the host according to the type of 
DHFR protein employed. If wild type DHFR protein is employed, it is preferable to select a host cell which 
is deficient in DHFR thus permitting the use of the DHFR coding sequence as marker for successful 
transfection in selective medium which lacks hypoxanthine, glycine, and thymidine (hgt~). An appropriate 
host cell in this case is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity, prepared and 

25 propagated as described by Urlaub and Chasin, 1980, "Proa, Nat'l. Acad. Sci." (USA) 77: 4216. 

On the other hand, if DNA encoding DHFR protein with low binding affinity for methotrexate (MTX) is 
used as the controlling sequence, it is not necessary to use DHFR resistant cells. Because the mutant 
DHFR is resistant to MTX, MTX containing media can be used as a means of selection provided that the 
host cells are themselves MTX sensitive. Most eukaryotic cells which are capable of absorbing MTX appear 

30 to be methotrexate sensitive. One such useful cell line is a CHO line, CHO-KI (ATCC No. CCL 61). 
Preferably, transformants are first selected for neomycin resistance (the transfection is conducted together 
with DNA encoding the neomycin resistance gene), followed by MTX amplification of the o and/or p chain 
expression as the case may be. See Kim et al., "Cell" 42: 129-138 (1985) and EP 160,457A. 

Other methods suitable for adaptation to the synthesis of a and/or 0 chains in recombinant vertebrate 

35 cell culture are described in M-J. Gething et al., "Nature" 293: 620-625 (1981); N. Mantei et al., "Nature" 
281 : 40-46; and A. Levinson et al., EP 1 17.060A and 1 17,058A. 

The inhibin a chain is expressed in recombinant cell culture with or without either of the /3-chain 
molecules. Similarly, host cells are transformed with DNA encoding either or both of the mature 0-chains. 
Based on analogy to TGF-0, the mature ^-chains are capable of forming homodimers or 0 a /&b 

40 heterodimers upon expression in recombinant culture. These structures are not inhibin and will be referred 
to herein as /8-chain dimers or activin. These are useful in the preparation of active inhibin, serving as 
sources of the j9-chain, or are used as gel electrophoresis standards to detect the diversion into j8-chain 
dimers of 0-chains synthesized in a and j8 chain cotransformants. As will be seen in Example 4, this is not 
a hypothetical problem. Of course, the dimers are also useful in modulating reproduction as noted above. 

45 jS-chain hetero or homodimers are separated by in vitro unfolding of the individual chains followed by 
oxidative disulfide bond formation with the a-chain in accord with processes generally known per se. 
Preferably, however, in preparing mature inhibin the recombinant host is transformed with DNA encoding 
both the a and either of the 0-chains. The intact hormonally active molecule is then assembled by the host 
cell in vivo, and it is thus unnecessary to combing the two chains by in vitro processing. The DNA encoding 

50 the a and 0-chains is preferably located on the same vector, and under the control of the same promoter, 
but this is not essential. 

Certain 0-chain amino acid sequence variants identified in the screening procedure will not bind to 
pituitary cell surface receptors nor as a consequence will they exhibit hormonal activity. Such variants, when 
expressed as homodimers in recombinant cell culture, are useful in immunoassays for activin when they 
55 bear immunological epitopes cross-reactive with the native 0-chain. In addition, such variants are coexpres- 
sed with DNA encoding hormonally active 0-chain to yield a hybrid bearing native and variant 0-chain. In 
this case the variant serves to stabilize the structure of the native £-chain. This form of j9-chain heterodimer 
is useful, like the homodimer, in immunoassays for activin. It may also function as an activin antagonist. 



13 



EP 0 222 491 B1 



The activin/inhibin ^-chains also are coexpressed with TGF-/S in order to produce 0-chain/TGF-0 
hybrids. Vectors and methods for the expression of TGF-0 are known. For example, see Derynck et al., 
Human Transforming Growth Factor-^ Complementary DNA Sequence and Expression in Normal and 
Transformed Cells "Nature" 316: 701-705 (1985). Contransformation of mammalian host cells by vectors 

5 bearing the TGF-j9 gene as described by Derynck et al. together with the 0 A or £ B chains of activin/inhibin 
will result in secretion of a proportion of a 0-chain/TGF-j8 hybrid dimers. This hybrid is useful in preparing 
TGF-£/j9-chain immunogens or in immunoassays. 

Inhibin, activin or prodomain sequences are recovered from transformed cells in accord with per se 
known procedures. When a polypeptide is expressed in recombinant bacteria as a retractile body, the 

70 desired polypeptide is recovered and refolded by conventional methods. Alternatively, the culture super- 
natants from transformed cells that secrete activin or inhibin, preferably mammalian cells, are simply 
separated from the cells by centrifugation. Then the inhibin generally is purified by successive purification 
procedures that include heparin-Sepharose affinity chromatography, gel filtration and at least one and 
preferably several RP-HPLC (reverse phase high pressure liquid chromatography) steps using different 

rs conditions in the stationary phase and/or mobile phase. Prodomain sequences produced by in vitro 
synthesis will be purified by conventional methods. 

The prodomain polypeptides that are preferred for use herein are recovered from the culture media of 
recombinant cells transformed to synthesize the a and/or $ chains as appropriate for the desired 
prodomain. Specifically, they are recovered by separating the culture medium polypeptides on native 

20 electrophoresis gel, excising bands having the predicted molecular weight and thereafter purifying the 
eluted polypeptides further, for example by FPLC or HPLC, followed by amino acid sequence determination 
for the substantially homogeneous separated polypeptides. Purified prodomain polypeptides then are used 
to raise antibodies, e.g., in rabbits, which when used in immunoaffinity purification will simplify the recovery 
of the prodomains. 

25 In the preferred procedure for isolating porcine hormonally active inhibin, clarified transformant culture 
supernatant or cell lysate is first purified by heparin-Sepharose® affinity chromatography, next by gel 
filtration on Sephacryl® S-200 gel and then with four successive RP-HPLCs using different mobile phase 
gradients and/or derivatized silica supports. Preferably, stationary phases having relatively low hydrophobic- 
ity are used, with C3-C8 columns being preferred and C3-C5 and phenyl columns being particularly 

so preferred. Solute specificity of the mobile phase is preferably adjusted by varying the concentration of an 
organic component, particularly acetonitrile. Although a single RP-HPLC fractionation significantly increases 
the purity relative to the gel-filtrated material, two or more, and preferably four, RP-HPLC purifications are 
generally performed subsequent to successive treatment by heparin-Sepharose chromatography and gel 
filtration. This method has been found to be adaptable to the purification of human inhibin from recombinant 

35 cell culture as well. 

The first step of the purification is heparin-Sepharose affinity chromatography, in which the protein is 
adsorbed to the Sepharose-bound heparin moieties under application conditions, and the adsorbed inhibin 
material is recovered by 1 M NaCI elution. This step greatly expedites the purification procedure for crude 
extracts because it allows a relatively large volume of a crude extract to be processed fairly rapidly while 
40 recovering an amount of protein exhibiting total inhibin activity equal to at least 90% of that of the crude 
extract. 

For the detection of inhibin activity in the various column fractions, aliquots ranging from 0.01% to 0.1% 
by volume are removed, and after added 100 ug human serum albumin in 100 ul water, the solvents were 
evaporated in a Speed-Vac concentrator (Savant, Hicksville, N.Y.). The residue as redissolved in 3 ml 1% 

45 fetal bovine serum in HDMEM, filtered through a Millex-GS 0.22 urn filter (Millipore Corp., Bedford, MA) and 
assayed in duplicate. To speed up the bioassays during the purification process, only basal inhibition of 
FSH secretion exerted by the inhibin activity is determined and plotted in the region where the inhibin 
proteins were expected to migrate in the chromatograms. 

To perform the heparin-Sepharose affinity chromatography, cell debris is spun down in a Beckman J2- 

50 21 centrifuge (Beckman Instruments, Inc., Palo Alto, CA.) using a JA-20 rotor at 10,000 rpm for 30 minutes. 
One half of the supernatant is diluted in 10 times its volume by the addition of 0.01 M Tris-HCI containing 
0.1 M NaCI, pH 7, in an Erlenmeyer flask and pumped simultaneously via silastic tubes (0.76 mm ID) into 
heparin-Sepharose® (Pharmacia Fine Chemicals, Piscataway, N.J.) columns (3.5 x 9 cm) by two Rabbit 4- 
channel peristaltic pumps (Rainin Instrument Co., Inc., Emeryville, CA) at 40 ml/hr per column. After all the 

55 fluid has been pumped through the heparin-Sepharose, the eight columns are washed simultaneously with 
0.01 M Tris-HCI, pH 7, containing 0.1 M NaCI in the same manner. The adsorbed proteins with inhibin 
activity are removed by washing the eight columns simultaneously with 0.01 M Tris-HCI containing 1 M 
NaCI, pH 7, as above, and the wash is collected into fractions. The inhibin activity is monitored by the in 



14 



EP 0 222 491 B1 



vitro bioassay described above. The columns are regenerated by further washing with 2M NaCI in 0.01 M 
Tris-HCI, pH 7, and re-equilibrated with 0.01 M Tris-HCI containing 0.1 M NaClfor purification of remaining 
extract. 

Next, the material is fractionated by gel filtration to separate proteins generally according to their 

5 molecular weights. The fractions having inhibin activity extracted by the heparin-Sepharose columns were 
pooled and dialyzed overnight to remove NaCI in a 28.6 mm cylinder diameter Spectrapor No. 3 membrane 
tubing with M r cutoff at 3,500 (Spectrum Medical Industries, Inc., Los Angeles, CA.) against 30% acetic 
acid. The retained fluid is centrifuged, as above, to remove a white precipitate, and the supernatant is 
divided into equal portions for applying to 5 x 100 cm Sephacryl S-200 superfine columns (Pharmacia Fine 

io Chemicals, Piscataway, N.J.). Each column is eluted with 30% acetic acid at 20 ml for 22 min., and the 
column fractions are monitored by UV absorption at 280 nm and by bioassay. 

The bioassay-positive protein from the S-200 columns is pooled and lyophilized. The lyophilized 
material is dissolved in 0.2N acetic acid (1 ml/ml) and filtered through a Millex-HA 0.45 urn filter (Millipore 
Corp., Bedford, MA.). The filtrate is applied directly onto a 1 x 25 cm Vydac 5-um particle-size C4 column 

75 (The Separations Group Hesperia, CA.) and developed with a gradient of TEAP buffer. In the TEAP system, 
buffer A consists of 0.25 N triethylammonium phosphate pH 3, and buffer B is 80% acetonitrile in buffer A. 
After ail the filtrate had been loaded, the column is washed with the aqueous buffer A until the UV 
absorption reached baseline. The fractions exhibiting inhibin activity are separated in a Beckman 332 
gradient liquid chromatography system (Beckman Instruments, Inc., Berkeley, CA.) equipped with a 

20 Spectroflow 757 UV detector (Kratos Analytical Instruments, Ramsey, N.J.), a Soltec 220 recorder (Soltec 
Corp,, Sun Valley, CA.) and a Redirac 2112 fraction collector (LKB Instruments, Inc., Gathersburg, MD). 
Zones of inhibin activity are detected by bioassay. 

Inhibin protein containing the j8 B chain is further purified free of inhibin containing the £ A species, if 
desired, by two more RP-HPLC steps. The first step uses a 1 x 25 cm Vydac 5-um-particle-size C4 column 

25 and a trifluoroacetic acid (TFA) buffer system and the second step employs a 1 x 25 cm Vydac 5-um- 
particle-size Phenyl column and the TEAP buffer system. In the TFA system, buffer A contains 1 ml 
trifluoroacetic acid in 999 ml water and buffer B is 1 ml trifluoroacetic acid in 199 ml water and 800 ml 
acetonitrile. The two inhibin species elute separately. Inhibin accumulated from a few batches was 
concentrated by RP-HPLC using a 0.46 x 25 cm Aquapore RF-300 10 um-particle-size column (Brownlee 

30 Labs.. Santa Clara, CA.) and the TFA buffer system. Ordinarily, however, this purification step will not be 
used with cell culture supernatants from transformants with DNA encoding only the 0 A or 0 B chains. 

Inhibin, activin, prodomain sequences or their variants are administered in the form of pharmaceutical^ 
acceptable nontoxic salts, such as acid addition salts or metal complexes e.g., with zinc, iron or the like 
(which are considered as salts for purposes of this application). Illustrative of such acid addition salts are 

35 hydrochloride, hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, 
ascorbate, tartrate and the like. Intravenous administration in isotonic saline, phosphate buffer solutions or 
the like is suitable. 

The polypeptide herein should be administered under the guidance of a physician, and pharmaceutical 
compositions will usually contain an effective amount of the peptide in conjunction with a conventional, 

40 pharmaceutically-acceptable carrier. The dosage will vary depending upon the specific purpose for which 
the protein is being administered, and dosage levels in the range of about 0.1 to about 1 milligram per Kg. 
of body weight may be used when inhibin is administered on a regular basis as a male contraceptive. 

Inhibin, activin, prodomain sequences or their variants desirably are administered from an implantable 
or skin-adhesive sustained-release article. Examples of suitable systems include copolymers of L-giutamic 

45 acid and gamma ethyl-L-glutamate (U. Sidman et al., 1983, "Biopolymers" 22(1): 547-556), poly <2- 
hydroxyethyl-methacrylate) (R. Langer et al., 1981, "J. Biomed. Mater. Res." 15: 167-277 and R. Langer, 
1982, "Chem. Tech." 12: 98-105) ethylene vinyl acetate (R. Langer et al., Id.), or poly-D-(-)-3- Hydrox- 
ybutyric acid (EP 133.988A). Such articles are implanted subcutaneously or are placed into contact with the 
skin or mucous membranes. 

50 In order to simplify the Examples certain frequently occurring methods will be referenced by shorthand 
phrases. 

Plasmids are designated by a low case p preceded and/or followed by capital letters and/or numbers. 
The starting plasmids herein are commercially available, are publicly available on an unrestricted basis, or 
can be constructed from publicly available plasmids or DNA in accord with published procedures. In 
55 addition, other equivalent plasmids are known in the art and wit) be apparent to the ordinary artisan. 

"Digestion" of DNA refers to catalytic cleavage of the DNA with an enzyme that acts only at certain 
locations in the DNA. Such enzymes are called restriction enzymes, and the sites for which each is specific 
is called a restriction site. "Partial" digestion refers to incomplete digestion by a restriction enzyme, i.e, 



15 



EP 0 222 491 B1 



conditions are chosen that result in cleavage of some but not all of the sites for a given restriction 
endonuclease in a DNA substrate. The various restriction enzymes used herein are commercially available 
and their reaction conditions, cofactors and other requirements as established by the enzyme suppliers 
were used. Restriction enzymes commonly are designed by abbreviations composed of a capital letter 

5 followed by other letters and then, generally, a number representing the microorganism from which each 
restriction enzyme originally was obtained. In generally, about 1 ug of plasmid or DNA fragment is used 
with about 1 unit of enzyme in about 20 ui of buffer solution. Appropriate buffers and substrate amounts for 
particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37 °C 
are ordinarily used, but may vary in accordance with the supplier's instructions. After incubation, protein is 

10 removed by extraction with phenol and chloroform, and the digested nucleic acid is recovered from the 
aqueous fraction by precipitation with ethanol. Digestion with a restriction enzyme infrequently is followed 
with bacterial alkaline phosphatase hydrolysis of the terminal 5' phosphates to prevent the two restriction 
cleaved ends of a DNA fragment from "circularizing" or forming a closed loop that would impede insertion 
of another DNA fragment at the restriction site. Unless otherwise stated, digestion of plasmids is not 

75 followed by 5' terminal dephosphorylation. Procedures and reagents for dephosphorylation are conventional 
(T. Maniatis et al., 1982, Molecular Cloning pp. 133-134). 

"Recovery" or "isolation" of a given fragment of DNA from a restriction digest means separation of the 
digest on polyacrylamide gel electrophoresis, identification of the fragment of interest by comparison of its 
mobility versus that of market DNA fragments of known molecular weight, removal of the gel section 

20 containing the desired fragment, and separation of the gel from DNA. This procedure is known generally. 
For example, see R. Lawn et al., 1981, "Nucleic Acids Res." 9: 6103-6114, and D. Goeddel et al., 1980, 
"Nucleic Acids Res." 8: 4057. 

"Southern Analysis" is a method by which the presence of DNA sequences in a digest or DNA- 
containing composition is confirmed by hybridization to a known, labelled oligonucleotide or DNA fragment. 

25 For the purposes herein, unless otherwise provided, Southern analysis shall mean separation of digests on 
1 percent agarose, denaturation and transfer to nitrocellulose by the method of E. Southern, 1975, "J. Mol. 
Biol." 98: 503-517, and hybridization as described by T. Maniatis et al., 1978, "Cell" 15: 687-701. 

"Transformation" means introducing DNA into an organism so that the DNA is replicable, either as an 
extrachromosomal element or chromosomal integrant. Unless otherwise provided, the method used herein 

30 for transformation of E. coli is the CaCfe method of Mandel et al., 1970, "J. Mol. Biol." 53: 154. 

"Ligation" refers to the process of forming phosphodiester bonds between two double stranded nucleic 
acid fragments (T. Maniatis et al., Id., p. 146). Unless otherwise provided, ligation may be accomplished 
using known buffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5 ug of approximately 
equimolar amounts of the DNA fragments to be ligated. 

35 "Preparation" of DNA from transformants means isolating plasmid DNA from microbial culture. Unless 
otherwise provided, the alkaline/SDS method of Maniatis et al., Id. p. 90., may be used. 

"Oligonucleotides" are short length single or double stranded polydeoxynucleotides which are chemi- 
cally synthesized by known methods and then purified on polyacrylamide gels. 

40 EXAMPLE 1 

Isolation of Cloned Inhibin o-Subunit cDNAs 

The strategy for identification of clones containing coding sequences for the porcine inhibin subunits 
45 was based on the "long-probe" approach, successful in some previous instances (Anderson et al., 1983, 
"Proc. Nat. Acad. Sci. USA" 80:6836-6842 and Ullrich et al., 1984, "Nature" 309:418-425). Briefly, a high- 
complexity cDNA library constructed in Xgt10 and derived from porcine ovarian mRNA by oligo-dT-primed 
cDNA synthesis was screened with a single 64-base-long synthetic oligodeoxynucleotide directed against 
the N-terminal amino acid sequence of the a-chain of porcine inhibin. It was found that the library is to be 
so prepared from fresh ovarian tissue because the inhibin chain mRNA was apparently quite labile. Approxi- 
mately 1 in 2,000 plaques hybridized with this probe, and sequence analysis of several hybridizing cloned 
cDNAs confirmed correct probe identification. This analysis revealed that none of the characterized cDNAs 
contained sufficient sequence information to predict the complete structure of the a-chain precursor protein. 
Rather than analyzing more clones from the same cDNA library, a second library was constructed by 3* 
55 extension on ovarian mRNA of a synthetic oligodeoxynucleotide complementary to a sequenced region 
encoding a precursor residues 60-64 (Fig. 1A). This library was screened with a suitable restriction fragment 
from a previously analyzed cDNA and yielded several isolates which specified the remainder of the DNA 
sequences encoding the N-terminal region of the a precursor. Completeness of the coding sequence was 



16 



EP 0 222 491 B1 



judged from the presence of a long reading frame which specifies the porcine a-chain peptide sequence 
and starts with a methionine codon preceded by an in-frame stop codon and followed by a hydrophobic 
sequence bearing the hallmarks of a signal peptide. The full sequences for the precursor protein and its 
cDNA are shown in Fig. 1 B. The complete protein including signal peptide has an Mr of ~40K consisting of 
5 364 amino acids, of which the C-terminal 134 (M r ~14.5K) constitute the porcine inhibin a-chain. There are 
several Arg-Arg sequences in the proregion of the precursor, one of them directly preceding the a subunit. 
We believe that this latter pair of basic residues is the processing site for the proteolytic release of the a 
peptide. The deduced precursor sequence predicts two N-linked glycosylation sites, one within the a chain 
proper. 

70 In addition to the coding region, the cDNA sequence contains a 3'-untranslated sequence of 167 
nucleotides, including the canonical AATAAA polyadenylation signal, and a 5*-untranslated region, the 
proper length of which is presently unknown. 
The detailed method was as follows: . 

Polyadenylated mRNA was prepared from freshly frozen porcine ovaries (Kaplan et al., "J. Biochem." 
75 183: 181-184) An oligo-dT-primed cDNA library of "6x1 0 6 clones in Xgt10 (Huynh et al., 1984, DNA Cloning 
Techniques ., Ed. D. Clover) was prepared form 5 ug polyA+ mRNA as described by Wood et al., "Nature" 
312 : 330-337 (1984), except that the EcoRI adaptors used had the sequence 

20 5 ' - AATTCACTCGAGACGC - 3 9 

' 3 9 - CTGAGCTCTCCG - 5 9 P . 

Approximately 1 x 10 6 unamplified cDNA clones were screened with 5 a-subunit oligonucleotide 

25 

S* - AC CG C C C C TTTGCCTTGG CCTTGGTCCCCTGCTGCTCTGAGACTGCTGCAGAGACCTCCTGAGG -V> 

based on the amino acid sequence underlined in Fig. 1B. Hybridization was carried out with the 
30 phosphorylated 32 P-labelled probe in SxSSC, 40% formamide at 37 • C. Filters were washed at 50 ° C with 
ixSSC, 0.1% SDS. Approximately 500 hybridization positive clones were obtained, twelve of which were 
purified and examined for insert size. The Eco RI inserts of five of these (\PIN-a2, -a5A, -a5, -a9, -a 10) 
were subcloned into M13 derivatives (Messing et al., 1981 "Nucl. Acids Res." 9:309-321) and sequenced 
by the dideoxy chain termination method of Sanger et al.,"Proc. Nat. Acad. Sci. USA" 74:5463-5467 (1977). 
35 A specifically primed library was prepared by priming 5 ug of polyA* mRNA with the oligonucleotide 5'- 
CCCCACAGCATGTCTT-3' (complementary to nucleotides 248-263) and subsequent cloning into Xgt10. 
Approximately 2x1 0 5 clones of the 1x10 s clones obtained were screened with the 5' 100bp EcoRI- Bam HI 
fragment prepared from XPIN-o2. Twelve of the 170 hybridization positive clones obtained were purified and 
two (XPIN-S12s, -S4s) were sequenced by the dideoxy method. The complete nucleotide sequences of the 
40 a-subunit cDNAs were obtained by subcloning various restriction fragments with the different X isolates into 
the M13 phage derivatives. Compressions were resolved by the use of deoxyinosine mixes in combination 
with the E. coli single stranded binding protein (Pharmacia). 

Isolation of Cloned Inhibin 0 Subunit cDNAS 

45 

The cDNA sequences encoding the precursors of the inhibin 0 subunits were obtained from the same 
cDNA libraries used for the a subunit. Overlapping cDNA clones were isolated by screening first with single 
long synthetic oligodeoxynucleotide probes based on the two N-terminal & subunit sequences and 
subsequently with suitable restriction fragments derived from characterized cDNA clones which served as 
50 probes for "walking" in both 5' and 3' directions (Fig. 2A). 

In more detail, approximately 2x10 s oligo-dT primed ovarian cDNA clones were screened with the 5' 
end labelled £ A oligonucleotide, 

66 5 ' -AAGAAGCACTTCTTTGTGTCCTTCAAGGACATTGGCTCGAATGACTGGATCATTGC-3' 

based on the amino acid sequence of residues 321-339. Five hybridization positives were obtained, of 
which three proved to contain /8 A coding sequences (XP1N-0A2, -jS A 4, -£ A 8). A 5' end 154 bp EcoRI- Hind lll 



17 



EP 0 222 491 B1 



(nucleotides 158-297) fragment and a 3' end 213 bp EcoRI-Pst fragment (nucleotides 1679-1892) derived 
from XPIN0A2 were used to screen 2x1 0 6 oligo-dT primed cDNA clones and 2x1 0 5 clones from the a-chain 
specifically primed library. Out of the sixteen clones analyzed in detail two were found to have longer 5' 
ends (XPIN-jS A 5s ( -0 A 22) and one clone XPIN-£ A 21 contained the entire 3'-untranslated region. Porcine 
5 inhibin /i B subunit cDNA clones were isolated by screening 2x10 s clones from the specifically primed library 
with the j9 B oligonucleotide 

A* - GGCCTGGAGTCTGATGGGAGAACCAACCTCT^ 

w 

which was based on the NH 2 -terminal sequence described in Fig. 1 A. Positive clones were further screened 
with the oligonucleotide inosine probe 5'-AAITCTATIAAIAAc TG C -3' ("I" in this sequence stands for 
inosine), which covers all the possibilities in the non-coding strand for the amino acid sequence QQFFIDF. 
Two clones (XPIN0 b -1s, -2s) were isolated and sequenced and found to code for the 0 B subunit. A 230 bp 

15 EcoRI- Sma (nucleotides 21-251) fragment was isolated from XPIN0 B -l and used as a hybridization probe to 
screen 2x1 0 6 oligo-dT primed cDNA clones. Two positives were obtained (XPIN0 B -3,4). The nucleotide 
sequence of these overlapping clones was used to construct the sequence shown. All sequences were 
obtained by subcloning specific fragments into M13 phage vectors (Messing et al., op cit .). The Eco Rl 
restriction sites referred to above are all contained within the cDNA adaptor fragment, and do not refer to 

20 sequences present in the cDNA. 

We noted that only very few clones from the oligo-dT-primed library (4 out of 2x1 0 5 ) hybridized with the 
synthetic probe for the /8-subunit of inhibin A. Although most of these proved correct by DNA sequence 
analysis, none contained a full 3'-untranslated region, as judged by the absence of a polyA homopolymer at 
their 3' ends. Absence of polyA tails suggested the existence of a very long 3'-untranslated sequence in 

25 this mRNA species and/or structural region(s) which prove difficult to copy by the polymerases used for 
library construction. Unexpectedly, a higher abundance ("10-fold) of inhibin 0 A subunit coding sequences 
was found in the cDNA library made by specific priming on a-subunit mRNA. This library was screened 
with the synthetic probe for the j8-chain of inhibin A on the subsequently refuted theory that the a precursor 
mRNA might also encode the subunit. The high abundance of inhibin jS A cDNA in this library was later 

30 traced to fortuitous complementarity of the specific a chain primer to a region in the 3'-untranslated portion 
of the corresponding mRNA. 

Only four cloned cDNAs encoding the 0 subunit of inhibin B were found in our libraries. The sequence 
information obtained from these clones failed to reveal the complete structure of the corresponding 
precursor protein and its cDNA. The sequences of cDNAs and deduced protein structures for the 

35 precursors of the & subunits are compared in Fig. 2B. The nucleotide sequence of inhibin £ A subunit cDNA 
is 3.6 kb in length and contains an open reading frame for a protein of 425 amino acids (Mr ~46K), the C- 
terminal 116 residues of which represent the /S subunit proper (Mr ~13K). This reading frame begins with a 
methionine codon followed by a sequence that codes for a characteristic signal peptide, the true length of 
which is believed to be 29 residues. The encoded 0 subunit is preceded by a string of 5 arginines at which 

40 it is presumably proteolytically cleaved from the precursor. Similar to the a subunit precursor, this p 
precursor contains several additional pairs of basic residues at which hitherto unknown biologically active 
peptide entities are believed to be released. It also contains one possible site of N-linked glycosylation in 
the proregion (Ash, residue 165). 

The deduced protein sequence for the 0 subunit of inhibin B shows high homology with the j8 A subunit 

45 sequence. 71 amino acid residues are identical and most changes are conservative in nature. Sequence 
homology, although of a lesser degree, is also found in the proregion of both 0 subunit precursors. 
Interestingly, an extremely purine-rich sequence rarely seen in coding regions but present in the cDNA 
encoding the inhibin 0 A precursor and resulting in a curious amino acid sequence is not found in the cDNA 
which codes for the homologous 0 B precursor. This results in a gap of 22 amino acid residues from the 0 B 

so precursor of inhibin when protein sequences are aligned for maximal homology. Such alignment also brings 
about a perfect match in the cysteine positions of both precursors (see Fig. 2B). 

Northern Analysis of a and j8 chain Precursor mRNAS 

56 Ovarian total and polyadenylated RNAs were analyzed by the Northern procedure using the sequenced 
cDNAs as probes to assess size and relative abundance of the mRNAs which encode the peptide subunits 
a and 0 and £ B of the heterodimeric inhibin molecule. Polyadenylated mRNA (2 ug: lanes a, b, c, and f; 8 
ug: lane d) and total flNA (10 ug: lanes e and g) were electrophoresed into a formaldehyde 1.2% agarose 



18 



EP 0 222 491 B1 



gel and blotted onto nitro-celiulose filters. The following 32 P-labelled cDNA fragments were used as 
hybridization probes under stringent conditions. Lane a: 240 bp EcoRI- Sma l (nucleotides 134-371) from a 
subunit cDNA; b: 154 pb EcoRI- Hind lll (nucleotides 158-297) from jSA subunit cDNA; c: 230 bp EcoRl- Sma 
(nucleotides 21-251) from 0 B subunit cDNA; d and e: EcoRI insert of \PIN-a2; f and g: EcoRI insert of XPIN- 

5 jS A 5. Filters were washed for 2 hours with 3 changes of O.lxSSC, 0.1% SDS at 60 *C. 

Analysis showed (Fig. 3) that a and jS mRNAs are of different size and abundance, as indicated by 
results obtained from cDNA cloning. From their respective band intensities the a precursor mRNA is 
estimated to be at least of 10-fold higher abundance than the mRNA for the j8 A precursor, and approxi- 
mately 20-fold higher than the mRNA for the 0 B precursor. 

70 Using ribosomal RNAs as size standards, the a precursor mRNA, which is a single species, is ~1500 
nucleotides in length, a size in good agreement with the cloned cDNA sequence (Fig. 1B). 0 A precursor 
mRNA sequences are represented by two main species of ~4.5 and "7.2 kb in length. The relatively higher 
intensity of both species in polyadenylated than total RNA suggests that the 4.5 kb species does not 
represent 28S RNA which hybridized to the cDNA probe. Thus, the £ precursor cDNA sequences shown in 

75 Fig. 2B are thought to represent the 4.5 kb mRNA, suggesting that the 5' untranslated region for the £ A 
. mRNA is approximately 900 nucleotides long. The fa precursor is encoded on one mRNA, of approximately 
4.5 kb in size, which is present at roughly half the level of the two j8 A mRNAs. Since the two 0 mRNAs are 
closely related, one can predict that both mRNAs have a similar structure and thus the £ B mRNA 
presumably possesses a long 5' and 3' untranslated region equivalent to that shown for the j8 A mRNA. 

20 Choice of a different polyadenylation signal might explain the existence of the 7.2 kb species. 

Homology To Transforming Growth Factor-ff 

The mature a and /S inhibin subunits contain seven and nine cysteine residues respectively. Upon 
25 alignment of the cysteine residues it is apparent that the two subunits share a similar cysteine distribution 
and some sequence homology exists around these residues (Fig. 4), suggesting that both types of subunits 
derive from one ancestral gene. Surprisingly, significant homology was found between the £ chain and the 
primary structure of human TGF-0 recently determined. As outlined in Fig. 4, both peptides are of nearly 
equal length (inhibin 0 A subunit, 116, j9 B subunit 115; TGFS, 116 residues) and show a strikingly similar 
30 distribution of their nine cysteine residues. Using this cysteine "skeleton" for alignment, the 0 A and TGF-£ 
sequences have an additional 31 residues in identical positions and show conservative changes in nine 
homologous places. Similar high homologies are seen upon comparison of the /S B and 0-TGF. Some gaps 
were introduced for better alignment (Fig. 4). The overall homology reaches 35%, but approaches 60% in 
certain sections (cf. porcine inhibin 0 A chain residues 1 1-45 and TGF residues 1 5-49), a very high degree of 
35 homology considering the difference in species. Interestingly, this homology extends beyond the termina- 
tion codon for protein synthesis in the respective cDNAs. Thus, the cDNAs for TGF-/S and both inhibin 0 
subunits contain a highly G and C rich sequence in this region, and they also possess unusually long 5' and 
3* untranslated regions. 

One can discount the suggestion that the £ subunit of inhibin is the porcine equivalent of human TGF-0, 
40 since there is almost absolute homology between human and murine 0-TGFs. These findings strongly 
indicate that both inhibin subunits and TGF-/8 have a common ancestor and belong to one gene family. All 
three peptides are derived from similarly-sized precursors (M r ~40K) where they occupy the C-terminal 110 
or so residues and are released by proteolytic cleavage at pairs of arginines. They form homo- or 
heterodimers, and subunits in the biologically active complex are linked by disulfide bridges. However, 
45 there is little sequence homology between TGF-j8 and the 0 subunits in the pro parts of their precursors, 
although the regions comprising the odd residues which precede the j8 subunit and TGF peptides display 
limited but significant sequence relatedness. 

EXAMPLE 2 

50 

Recombinant Synthesis of Porcine Inhibin 

The plasmid used for recombinant synthesis of porcine inhibin was pSVE-PaB A lnh-DHFR. The proce- 
dure to construct this plasmid is shown in Fig. 5. This plasmid was constructed as follows: 
55 pHBS348-E (EP 0073656A) was partially digested with EcoRI, blunted with E. coli DNA polymerase I 
(Klenow fragment) and the four dNTPs, ligated and the ligation mixture was transformed into E. coli 294 
(ATCC 31446). The transformed culture was plated on ampicillin media plates and resistant colonies 
selected. Plasmids were screened for the loss of the EcoRI site preceding the SV40 early promoter. A 



19 



EP 0 222 491 B1 



plasmid having the site deleted is referred to as pHBS348-EII. 

pHBS348-EII was digested with Eco RI and Eco RI to produce two fragments, fragment I containing the 

SV40 early promoter, pml_-Amp r sequences and the HBsAg 3' untranslated region and fragment 2 

containing the HBsAg (hepatitis B antigen) coding sequences. 
5 XPINj9 A 5 s containing the coding region for the porcine inhibin 0 A subunit was digested with EcoRI and 

Sma l and the 1335 bp fragment (fragment 3) containing the 0 A coding region recovered by polyacrylamide 

gel electrophoresis. Fragment I, recovered by agarose gel electrophoresis, was ligated to fragment 3 and 
. the ligation mixture transformed into E. coli strain 294 (ATCC 31446). The transformed culture was plated 

on ampicillin media plates and resistant colonies selected. Plasmid DNA was prepared from transformants 
70 and checked by restriction analysis for the presence of the correct DNA fragments. This plasmid is referred 

to as pSVE-p0 A lnh. 

pHBS348-E (EP 0073656A) was partially digested with EcoRI, blunted with E. coli DNA polymerase I 
(Klenow fragment) and the four dNTPs, ligated to the synthetic oligonucleotide 5' GGTCGACC-3 1 containing 
the Sail recognition site. The transformed culture was plated on ampicillin media plates and resistant 

75 colonies selected. Plasmids were screened for the presence of the extra Sail restriction site. Plasmid DNA 
is prepared from this construction (pHBS348-ESall). 

XPINa-12s and XPINa-2 were digested with EcoRI and Bam HI; A 104 b pEco RI- Bam HI fragment from 
XPINa-12s containing the 5' coding region and a 1246 bp Eco RI- Bam HI fragment from XPINa-2 containing 
the middle and 3' coding region were recovered and ligated together. The ligation mixture was digested 

20 with EcoRI, the enzyme heat denatured, and the mixture ligated to EcoRI-digested pUC9 (BRL). Recom- 
binants were selected and confirmed by restriction analysis. DNA was prepared from the correct plasmid 
(pPINa). 

pPINo, containing the complete coding region for porcine a-inhibin was digested with Nco l and Eco RI, 
filled in by Pol(l)K in the presence of 4dNTP f s, the 1280 bp fragment (fragment 4) was recovered by gel 

25 electrophoresis. pHBS348-ESall was digested with Sstll and Hindlll, filled in by Pol(l)K in the presence of 
4dNTP's, and fragment 5 containing the PML-Amp r region, SV40 early promoter and HBsAg 3' untranslated 
region was recovered by gel electrophoresis. Fragments 4 and 5 were ligated together and the ligation 
mixture used to transform E. coli 294 (ATCC 31446). Recombinants were selected by growing on Ampicillin 
media plates. The desired recombinant is called pSVE-Palnh. 

30 pHBS348-ESall was digested with Sail and Hindlll and fragment 6 containing the pML-Amp r , and SV40 
early promoter was recovered by gel electrophoresis. pFD II (EP 117,060A) was digested with Sail and 
Hind lll and fragment 7 was recovered which contains the normal mouse DHFR gene fused to the HBsAg 3' 
untranslated region. Fragments 6 and 7 were ligated, and the ligation mixture transformed into E. coli strain 
294 (ATCC 31446). The transformed culture was plated on ampicillin media plates and resistant colonies 

35 selected. Plasmid DNA was prepared from transformants and checked by restriction analysis for the 
presence of the correct DNA fragments. This construction is referred to as pFDII-Sah. 

pSVE-Palnh was digested with Sail and fragment 8 was recovered which contains the SV40 early 
promoter and .the a-inhibin coding region fused to the HBsAg 3'-untranslated region. pFDII-Sall was 
digested with Sail and fragment 9 containing the SV40 early promoter and the mouse DHFR coding region 

40 linked to the HBsAg 3'-untranslated region was recovered. pSVE-£ A lnh was linearized by Sail digestion and 
ligated to fragments 8 and 9 in a three part ligation. The ligation mixture was transformed into E. coli strain 
294 (ATCC 31446). The transformed culture is plated on ampcillin media plates and resistant colonies 
selected. Transformants were screened for the presence of fragments 8 and 9 in the correct orientation 
such that transcription from the three SV40 early promoters will proceed in the same direction. This final 

45 plasmid is designated pSVE-Pa/S A lnh-DHFR. 

Plasmid pSVE-Pa£ A lnh-DHFR was transfected into DHFR deficient CHO cells (Urlaub and Chasin, 
1980, PNAS 77,4216-4220). However, any DHFR" mammalian host cell is suitable for use with this plasmid. 
Alternatively, any mammalian host cell is useful when the host cell is cotransformed with a plasmid 
encoding neomycin resistance, and transformants identified by their ability to grow in neomycin-containing 

so medium. 

The transfected CHO cells were selected by culturing in 15 HGT~ medium. The cells were allowed to 
grow to confluency in 15cm diameter plates. The cells thereafter were cultured in serum free medium for 48 
hours prior to harvest. 50ml of supernatant medium was lyophilized after the addition of 100mg human 
serum albumin. The residue was redissolved in 3ml 1% fetal bovine serum in HDMEM (GIBCO Laborato- 
55 ries, Santa Clara, CA), filtered through a Millex-GS 0.22mM filter (Millipore Corp., Bedford, MA) and assayed 
in duplicate. 

The inhibin hormonal activity in the transformant supernatants was determined by an in vitro bioassay 
using rat anterior pituitary monolayer culture, Vale, W. et aj. Endocrinology , 91, 562-572 (1972). In brief, 21- 



20 



EP 0 222 491 B1 



day-old female rat anterior pituitaries were collected, enzymatically dispersed and plated in 10% fetal 
bovine serum in HDMEM (GIBCO Laboratories, Santa Clara, CA) into 24-well tissue culture plates (Falcon 
Plastic, Oxnard, CA) on day 1. On day 2, the medium was changed to 1% fetal bovine serum in HDMEM, 
and the transformant medium sample was added. Incubation was continued for another 48 hours. The 

5 monolayer medium was then harvested, and the LH and FSH contents were determined by radio- 
immunoassay (RIA) using materials provided by The Pituitary Hormone Program of NIADDKD. In this assay, 
the inhibin-containing CHO cell culture inhibits the basal release of FSH but not LH t as compared to control 
pituitary cells that received the incubation medium only. The amount of porcine inhibin detected in 
transformant supernatants was 20 ng/ml and exhibited a dose response curve parallel to that obtained with 

70 pure porcine ovarian inhibin. 

Immunological cross-reactivity is assayed by a sandwich-type radioimmunoassay. Rabbit antisera are 
raised against purified porcine follicular inhibin by s.c. immunization of rabbits with the porcine inhibin in 
Freund's complete adjuvant. The presence of anti-inhibin in the antiserum is detected by incubation of the 
antiserum with purified porcine inhibin and assaying for the formation of an immune complex by conven- 

75 tional techniques, e.g. gel filtration. An aliquot of the antisera is coated onto goat-anti-rabbit IgG precoated 
polystyrene test tubes. The recombinant culture supernatant or extract is diluted into phosphate buffered 
saline and added to the coated tubes, incubated overnight and washed. Another aliquot of the rabbit 
antiserum is added to the test tubes, incubated and washed. Radioiodinated goat antirabbit IgG is added to 
the tubes, incubated and unbound goat antiserum removed by washing. The recombinant produced inhibin 

20 cross-reacts with the rabbit antiserum, as evidenced by bound counts on the test tubes which exceed those 
of controls incubated with culture medium or extracts from untransformed host cells. 

EXAMPLE 3 

25 Construction of Human Inhibin Vector and Expression of Human Inhibin in Recombinant Cell Culture-I 

Expression of human inhibin cr£ A facilitated by the discovery that the mature porcine and human £ A 
chains are identical. Thus, construction of a vector for the expression of human inhibin can proceed from 
plasmid pSVE-j8 A -lnh from Example 1 , which contains the porcine £ A -encoding cDNA. 

30 A Xgt 10 library of human ovarian cDNA made from 10 ug of ovarian mRNA was subjected to Southern 
analysis using radiophosphate labelled porcine cDNA encoding a, £ A and £b chains. XHINq-2 was identified 
as containing coding regions for the human a inhibin chain. The prevalence of hybridizing clones in the 
case of human a inhibin was considerably less than that found for porcine a inhibin, on the order of 1 in 
100,000 human clones hybridized to the 685 bp Sma l fragment of the porcine cDNA for alnh. The 0 chain 

35 clones were also rare, with the jS B clones being present at about 3 times the level of 0 A (1 and 3 out of 
about 1 ,000,000 clones, respectively). None of the & chain clones were full length. They were supplemented 
with a primed cDNA library and assembled generally as described above for the porcine cDNA. The X 
inserts were recovered by EcoRI digestion. 

Plasmids pHINa-2 is digested with Nco l and Sma l, and the 1049 bp 15 fragment (fragment 10) is 

40 recovered by gel electrophoresis. pPina (Example 2) is digested with EcoRI and Pvull. The 98 bp fragment 
(fragment 11) is recovered by gel electrophoresis. Fragments 10 and 11 are ligated to adaptor I 

S 9 - CTGCTCCTCTTGCTGTTGGCCCCACGCAGTGCGCATGGCTCCCAGGCCCCGGAGCTGGACC- 3 ' , 

45 

in combination with adaptor II which is the complement of adaptor I. The resulting 1208 bp fragment 
(fragment 12) is treated with Klenow fragment of Pol(l) and the 4 dNTP's and ligated to pHBS348-ESall 
which has been restricted with Hindtll and Sac 1 1 and blunt-ended as described in Example 1. Alternatively, 
pPina was digested with EcoRI and Hpa ll with the fragment encoding upstream from the Hpa ll site (that is, 
so the first 21 residues of the porcine sequence) being recovered. The adaptor used in this alternative 
approach was 

5'CGGAGCTCGACC 3' 
55 3' CTCGAGCTGG 5'. 



21 



EP 0 222 491 B1 



A plasmid pSVE-Halnh having the correct orientation of fragment 12 is identified by sequence analysis of 
transformants. This construction (pSVE-Halnh) thus contains the first 24 residues of the porcine signal 
sequence with the remainder being prepro human inhibin. Plasmid pSVE-Halnh is digested with Sail. The 
fragment containing the SV40 promoter and human inhibin sequence is ligated to fragment 9 and Sail 
5 digested pSVE-0 A lnh (Example 2). This final plasmid designated pSVE-ha£ A lnh-DHFR1 is transfected into 
DHFR-deficient CHO cells and selected as described in Example 2. The culture supernatant contains 
hormonally active human inhibin. 

EXAMPLE 4 

70 

Construction of Human Inhibin Vector and Expression of Human Inhibin in Recombinant Cell Culture-ll 

This example is similar to Example 3 except that the pro sequence of human inhibin 0 B was employed 
in the place of the porcine jS B prepro domain. 

75 The lambda gttO library of Example 3 yielded XHINa2, as described in example 3, together with 
XHIN0 A -5 and -14. The latter two phage were employed to construct the full length & A coding cDNA by 
ligating the 311 bp EcoRI- Hind lll fragment (fragment 13) of XHIN0 A -5 to the 1101 bp Hind lll-Hpal fragment 
(fragment 14) of XHIN/S A -14 and ligating this mixture in an Eco R1- Sma l digested mp18 vector (Biolabs). 
Clones were selected and screened for the appropriate sized insert. An mp18 vector containing the correct 

20 insert was treated with DNA polymerase(l) and the four dNTPs in order to render it double stranded, and 
thereafter digested with Xba l (which cleaves in the mp18 polylinker sequence), blunted with DNA poly- 
merase I and the four dNTPs, and digested with EcoR1. A 1320 bp fragment (fragment 15) was ligated to 
the Eco R1-EcoRV fragment 1 from Example 2. This ligation mixture was used to transform E. coli 294 cells. 
Clones were screened by Southern Hybridization and confirmed by restriction analysis. The clone contain- 

25 ing the hlnh£ A coding sequence was designated pSVE-hum/? A lnh. A plasmid containing the human j8 A 
coding sequences and the human a-inhibin sequences together with the DHFR gene is constructed from 
plasmids pSVE-hum£ A lnh, pSVE-Holnh and pFDIISall as outlined above. Specifically, the Sal fragments 
from pSVE-Halnh and pFDIISall which contain the human alpha inhibin and the DHFR genes were ligated 
with Sail digested pSVE-hum0 A lnh and a clone containing all three genes was identified. This plasmid, 

30 designated pSVE-huma0 A lnh-DHFR2 was transfected into DHFR" CHO cells and selected by culture in 
ght" medium. 24 clones were picked, grown to confluency in ght~ medium under conditions conventional 
for CHO ceils for two days, allowed to rest for 2 more days and thereafter the culture media were assayed 
for inhibin and activin activity using the rat pituitary cell assay described above. 4 clones were found to 
secrete significant levels of human a£ A inhibin (ho/S A -8, 12, 14, and 18). The levels in the culture medium 

35 for each clone were, respectively, 125, 125, 200 and 250 ng/ml. Another clone (ha0 A -11) produced activin 
as the /8 A j8 A homodimer, but no detectable inhibin, as determined by biological activity and the lack of a 
chain immunoreactivity in the culture medium for this clone. Clone ha0 A -16 secreted only a chain and was 
devoid of activin or inhibin activity. 

40 EXAMPLE 5 

Recombinant Expression of Human Activin 

As reported by Vale et al. (Id.) and Ling et al. (Id.), homodimers and heterodimers of the & chains A 
45 and/or B have the opposite effect of inhibin on the pituitary, introducing rather than inhibiting FSH secretion. 
These proteins, collectively termed activin, are made in a and 0 chain cotransformants as described in 
Example 4. However, somewhat less screening for an appropriate transformant is needed if the initial 
transfection is conducted with a vector or vectors that do not contain the a chain gene. A suitable vector is 
readily constructed from the above-described vectors toy excising the o chain gene. Plasmid pSVE- 
50 hum£ A lnh from Example 4 is digested with Sail and ligated to fragment 9 (Example 2) containing the DHFR 
gene. The ligation mixture was used to transfect E. coli 294 cells and colonies selected on the basis of 
failure to hybridize to the a chain sequence but which did hybridize to the 0 chain DNA. A clone pSVE- 
hurr»0 A lnh-DHFR was identified from which the o chain DNA has been deleted. This clone is transfected into 
DHFR - CHO cells as described above. Transformants are identified that secrete activin into the culture 
55 medium. Similarly, an expression vector containing a /3b coding sequence (reconstituted by ligating DNA 
encoding the first 34 amino acids of human ft A to the remaining coding sequence of the human fi B chain) is 
readily constructed and contransfected with pSVE-hum0 A lnh-DHFR to produce the heterodimer. The 
reconstructed human 0 B gene also is used in the forgoing plasmids in order to produce a0 B -inhibin which, 



22 



EP 0 222 491 B1 



in the in vitro bioassay has essentially equivalent biological potency to the q/3 a form of inhibin. 

Although the invention has been described with regard to its preferred embodiments, which constitute 
the best mode presently known to the inventors, it should be understood that various changes and 
modifications as would be obvious to one having the ordinary skill in the art may be made without departing 
5 from the scope of the invention which is set forth in claims appended hereto. 

Claims 

Claims for the following Contracting States : BE, CH, DE, FR, GB, IT, LI, NL, SE 

io 1. A method comprising culturing a host cell transformed with a vector which includes nucleic acid 
encoding a human or porcine inhibin a chain and/or a human or porcine inhibin p chain the amino acid 
sequences of which are as depicted in Fig 1B (porcine a chain), Fig 2B (porcine jS chains), Fig 6A 
(human o chain) and Figs 8 and 9 (human 0 chains), or an amino acid sequence variant by way of 
insertion, deletion or substitution of a said depicted sequence, the variant being substantially homolo- 

75 gous with a polypeptide of a depicted sequence but excluding bovine inhibin a chain and the partial 
bovine inhibin 0 chain of the sequence 









Gly-Leu 


-Glu-Cys-Asp-Gly-Lys-Val 


-Asn-Ile-Cys 


Cys- 


-Lys- 


Lys- 


-Gln-Phe- 


Phe-Val -Ser- Phe-Lys- Asp- 


Ile-Gly-Trp- 


Asrv 


-Asp- 


Trp- 


-Ile-Ile- 


Ala -Pro- Ser-Gly-Tyr-Hi s- 


Ala-Asn-Tyr- 


Cys 


-Glu- 


Gly- 


-Glu-Cys- 


Pro-Ser-His-Ile-Ala-Gly- 


Thr-Ser-Gly- 


Ser 


-Ser- 


■Leu- 


-Ser-Phe- 


His-Ser-Thr-Val-Ile-Asn- 


His-Tyr-Arg- 


Met 


- Arg- 


-Gly- 


-His-Ser 







30 

and 

1) being cross reactive with antibodies raised against a polypeptide of a depicted sequence; or 2) 
being cross reactive with cell surface receptors for a polypeptide of a depicted sequence; or 3) having 
like hormonal activity to a polypeptide of a depicted sequence. 

35 

2. A method according to claim 1 wherein the nucleic acid encodes a human or porcine inhibin a chain 
and/or a human or porcine 0 chain the amino acid sequences of which are as depicted in Fig 1B 
(porcine o chain), Fig 2B (porcine 0 chains), Fig 6A (human a chain) and Figs 8 and 9 (human 0 
chains). 

40 

3. A method according to Claim 1 wherein the vector encodes an inhibin 0 chain, or an amino acid 
sequence variant thereof, other than native human or porcine 0 A or the partial bovine inhibin 0 chain 
specified in claim 1 . 

45 4. A method according to claim 1 wherein the nucleic acid encodes a variant selected from: porcine 
inhibin derivatives (Asn2&6— GlnJInha; [CVS325 or Cys324-»A]lnha; [Cyssei or CyS36 3-*A]lnha; [LyS32i 
or Lys322-*A]lnh/S A ;[LyS322-*His or Ser]lnhj9 A ; [Lys 3 i5- , Arg; Valsu— Thr] lnh/? A ;[Cys 3 88 or Cyssso—A]- 
lnhj9 A , [LyS4ii—Gln]lnh0 A ; [Arg 3 i5— Lys; Val 3 i6->Thr]lnh0 B ; [Cys 3 i 9 or CyS32o- , A]lnh/S B ; [Pro 3 si 
GIV382— Pro Phe Gly]lnh/9 B ; [Arg 3 g5— Gln]lnh0 B » wherein Inh is an abbreviation of inhibin and the 

50 residue numbers for lnhj8 B are those used for the corresponding lnh/9 A residue (see Fig. 2B); human 
inhibin & A chain variants having variations which are substitution or deletion at, or insertion next to, a 
residue selected from residues 293-297, 364-376 and 387-398 of the sequence depicted in Fig. 8; and 
variants of human a chain which are greater than 90% homologous with the sequence depicted in Fig. 
6A. 

55 

5. A method according to claim 1 wherein the nucleic acid encodes a variant of a human 0 A inhibin chain 
selected from Phe 3 o2^lle or Leu; Gln 2 97— Asp or Lys; Trp 3 o7— Tyr or Phe; Trp 3 io-*Tyr or Phe; 
Mean— Phe or Val; Tyr 3 i7-*Trp or Thr; HiS3is-*Lys; Alasig— Ser; Asn 3 2o— Gin, Tyr or His; Tyrs 2 i— Thr 



23 



EP 0 222 491 B1 



or Asp, Phe34o-*Tyr; Hiss53-*Asp; HiS353"*Lys (a 0 A /0 B hybrid); Phesss^Tyr; Val364—Phe; 
Val364-*Leu; Tyrs75-*Thr; Tyr 37 6->Trp; Asn3 B 9—Gln, His or Lys; He39i-*Leu or Thr; MeU9o ^Leu or 
Ser; Val392~*Phe, Glu, Thr or ile; or comparably modified human 0 B chain 

5 6. The method of any one of the preceding claims wherein the nucleic acid encoding the inhibin a chain 
and/or & chain is operably linked to a promoter recognized by the host cell and including the further 
step of recovering inhibin or a /3 chain dimer from the culture medium. 

7. The method of any one of the preceding claims wherein the cell is a prokaryote. 

70 

8. The method of any preceding claim wherein the vector comprises nucleic acid encoding the prepro 
form of an inhibin a chain or an inhibin 0 chain. 

9. The method of claim 8 wherein the vector comprises nucleic acid encoding the prepro form of both the 
75 inhibin a chain and an inhibin jS chain. 

10. The method of any one of claims 1 to 6, 8 or 9 wherein the cell is a cell from a multicellular organism 
and hormonally active inhibin is produced. 

20 11. The method of claim 6 wherein the promoter is a viral promoter. 

12. The method of claim 11 wherein the promoter is an SV40 promoter. 

13. The method of any preceding claim wherein mature porcine or human inhibin is recovered. 

25 

14. The method of any of claims 1 to 7 or 10 to 12 wherein the vector comprises nucleic acid encoding the 
prepro form of an inhibin & chain and a mature jS-chain dimer is recovered free of the a -chain. 

15. The method of claim 10 or 13 wherein the 0 chain is the 0 A chain and the inhibin is present in a 
30 concentration greater than about 20 ng/ml in the culture medium. 

16. A composition comprising human or porcine inhibin made up of an a and a chain, the amino acid 
sequences of said a and 0 chains being selected from those depicted in Fig 1 B (porcine a chain), Fig 
2B (porcine 0 chains), Fig 6A (human a chain) and Figs 8 and 9 (human 0 chains), and amino acid 

35 sequence variants by way of insertion, deletion or substitution of a polypeptide of a said depicted 
sequence, which variants are substantially homologous with a polypeptide of a depicted sequence, but 
excluding bovine inhibin a chain and 1) are cross reactive with antibodies raised against a polypeptide 
of a depicted sequence; 2) are cross reactive with cell surface receptors for a polypeptide of a depicted 
sequence; or 3) have like hormonal activity to a polypeptide of a depicted sequence; which composition 

40 is completely free of unidentified human or porcine proteins. 

17. A composition according to Claim 16 wherein the sequences of said q and chains are selected from 
those depicted in Fig 1B (porcine a chain), Fig 2B (porcine 0 chains), Fig 6A (human a chain) and Figs 
8 and 9 (human 0 chains). 

45 

18. A composition according to claim 16 wherein said variants are selected from: porcine inhibin derivatives 
(Asn 2 t6^ln]Glnha; [Cys 3 25 or CyS324-»A]lnha; [CyS36i or CVS3&3— A]lnha; [LVS321 or Lys3 2 2-*A]lnh/S A ; 
[LyS322~*His or Ser]lnh0 A ; [LVS315— Arg; Val3i6~*Thr] lnh/3 A ; [CyS388 or Cys39o-*A]lnhj8 A , 
[Lys4n— Gln]lnh)S A ; [Arg 3 i5— Lys; Val3i6-*Thr]lnhj8 B ; [Cys 3 ig or Cyss2o— A]lnh0 B [Prossi Gly382^ P'o 

so Phe Gly]lnh£ B ; [Argsgs— Gln]1nhjS B , wherein Inh is an abbreviation of inhibin and the residue numbers 
for lnh/3 B are those used for the corresponding lnh)3 A residue (see Fig. 2B); human inhibin 0 A chain 
variants having variations which are substitution or deletion at, or insertion next to, a residue selected 
from residues 293-297, 364-376 and 387-398 of the sequence depicted in Fig. 8; and variants of human 
a chain which are greater than 90% homologous with the sequence depicted in Fig. 6A. 

55 

19. A composition according to claim 16 including a variant of a human 0 A inhibin chain selected from 
Phe302— He or Leu; Gln297-*Asp or Lys; Trp307-*Tyr or Phe; Trp 3 io^Tyr or Phe; lle3n-*Phe or Val; 
Tyr 317 -*Trp or Thr; Hissis-^Lys; Ala 3 i 9 — Ser; Asns2o— <3ln, Tyr or His; Tyr 32 i-*Thr or Asp, 



24 



EP 0 222 491 B1 



Phe34o—Tyr; Hisss3— Asp; HiS3sa— Lys (a ft A /ft B hybrid); Phe 3 5&-*Tyr; Va^—Phe; Val3&4— Leu; 
Tyr 3 75— Thr;. Tyr376-*Trp; Asn389-*Gln, His or Lys; lle 3 9i-*Leu or Thr; Met39o -*Leu or Ser; 
Vabg2— Phe, Glu, Thr or He; or comparably modified human /S B chain. 

5 20. A composition comprising a prodomain of human or porcine a or ft B inhibin as depicted in Fig 1B 
(porcine a chain), Fig 2B (porcine ft& chain), Fig 6A (human a chain), and Fig 9 (human ft Q chain) 
unassociated with native glycosylation. 

21- A composition comprising a homodimer of mature human or porcine inhibin £ A or jS B chains, said 
70 chains being as depicted in Fig 2B (porcine ft chains) and Figs 8 and 9 (human ft chains) or of an 
amino acid sequence variant by way of insertion, deletion or substitution of a polypeptide of a said 
depicted sequence the variant being substantially homologous with a polypeptide of a depicted 
sequence and 1) being cross reactive with antibodies raised against a polypeptide of a depicted 
sequence; 2) being cross reactive with ceil surface receptors for a polypeptide of a depicted sequence; 
75 or 3) having like hormonal activity to a polypeptide of a depicted sequence; which composition is free 
of the inhibin a chain. 

22. A composition according to Claim 21 which is a homodimer of mature human or porcine 0 B chains or a 
said amino acid sequence variant thereof. 

20 

23. A composition comprising a heterodimer of mature human or porcine inhibin ft A with mature human or 
porcine inhibin 0 B , said chains being as depicted in Fig 2B (porcine ft chains) and Figs 8 and 9 (human 
ft chains) or of an amino acid sequence variant by way of insertion, deletion or substitution of a said 
depicted sequence the variant being substantially homologous with a polypeptide of a depicted 

25 sequence and 1) being cross reactive with antibodies raised against a polypeptide of a depicted 
sequence; 2) being cross reactive with cell surface receptors for a polypeptide of a depicted sequence; 
or 3) having like hormonal activity to a polypeptide of a depicted sequence; which composition is free 
of the inhibin a chain. 

30 24. Non-chromosomal DNA encoding a human or porcine inhibin a or a human or porcine inhibin ft chain 
the amino acid sequences of which are as depicted in Fig 1B (porcine o chain), Fig 2B (porcine ft 
chains), Fig 6A (human a chain) and Figs 8 and 9 (human ft chains) or an amino acid sequence variant 
by way of insertion, deletion or substitution of a polypeptide of a said depicted sequence, the variant 
being substantially homologous with a polypeptide of a depicted sequence excluding bovine inhibin a 

35 chain and the partial bovine inhibin ft chain of the sequence 

Gly-Leu-Glu-Cys-Asp- 



Gly- 


Lys- 


-Val- 


Asn- 


-Ile- 


-Cys- 


-Cys- 


Lys- 


Lys- 


-Gln- 


Phe- 


Phe- 


-Val- 


-Ser- 


Phe- 


Lys- 


- Asp- 


■Ile- 


-Gly- 


-Trp- 


-Asn- 


Asp- 


Trp- 


-Ile- 


Ile- 


•Ala- 


-Pro- 


-Ser- 


Gly- 


■Tyr- 


-His- 


Ala- 


- Asn- 


-Tyr- 


-Cys- 


■Glu- 


■Gly- 


-Glu- 


■Cys- 


-Pro- 


-Ser- 


-His- 


Ile- 


Ala 


-Gly- 


-Thr 


-Ser- 


-Gly 


-Ser- 


-Ser- 


■Leu- 


-Ser- 


■Phe- 


■His 


-Ser- 


-Thr- 


Val- 


■He' 


-Asn- 


-His 


-Tyr- 


-Arg 


-Met- 


-Arg- 


-Gly- 


-His- 


■Ser 









so and 

1) being cross reactive with antibodies raised against a polypeptide of a depicted sequence; or 2) 
being cross reactive with cell surface receptors for a polypeptide of a depicted sequence; or 3) having 
like hormonal activity to a polypeptide of a depicted sequence. 

55 25. The DNA of claim 24 encoding a human or porcine inhibin o or a human or porcine inhibin ft chain 
whose amino acid sequence is as depicted in Fig 1 B (porcine a chain), Fig 2B (porcine ft chains), Fig 
6A (human a chain) and Figs 8 and 9 (human ft chains). 



25 



EP 0 222 491 B1 



26. The DNA of claim 24 which encodes a variant selected from: porcine inhibin derivatives (Asn266~*Gln]- 
lnha;[CyS325 or Cys324—A]lnha; [Cyssti or Cys3t3-*A]lnha; [LVS321 or LyS322-*A]lnhj9 A ; [Lys322-*His 
or Ser]lnh£ A ; [Lys 3 i5-Arg; Val 3 u— Thr] lnh/3 A ; [Cys 38 a or Cys 3 9o-A]lnhjS A ,[Lys4i Gln]lnhj8 A ; 
[Arg 3 is-Lys; Val 3 i&-Thr]lnh0 B ; [Cyssig or Cys32o— A]lnh0 B ; [Pro 38 i Gly 3 82- Pro Phe Gly]- 

5 lnhj8 B ;[Arg 3 95-*Gln]lnh0 B , wherein Inh is an abbreviation of inhibin and the residue numbers for lnh£ B 
are those used for the corresponding lnhj9 A residue (see Fig. 2B); human inhibin /8 A chain variants 
having variations which are substitution or deletion at, or insertion next to, a residue selected from 
residues 293-297, 364-376 and 387^398 of the sequence depicted in Fig. 8; and variants of human a 
chain which are greater than 90% homologous with the sequence depicted in Fig. 6A. 

io 

27. The DNA of claim 24 which encodes a variant of a human 0 inhibin chain selected from Phe 3 o 2 -Hle or 
Leu; Gln 2 97— Asp or Lys; Trp 30 7-*Tyr or Phe; Trp 3 io-*Tyr or Phe; lle^i—Phe or Val; Tyrai7-*Trp or 
Thr; Hissis—Lys; Alasig— Ser; Asn 32 o— Gin, Tyr or His; Ty^i—Thr or Asp, Phea4o-*Tyr; His 3 s3-*Asp; 
HiS353-*Lys (a 0 A /j8 B hybrid); Phesse— Tyr; Vabe^—Phe; Val 36 4— Leu; Tyr 3 7s— Thr; Tyr 3 76-Trp; 

rs Asn 38 9-*Gln, His or Lys; llesgt-^Leu or Thr; Mebgo —Leu or Ser; Val392-*Phe, Glu, Thr or lie; or 
comparable modified human 0 B chain 

28. The DNA of any of claims 24 to 27 which is free of intervening untranslated sequences. 
20 29. The DNA of any one of claims 24 to 28 which is labelled with a detectable moiety. 

30. A replicable vector comprising a DNA of any of claims 21 to 27. 

31. The vector of claim 30 comprising a viral promoter operably linked to the DNA encoding the inhibin a 
25 and/or j9 chains. 

32. The vector of claim 30 or 31 which contains DNA encoding both an inhibin a and an inhibin & chain. 

33. The vector of claim 30 or 31 which contains DNA encoding an inhibin 0 chain but not the inhibin a 
30 chain. 

34. A host cell transformed with a replicable vector comprising DNA encoding the human or porcine inhibin 
a and/or an inhibin j8 chain the amino acid sequences of which are as depicted in Fig 1 B (porcine o 
chain), Fig 2B (porcine & chains), Fig 6A (human a chain) and Figs 8 and 9 (human /9 chains) or an 

35 amino acid sequence variant by way of insertion, deletion or substitution of a polypeptide of a said 
depicted sequence, the variant being substantially homologous with a polypeptide of a depicted 
sequence, excluding bovine inhibin a chain, and the partial bovine inhibin 0 chain of the sequence 

Gly-Leu-Glu-Cys-Asp-Gly- 

40 

Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe-Phe-Val-Ser-Phe- 
Lys-Asp-Ile-Gly-Trp-Asn-Asp-Trp-Ile-Ile-Ala-Pro-Ser-Gly- 

45 Tyr-His-Ala-Asn-Tyr-Cys-Glu-Gly-Glu-Cys-Pro-Ser-His-Ile- 
Ala-Gly-Thr-Ser-Gly-Ser-Ser-Leu-Ser-Phe-His-Ser-Thr-Val^ 

5o He-Asn-His-Tyr-Arg-Met-Arg-Gly-His-Ser 

and 

1) being cross reactive with antibodies raised against a polypeptide of a depicted sequence; or 2) 
being cross reactive with cell surface receptors for a polypeptide of a depicted sequence; or 3) having 
56 like hormonal activity to a polypeptide of a depicted sequence. 

35. A host cell according to claim 34 wherein the DNA encodes a human or porcine inhibin a and/or an 
inhibin & chain, the amino acid sequences of which are as depicted in Fig 1 B (porcine o chain), Fig 2B 



26 



EP 0 222 491 B1 



(porcine ft chains), Fig 6A (human a chain) and Figs 8 and 9 (human ft chains). 

36. A host cell according to claim 34 wherein the DNA encodes a variant selected from: porcine inhibin 
derivatives (Asn 2 66- ,, Gln]lnha; [CVS325 or CyS324-*A]lnha; [CVS361 or CyS3&3-*A]lnha; [Lys32i or 

5 Lys322-*A]lnh0 A ;[LyS322-*His or Ser]lnhj8 A ; [Lyssis— Arg; Valsu— Thr] Inh^AllCyssss or Cyssso— A]- 

Inh^A, [Lys 411— Gln]lnh0 A ; [Arg 3 i5— Lys; Valsu—Thrllnhfo; [CyS3ia or CySs2o— A]lnh£ B ; [PrbsBi 
Gly382~* Pro Phe Gly]lnh0 B ; tArgsgs—Glnllnhfo, wherein Inh is an abbreviation of inhibin and the 
residue numbers for \nhft B are those used for the corresponding lnh£ A residue (see Fig. 2B); human 
inhibin ft A chain variants having variations which are substitution or deletion at, or insertion next to, a 

70 residue selected from residues 293-297, 364-376 and 387-398 of the sequence depicted in Fig. 8; and 
variants of human a chain which are greater than 90% homologous with the sequence depicted in Fig. 
6A. 

37. A host cell according to claim 34 wherein the DNA encodes a variant of a human ftp, inhibin chain 
75 selected from Phe302-*He or Leu; Gln 2 97^Asp or Lys; Trpsc^—Tyr or Phe; Trpsic— Tyr or Phe; 

Ile3n —Phe or Val; Tyr3i7— Trp or Thr; HiS3i8~*Lys; Ala3ig— Ser; Asns 2 o— Gin, Tyr or His; Tyr32i— Thr 
or Asp, Phesio-Tyr; His353—Asp; HtS353—Lys (a ft A /ft B hybrid); Phe35&—Tyr; Vals^— Phe; 
Vals64-*Leu; Tyr375~*Thr; Tyr37&-*Trp; Asn389-*Gln, His or Lys; 116391— Leu or Thr; Metsao -*Leu or 
Ser; Val3s 2 — Phe, Glu t Thr or He; or comparably modified human ft B chain 

20 

38. The cell of any of claims 34 to 37 which is a eukaryotic cell. 

39. A cell-free composition that is free of mature o chain polypeptide containing a human or porcine inhibin 
a chain prodomain polypeptide sequence as depicted in Fig 1 B (porcine a chain) or Fig 6A (human a 

25 chain) or an amino acid sequence variant by way of insertion, deletion or substitution of a said 
prodomain sequence, the variant being substantially homologous with a polypeptide of a said sequence 
and being cross reactive with antibodies raised against a polypeptide of a depicted sequence. 

40. A cell-free composition containing 

30 a) a polypeptide comprising the human inhibin ft* chain prodomain sequence 

HSAAPDCPSCALAALPKDVPNSQPEMVEAVKKHILNMLHL (Amino acids 1-4 0 
35 of Fig 8), PDVTQPVPKAALLNA I RKLHVGKVGENG YVE I EDD I G (amino 

acids 44-80 of Fig 8.), 
4Q AEMNELMEQTSEI ITFAESGTARKTLHFEISKEGSDLSWERAEWLFLKVPKAN- 

RTRTKVTI RLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKWDA ( amino 

acids 83-185 of Fig 8), STWHVFPVSSSIQRLLD- 

45 

QGKSSLDVRIACEQCQESGASLVLLG (amino acids 188-230 of Fig 8) , 
or naturally occurring mammalian amino acid sequence variants thereof; 

50 



55 



27 



EP 0 222 491 B1 



b) a polypeptide comprising the human inhibin 0b chain prodomain sequence 

CTSCGGFRRPEELGRVDGDFLEAV , (amino acids 

7-30 of Fig 9) , 

HILSRLQMRGRPNITHAVPKAAMVTALRKLHAGKVREDGRVEIPHLDGHASPGAD- 
GQERVSEIISFAETDGLASSRVRLYFFISNEGNQNLFWQASLWLYLKLLPYVLEKGS 
(amino acids 33-145 of Fig 9) , 

VRVKVYFQEQGHGDRWNMVEKRVDLKRSGWHTFPLTEAIQALFERGE (amino 
acids 149 - 195 of Fig 9) , 

LNLDVQCDSCQELAWPVFVDPGEESHRPFVWQARLGDSRHRI (amino acids 
198-241 of Fig 9) , 

or naturally occurring mammalian amino acid sequence variants thereof; or 

c) a polypeptide free of the mature a chain amino acid sequence comprising the human inhibin a 
chain prodomain sequences 

KVRALFLDALGPPAVTREGGDPGV (amino 

acids 1 - 24 of Fig 6) , 

HALGGFTHRGSEPEEEEDVSQAILFPATDASCEDKSAARGLAQEAEEGLFRYMFR- 
PSQHTRSRQVTSAQLWFHTGLDRQGTAASNSS E PLLGLLALS PGGPVAVPMSUGH - 
APPHWAVLHLATSALSLLTHPVLVLLLRCPLCTCSARPEATPFLVAHTRTRPPSGGERA 
(amino acids 32 - 199 of Fig 6) , 

or naturally occurring mammalian amino acid sequence variants thereof. 

41. A cell-free composition containing a human or porcine inhibin £ chain prodomain polypeptide of a 
sequence as depicted in Fig 2B (porcine 0 chains) and Figs 8 and 9 (human p chains) or an amino 
acid sequence variant by way of insertion, deletion, or substitution of a said prodomain sequence, the 
variant being substantially homologous with a polypeptide of a said sequence, and being cross reactive 
with antibodies raised against a polypeptide of a depicted sequence. 

42. The composition of claim 41 wherein the $ chain is the /3 A chain and the composition is free of mature 
0 A chain sequence. 

43. The composition of claim 41 wherein the & chain is the £ B chain and the composition is free of mature 
0b chain sequence. 



28 



EP 0 222 491 B1 



44. The composition of claim 40 wherein the variant is the corresponding porcine amino acid sequence, the 
composition containing: 

a) a polypeptide comprising the porcine inhibin j8 A chain prodomain sequence depicted at: amino 
acids 28 to 58; amino acids 61 to 87; amino acids 90 to 108; amino acids 111 to 179; amino acids 

5 182 to 213; amino acids 216 to 258; or amino acids 28 to 87 of Fig 2B; or 

b) a polypeptide free of the mature a chain amino acid sequence comprising the porcine inhibin a 
chain prodomain polypeptide sequences depicted at: amino acids 20 to 54; or amino acids 70 to 228 
of Fig 1B. 

io 45. The composition of any of claims 39 to 44 wherein the polypeptide is unaccompanied by native 
glycosylation. 

46. The composition of any of claims 39 to 45 which is sterile and wherein the polypeptide further 
comprises an immunogenic polypeptide. 

75 

47. The use of a composition of any of claims 39 to 46 in the preparation of an antibody capable of binding 
said polypeptide. 

48. The composition of any of claims 39 to 46 wherein the polypeptide is conjugated to a detectable group. 

20 

49,. The composition of claim 48 wherein the group is an enzyme, fluorophore or radioisotope. 

50. The composition of any of claims 39 to 44, 48 or 49 which is insolubilized by non-covalent absorption 
or covalent cross-linking to a water insoluble support. 

25 

51. The composition of any of claims 39 to 46, 48 or 49 further comprising a physiologically acceptable 
implantable matrix for controlled release of the polypeptide into the tissues of an animal. 

Claims for the following Contracting State : LU 

30 

1. A method comprising culturing a host cell transformed with a vector which includes nucleic acid 
encoding a human or porcine inhibin o chain and/or a human or porcine inhibin 0 chain the amino acid 
sequences of which are as depicted in Fig 1B (porcine a chain), Fig 2B (porcine & chains), Fig 6A 
(human a chain) and Figs 8 and 9 (human 0 chains), or an amino acid sequence variant by way of 

35 insertion, deletion or substitution of a said depicted sequence, the variant being substantially homolo- 
gous with a polypeptide of a depicted sequence but excluding bovine inhibin a chain and 1) being 
cross reactive with antibodies raised against a polypeptide of a depicted sequence; or 2) being cross 
reactive with cell surface receptors for a polypeptide of a depicted sequence; or 3) having like 
hormonal activity to a polypeptide of a depicted sequence. 

40 

2. A method according to Claim 1 wherein the nucleic acid encodes a human or porcine inhibin a chain 
and/or a human or porcine 0 chain the amino acid sequences of which are as depicted in Fig 1B 
(porcine o chain), Fig 2B (porcine & chains), Fig 6A (human a chain) and Figs 8 and 9 (human & 
chains). 

45 

3. A method according to Claim 1 wherein the vector encodes an inhibin 0 chain, or an amino acid 
sequence variant thereof, other than native human or porcine 0 A . 

4. A method according to claim 1 wherein the nucleic acid encodes a variant selected from: porcine 
so inhibin derivatives (Asnsse-^GInllnhor; [CVS325 or CyS324~*A]lnha; [CVS3&1 or CyS36 3^A]Inha; (Lyss2i 

or Lys322-*A]lnh£ A ;[Lys322-*His or Ser]lnh£ A ; [Lyssis^Arg; Val 3 i6-*Thr] lnhjS A ;[Cys 3 88 or Cysaso^A]- 
inh0 A , [Lys4n->Gln]lnh/9 A ; [Arg 3 is— Lys; Val3i6-*Thr]lnh0 B ; tCys3is or Cys 3 2o—A]lnh0 B ; [Pro 3 si 
Gly 38 2— Pro Phe Gly]lnhj8 B ; [Arg 39 s->Gln]lnh0 B( wherein Inh is an abbreviation of inhibin and the 
residue numbers for lnh/3 B are those used for the corresponding lnh£ A residue (see Fig. 2B); human 
55 inhibin 0 A chain variants having variations which are substitution or deletion at, or insertion next to, a 
residue selected from residues 293-297, 364-376 and 387-398 of the sequence depicted in Fig. 8; and 
variants of human a chain which are greater than 90% homologous with the sequence depicted in Fig. 
6A. 



29 



EP 0 222 491 B1 



5. A method according to claim 1 wherein the nucleic acid encodes a variant of a human j8 A inhibin chain 
selected from Phe3o 2 —He or Leu; Gln 2 s7— Asp or Lys; Trp 3 o7—Tyr or Phe; Trpaio—Tyr or. Phe; 
lle 3 n— Phe or Va!; Tyr 3 i7-»Trp or Thr; His 3 i8~*Lys; Ala 3 ig->Ser; Asn^o—GIn, Tyr or His; Tyr 3 2i — Thr 
or Asp, Phe 3 *o-Tyr; His 35 3-»Asp; His^-Lys (a 0 A /0 B hybrid); Phe 3 56— Tyr; VabG^Phe; 

5 Val 3 &4— Leu; Tyr 37 5-*Thr; Tyr 37 6-*Trp; Asn 38 9-*Gln, His or Lys; lle 3 gi-*Leu or Thr; Met39o -*Leu or 
Ser; Va^—Phe, Glu, Thr or lie; or comparably modified human 0 B chain 

6. The method of any one of the preceding claims wherein the nucleic acid encoding the inhibin a chain 
and/or $ chain is operably linked to a promoter recognized by the host cell and including the further 

w step of recovering inhibin or a 0 chain dimer from the culture medium. 

7. The method of any one of the preceding claims wherein the cell is a prokaryote. 

8. The method of any preceding claim wherein the vector comprises nucleic acid encoding the prepro 
15 form of an inhibin a chain or an inhibin 0 chain. 

9. The method of claim 8 wherein the vector comprises nucleic acid encoding the prepro form of both the 
inhibin a chain and an inhibin & chain. 

20 10. The method of any one of claims 1 to 6, 8 or 9 wherein the cell is a cell from a multicellular organism 
and hormonally active inhibin is produced. 

11. The method of claim 6 wherein the promoter is a viral promoter. 

25 12. The method of claim 11 wherein the promoter is an SV40 promoter. 

13. The method of any preceding claim wherein mature porcine or human inhibin is recovered. 

14. The method of any of claims 1 to 7 or 10 to 12 wherein the vector comprises nucleic acid encoding the 
30 prepro form of an inhibin j8 chain and a mature /S-chain dimer is recovered free of the o -chain. 

15. The method of claim 10 or 13 wherein the & chain is the £ A chain and the inhibin is present in a 
concentration greater than about 20 ng/ml in the culture medium. 

35 16. A composition comprising human or porcine inhibin made up of an a and a £ chain, the amino acid 
sequences of said a and & chains being selected from those depicted in Fig 1 B (porcine a chain), Fig 
2B (porcine & chains), Fig 6A (human a chain) and Figs 8 and 9 (human 0 chains), and amino acid 
sequence variants by way of insertion, deletion or substitution of a polypeptide of a said depicted 
sequence, which variants are substantially homologous with a polypeptide of a depicted sequence, but 

40 excluding bovine inhibin a chain and 1) are cross reactive with antibodies raised against a polypeptide 
of a depicted sequence; 2) are cross reactive with cell surface receptors for a polypeptide of a depicted 
sequence; or 3) have like hormonal activity to a polypeptide of a depicted sequence; which composition 
is completely free of unidentified human or porcine proteins. 

45 17. A composition according to Claim 16 wherein the sequences of said a and 0 chains are selected from 
those depicted in Fig 1 B (porcine a chain), Fig 2B (porcine £ chains), Fig 6A (human a chain) and Figs 
8 and 9 (human 0 chains). 

18. A composition according to claim 16 wherein said variants are selected from: porcine inhibin derivatives 
so (Asn 2 66-*ln]Glnha; [Cys 3 25 or Cys 32 4—A]lnha; [Cys 3 6i or Cys 3 6 3 -*A]lnha; [Lys 3 2i or Lys 3 22- , *A]lnh/8 A ; 
[Lys322—His or Ser]lnh0 A ; [Lys 3 is^Arg; Val 3 u— Thr] lnhjS A ; [Cys 3 g8 or Cys 39 o— A]lnhjS A , 
[Lys*ii— Gln]lnh0 A ; [Arg 3 i 5 — Lys; Val 3 i6— Thr]lnhjS B ; [Cys 3 ig or Cys 3 2o— A]lnhjS B [Pro 3 si Gly 3 82— Pro 
Phe Gly]lnh£ B ; [Arg 33 5— Gln]lnhjS B , wherein Inh is an abbreviation of inhibin and the residue numbers 
for Inhfo are those used for the corresponding lnh0 A residue (see Fig. 2B); human inhibin /8 A chain 
55 variants having variations which are substitution or deletion at, or insertion next to, a residue selected 
from residues 293-297, 364-376 and 387-398 of the sequence depicted in Fig. 8; and variants of human 
a chain which are greater than 90% homologous with the sequence depicted in Fig. 6A. 



30 



EP 0 222 491 B1 



19. A composition according to claim 16 including a variant of a human 0 A inhibin chain selected from 
Phe302— He or Leu; Gln 2 97— Asp or Lys; Trp 3 c7-»Tyr or Phe; Trp 3 i 0 — Tyr or Phe; lle 3 u—Phe or Val; 
Tyr 3 i7— Trp or Thr; His 3 i 8 — Lys; Ala 3 is— Ser; Asn 3 2o— Gin, Tyr or His; Tyr 3 2i— Thr or Asp, 
Phe 3 4o-*Tyr; His 3 5 3 —Asp; HiS3S3— Lys (a 0 A /£ B hybrid); Phe35&->Tyr; Val3&4—Phe; Vab&^Leu; 

5 Tyr 3 75-*Thr; Tyr 3 7&— Trp; Asn 38 9— Gin, His or Lys; lle 3 gi— Leu or Thr; Metsso —Leu or Ser; 
Val 3 92-*Phe, Glu, Thr or He; or comparably modified human 0 B chain. 

20. A composition comprising a prodomain of human or porcine a or 0 B inhibin as depicted in Fig 1B 
(porcine a chain), Fig 2B (porcine 0 B chain), Fig 6A (human a chain), and Fig 9 (human jS B chain) 

70 unassociated with native glycosylation. 

21. A composition comprising a homodimer of mature human or porcine inhibin 0 A or 0 B chains, said 
chains being as depicted in Fig 2B (porcine 0 chains) and Figs 8 and 9 (human 0 chains) or of an 
amino acid sequence variant by way of insertion, deletion or substitution of a polypeptide of a said 

75 depicted sequence the variant being substantially homologous with a polypeptide of a depicted 
sequence and 1) being cross reactive with antibodies raised against a polypeptide of a depicted 
sequence; 2) being cross reactive with cell surface receptors for a polypeptide of a depicted sequence; 
or 3) having like hormonal activity to a polypeptide of a depicted sequence; which composition is free 
of the inhibin a chain. 

20 

22. A composition according to Claim 21 which is a homodimer of mature human or porcine fa chains or a 
said amino acid sequence variant thereof 

23. A composition comprising a heterodimer of mature human or porcine inhibin /S A with mature human or 
25 porcine inhibin 0 B , said chains being as depicted in Fig 2B (porcine 0 chains) and Figs 8 and 9 (human 

jS chains) or of an amino acid sequence variant by way of insertion, deletion or substitution of a said 
depicted sequence the variant being substantially homologous with a polypeptide of a depicted 
sequence and 1 ) being cross reactive with antibodies raised against a polypeptide of a depicted 
sequence; 2) being cross reactive with cell surface receptors for a polypeptide of a depicted sequence; 
30 or 3) having like hormonal activity to a polypeptide of a depicted sequence; which composition is free 
of the inhibin a chain. 

24. Non-chromosomal DNA encoding a human or porcine inhibin a or a human or porcine inhibin 0 chain 
the amino acid sequences of which are as depicted in Fig 1B (porcine a chain), Fig 2B (porcine /S 

35 chains), Fig 6A (human a chain) and Figs 8 and 9 (human £ chains) or an amino acid sequence variant 
by way of insertion, deletion or substitution of a polypeptide of a said depicted sequence, the variant 
being substantially homologous with a polypeptide of a depicted sequence excluding bovine inhibin a 
chain and 1) being cross reactive with antibodies raised against a polypeptide of a depicted sequence; 
or 2) being cross reactive with cell surface receptors for a polypeptide of a depicted sequence; or 3) 

40 having like hormonal activity to a polypeptide of depicted sequence. 

25. The DNA of claim 24 encoding a human or porcine inhibin a or a human or porcine inhibin 0 chain 
whose amino acid sequence is as depicted in Fig 1 B (porcine a chain), Fig 2B (porcine 0 chains), Fig 
6A (human a chain) and Figs 8 and 9 (human chains). 

45 

26. The DNA of claim 24 which encodes a variant selected from: porcine inhibin derivatives (Asn 2 6&-*Gln]- 
lnha;[CyS325 or Cys 32 4— A]lnho; [Cys 3 6i or Cys36 3 -*A]lnha; [Lys 3 2i or Lys 3 22— f A]lnh/S A ; [Lys 32 2— His 
or Ser]lnh£ A ; [Lys 3 i 5 —Arg; Valsu— Thr] lnh£ A ; [Cys 3 s8 or Cys 390 — A]lnh0 A ,[LyS4i 1— Gln]lnh0 A ; 
[Arg 3 is— Lys; Valine — Thr]lnh0 B ; [CVS319 or CyS32o— A]lnhjS B ; [Pro 3 si Gly 3B 2— Pro Phe Gly]- 

50 lnh£ B ;[Arg395-*Gln]lnhj9 B , wherein Inh is an abbreviation of inhibin and the residue numbers for lnh/3 B 
are those used for the corresponding lnh/S A residue (see Fig. 2B); human inhibin 0 A chain variants 
having variations which are substitution or deletion at, or insertion next to, a residue selected from 
residues 293-297, 364-376 and 387-398 of the sequence depicted in Fig. 8; and variants of human a 
chain which are greater than 90% homologous with the sequence depicted in Fig. 6A. 

55 

27. The DNA of claim 24 which encodes a variant of a human 0 inhibin chain selected from Pheso2-*lle or 
Leu; Gln 29 7^Asp or Lys; Trp 3D 7-*Tyr or Phe; Trp 3 i 0 -*Tyr or Phe; lle 3 n-*Phe or Val; Tyr 3 i 7 — Trp or 
Thr; His 3 ie— Lys; A!a 3 i9—Ser; Asn 3 2o— Gin, Tyr or His; Tyrs2i-Thr or Asp, Phe 3 4o— Tyr; His 3 s 3 — Asp; 



31 



EP 0 222 491 B1 



Hisasa— Lys (a 0 A //3 B hybrid); Phease—Tyr; Val 36 4— Phe; Vabe*— Leu; Tyr 3 7s—Thr; Tyr 376 — Trp; 
Asn 3 89— Gin, His or Lys; lle 3 gi—Leu or Thr; Met39o —Leu or Ser; Val 39 2— Phe, GIu, Thr or He; or 
comparable modified human 0 B chain 

5 28. The DNA of any of claims 24 to 27 which is free of intervening untranslated sequences. 

29. The DNA of any one of claims 24 to 28 which is labelled with a detectable moiety. 

30. A replicable vector comprising a DNA of any of claims 21 to 27. 

10 

31. The vector of claim 30 comprising a viral promoter operably linked to the DNA encoding the inhibin a 
and/or £ chains. 

32. The vector of claim 30 or 31 which contains DNA encoding both an inhibin a and an inhibin j9 chain. 

75 

33. The vector of claim 30 or 31 which contains DNA encoding an inhibin & chain but not the inhibin a 
chain. 

34. A host cell transformed with a replicable vector comprising DNA encoding the human or porcine inhibin 
20 a and/or an inhibin & chain the amino acid sequences of which are as depicted in Fig 1 B (porcine a 

chain), Fig 2B (porcine £ chains), Fig 6A (human o chain) and Figs 8 and 9 (human 0 chains) or an 
amino acid sequence variant by way of insertion, deletion or substitution of a polypeptide of a said 
depicted sequence, the variant being substantially homologous with a polypeptide of a depicted 
sequence, excluding bovine inhibin a chain, and 1) being cross reactive with antibodies raised against a 
25 polypeptide of a depicted sequence; or 2) being cross reactive with cell surface receptors for a 
polypeptide of a depicted sequence; or 3) having like hormonal activity to a polypeptide of a depicted 
sequence. 

35. A host cell according to claim 34 wherein the DNA encodes a human or porcine inhibin a and/or an 
so inhibin $ chain, the amino acid sequences of which are as depicted in Fig 1B (porcine a chain), Fig 2B 

(porcine 0 chains), Fig 6A (human a chain) and Figs 8 and 9 (human 0 chains). 

36. A host cell according to claim 34 wherein the DNA encodes a variant selected from: porcine inhibin 
derivatives (Asn266— Gln]lnha; [Cys 3 25 or Cys 3 24-*A]lnha; [Cys 3 &i or Cys 3 G3— AJInha; [Lys 3 2i or 

35 Lys 3 22-*A]lnh/S A ;[Lys 3 22-*His or Ser]lnh0 A ; [Lys 3 is-»Arg; Val 3 i6— Thr] lnh/S A ;[Cys 3 88 or Cys 3 9o— A]- 
lnhj3 A) [Lys 41 i-*Gln]lnh0 A ; [Arg 3 i 5 — Lys; Val 3 u-*Thr]lnhjS B ; [Cys 3 i 9 or Cys 3 2o— A]lnhj9 B ; [Pro 3 ai 
GIV382- ,> Pro Phe Gly]lnh0 B ; [Arg 39S — Gln]lnhj8 B , wherein Inh is an abbreviation of inhibin and the 
residue numbers for lnh0 B are those used for the corresponding lnhj8 A residue (see Fig. 2B); human 
inhibin j8 A chain variants having variations which are substitution or deletion at, or insertion next to, a 

40 residue selected from residues 293-297, 364-376 and 387-398 of the sequence depicted in Fig. 8; and 
variants of human o chain which are greater than 90% homologous with the sequence depicted in Fig. 
6A. 

37. A host cell according to claim 34 wherein the DNA encodes a variant of a human 0 A inhibin chain 
45 selected from Phe302-*He or Leu; Gln29 7-*Asp or Lys; Trp 3 o7~*Tyr or Phe; Trp 3 io-Tyr or Phe; 

lle 3 n— Phe or Val; Tyrai7— f Trp or Thr; His 3 is— Lys; Ala3is—Ser; Asn 3 2o->Gln, Tyr or His; Tyr 3 2i— Thr 
or -Asp, Phe 3 4o—Tyr; His 3 5 3 — Asp; His 3 s 3 -*Lys (a 0 A /0 B hybrid); Phe 3 56— Tyr; Val 3 6*-*Phe; 
Val3&4-*Leu; Tyr 3 75—Thr; Tyr 37 6-*Trp; Asn 3 89-*Gln, His or Lys; lle 39 i-^Leu or Thr; Mefego —Leu or 
Ser; Val 3 92-*Phe, Gtu, Thr or He; or comparably modified human /5 B chain 

50 

38. The cell of any of claims 34 to 37 which is a eukaryotic cell. 

39. A cell-free composition that is free of mature a chain polypeptide containing a human or porcine inhibin 
a chain prodomain polypeptide sequence as depicted in Fig 1 B (porcine o chain) or Fig 6A (human a 

55 chain) or an amino acid sequence variant by way of insertion, deletion or substitution of a said 
prodomain sequence, the variant being substantially homologous with a polypeptide of a said sequence 
and being cross reactive with antibodies raised against a polypeptide of a depicted sequence. 



32 



EP 0 222 491 B1 

40. A cell-free composition containing 

a) a polypeptide comprising the human inhibin 0 A chain prodomain sequence 

HSAAPDCPSCALAALPKDVPNSQPEMVEAVKKHILNMLHL (Amino acids 1-40 
of Fig 8) , PDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIG (amino 
acids 44-80 of Fig 8.), 

AEMNELMEQTSEIITFAESGTARKTLHFEISKEGSDLSWERAEVWLFLKVPKAN- 
RTRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKWDA (amino 
acids 83-185 of Fig 8), STWHVFPVSSSIQRLLD- 
QGKSSLDVRIACEQCQESGASLVLLG (amino acids 188-230 of Fig 8) , 

or naturally occurring mammalian amino acid sequence variants thereof; 

b) a polypeptide comprising the human inhibin j3 B chain prodomain sequence 

CTSCGGFRRPEELGRVDGDFLEAV , (amino acids 

7-30 of Fig 9) , 

HILSRLQMRGRPNITHAVPKAAMVTALRKLHAGKVREDGRVEIPHLDGHASPGAD- 
GQERVSEIISFAETDGLASSRVRLYFFISNEGNQNLFWQASLWLYLKLLPYVLEKGS 
(amino acids 33-145- of Fig 9) , 

WVK\T^FQEQGHGDRWNMVEKRVDLKRSGWHTFPLTEAI-QALFERGE (amino 
acids 149 - 195 of Fig 9), 

LNLDVQCDSCQELAWPVFVDPGEESHRPFVWQARLGDSRHRI (amino acids 
198-241 of Fig 9) , 

or naturally occurring mammalian amino acid sequence variants thereof; or 

c) a polypeptide free of the mature a chain amino acid sequence comprising the human inhibin a 
chain prodomain sequences 

KVRALFLDALGPPAVTREGGDPGV ( amino 

acids 1 - 24 of Fig 6) , 



33 



EP 0 222 491 B1 



HALGGFTHRGSEPEEEEDVSQAILFPATDASCEDKSAARGLAQEAEEGLFRYMFR- 
PSQHTRSRQVTSAQLWFHTGLDRQGTAASNSSEPLLGLLALSPGGPVAVPMSLGH- 

5 

APPHWAVLHLATSALSLLTHPVLVLLLRCPLCTCSARPEATPFLVAHTRTRPPSGGERA 
(amino acids 32 - 199 of Fig 6), 

w 

or naturally occurring mammalian amino acid sequence variants thereof. 

41. A cell-free composition containing a human or porcine inhibin j9 chain prodomain polypeptide of a 
sequence as depicted in Fig 2B (porcine 0 chains) and Figs 8 and 9 (human & chains) or an amino 

75 acid sequence variant by way of insertion, deletion, or substitution of a said prodomain sequence, the 
variant being substantially homologous with a polypeptide of a said sequence, and being cross reactive 
with antibodies raised against a polypeptide of a depicted sequence. 

42. The composition of claim 41 wherein the £ chain is the /8 A chain and the composition is free of mature 
20 £a chain sequence. 

43. The composition of claim 41 wherein the 0 chain is the j8 B chain and the composition is free of mature 
0 B chain sequence. 

25 44. The composition of claim 40 wherein the variant is the corresponding porcine amino acid sequence, the 
composition containing: 

a) a polypeptide comprising the porcine inhibin 0 A chain prodomain sequence depicted at: amino 
acids 28 to 58; amino acids 61 to 87; amino acids 90 to 108; amino acids 111 to 179; amino acids 
182 to 213; amino acids 216 to 258; or amino acids 28 to 87 of Fig 2B; or 
30 b) a polypeptide free of the mature a chain amino acid sequence comprising the porcine inhibin a 

chain prodomain polypeptide sequences depicted at: amino acids 20 to 54; or amino acids 70 to 228 
ofFiglB. 

45. The composition of any of claims 39 to 44 wherein the polypeptide is unaccompanied by native 
35 glycosylation. 

46. The composition of any of claims 39 to 45 which is sterile and wherein the polypeptide further 
comprises an immunogenic polypeptide. 

40 47. The use of a composition of any of claims 39 to 46 in the preparation of an antibody capable of binding 
said polypeptide. 

48. The composition of any of claims 39 to 46 wherein the polypeptide is conjugated to a detectable group. 

45 49. The composition of claim 48 wherein the group is an enzyme, fluorophore or radioisotope. 

50. The composition of any of claims 39 to 44, 48 or 49 which is insolubilized by non-covalent absorption 
or covalent cross-linking to a water insoluble support. 

so 51. The composition of any of claims 39 to 46, 48 or 49 further comprising a physiologically acceptable 
implantable matrix for controlled release of the polypeptide into the tissues of an animal. 

Claims for the following Contracting State : GR 

55 1. A method comprising culturing a host cell transformed with a vector which includes nucleic acid 
encoding a human or porcine inhibin a chain and/or a human or porcine inhibin 0 chain the amino acid 
sequences of which are as depicted in Fig 1B (porcine a chain), Fig 2B (porcine 0 chains), Fig 6A 
(human a chain) and Figs 8 and 9 (human j8 chains), or an amino acid sequence variant by way of 



34 



EP 0 222 491 B1 



insertion, deletion or substitution of a said depicted sequence, the variant being substantially homolo- 
gous with a polypeptide of a depicted sequence and 1) being cross, reactive with antibodies raised 
against a polypeptide of a depicted sequence; or 2) being cross reactive with cell surface receptors for 
a polypeptide of a depicted sequence; or 3) having like hormonal activity to a polypeptide of a depicted 
5 sequence. 

2. A method according to Claim 1 wherein the nucleic acid encodes a human or porcine inhibin a chain 
and/or a human or porcine 0 chain the amino acid sequences of which are as depicted in Fig 1B 
(porcine a chain), Fig 2B (porcine 0 chains), Fig 6A (human a chain) and Figs 8 and 9 (human /S 

70 chains). 

3. A method according to Claim 1 wherein the vector encodes an allelic variant of human or porcine 
inhibin a and/or £ chain. 

75 4. The method of any one of the preceding claims wherein the nucleic acid encoding the inhibin a chain 
and/or & chain is operably linked to a prompter recognized by the host cell and including the further 
step of recovering inhibin a chain, inhibin £ chain, inhibin or a & chain dimer from the culture medium. 

5. The method of any one of the preceding claims wherein the cell is a prokaryote. 

20 

6. The method of any one of the preceding claims wherein the vector comprises nucleic acid encoding 
the prepro form of an inhibin a chain or an inhibin & chain. 

7. A method according to claim 6 wherein the vector comprises nucleic acid encoding the prepro forms of 
25 both the inhibin a chain and an inhibin j3 chain. 

8. The method of any one of claim 1 to 4, 6 or 7 wherein the cell is a cell from a multicellular organism 
and hormonally active inhibin is produced. 

30 9. The method of claim 4 wherein the promoter is a viral promoter. 

10. The method of claim 9 wherein the promoter is an SV40 promoter. 

11. The method of any preceding claims wherein mature porcine or human inhibin is recovered. 

35 

12. The method of any of claims 1 to 5 or 8 to 10 wherein the vector comprises nucleic acid encoding the 
prepro form of an inhibin & chain and a mature 0-chain dimer is recovered free of the a-chain. 

13. The method of claim 8 or 11 wherein the 0 chain is the 0 A chain and the inhibin is present in a 
40 concentration greater than about 20 ng/ml in the culture medium. 

14. A method according to Claim 4 wherein the inhibin is human or porcine inhibin made up of an a and a 
jS chain, amino acid sequences of said a and £ chain being selected from those depicted in Fig 1B 
(porcine a chain), Fig 2B (porcine 0 chains), Fig 6A (human a chain) and Figs 8 and 9 (human 0 

45 chains), and amino acid sequence variants by way of insertion, deletion or substitution of a polypeptide 
of a said depicted sequence, which variants are substantially homologous with a polypeptide of a 
depicted sequence and 1 ) are cross reactive with antibodies raised against a polypeptide of a depicted 
sequence; 2) are cross reactive with cell surface receptors for a polypeptide of a depicted sequence; or 
3) have like hormonal activity to a polypeptide of a depicted sequence; which composition is 

so completely free of unidentified human or porcine proteins. 

15. A method according to Claim 14 wherein the inhibin is human or porcine inhibin made up of an o and a 
0 chain, the amino acid sequences of said a and /3 chain being selected from those depicted in Fig 1B 
(porcine a chain), Fig 2B (porcine 0 chains), *Fig 6A (human a chain) and Figs 8 and 9 (human $ 

55 chains). 

16. A method according to Claim 4 wherein the recovered inhibin chain is a prodomain of human or 
porcine a or 0 B inhibin as depicted in Fig 1B (porcine a chain), Fig 2B (porcine 0 B chain), Fig 6A 



35 



EP 0 222 491 B1 



(human a chain), and Fig 9 (human jS B chain) unassociated with native glycosylation. 

17, A method according to Claim 4 wherein a £ chain dimer is recovered which is a homodimer of mature 
human or porcine inhibin & A or 0 B chains, said chains being as depicted in Fig 2B (porcine 0 chains) 

5 and Figs 8 and 9 (human £ chains) or an amino acid sequence variant by way of insertion, deletion or 
substitution of a polypeptide of a said depicted sequence the variant being substantially homologous 
with a polypeptide of a depicted sequence and 1) cross reactive with antibodies raised against a 
polypeptide of a depicted sequence; 2) being cross reactive with cell surface receptors for a 
polypeptide of a depicted sequence; or 3) having like hormonal activity to a polypeptide of a depicted 

10 sequence; which dimer is free of the inhibin a chain. 

18. A method according to Claim 17 wherein a homodimer of mature human or porcine j3 B chains or a said 
amino acid sequence variant thereof is recovered. 

75 19. A method according to Claim 4 wherein a jS chain dimer is recovered which is a heterodimer of mature 
human or porcine inhibin j9 A with mature human or porcine inhibin 0 B . said chains being as depicted in 
Fig 2B (porcine £ chains) and Figs 8 and 9 (human £ chains) or an amino acid sequence variant by 
way of insertion, deletion or substitution of a polypeptide of a said depicted sequence the variant being 
substantially homologous with a polypeptide of a depicted sequence and 1) being cross reactive with 

20 antibodies raised against a polypeptide of a depicted sequence; 2) being cross reactive with cell 
surface receptors for a polypeptide of a depicted sequence; or 3) having like hormonal activity to a 
polypeptide of a depicted sequence; which dimer is free of the inhibin a chain. 

20. Non chromosomal DNA encoding a human or porcine inhibin a or a human or porcine inhibin £ chain 
25 the amino acid sequences of which are as depicted in Fig 1B (porcine o chain), Fig 2B (porcine /S 

chains), Fig 6A (human a chain) and Figs 8 and 9 (human £ chains) or an amino acid sequence variant 
by way of insertion, deletion or substitution of a polypeptide of a said depicted sequence, the variant 
being substantially homologous with a polypeptide of a depicted sequence and 1) being cross reactive 
with antibodies raised against a polypeptide of a depicted sequence; or 2) being cross reactive with cell 
30 surface receptors for a polypeptide of a depicted sequence; or 3) having like hormonal activity to a 
polypeptide of a depicted sequence. 

21. A DNA according to Claim 20 encoding a human or porcine inhibin a or a human or porcine inhibin 0 
chain whose amino acid sequence is as depicted in Fig 1B (porcine a chain), Fig 2B (porcine & 

35 chains), Fig 6A (human a chain) and Figs 8 and 9 (human 0 chains). 

22. The DNA of claim 20 or 21 which is free of intervening untranslated sequences. 
2a The DNA of any one of Claims 20 to 22 which is labelled with a detectable moiety. 

40 

24. A replicable vector comprising a DNA of claim 20 or 21 . 

25. The vector of Claim 24 comprising a viral promoter operably linked to the DNA encoding the inhibin a 
and/or 0 chains. 

45 

26. The vector of claim 24 or 25 which contains DNA encoding both an inhibin a and an inhibin 0 chain. 

27. The vector of claim 24 or 25 which contains DNA encoding an inhibin 0 chain but not the inhibin a 
chain. 

50 

28. A host cell transformed with a replicable vector comprising DNA encoding the human or porcine inhibin 
a and/or an inhibin £ chain the amino acid sequences of which are as depicted in Fig 1B (porcine a 
chain), Fig 2B (porcine £ chains), Fig 6A (human a chain) and Figs 8 and 9 (human 0 chains) or an 
amino acid sequence variant by way of insertion, deletion or substitution of a polypeptide of a said 

55 depicted sequence, the variant being substantially homologous with a polypeptide of a depicted 
sequence and 1) being cross reactive with antibodies raised against a polypeptide of a depicted 
sequence; or 2) being cross reactive with cell surface receptors for a depicted sequence; or 3) having 
like hormonal activity to a depicted sequence. 



36 



EP 0 222 491 B1 



29. A host cell according to Claim 28 wherein the DNA encodes a human or porcine inhibin a and/or an 
inhibin 0 chain, the amino acid sequences of which are as depicted in Fig 1 B (porcine a chain), Fig 2B 
(porcine £ chains), Fig 6A (human a chain) and Figs 8 and 9 (human & chains). 

30. The cell of claim 28 or 29 which is a eukaryotic cell. 

31. A method according to Claim 4 wherein the recovered inhibin chain is a cell-free composition, free of 
mature a chain sequence, containing a human or porcine inhibin a chain prodomain polypeptide 
sequence as depicted in Fig 1B (porcine a chain) or Fig 6A (human a chain) or an amino acid 
sequence variant by way of insertion, deletion or substitution of a said prodomain sequence, the variant 
being substantially homologous with a polypeptide of a a said sequence and being cross reactive with 
antibodies raised against a polypeptide of a depicted sequence. 

32. A method according to claim 4 wherein the recovered inhibin chain is a cell-free composition containing 

a) a polypeptide comprising the human inhibin 0 A chain prodomain sequence 

HSAAPDCPSCALAALPKDVPNSQPEMVEAVKKHI LNMLHL ( amino acids 1- 

40 Of Fig 8), PDVTQPVPKAALLNAIRKLHVGKVGENGYVEI EDDIG 
(amino acids 44-80 of Fig 8). 

AEMNELMEQTSEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKAN- 
RTRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDA (amino 

acids 83-185 of Fig 8), 

STWHVFPVSSSIQRLLDQGKSSLDVRIACEQCQESGASLVLLG (amino acids 
188-230 of Fig 8), 

or naturally occurring mammalian amino acid sequence variants thereof; 

b) a polypeptide comprising the human inhibin 0 B chain prodomain sequence 

CTSCGGFRRPEELGRVDGDFLEAV, ( amino 

acids 7-30 of Fig 9), 

HILSRLQMRGRPNITHAVPKAAMVTALRKLHAGKVREDGRVEIPHLDGHASPGAD- 
GQERVSEIISFAETDGLASSRVRLYFFISNEGNQNLFVVQASLWLYLKLLPYV- 
LEKGS (amino acids 33-145 of Fig 9), 

VRVKVYFQEQGHDRWNMVEKRVDLKRSGWHTFPLTEAIQALFERGE ( amino 
acids 149 - 195 of Fig 9), 

LNLDVQCDSCQELAVVPVFVDPGEESHRPFVVVQARLGDSRHRI (amino 
acids 198-241 of Fig 9), 

or naturally occurring mammalian amino acid sequence variants thereof; or 

37 



EP 0 222 491 B1 



c) a polypeptide free of the mature a chain amino acid sequence comprising the human inhibin a 
chain prodomain sequences 

KVRALFLDALGPPAVTREGGPGV ( amino acids 

1 - 24 of Fig 6), 

HALGGFTHRGSEPEEEEDVSQAILFPATDASCEDKSAARGLAQEAEEGLFRYMFR- 
PSQHTRSRQVTSAQLWFHTGLDRQGTAASNSSEPLLGLLALSPGGPVAVPMSLGH- 
APPHWAVLHLATSALSLLTHPVLVLLLRCPLCTCSARPEATPFLVAHTRTRPPSG- 



GERA (amino acids 32 - 199 of Fig 6), 

or naturally occurring mammalian amino acid sequence variants thereof. 

33. A method according to Claim 4 wherein the recovered inhibin chain is a cell-free composition 
containing a human or porcine inhibin 0 chain prodomain polypeptide of a sequence as depicted in Fig 
2B (porcine jS chains) and Figs 8 and 9 (human chains) or an amino acid sequence variant by way of 
insertion, deletion, or substitution of a said prodomain polypeptide of a sequence, the variant being 
substantially homologous with a polypeptide of a said sequence and being cross reactive with 
antibodies raise against a polypeptide of a depicted sequence. 

34. The method of claim 33 wherein the 0 chain is the & A chain and the composition is free of mature /5 A 
chain sequence. 

35. The method of claim 33 wherein the 0 chain is the jS B chain and the composition is free of mature jS B 
chain sequence. 

36. The method of claim 32 wherein the variant is the corresponding porcine amino acid sequence, the 
composition containing: 

a) a polypeptide comprising the porcine inhibin & A chain prodomain sequence depicted at: amino 
acids 28 to 58; amino acids 61 to 87; amino acids 90 to 108; amino acids 111 to 179; amino acids 
182 to 213; amino acids 216 to 258; or amino acids 28 to 87 of Fig 2B; or 

b) a polypeptide free of the mature a chain amino acid sequence comprising the porcine inhibin a 
chain prodomain polypeptide sequences depicted at: amino acids 20 to 54; or amino acids 70 to 228 
of Fig 1B. 

37. The method of any of claims 31 to 36 wherein the polypeptide is unaccompanied by native 
glycosylation. 

38. The method of any one of claims 31 to 37 wherein the composition is sterile and further comprising 
coupling the polypeptide with an immunogenic polypeptide. 

39. The use of a composition of any of claims 31 to 38 in the preparation of an antibody capable of binding 
said polypeptide. 

40. The method of any of claims 31 to 38 further comprising conjugating the polypeptide to a detectable 
group. 

41. The method of claim 40 wherein the group is an enzyme, fluorophore or radioisotope. 

42. The method of any of claims 31 to 38, 40, or 41 wherein the composition is insolubilized by non- 
covalent absorption or covalent cross-linking to a water insoluble support. 



38 



EP 0 222 491 B1 



43. The method of any of claims 31 to 38 or 40 to 41 further comprising mixing the composition with a 
physiologically acceptable implantable matrix for controlled release of the polypeptide into the tissues 
of an animal. 

5 Claims for the following Contracting States : AT, ES 

1. A method comprising culturing a host cell transformed with a vector which includes nucleic acid 
encoding a human or porcine inhibin a chain and/or a human or porcine inhibin jS chain the amino acid 
sequences of which are as depicted in Fig 1B (porcine a chain), Fig 2B (porcine 0 chains), Fig 6A 

io (human a chain) and Figs 8 and 9 (human 0 chains), or an amino acid sequence variant by way of 
insertion, deletion or substitution of a said depicted sequence, the variant being substantially homolo- 
gous with a polypeptide of a depicted sequence and 1) being cross reactive with antibodies raised 
against a polypeptide of a depicted sequence; or 2) being cross reactive with cell surface receptors for 
a polypeptide of a depicted sequence; or 3) having like hormonal activity to a polypeptide of a depicted 

75 sequence. 

2. A method according to claim 1 wherein the nucleic acid encodes a human or porcine inhibin o chain 
and/or a human or porcine j9 chain the amino acid sequences of which are as depicted in Fig 1B 
(porcine a chain), Fig 2B (porcine & chains), Fig 6A (human a chain) and Figs 8 and 9 (human 0 

20 chains). 

3. A method according to claim 1 wherein the vector encodes an allelic variant of human or porcine 
inhibin a and/or & chain. 

25 4. The method of any one of the preceding claims wherein the nucleic acid encoding the inhibin a chain 
and/or 0 chain is operably linked to a promoter recognized by the host cell and including the further 
step of recovering inhibin o chain, inhibin 0 chain, inhibin or a 0 chain dimer from the culture medium. 

5. The method of any one of the preceding claims wherein the cell is a prokaryote. 

30 

6. The method of any one of the preceding claims wherein the vector comprises nucleic acid encoding 
the prepro form of an inhibin a chain or an inhibin & chain. 

7. A method according to claim 6 wherein the vector comprises nucleic acid encoding the prepro forms of 
35 both the inhibin a chain and an inhibin 0 chain. 

8. The method of any one of claim 1 to 4, 6 or 7 wherein the cell is a cell from a multicellular organism 
and hormonally active inhibin is produced. 

40 9. The method of claim 4 wherein the promoter is a viral promoter. 

10. The method of claim 9 wherein the promoter is an SV40 promoter. 

11. The method of any preceding claims wherein mature porcine or human inhibin is recovered. 

45 

12. The method of any of claims 1 to 5 or 8 to 10 wherein the vector comprises nucleic acid encoding the 
prepro form of an inhibin 0 chain and a mature 0-chain dimer is recovered free of the a-chain. 

13. The method of claim 8 or 11 wherein the 0 chain is the /J A chain and the inhibin is present in a 
50 concentration greater than about 20 ng/ml in the culture medium. 

14. A method according to claim 4 wherein the inhibin is human or porcine inhibin made up of an a and a 
£ chain, the amino acid sequences of said a and £ chain being selected from those depicted in Fig 1B 
(porcine a chain), Fig 2B (porcine £ chains), Fig 6A (human a chain) and Figs 8 and 9 (human 0 

55 chains), and amino acid sequence variants by way of insertion, deletion or substitution of a polypeptide 
of a said depicted sequence, which variants are substantially homologous with a polypeptide of a 
depicted sequence and 1 ) are cross reactive with antibodies raised against a polypeptide of a depicted 
sequence; 2) are cross reactive with cell surface receptors for a polypeptide of a depicted sequence; or 



39 



EP 0 222 491 B1 



3) have like hormonal activity to a polypeptide of a depicted sequence; which composition is 
completely free of unidentified human or porcine proteins. 

15. A method according to claim 14 wherein the inhibin is human or porcine inhibin made up of an a and a 
6 /8 chain, the amino acid sequences of said a and j8 chain being selected from those depicted in Fig 1 B 

(porcine a chain), Fig 2B (porcine $ chains), Fig 6A (human a chain) and Figs 8 and 9 (human 0 
chains). 

16. A method according to claim 4 wherein the recovered inhibin chain is a prodomain of human or porcine 
io a or fa inhibin as depicted in Fig 1B (porcine a chain), Fig 2B (porcine 0 B chain), Fig 6A (human o 

chain), and Fig 9 (human 0 B chain) unassociated with native glycosylation. 

17. A method according to claim 4 wherein a j8 chain dimer is recovered which is a homodimer of mature 
human or porcine inhibin fi A or $ B chains, said chains being as depicted in Fig 2B (porcine & chains) 

15 and Figs 8 and 9 (human j8 chains) or an amino acid sequence variant by way of insertion, deletion or 
substitution of a polypeptide of a said depicted sequence the variant being substantially homologous 
with a polypeptide of a depicted sequence and 1) being cross reactive with antibodies raised against a 
polypeptide of a depicted sequence; 2) being cross reactive with cell surface receptors for a 
polypeptide of a depicted sequence; or 3) having like hormonal activity to a polypeptide of a depicted 

20 sequence; which dimer is free of the inhibin a chain. 

18. A method according to claim 17 wherein a homodimer of mature human or porcine 0 B chains or a said 
amino acid sequence variant thereof is recovered. 

25 19. A method according to claim 4 wherein a j8 chain dimer is recovered which is a heterodimer of mature 
human or porcine inhibin jS A with mature human or porcine inhibin 0 Bt said chains being as depicted in 
Fig 2B (porcine 0 chains) and Figs 8 and 9 (human & chains) or an amino acid sequence variant by 
way of insertion, deletion or substitution of a polypeptide of a said depicted sequence the variant being 
substantially homologous with a polypeptide of a depicted sequence and 1) being cross reactive with 

30 antibodies raised against a polypeptide of a depicted sequence; 2) being cross reactive with cell 
surface receptors for a polypeptide of a depicted sequence; or 3) having like hormonal activity to a 
polypeptide of a depicted sequence; which heterodimer is free of the inhibin a chain. 

20. A method for the production of non chromosomal DNA encoding a human or porcine inhibin a or a 
as human or porcine inhibin 0 chain as depicted in Fig 1B (porcine a chain), Fig 2B (porcine 0 chains), 
Fig 6A (human a chain) and Figs 8 and 9 (human 0 chains) or an amino acid sequence variant by way 
of insertion, deletion or substitution of a polypeptide of a said depicted sequence, the variant being 
substantially homologous with a polypeptide of a depicted sequence and 1) being cross reactive with 
antibodies raised against a polypeptide of a depicted sequence; or 2) being cross reactive with cell 
40 surface receptors for a polypeptide of a depicted sequence; or 3) having like hormonal activity to a 
polypeptide of a depicted sequence; the method comprising chemical synthesis, screening mRNA from 
ovary, or screening genomic libraries of any cell. 

A method according to claim 20 wherein the DNA encodes a human or porcine inhibin a or a human or 
porcine inhibin 0 chain whose amino acid sequence is as depicted in Fig 1 B (porcine a chain), Fig 2B 
(porcine j8 chains), Fig 6A (human a chain) and Figs 8 and 9 (human & chains). 

22. The method of claim 20 or 21 wherein the non-chromosomal DNA is free of intervening untranslated 
sequences. 

50 

23. The method of any one of claims 20 to 22 further comprising the step of labelling the non-chromosomal 
DNA with a detectable moiety. 

24. A method for the production of a replicable vector, the method comprising inserting a DNA of claim 20 
55 or 21 in a cloning vector. 

25. The method of claim 24 wherein the DNA is inserted into a vector which has a viral promoter operably 
linked to the DNA encoding the inhibin a and/or 0 chains thereby to produce an expression vector. 



21. 

45 



40 



EP 0 222 491 B1 



26. The method of claim 24 or 25 wherein DNA encoding both an inhibin o and an inhibin & chain is 
inserted into the vector. 

27. The method of claim 24 or 25 wherein DNA encoding an inhibin & chain but not the inhibin a chain is 
5 inserted into the vector. 

28. A host cell transformed with a replicable vector comprising DNA encoding the human or porcine inhibin 
a and/or an inhibin & chain the amino acid sequences of which are as depicted in Fig 1B (porcine a 
chain), Fig 2B (porcine £ chains), Fig 6A (human a chain) and Figs 8 and 9 (human 0 chains) or an 

70 amino acid sequence variant by way of insertion, deletion or substitution of a polypeptide of a said 
depicted sequence, the variant being substantially homologous with a polypeptide of a depicted 
sequence and 1) being cross reactive with antibodies raised against a polypeptide of a depicted 
sequence; or 2) being cross reactive with cell surface receptors for a polypeptide of a depicted 
sequence; or 3) having like hormonal activity to a polypeptide of a depicted sequence. 

75 

29. A host cell according to claim 28 wherein the DNA encodes a human or porcine inhibin a and/or an 
inhibin & chain, the amino acid sequences of which are as depicted in Fig 1 B (porcine a chain), Fig 2B 
(porcine jS chains), Fig 6A (human a chain) and Figs 8 and 9 (human 0 chains). 

20 30. The cell of claim 28 or 29 which is a eukaryotic cell. 

31. A method according to claim 4 wherein the recovered inhibin chain is a cell-free composition free of 
mature o chain sequences containing a human or porcine inhibin a chain prodomain polypeptide 
sequence as depicted in Fig 1B (porcine a chain) or Fig 6A (human a chain) or an amino acid 

25 sequence variant of a said prodomain sequence, the variant being substantially homologous with a 
polypeptide of a said sequence and being cross reactive with antibodies raised against a polypeptide of 
a depicted sequence. 

32. A method according to claim 4 wherein the recovered inhibin chain is a cell-free composition containing 
30 a) a polypeptide comprising the human inhibin & A chain prodomain sequence 

HSAAPDCPSCALAALPKDVPNSQPEMVEAVKKHILNMLHL ( amino acids 1- 
40 of Fig 8, PDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIG (amino 

35 

acids 44-80 of Fig 8). 

AEMNELMEQTSEI ITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKAN- 
40 TRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDA, amino 
acids 83-185 of Fig 8), 
STWHVFPVSSSIQRLLDQGKSSLDVRIACEQCQESGASLVLLG (amino acids 

45 

188-230 of Fig 8), 

or naturally occurring mammalian amino acid sequence variants thereof; 

so 



55 



41 



EP 0 222 491 B1 



b) a polypeptide comprising the human inhibin 0b chain prodomain sequence 

CTSCGGFRRPEELGRVDGDFLEAV, ( amino 

acids 7-30 of Fig 9), 

HILSRLQMRGRPNITHAVPKAAMVTALRKLHAGKVREDGRVEIPHLDGHASPGAD- 
GQERVSEIISFAETDGLASSRVRLYFFISNEGNQNLFVVQASLWLYLKLLPYV- 
_LEKGS (amino acids 33-145 of Fig 9), 

VRVKVYFQEQGHDRWNMVEKRVDLKRSGWHTFPLTEAIQALFERGE ( amino 
acids 149 - 195 of Fig 9), 

LNLDVQCDSCQELAVVPVFVDPGEESHRPFVVVQARLGDSRHRI ( amino 
acids 198-241 of Fig 9), 

or naturally occurring mammalian amino acid sequence variants thereof; or 

c) a polypeptide free of the mature a chain amino acid sequence comprising the human inhibin a 
chain prodomain sequences 

KVRALFLDALGPPAVTREGGPGV (amino acids 

1 - 24 of Fig 6), 

HALGGFTHRGSEPEEEEDVSQAILFPATDASCEDKSAARGLAQEAEEGLFRYMFR- 
PSQHTRSRQVTSAQLWFHTGLDRQGTAASNSSEPLLGLLALSPGGPVAVPMSLGH- 
APPHWAVLHLATSALSLLTHPVLVLLLRCPLCTCSARPEATPFLVAHTRTRPPSG- 
GERA (amino acids 32 - 199 of Fig 6), 

or naturally occurring mammalian amino acid sequence variants thereof. 

33. A method according to claim 4 wherein the recovered inhibin chain is a cell-free composition containing 
a human or porcine inhibin 0 chain prodomain polypeptide of a sequence as depicted in Fig 2B 
(porcine j8 chains) and Figs 8 and 9 (human 0 chains) or an amino acid sequence variant by way of 
insertion, deletion or substitution of a said prodomain polypeptide of a sequence, the variant being 
substantially homologous with a polypeptide of a said sequence and being cross reactive with 
antibodies raised against a polypeptide of a depicted sequence. 

34. The method of claim 33 wherein the 0 chain is the & A chain and the composition is free of mature 0 A 
chain sequence. 

35. The method of claim 33 wherein the & chain is the jS B chain and the composition is free of mature 0 B 
chain sequence. 

36. The method of claim 32 wherein the variant is the corresponding porcine amino acid sequence, the 
composition containing: 

a) a polypeptide comprising the porcine inhibin 0 A chain prodomain sequence depicted at: amino 
acids 28 to 58; amino acids 61 to 87; amino acids 90 to 108; amino acids 11 to 179; amino acids 
182 to 213; amino acids 216 to 258; or amino acids 28 to 87 of Fig 2B; or 



42 



EP 0 222 491 B1 



b) a polypeptide free of the mature a chain amino acid sequence comprising the porcine inhibin a 
chain prodomain polypeptide sequences depicted at: amino acids 20 to 54; or amino acids 70 to 228 
of Fig 1B. 

5 37. The method of any of claims 31 to 36 wherein the polypeptide is unaccompanied by native 
glycosylation. 

38. The method of any one of claims 31 to 37 wherein the composition is sterile and further comprising 
coupling the polypeptide to an immunogenic polypeptide. 

70 

39. The use of a polypeptide of any of claims 31 to 38 in the preparation of an antibody capable of binding 
said polypeptide. 

40. The method of any of claims 31 to 38 further comprising conjugating the polypeptide to a detectable 
76 group. 

41. The method of claim 40 wherein the group is an enzyme, fluorophore or radioisotope. 

42. The method of any of claims 31 to 38, 40 or 41 wherein the composition is insolubilized by non- 
20 covalent absorption or covalent cross-linking to a water insoluble support. 

43. The method of any of claims 31 to 38 or 40 or 41 further comprising mixing the composition with a 
physiologically acceptable implantable matrix for controlled release of the polypeptide into the tissues 
of an animal. 

25 

PatentansprUche 

Patentanspruche fur folgende Vertragsstaaten : BE, CH, DE, FR, GB, IT, LI, NL, SE 

1. Verfahren, umfassend das Kultivieren einer Wirtszelle, die mit einem Vektor transformiert wurde, der 
30 Nukleinsaure enthalt, die fur eine menschliche Oder Schweine-lnhibin-a-Kette und/oder eine menschli- 
che Oder Scnweine-lnhibin-0-Kette, deren Aminosauresequenzen wie in Fig. 1B (Schweine-a-Kette), 
Fig. 2B (Schweine-/8-Ketten), Fig. 6A (menschliche a-Kette) und Fig. 8 und 9 (menschliche £-Ketten) 
dargestellt sind, Oder fur eine durch Einfugung, Loschung Oder Substitution einer der dargestellten 
Sequenzen erhaltene Aminosauresequenz-Variante kodiert, wobei die Variante im wesentlichen homo- 
35 log zu einem Polypeptid mit einer dargestellten Sequenz ist, jedoch unter AusschluB der Rinder-lnhibin- 
a-Kette und der partiellen Rinder-lnhibin-0-Kette mit der Sequenz 









Gly-Leu 


-Glu 


-Cys-Asp-Gly-Lys-Val-Asn-Ile-Cys 


Cys- 


-Lys- 


Lys- 


-Gln-Phe- 


Phe- 


Val-Ser-Phe-Lys-Asp-Ile-Gly-Trp- 


Asn 


-Asp- 


Trp- 


-Ile-Ile- 


Ala- 


Pro-Ser-Gly-Tyr-His-Ala-Asn-Tyr- 


Cys 


-Glu- 


Gly- 


-Glu-Cys- 


Pro- 


Ser-His-Ile-Ala-Gly-Thr-Ser-Gly- 


Ser 


-Ser- 


•Leu' 


-Ser-Phe- 


His- 


Ser-Thr-Val-Ile-Asn-His-Tyr-Arg- 


Met 


- Arg- 


■Gly 


-His-Ser 







50 

und wobei die Variante 

1) kreuzreaktiv mit gegen ein Polypeptid mit einer dargestellten Sequenz gebildeten Antikorpern 
ist; Oder 2) kreuzreaktiv mit Zelloberflachen-Rezeptoren fur ein Polypeptid mit einer dargestellten 
Sequenz ist; Oder 3) Shnliche hormonelle Aktivitat wie ein Polypeptid mit einer dargestellten Sequenz 
55 besitzt. 

2. Verfahren nach Anspruch 1 , worin die Nukleinsaure fur eine menschliche Oder Schweine-lnhibin-a-Kette 
und/oder eine menschliche Oder Schweine-0-Kette kodiert, deren Aminosauresequenzen wie in Fig. 1B 



43 



EP 0 222 491 B1 



(Schweine-a-Kette), Fig. 2B (Schweine-j9-Ketten), Fig. 6A (menschliche a-Kette) und Fig. 8 und 9 
(menschliche /3-Ketten) dargestellt sind. 

3. Verfahren nach Anspruch 1, worin der Vektor fur eine lnhibin-£-Kette oder fur eine Aminosaurese- 
5 quenz-Variante davon kodiert, die von der nativen menschlichen oder Schweine-/J A -Kette oder der in 

Anspruch 1 spezifizierten, partiellen Rinder-lnhibin-j8-Kette verschieden ist. 

4. Verfahren nach Anspruch 1, worin die Nukleinsaure fur eine Variante kodiert, ausgewahlt aus: den 
Schweine-lnhibinderivaten (Asn266-*Gln]lnha; [Cys32& oder Cys324->A]lnha; [Cys36i oder CyS363-*A]- 

70 Inha; [Lyss2i oder LyS322—A]ln0 A ;[Lys322-*His °cler Ser]lnh/9 A ; [Lyssis—Arg; Vahie—Thr] 
lnh0 A ;[Cys 3 88 oder CyS3 S o-A]lnh0 A , [Ly&n i-Gln]lnh/S A ; [Arg 3 i 5 -Lys; Val 3 i6-Thr]lnh0 B ; [Cys 3 i 9 
oder Cys 3 2o-*A]lnhj8 B ; [Pr038i Gly382— Pro Phe Gly]lnhj9 B ; [Arg 39 5-*Gln]lnh/3 B , worin Inh eine AbkOr- 
zung fur Inhibin ist und die Nummern der Reste fOr Inhj3 B die fGr den entsprechenden lnh0 A -Rest 
verwendeten sind (siehe Fig. 2B); den menschliche lnhibin-/S A -Ketten-Varianten, die Variationen aufwei- 

75 sen, die Substitution oder Loschung eines Restes oder Einfugung neben einem Rest, ausgewahlt aus 
den Resten 293 - 297, 364 - 376 und 387 - 398, der in Fig. 8 dargestellten Sequenz sind; und den 
Varianten menschlicher a-Ketten, die zu mehr als 90% homolog zu der in Fig. 6A dargestellten 
Sequenz sind. 

20 5. Verfahren nach Anspruch 1, worin die Nukleinsaure fur eine Variante einer menschlichen 0 A -lnhibin- 
Kette, ausgewShlt aus Phe302-*He oder Leu; Gln297- |, Asp oder Lys; Trp307~*Tyr oder Phe; Trp3io-*Tyr 
oder Phe; Ilea n -♦Phe oder Val; Tyr 3 i7-*Trp oder Thr; HiS3is-*Lys; Alaa^—Sen Asn32o-*Gln, Tyr oder 
His; Tyr32i—Thr oder Asp, Phe34o~*Tyr; HiS353— 'Asp; His3S3—Lys (a jS A //9 B hybrid); Phe356-*Tyr; 
Val364-*Phe; Val3S4-*Leu; Tyr375— Thr; Tyr376—Trp; Asn389—Gln, His Oder Lys; He39i-*Leu oder Thr; 

25 Met3so -*Leu oder Ser; Val392 -,, Phe 1 Glu, Thr oder He; oder eine vergleichbar modifizierte menschliche 
j8 B -Kette kodiert. 

6. Verfahren nach irgendeinem der vorangegangenen AnsprUche, worin die Nukleinsaure, die fur die 
Inhibin-a-Kette und/oder -£-Kette kodiert, mit einem von der Wirtszelle erkannten Promotor operabel 

30 verbunden ist, und das den weiteren Schritt der Gewinnung von Inhibin oder einem £-Ketten-Dimer aus 
dem Kulturmedium umfaBt. 

7. Verfahren nach irgendeinem der vorangegangenen Anspruche, worin die Zelle ein Prokaryot ist. 

35 8. Verfahren nach irgendeinem der vorangegangenen AnsprOche, worin der Vektor Nukleinsaure umfaBt, 
die fur die Prapro-Form einer Inhibin-a-Kette oder einer lnhibin-0-Kette kodiert. 

9. Verfahren nach Anspruch 8, worin der Vektor eine Nukleinsaure umfaBt, die sowohl fur die Prapro-Form 
einer Inhibin-a-Kette als auch einer lnhibin-0-Kette kodiert. 

40 

10. Verfahren nach irgendeinem der Anspruche 1 - 6, 8 oder 9, worin die Zelle eine Zelle aus einem 
vielzelligen Organismus ist und hormonal aktives Inhibin produziert wird. 

11. Verfahren nach Anspruch 6, worin der Promotor ein viraler Promotor ist. 

45 

12. Verfahren nach Anspruch 11, worin der Promotor ein SV40-Promotor ist. 

13. Verfahren nach irgendeinem der vorangegangenen Anspruche, worin reifes Schweine- oder menschli- 
ches Inhibin gewonnen wird. 

50 

14. Verfahren nach irgendeinem der Anspruche 1 - 7 oder 10 - 12, worin der Vektor Nukleinsaure umfaBt, 
die fur die Prapro-Form einer lnhibin-0-Kette kodiert, und ein reifes £-Ketten-Dimer frei von der a-Kette 
gewonnen wird. 

55 15. Verfahren nach Anspruch 10 oder 13, worin die £-Kette die /3 A -Kette ist und das Inhibin in einer 
Konzentration von uber etwa 20 ng/ml im Kulturmedium vorliegt. 



44 



EP 0 222 491 B1 



16. Zusammensetzung, umfassend menschliches Oder Schweine-lnhibin, das aus einer a- und einer &• 
Kette besteht, wobet die Aminosauresequenzen der a- und 0-Ketten aus jenen in Fig. 1B (Schweine-a- 
Kette), Fig. 2B (Schweine-/3-Ketten), Fig. 6A (menschliche a-Kette) und Fig. 8 und 9 (menschliche &• 
Ketten) dargestellten ausgewahlt werden, und durch EinfOgung, Loschung Oder Substitution eines 

5 Polypeptids mit einer der dargestellten Sequenzen erhaltenen Aminosauresequenz- Varianten, wobei die 
Varianten im wesentltchen homolog zu einem Polypeptid mit einer dargestellten Sequenz sind, jedoch 
unter AusschluB der Rinder-lnhibin-a-Kette, und 1) kreuzreaktiv mit gegen ein Polypeptid mit einer 
dargestellten Sequenz gebildeten Antikorpern; 2) kreuzreaktiv mit Zelloberflachen-Rezeptoren fur ein 
Polypeptid mit einer dargestellten Sequenz sind; Oder 3) ahnliche hormonelle Aktivitat wie ein 

70 Polypeptid mit einer dargestellten Sequenz besitzen; wobei die Zusammensetzung vollstandig frei von 
nichtidentifizierten menschlichen Oder Schweine-Proteinen ist. 

17. Zusammensetzung nach Anspruch 16 t worin die Sequenzen der a- und £-Ketten aus jenen ausgewShlt 
werden, die in Fig. 1B (Schweine-a-Kette), Fig. 2B (Schweine-0-Ketten), Fig. 6A (menschliche a-Kette) 

75 und Fig. 8 und 9 (menschliche 0-Ketten) dargestellt sind. 

18. Zusammensetzung nach Anspruch 16, worin die Varianten ausgewahlt werden aus: den Schweine- 
Inhibinderivaten (Asn26&-*ln]Glnha; [Cys325 Oder Cys 32 4— A]lnha; (Cys 36 i oder Cys363-*A]lnha; 
[LyS32i oder Lys322— A]InhjS A ; [Lys 3 22— His Oder Ser]lnhj8 A ; [Lyssis—Arg; Valsu— Thr] lnhjS A ; [CyS3BB 

20 oder Cys 39 o— A]lnhj8 A , [Lys4 1 1 — Gln]lnhj6 A ; [Arg 3 i 5 — Lys; Val 3 i 6 -*Thr]lnh0 B ; [Cys3ig oder Cys 32 o—A]- 
Inh^s [Prossi G!y382-> Pro Phe Gly]lnhj8 B ; [Arg 3 95— Gln]lnh/3 B , worin Inh eine Abkurzung fUr Inhibin ist 
und die Nummern der Reste fur lnh0 B die fur den entsprechenden lnh/8 A -Rest verwendeten sind (siehe 
Fig. 2B); den menschlichen lnhibin-0 A -Ketten-Varianten, die Variationen aufweisen, die Substitution oder 
Loschung eines Restes oder EinfOgung neben einem Rest, ausgewahlt aus den Resten 293 - 297, 364 

25 - 376 und 387 - 398, der in Fig. 8 dargestellten Sequenz, sind; und den Varianten menschlicher o- 
Ketten, die zu mehr als 90% homolog zu der in Fig. 6A dargestellten Sequenz sind. 

19. Zusammensetzung nach Anspruch 16, umfassend eine Variante einer menschlichen £ A -lnhibin-Kette, 
ausgewahlt aus Phe 3 o2-*He Oder Leu; Gln297~*Asp oder Lys; Trp 30 7-*Tyr oder Phe; Trp 3 io-*Tyr oder 

30 Phe; lleau-^Phe oder Val; Tyr3i7-*Trp oder Thr; His 3 io— Lys; Alasis—Ser; Asn32o— -Gin, Tyr oder His; 
Tyr32i->Thr oder Asp, Phe 3 4o— Tyr; His 3 5 3 — Asp; His35 3-*Lys (a £ A /jS B hybrid); Phe3S6-*Tyr; 
Val 3 64^Phe; Val 3 64-*Leu; Tyrszs—Thr; Tyr 3 76—Trp; Asn 3 8s— Gin, His oder Lys; Ile39i— 'Leu oder Thr; 
Met39o —Leu oder Ser; Val 3 9 2-*Phe, Glu, Thr oder He; oder eine vergleichbar modifizierte menschliche 
j8 B -Kette. 

35 

20. Zusammensetzung, umfassend eine Prodomane von menschlichem oder Schweine-o- oder -£ B -lnhibin, 
wie in Fig. 1B (Schweine-a-Kette), Fig. 2B (Schweine-0 B -Ketten), Fig. 6A (menschliche a-Kette) und Fig. 
9 (menschliche /S B -Kette) dargestellt, die nicht mit nativer Glykosylierung assoziiert ist. 

40 21. Zusammensetzung, umfassend ein Homodimer von reifen menschlichen oder Schweine-lnhibin-/8 A - 
oder -/9 B -Ketten, wobei die Ketten wie in Fig. 2B (Schweine-0-Ketten) und Fig. 8 und 9 (menschliche $• 
Ketten) dargestellt sind, oder einer durch EinfOgung, Loschung oder Substitution eines Polypeptids mit 
einer der dargestellten Sequenzen erhaltene Aminosauresequenz-Variante, wobei die Variante im 
wesentlichen homolog zu einem Polypeptid mit einer dargestellten Sequenz und 1) kreuzreaktiv mit 

45 gegen ein Polypeptid mit einer dargestellten Sequenz. gebildeten Antikorpern; 2) kreuzreaktiv mit 
Zelloberflachen-Rezeptoren fOr ein Polypeptid mit einer dargestellten Sequenz ist; oder 3) ahnliche 
hormonelle Aktivitat wie ein Polypeptid mit einer dargestellten Sequenz besitzt; wobei die Zusammen- 
setzung vollstandig frei von der Inhibin-a-Kette ist. 

so 22. Zusammensetzung nach Anspruch 21 , die ein Homodimer von reifen menschlichen oder Schweine-0 B - 
Ketten oder einer der Aminosauresequenz-Varianten davon ist. 

23. Zusammensetzung, umfassend ein Heterodimer von reifem menschlichem oder Schweine-Inhibin-0 A 
mit reifem menschlichem oder Schweine-Inhibin-0 B , wobei die Ketten wie in Fig. 2B (Schweine-i9- 
55 Ketten) und Fig. 8 und 9 (menschliche 0-Ketten) dargestellt sind, oder einer durch EinfOgung, 
Loschung oder Substitution eines Polypeptids mit einer der dargestellten Sequenzen erhaltene Amino- 
sauresequenz-Variante, wobei die Variante im wesentlichen homolog zu einem Polypeptid mit einer 
dargestellten Sequenz und 1) kreuzreaktiv mit gegen ein Polypeptid mit einer dargestellten Sequenz 



45 



EP 0 222 491 B1 



gebildeten Antikorpern; 2) kreuzreaktiv mit Zelloberflachen-Rezeptoren fur ein Polypeptid mit einer 
dargestellten Sequenz ist; Oder 3) Shnliche hormonelle AktivitSt wie ein Polypeptid mit einer dargestell- 
ten Sequenz besitzt; wobei die Zusammensetzung frei von der Inhibin-a-Kette ist. 

5 24. Nicht-chromosomale DNA, die fur eine menschliche oder Schweine-lnhibin-a- Oder eine menschliche 
oder Schweine-lnhibin-jS-Kette, deren Aminosauresequenzen wie in Fig. 1B (Schweine-a-Kette), Fig. 2B 
(Schweine-0-Ketten), Fig. 6A (menschliche a-Kette) und Fig. 8 und 9 (menschliche £-Ketten) dargestellt 
sind, oder fur eine durch Einfugung, Loschung oder Substitution eines Polypeptids mit einer der 
dargestellten Sequenzen erhaltene Aminosauresequenz-Variante kodiert, wobei die Variante im wesent- 

w lichen homolog zu einem Polypeptid mit einer der dargestellten Sequenzen ist, jedoch unter AusschluB 
der Rinder-lnhibin-a-Kette und der partiellen Rinder-lnhibin-0-Kette mit der Sequenz . 

Gly-Leu-Glu-Cys-Asp- 



Gly- 


Lys 


-Val- 


Asn- 


Ile- 


-Cys- 


Cys 


-Lys- 


■Lys- 


■Gln- 


Phe- 


•Phe- 


-Val- 


Ser- 


Phe- 


Lys 


- ASD- 


Ile- 


■Gly- 


-Trp- 


■Asrv 


-Asp- 


Trp- 


Ile- 


Ile- 


•Ala 


-Pro- 


Ser* 


Gly- 


Tyr 


-His- 


Ala- 


Asn 


-Tyr- 


Cys 


-Glu- 


Gly- 


•Glu- 


Cys- 


Pro 


-Ser- 


His- 


Ile- 


Ala 


-Gly- 


Thr- 


■Ser- 


-Gly- 


■Ser 


-Ser- 


Leu- 


Ser- 


Phe- 


•His 


-Ser- 


Thr- 


Val- 


lie 


►Asn- 


His- 


Tyr 


- Arg- 


■Met 


-Arg- 


Gly- 


His- 


Ser 









25 

und wobei die Variante 

1) kreuzreaktiv mit gegen ein Polypeptid mit einer dargestellten Sequenz gebildeten Antik6rpern 
ist; oder 2) kreuzreaktiv mit Zelloberflachen-Rezeptoren fur ein Polypeptid mit einer dargestellten 
30 Sequenz ist; oder 3) ahnliche hormonelle Aktivitat wie ein Polypeptid mit einer dargestellten Sequenz 
besitzt. 

25. DNA nach Anspruch 24, die fiir eine menschliche oder Schweine-lnhibin-a- oder eine menschliche oder 
Schweine-lnnibin-0-Kette kodiert, deren Aminosauresequenz wie in Fig. 1B (Schweine-a-Kette), Fig. 2B 

35 (Schweine-0-Ketten), Fig. 6A (menschliche a-Kette) und Fig. 8 und 9 (menschliche j8-Ketten) dargestellt 
ist. 

26. DNA nach Anspruch 24, die fiir eine Variante kodiert, ausgewahlt aus: den Schweine-lnhibinderivaten 
(Asn266~*Gln]lnha; [CyS325 oder CyS324—A]lnha; [Cyss&i oder Cys36 3— AJInha; {LVS321 Oder 

40 Lys322— A]lnh/3 A ; [Lys 322 -*His oder Ser]lnh0 A ; [Lys 3 i5-*Arg; Vabi6-*Thr] lnh£ A ; [CyS388 oder 
Cyssso— A]lnh/9 A ,[LyS4n- ,, Gln]lnh^ A ; [Arg 3 i5—Lys; Vabu— Thr]lnh0 B ; {Cyssi9 Oder CyS32o-*A]lnh/3 e ; 
[Prossi Gly382~* Pro Phe Gly]lnh0B;[Arg395-*Gln]lnh/iB. worin Inh eine Abkurzung fiir Inhibin ist und die 
Nummern der Reste fur lnh£ B die fiir den entsprechenden lnhjS A -Rest verwendeten sind (siehe Fig. 
2B); den menschlichen lnhibin-0 A -Ketten-Varianten, die Variationen aufweisen, die Substitution oder 

45 Loschung eines Restes oder Einfugung neben einem Rest, ausgewahlt aus den Resten 293 - 297, 364 
- 376 und 387 - 398, der in Fig. 8 dargestellten Sequenz sind; und den Varianten menschlicher a- 
Ketten, die zu mehr als 90% homolog zu der in Fig. 6A dargestellten Sequenz sind. 

27. DNA nach Anspruch 24, die fur eine Variante einer menschlichen jS-lnhibin-Kette, ausgewahlt aus 
so Phe 3 o2— 'He oder Leu; Gln297-*Asp oder Lys; Trp3 0 7—Tyr oder Phe; Trp3io- ,, Tyr oder Phe; llesn-^Phe 

oder Val; Tyr3i7—Trp oder Thr; HiS3i8—Lys; Alasis-'Ser; Asn32o-*G1n, Tyr oder His; Tyr32i~*Thr Oder 
Asp, Phe34o-»Tyr; HiS3ss— Asp; Hiss53— Lys (a £ a /0b hybrid); Phesse—Tyr; Val364—Phe; Vabc*— Leu; 
Tyr 3 75"*Thr; Tyr 3 76-»Trp; Asn389— Gin, His Oder Lys; He39i-*Leu oder Thr; Met39o -*Leu oder Ser; 
Vals92-*Phe ( Glu, Thr oder lie; oder eine vergleichbar modifizierte menschliche fo-Kette kodiert. 

55 

28. DNA nach irgendeinem der Anspruche 24 - 27, die frei von ihtervenierenden untranslatierten Sequen- 
zen ist. 



46 



EP 0 222 491 B1 



29. DNA nach irgendeinem der Ansprtiche 24 - 28, die mit einer feststellbaren Gruppe markiert ist. 

30. Replizierbarer Vektor, umfassend eine DNA nach irgendeinem der Ansprtiche 21 - 27. 

31. Vektor nach Anspruch 30, umfassend einen viralen Promoter, der mit der fur die Inhibin-o- oder -0- 
Ketten kodierenden DNA operabel verbunden ist. 

32. Vektor nach Anspruch 30 Oder 31 , der DNA enthalt, die sowohl fur eine Inhibin-a- als auch eine Inhibin- 
0-Kette kodiert. 

33. Vektor nach Anspruch 30 Oder 31, der DNA enthalt, die fOr eine lnhibin-0-Kette, jedoch nicht fur eine 
eine Inhibin-o-Kette kodiert. 

34. Wirtszelle, die mit einem replizierbaren Vektor transformiert wurde, der DNA enthalt, die fur eine 
menschliche oder Schweine-lnhibin-a-Kette und/oder eine menschliche Oder Scnweine-lnhibin-0-Kette, 
deren Aminosauresequenzen wie in Fig. 1B (Schweine-a-Kette), Fig. 2B (Schweine-0-Ketten), Fig. 6A 
(menschliche a-Kette) und Fig. 8 und 9 (menschliche £-Ketten) dargestellt sind, Oder eine durch 
Einfugung, Loschung oder Substitution eines Polypeptids mit einer der dargestellten Sequenzen 
erhaltene Aminosauresequenz- Variante kodiert, wobei die Variante im wesentlichen homolog zu einem 
Polypeptid mit einer dargestellten Sequenz ist, jedoch unter Ausschlufi der Rrnder-lnhibin-a-Kette und 
der partiellen Rinder-lnhibin-j8-Kette mit der Sequenz 



Gly-Leu-Glu-Cys-Asp-Gly- 
Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe-Phe-Val-Ser«Phe- 
Lys-Asp-Ile-Gly-Trp-Asn-Asp-Trp-lle-Ile"Ala-Pro-Ser-Gly« 
Tyr-His-Ala-Asn-Tyr-Cys-Glu-Gly-Glu-Cys-Pro-Ser-His-Ile- 
Ala-Gly-Thr-Ser-Gly-Ser-Ser-Leu-Ser-Phe-His-Ser-Thr-Val- 
Ile-Asn-His-Tyr-Arg-Met-Arg-Gly-His-Ser 



und wobei die Variante 

1) kreuzreaktiv mit gegen ein Polypeptid mit einer dargestellten Sequenz gebildeten Antikorpern 
ist; oder 2) kreuzreaktiv mit Zelloberflachen-Rezeptoren fur ein Polypeptid mit einer dargestellten 
Sequenz ist; oder 3) ahniiche hormonelle Aktivitat wie ein Polypeptid mit einer dargestellten Sequenz 
besitzt 

35. Wirtszelle nach Anspruch 34, worin die DNA fur eine menschliche Oder Schweine-lnhibin-a- und/oder 
eine -lnhibin-j8-Kette kodiert, deren Aminosauresequenzen wie in Fig. 1B (Schweine-a-Kette), Fig. 2B 
(Schwein-0-Ketten), Fig. 6A (menschliche a-Kette) und Fig. 8 und 9 (menschliche £-Ketten) dargestellt 
sind. 

36. Wirtszelle nach Anspruch 34, worin die DNA fur eine Variante kodiert, ausgewahlt aus: den Schweine- 
Inhibinderivaten (Asn266~*Gln]lnha; [Cys325 oder Cys324A]lnha; [CyS36i oder CyS363^A]lnha; *{Lys32i 
Oder Lys322- f A]lnh)SA;[LyS322-*His Oder Ser]lnh0 A ; [Lyssis—Arg; Vabu— Thr] lnhj8 A ;[CyS388 oder 
Cyssso—AJInhtfA, [Lys 4 i 1— Gln]lnh0 A ; [Arg 3 i5—Lys; Vabi6->ThrJlnhjS B ; [Cys3i9 oder CyS32o—A]lnhi9B; 
[Proagi 6^382— Pro Phe Gly]lnh/S B ; [Arg 3 9 5 — Gln]lnhj8 B , worin Inh eine Abkurzung fur Inhibin ist und 
die Nummern der Reste fur lnh£ B die fiir den entsprechenden lnh0 A -Rest verwendeten sind <siehe Fig. 
2B); den menschlichen lnhibin-0A-Ketten-Varianten, die Variationen aufweisen, die Substitution oder 
Loschung eines Restes oder Einfugung neben einem Rest, ausgewahlt aus den Resten 293 - 297, 364 
- 376 und 387 - 398, der in Fig. 8 dargestellten Sequenz sind; und den Varianten menschlicher a- 
Ketten, die zu mehr als 90% homolog zu der in Fig. 6A dargestellten Sequenz sind. 



47 



EP 0 222 491 B1 



37. Wirtszelle nach Anspruch 34, worin die DNA fur eine Variante einer menschlichen /3 A -lnhibin-Kette, 
ausgewMhlt aus: Phe302— He Oder Leu; Gln 2 97-*Asp Oder Lys; Trp307-*Tyr Oder Phe; Trp 3 io-^Tyr Oder 
Phe; Ileal i^Phe Oder Val; Tyr3i?-*Trp oder Thr; HiS3is-*Lys; Ala 3 i9^Ser; Asr^o^GIn, Tyr oder His; 
Tyr 3 2i—Thr oder Asp, Phe 3 4o-'Tyr; His 3 53— Asp; HiS353-*Lys (a 0 A //S B hybrid); Phe 3 56— Tyr; 
Val 3 64-*Phe; Vab64— Leu; Tyr 37 5^Thr; Tyr 3 7&— Trp; Asn 3 89-*Gln, His oder Lys; He39i—Leu oder Thr; 
Met 3 9o —Leu oder Ser; Val392-*Phe, Glu, Thr oder He; oder einer vergleichbar modifizierten menschli- 
chen j9 B -Kette kodiert. 

3a Zelle nach irgendeinem der AnsprUche 34 - 37, die eine eukaryotische Zelle ist. 

39. Zellfreie Zusammensetzung, die frei von reifem a-Ketten-Poiypeptid ist, die eine menschliche oder 
Schweine-lnhibin-a-Ketten-Prodomanen-Polypeptidsequenz, wie in Fig. 1B (Schweine-a-Kette) oder Fig. 
6A (menschliche a-Kette) dargestellt, oder eine durch Einfugung, Loschung oder Substitution einer 
dieser Prodomanen-Sequenzen erhaltene Aminosauresequenz-Variante enthalt, wobei die Variante im 
wesentlichen homolog zu einem Polypeptid mit einer der dargestellten Sequenzen und kreuzreaktiv mit 
gegen ein Polypeptid mit einer der dargestellten Sequenzen gebildeten Antikorpern ist. 

40. Zellfreie Zusammensetzung, umfassend 

a) ein Polypeptid, umfassend die menschliche lnhibin-£ A -Ketten-Prodomariensequenz 

HSAAPDCPSCALAALPKDVPNSQPEMVEAVKKHILNMLHL 
(Aminosauren 1 - 40 aus Fig. 8) 

PDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIG 
(Aminosauren 44 - 80 aus Fig. 8) 

AEMNELMEQTSEI ITFAESGTARKTLHFEISKEGSDLSWERAEVWLFLKVPKAN- 

RTRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKWDA 
(Aminosauren 83- 185 aus Fig. 8) STWHVFPVSSSIQRLLD- 

QGKS S LDVR I ACEQCQESGASLVLLG 
(Aminosauren 188 - 230 aus Fig. 8) 

oder naturlich vorkommende Saugetier-Aminosauresequenz-Varianten davon; 

b) ein Polypeptid, umfassend die menschliche lnhibin-0 B -Ketten-Prodomanensequenz 

CTSCGGFRRPEELGRVDGDFLEAV 
(Aminosauren 7 - 30 aus Fig. 9) 



48 



EP 0 222 491 B1 



HILSRLQMRGRPNITHAVPKAAMVTALRKLHAGKVREDGRVEIPHLDGHASPGAD- 

GQERVSE IIS F AETDGLAS S R VRL Y F F I SNEGNQNLFWQASLWLYLKLLPYVLEKGS 
(Aminosauren 33 - 145 aus Fig. 9) 

WVKVYFQEQGHGDRWNMVEKRVDLKRSGWHTFPLTEAIQALFERGE 
(Aminosauren 149 - 195 aus Fig. 9) 

LNLDVQCDSCQELAWPVFVDPGEESKRPFVWQARLGDSRHRI 
(Aminosauren 198 - 241 aus Fig. 9) 

Oder naturlich vorkommende Saugetier-Aminosauresequenz-Varianten davon; oder 

c) ein Polypeptid, das frei von der reifen a-Ketten-Aminosa'uresequenz ist, umfassend die menschli- 

chen Inhibin-a-Ketten-Prodomanensequenzen 



KVRALFLDALG ? P AVTREGGD PG V 

(Aminosauren 1 - 24 aus Fig. 6) 

HALGGFTHRGSEPEEEEDVSQAILFPATDASCEDKSAARGLAQEAEEGLFRYMFR- 

PSQHTRSRQVTSAQLWFHTGLDRQGTAASNSSEPLLGLLALSPGGPVAVPMSLGH- 

APPHWAVLHLATSALSLLTHPVLVLLLRCPLCTCSARPEATPFLVAHTRTRPPSGGERA 
(Aminosauren 32- 199 aus Fig. 6) 

oder naturlich vorkommende Saugetier-Aminosauresequenz-Varianten davon. 

41. Zellfreie Zusammensetzung, umfassend ein menschliches Oder Schweine-lnhibin-0-Ketten-Prodoma- 
nen-Polypeptid mit einer Sequenz, wie in Fig. 2B (Schweine-0-Ketten) und Fig. 8 und 9 (menschliche 
0-Ketten) dargestellt, oder eine durch Einfugung, Loschung oder Substitution einer dieser Prodomanen- 
Sequenzen erhaltene Aminosauresequenz-Variante, wobei die Variante im wesentlichen homolog zu 
etnem Polypeptid mit einer der dargestellten Sequenzen und kreuzreaktiv mit gegen ein Polypeptid mit 
einer der dargestellten Sequenzen gebildeten Antikorpern ist. 

42. Zusammensetzung nach Anspruch 41, worin die /8-Kette die 0 A -Kette und die Zusammensetzung frei 
von reifer 0 A -Ketten-Sequenz ist. 

43. Zusammensetzung nach Anspruch 41, worin die 0-Kette die £ B -Kette und die Zusammensetzung frei 
von reifer 0 B -Ketten-Sequenz ist. 

44. Zusammensetzung nach Anspruch 40, worin die Variante die entsprechende Schweine-Aminosaurese- 
quenz ist, wobei die Zusammensetzung enthalt: 

a) ein Polypeptid, umfassend die Schweine-lnhibin-/S A -Ketten-Prodomanensequenz, die dargestellt 
wird durch die Aminosauren: 28 - 58; 61 - 87; 90 - 108; 111 - 179; 182 - 213; 216 - 258; oder 28 - 
87 aus Fig. 2B; oder 

b) ein Polypeptid, das frei von der reifen o-Ketten-Aminosauresequenz ist, umfassend die Schweine- 
Inhibin-a-Ketten-Prodomanen-Polypeptidsequenzen, die dargestellt werden durch die Aminosauren: 
20 - 54 oder 70 - 228 aus Fig. 1 B. 

45. Zusammensetzung nach irgendeinem der Anspruche 39 - 44, worin das Polypeptid nicht von nativer 
Glykosylierung begleitet wird. 



49 



EP 0 222 491 B1 



46. Zusammensetzung nach irgendeinem der Anspruche 39 - 45, die steril ist, und worin das Polypeptid 
weiters ein immunogenes Polypeptid umfaBt. 

47. Verwendung einer Zusammensetzung nach irgendeinem der AnsprGche 39 - 46 zur Herstellung eines 
5 Antikorpers, der in der Lage ist, das Polypeptid zu binden. 

48. Zusammensetzung nach irgendeinem der Anspruche 39 - 46, worin das Polypeptid an eine feststellba- 
re Gruppe konjugiert ist. 

w 49. Zusammensetzung nach Anspruch 48, worin die Gruppe ein Enzym, Fluorophor oder Radioisotop ist. 

50. Zusammensetzung nach irgendeinem der Anspruche 39 - 44, 48 Oder 49, die durch nicht-kovalente 
Absorption Oder kovalente Vernetzung an einen wasserunloslichen Trager unloslich gemacht ist. 

75 51. Zusammensetzung nach irgendeinem der Anspruche 39 - 46, 48 Oder 49, weiters umfassend eine 
physiologisch annehmbare, implantierbare Matrix zur gesteuerten Freisetzung des Polypeptids in die 
Gewebe eines Tieres. 

Patentanspruche fur folgenden Vertragsstaat : LU 

20 

1. Verfahren, umfassend das Kultivieren einer Wirtszeile, die mit einem Vektor transformiert wurde, der 
Nukleinsaure enthalt, die fur eine menschliche Oder Schweine-lnhibin-a-Kette und/oder eine menschli- 
che Oder Schweine-lnhibin-0-Kette, deren Aminosauresequenzen wie in Fig. 1B (Schweine-a-Kette), 
Fig. 2B (Schweine-0-Ketten), Fig. 6 A (menschliche a-Kette) und Fig. 8 und 9 (menschliche £-Ketten) 

25 dargestellt sind, oder eine durch Einfugung, Loschung oder Substitution einer der dargestellten 
Sequenzen erhaltene Aminosauresequenz-Variante kodiert, wobei die Variante im wesentlichen homo- 
log zu einem Polypeptid mit einer dargestellten Sequenz ist, jedoch unter Ausschlufi der Rinder-lnhibin- 
a-Kette, und wobei die Variante 1) kreuzreaktiv mit gegen ein Polypeptid mit einer dargestellten 
Sequenz gebildeten Antikorpern ist; oder 2) kreuzreaktiv mit Zelloberflachen-Rezeptoren fur ein 

30 Polypeptid mit einer dargestellten Sequenz ist; oder 3) ahnliche hormonelle AktivitSt wie ein Polypeptid 
mit einer dargestellten Sequenz besitzt 

2. Verfahren nach Anspruch 1 , worin die Nukleinsaure fur eine menschliche oder Schweine-lnhibin-a-Kette 
und/oder eine menschliche oder Schweine-lnhibin-/3-Kette kodiert, deren Aminosauresequenzen wie in 

35 Fig. 1B (Schweine-a-Kette), Fig. 2B (Schweine-0-Ketten), Fig. 6A (menschliche a-Kette) und Fig. 8 und 
9 (menschliche 0-Ketten) dargestellt sind. 

3. Verfahren nach Anspruch 1 , worin der Vektor fur eine andere lnhibin-/8-Kette als die native menschliche 
oder Schweine-0 A -Kette oder fur eine Aminosauresequenz-Variante davon kodiert. 

40 

4. Verfahren nach Anspruch 1, worin die Nukleinsaure fur eine Variante, ausgewahlt aus: den Schweine- 
Inhibinderivaten (Asn 2 66— GlnJInha; [CyS325 oder Cys324^A]lnha; [Cys36i oder CyS36 3~*A]lnha; 
[Lys32t oder Lys322- |, A]lnh;8 A ;[Lys322— His oder Ser]lnh0 A ; [Lysms^Arg; Vabie-^Thr] lnh/3 A ;[Cys388 
oder Cys39o^A]lnhj8 A , [Lvs/m 1— Gln]lnh0 A ; [Arg 3 i S — Lys; Val 3 i6-*Thr]lnhjS B ; [Cys 3 ig Oder Cy$ 32 o^A]- 

45 Inhfo; [Pr038i Gly 3 82-* Pro Phe Gly]lnh0 B ; [Argsgs—GlnJInhfo, worin Inh eine Abkurzung fur Inhibin ist 
und die Nummern der Reste fur lnh£ B die ftir den entsprechenden lnh/S A -Rest verwendeten sind (siehe 
Fig. 2B); den menschlichen lnhibin-0 A -Ketten-Varianten, die Variationen aufweisen, die Substitution oder 
Loschung eines Restes oder Einfugung neben einem Rest, ausgewahlt aus den Resten 293 - 297, 364 
- 376 und 387 - 398, der in Fig. 8 dargestellten Sequenz sind; und den Varianten menschlicher a- 

so Ketten, die zu mehr als 90% homolog zu der in Fig. 6A dargestellten Sequenz sind, kodiert. 

5. Verfahren nach Anspruch 1, worin die Nukleinsaure fur eine Variante einer menschlichen /8 A -lnhibin- 
Kette, ausgewahlt aus Phe 3 o2-*lle oder Leu; Gln29 7~*Asp oder Lys; Trp 3 o7~*Tyr oder Phe; Trp3io-*Tyr 
oder Phe; llesn—Phe oder Val; Tyr 3 i7-*Trp oder Thr; His 3 is— Lys; Ala3i9-*Ser; Asn 3 2o^Gln, Tyr oder 

55 His; Tyr 32 i-»Thr oder Asp, Phe 3 4o->Tyr; His 3S3 — Asp; His 3 s3^Lys (a 0 A /0 B hybrid); Phe 3 56-»Tyr; 
Val 3 64—Phe; Val 3 &4—Leu; Tyr 3 75-Thr; Tyr 376 -*Trp; Asn 38 9-K3ln, His oder Lys; I!e39i— Leu oder Thr; 
Met39o -*Leu oder Ser; Val392-*Phe, Glu, Thr or He; oder eine vergleichbar modifizierte menschliche 
/S B -Kette kodiert. 



50 



EP 0 222 491 B1 



6. Verfahren nach irgendeinem der vorangegangenen Anspruche, worin die Nukleinsaure, die fur die 
Inhibin-a-Kette und/oder -0-Kette kodiert, mit einem von der Wirtszelle erkannten Promotor operabel 
verbunden ist, und das den weiteren Schritt der Gewinnung von Inhibin oder einem 0-Ketten-Dimer aus 
dem Kulturmedium umfaBt. 

5 

7. Verfahren nach irgendeinem der vorangegangenen Anspruche, worin die Zelle ein Prokaryot ist. 

8. Verfahren nach irgendeinem der vorangegangenen Anspruche, worin der Vektor Nukleinsaure umfaBt, 
die fUr die Prapro-Form einer Inhibin-a-Kette Oder einer lnhibin-0-Kette kodiert. 

70 

9. Verfahren nach Anspruch 8, worin der Vektor eine Nukleinsaure umfaBt, die sowohl fUr die Prapro-Form 
einer Inhibin-a-Kette als auch einer lnhibin-£-Kette kodiert. 

10. Verfahren nach irgendeinem der Anspruche 1 - 6, 8 oder 9, worin die Zelle eine Zelle aus einem 
75 vielzelligen Organismus ist und hormonell aktives Inhibin produziert wird. 

11. Verfahren nach Anspruch 6, worin der Promotor ein viraler Promotor ist. 

12. Verfahren nach Anspruch 11, worin der Promotor ein SV40-Promotor ist. 

20 

13. Verfahren nach irgendeinem der vorangegangenen Anspruche, worin reifes Schweine- oder menschli- 
ches Inhibin gewonnen wird. 

14. Verfahren nach irgendeinem der Anspruche 1 - 7 oder 10 - 12, worin der Vektor Nukleinsaure umfaBt, 
25 die fur die Prapro-Form einer lnhibin-0-Kette kodiert, und ein reifes j8-Ketten-Dimer frei von der a-Kette 

gewonnen wird. 

15. Verfahren nach Anspruch 10 oder 13, worin die 0-Kette die 0 A -Kette ist und das Inhibin in einer 
Konzentration von uber etwa 20 ng/ml im Kulturmedium vorliegt. 

30 

16. Zusammensetzung, umfassend menschliches oder Schweine-lnhibin, das aus einer a- und einer 0- 
Kette besteht, wobei die Aminosauresequenzen der a- und 0-Ketten aus jenen in Fig. 1B (Schweine-a- 
Kette), Fig. 2B (Schweine-0-Ketten), Fig. 6A (menschliche a-Kette) und Fig. 8 und 9 (menschliche &- 
Ketten) dargestellten ausgewahlt werden, und durch Einfugung, Loschung oder Substitution eines 

35 Polypeptids mit einer der dargestellten Sequenzen erhaltene Aminosauresequenz-Variante, wobei die 
Varianten im wesentlichen homolog zu einem Polypeptid mit einer dargestellten Sequenz, jedoch unter 
AusschluB der Rinder-lnhibin-a-Kette, und 1) kreuzreaktiv mit gegen ein Polypeptid mit einer dargestell- 
ten Sequenz gebildeten Antikorpern; 2) kreuzreaktiv mit Zelloberflachen-Rezeptoren fur ein Polypeptid 
mit einer dargestellten Sequenz sind; oder 3) ahnliche hormonelle Aktivitat wie ein Polypeptid mit einer 

40 dargestellten Sequenz besitzen; wobei die Zusammensetzung vollstandig frei von nichtidentifizierten 
menschlichen oder Schweine-Proteinen ist. 

17. Zusammensetzung nach Anspruch 16, worin die Sequenzen der a- und 0-Ketten aus jenen ausgewahlt 
werden, die in Fig. 1B (Schweine-a-Kette), Fig. 2B (Schweine-jS-Ketten), Fig. 6A (menschliche a-Kette) 

45 und Fig. 8 und 9 (menschliche 0-Ketten) dargestellt sind. 

18. Zusammensetzung nach Anspruch 16, worin die Varianten ausgewahlt werden aus: den Schwein- 
Inhibinderivaten (Asn 2 6&^ln]Glnha; [CyS325 oder CyS324-*A]lnha; [Cys36i Oder Cys363— A]lnha; 
[Lys 32 i Oder Lys322— A]lnh0 A ; [Lys 3 22->His oder Ser]lnh£ A ; [LyS3is— Arg; Val 3 i6— Thr] lnh/S A ; [CyS388 

so oder CyS39o— A]lnh0 A> [Lys4i 1— Gln]lnh0 A ; [Arg3i&— Lys; Val3i&^Thr]lnh/3 B ; [Cysai9 Oder CyS32o-*A]- 
Inh^e [Pr038i Gly38 2 — Pro Phe Gly]lnh^ B ; [Arg395— Gln]lnh0B, worin Inh eine Abkurzung fur Inhibin ist 
und die Nummern der Reste fur lnhj8 B die fUr den entsprechenden lnh^9 A -Rest verwendeten sind (siehe 
Fig. 2B); den menschlichen lnhibin-0 A -Ketten-Varianten t die Variationen aufweisen, die Substitution oder 
Loschung eines Restes oder Einfugung neben einem Rest, ausgewahlt aus den Resten 293 - 297, 364 

55 - 376 und 387 - 398, der in Fig. 8 dargestellten Sequenz sind; und den Varianten menschlicher a- 
Ketten, die zu mehr als 90% homolog zu der in Fig. 6A dargestellten Sequenz sind. 



51 



EP 0 222 491 B1 



19. Zusammensetzung nach Anspruch 16, umfassend eine Variante einer menschlichen 0 A -Inhibin-Kette, 
ausgewahlt aus Phe302— He oder Leu; Gln 2 97- Asp Oder Lys; Trp 30 7- Tyr oder Phe; Trp3io— Tyr oder 
Phe; 116311— Phe oder Val; Tyr 317 — Trp oder Thr; His 3 i 8 — Lys; Ala3i9— Ser; Asn 32 o- Gin, Tyr oder His; 
Tyr 32 i— Thr oder Asp, Phe34o— Tyr; His 3 53— Asp; HiS353— Lys (a £ A /0 B Hybrid); Phe 3 56- Tyr; 

5 Valse*— Phe; Val364— Leu; Tyr 37 5- Thr; Tyr 37 &- Trp; Asn 3 89— Gin, His oder Lys; lle 3 9i- Leu oder Thr; 
Met39o —Leu oder Ser; Val 3 92— Phe, Glu, Thr oder He; oder einer vergleichbar modifizierten menschli- 
chen 0 B -Kette. 

20. Zusammensetzung, umfassend eine Prodomane von menschlichem oder Schweine-a- oder -j8 B -lnhibin, 
70 wie in Fig. 1B (Schweine-a-Kette), Fig. 2B (Schweine-0 B -Kette), Fig. 6A (menschliche a-Kette) und Fig. 

9 (menschliche 0 B -Kette) dargestellt, die nicht mit nativer Glykosylierung assoziiert ist. 

21. Zusammensetzung, umfassend ein Homodimer von reifen menschlichen oder Schweine-Inhibin-0 A - 
oder -0 B -Ketten, wobei die Ketten wie in Fig. 2B <Schweine-0-Ketten) und Fig. 8 und 9 (menschliche 0- 

75 Ketten) dargestellt sind, oder einer durch EinfOgung, Loschung oder Substitution eines Polypeptids mit 
einer der dargestellten Sequenzen erhaltene Aminosauresequenz-Variante, wobei die Variante im 
wesentlichen homolog zu einem Polypeptid mit einer dargestellten Sequenz und 1) kreuzreaktiv mit 
gegen ein Polypeptid mit einer dargestellten Sequenz gebildeten Antikorpern; 2) kreuzreaktiv mit 
Zelloberflachen-Rezeptoren fur ein Polypeptid mit einer dargestellten Sequenz ist; oder 3) ahnliche 

20 hormonelle Aktivitat wie ein Polypeptid mit einer dargestellten Sequenz besitzt; wobei die Zusammen- 
setzung frei von der Inhibin-a-Kette ist. 

22. Zusammensetzung nach Anspruch 21 , die ein Homodimer von reifen menschlichen oder Schweine-j8 B - 
Ketten oder einer der Aminosauresequenz-Varianten davon ist. 

25 

23. Zusammensetzung, umfassend ein Heterodimer von reifem menschlichem oder Schweine-lnhibin-£ A 
mit reifem menschlichem oder Schweine-1nhibin-0 B , wobei die Ketten wie in Fig. 2B (Schweine-0- 
Ketten) und Fig. 8 und 9 (menschliche 0-Ketten) dargestellt sind, oder einer durch Einfugung, 
Loschung oder Substitution einer der dargestellten Sequenzen erhaltenen Aminosauresequenz-Varian- 

30 te, wobei die Variante im wesentlichen homolog zu einem Polypeptid mit einer dargestellten Sequenz 
und 1) kreuzreaktiv mit gegen ein Polypeptid mit einer dargestellten Sequenz gebildeten Antikorpern; 
2) kreuzreaktiv mit Zelloberflachen-Rezeptoren fur ein Polypeptid mit einer dargestellten Sequenz ist; 
oder 3) ahnliche hormonelle Aktivitat wie ein Polypeptid mit einer dargestellten Sequenz besitzt; wobei 
die Zusammensetzung frei von der Inhibin-a-Kette ist. 

35 

24. Nicht-chromosomale DNA, die fur eine menschliche oder Schweine-lnhibin-o- oder eine menschliche 
oder Schweine-lnhibin-0-Kette, deren Aminosauresequenzen wie in Fig. 1B (Schweine-a-Kette), Fig. 2B 
(Schweine-0- Ketten), Fig. 6A (menschliche a-Kette) und Fig. 8 und 9 (menschliche jS-Ketten) dargestellt 
sind, oder fUr eine durch Einfugung, Loschung oder Substitution eines Polypeptids mit einer der 

40 dargestellten Sequenzen erhaltene Aminosauresequenz-Variante kodiert, wobei die Variante im wesent- 
lichen homolog zu einem Polypeptid mit einer dargestellten Sequenz ist, jedoch unter Ausschlufi der 
RindeMnhibin-a-Kette, und wobei die Variante 1) kreuzreaktiv mit gegen ein Polypeptid mit einer 
dargestellten Sequenz gebildeten Antikorpern ist; oder 2) kreuzreaktiv mit Zelloberflachen-Rezeptoren 
fur ein Polypeptid mit einer dargestellten Sequenz ist; oder 3) ahnliche hormonelle Aktivitat wie ein 

45 Polypeptid mit einer dargestellten Sequenz besitzt. 

25. DNA nach Anspruch 24, die fur eine menschliche oder Schwein-lnhibin-a- oder eine menschliche oder 
Schwein-lnhibin-£-Kette kodiert, deren Aminosauresequenz wie in Fig. 1B (Schweine-a-Kette), Fig. 2B 
(Schweine-tf-Ketten), Fig. 6A (menschliche a-Kette) und Fig. 8 und 9 (menschliche jS-Ketten) dargestellt 

so ist. 

26. DNA nach Anspruch 24, die fur eine Variante kodiert, ausgewahlt aus: den Schweine-lnhibinderivaten 
(Asn266— Gln]lnha; [CVS325 oder CVS324— A]lnha; {Cyss6i oder Cys36 3— A]lnha; [Lys 3 2i oder 
Lys 3 22— A]lnh/3 A ; [Lyss22- His oder Ser]Inh0 A ; [Lyssu- Arg; Vabie— Thr] lnh/S A ; [CyS388 Oder 

55 Cys 3 9o— A]lnh0 A ,[Lys4ii— Gln]lnhjS A ; [Arg 3 i5-*Lys; Vabu— Thr]lnhj9 B ; [Cys 3 ig oder Cys 3 2 0 — A]lnh/S B ; 
[Pro38i Gly382— Pro Phe Gly]lnhj9 B ;[Arg 3 95— Gln]lnh0 B » worin Inh eine Abkurzung fur Tnhibin ist und die 
Nummern der Reste fur lnh0 B die fur den entsprechenden lnh£ A -Rest verwendeten sind (siehe Fig. 
2B); den menschlichen lnhibin-0 A -Ketten-Varianten, die Variationen aufweisen, die Substitution oder 



52 



EP 0 222 491 B1 



Loschung eines Restes Oder Einfugung neben einem Rest, ausgewahlt aus den Resten 293 - 297, 364 

- 376 und 387 - 398, der in Fig. 8 dargestellten Sequenz sind; und den Varianten menschlicher a- 
Ketten, die zu mehr als 90% homolog zu der in Fig. 6A dargestellten Sequenz sind. 

5 27. DNA nach Anspruch 24, die fur eine Variante einer menschlichen 0-lnhibin-Kette, ausgewahlt aus 
Phe302— He oder Leu; Gln 2 97-*Asp Oder Lys; Trp 30 7-*Tyr Oder Phe; Trp 3 io-*Tyr oder Phe; llesn-^Phe 
Oder Val; Tyr 3 i7-*Trp oder Thr; His 3 i8-*Lys; Ala 3 i 9 -*Ser; Asn 32 o-*Gln, Tyr oder His; Tyr 32 i— Thr oder 
- Asp, Phe 3 4o— Tyr; His 35 3—Asp; His 3 53— Lys (a /S A //S B hybrid); Phe 3 56— Tyr; Val^—Phe; Val 36 4— Leu; 
Tyr 3 75- f Thr; Tyrm—Trp; Asn 38 9->Gln, His oder Lys; lle 3 9i^Leu Oder Thr; Mebgo —Leu Oder Ser; 

70 Val 3 92-*Phe, Glu, Thr oder lie; oder einer vergleichbar modifizierten menschlichen 0 B -Kette kodiert. 

28. DNA nach irgendeinem der Anspruche 24 - 27, die frei von intervenierenden untranslatierten Sequen- 
zen ist. 

75 29. DNA nach irgendeinem der Anspruche 24 - 28, die mit einer feststellbaren Gruppe markiert ist. 

30. Replizierbarer Vektor, umfassend eine DNA nach irgendeinem der Anspruche 21 - 27. 

31. Vektor nach Anspruch 30, umfassend einen viralen Promoter, der mit der fiir die lnhibin-a- oder -0- 
20 Ketten kodierenden DNA operabel verbunden ist. 

32. Vektor nach Anspruch 30 oder 31, der DNA enthalt, die sowohl fur eine lnhibin-a- als auch eine Inhibin- 
0-Kette kodiert. 

25 33. Vektor nach Anspruch 30 oder 31, der DNA enthalt, die fur eine lnhibin-£-Kette, jedoch nicht fur eine 
eine Inhibin-o-Kette kodiert. 

34. Wirtszelle, die mit einem replizierbaren Vektor transformiert wurde, der DNA enthalt, die fUr eine 
menschliche oder Schweine-lnhibin-a- und/oder eine -lnhibin-£-Kette, deren Aminosauresequenzen wie 
30 in Fig. 1B (Schweine-a-Kette), Fig. 2B (Schweine-0-Ketten), Fig. 6A (menschliche a-Kette) und Fig. 8 
und 9 (menschliche 0-Ketten) dargestellt sind, oder eine durch Einfugung, Loschung oder Substitution 
eines Polypeptids mit einer der dargestellten Sequenzen erhaltene Aminosauresequenz-Variante ko- 
diert, wobei die Variante im wesentlichen homolog zu einem Polypeptid mit einer dargestellten 
Sequenz ist, jedoch unter AusschluS der Rinder-lnhibin-a-Kette, und wobei die Variante 1) kreuzreaktiv 
35 mit gegen ein Polypeptid mit einer dargestellten Sequenz gebildeten Antikorpern ist; Oder 2) kreuzreak- 
tiv mit Zelloberflachen-Rezeptoren fur ein Polypeptid mit einer dargestellten Sequenz ist; oder 3) 
ahnliche hormonelle Aktivitat wie ein Polypeptid mit einer dargestellten Sequenz besitzt 

Wirtszelle nach Anspruch 34, worin die DNA fiir eine menschliche oder Schweine-lnhibin-a- und/oder 
eine -lnhibin-0-Kette kodiert, deren Aminosauresequenzen wie in Fig. 1B (Schweine-a-Kette), Fig. 2B 
(Schweine-/S-Ketten), Fig. 6A (menschliche a-Kette) und Fig. 8 und 9 (menschliche /S-Ketten) dargestellt 
sind. 

36. Wirtszelle nach Anspruch 34, worin die DNA fur eine Variante kodiert, ausgewahlt aus: den Schweine- 
45 Inhibinderivaten (Asn 2 66—G!n]lnha; [Cys 3 25 oder Cys 32 4—A]lnha; [Cys 3 ei oder Cysses— A]lnha; 
[Lys 3 2i oder LVS322— A]lnh/3 A ;[Lys 3 22->His Oder Ser]lnh0 A ; [Lys 3 i5-*Arg; Vahie— Thr] lnhj8 A ;[Cys388 
oder Cys 39 o-*A]lnh0 A , [Lys 41 i^Gln]lnh0 A ; {Arg 3 i 5 -Lys; Vakm^Thrjlnh/Ss; [Cys 3 i 9 oder Cys 32 o-»A]- 
lnh0 B ; [Pr038i Gly 3 82— Pro Phe Gly]lnh/9 B ; IArg 39 5-*Gin]lnh£ B , worin Inh eine Abkurzung tur Inhibin ist 
und die Nurrimern der Reste fiir Inhfo die fur den entsprechenden lnh0 A -Rest verwendeten sind (siehe 
50 Fig. 2B); den menschlichen Inhibin- 0 A -Ketten- Varianten, die Variationen aufweisen, die Substitution oder 
Loschung eines Restes oder Einfugung neben einem Rest, ausgewahlt aus den Resten 293 - 297, 364 

- 376 und 387 - 398, der in Fig. 8 dargestellten Sequenz sind; und den Varianten menschlicher a- 
Ketten, die zu mehr als 90% homolog zu der in Fig. 6A dargestellten Sequenz sind. 

55 37. Wirtszelle nach Anspruch 34, worin die DNA fur eine Variante einer menschlichen £ A -lnhibin-Kette, 
ausgewahlt aus: Phe 3 o2^lle oder Leu; Gln297— 'Asp oder Lys; Trp 3 o7-*Tyr oder Phe; Trp 3 i 0 — Tyr oder 
Phe; lle 3 u-*Phe oder Val; Tyr 3 i7~*Trp oder Thr; HiS3is-*Lys; Ala 3 i9—Ser; Asn 32 o-*Gln, Tyr oder His; 
Tyr 32 i-Thr oder Asp, Phe 34 o— Tyr; His 3 53-*Asp; His 353 -*Lys (a 0 A /0 B hybrid); Phe 35 6^Tyr; 



35. 

40 



53 



EP 0 222 491 B1 



Val3&«-*Phe; Val 3 64-*Leu; Tyr 37 5— Thr; Tyr 37 6-*Trp; Asn 38 9— Gin, His Oder Lys; Ile3si— Leu Oder Thr; 
Met39o -*Leu Oder Ser; Val392-*Phe, Glu t Thr Oder He; oder eine vergleichbar modifizierte menschliche 
/3 B -Kette kodiert. 

38. Zelle nach irgendeinem der Anspruche 34 - 37, die eine eukaryotische Zelle ist. 

39. Zellfreie Zusammensetzung, die frei von reifem a-Ketten-Polypeptid ist, die eine menschliche Oder 
Schweine-lnhibin-a-Ketten-Prodomanen-Polypeptidsequenz, wie in Fig. 1B (Schweine-a-Kette) Oder Fig. 
6A (menschliche a-Kette) dargestellt, Oder eine durch EinfOgung, Loschung Oder Substitution einer 
dieser Prodomanen-Sequenzen erhaltene Aminosauresequenz-Variante enthalt, wobei die Variante im 
wesentlichen homolog zu einem Polypeptid mit einer der dargestellten Sequenzen und kreuzreaktiv mit 
gegen ein Polypeptid mit einer der dargestellten Sequenzen gebildeten Anttkorpern ist. 

40. Zellfreie Zusammensetzung, umfassend 

a) ein Polypeptid, umfassend die menschliche lnhibin-0 A -Ketten-Prodomanensequenz 

HSAAPDCPSCALAALPKDVPNSQPEMVEAVKKHILNMLHL 
(Aminosauren 1 - 40 aus Fig. 8) 

PDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIG 
(Aminosauren 44 - 80 aus Fig. 8) 

AEMNELMEQTSEIITFAESGTARKTLHFEISKEGSDLSWERAEVWLFLKVPKAN- 

RTRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKWDA 
(Aminosauren 83 - 185 aus Fig. 8) STWHVFPVSSSIQRLLD- 

Q GKS S LD VR I ACEQ CQ ESG AS L VLLG 

(Aminosauren 188 - 230 aus Fig. 8) 

Oder naturlich vorkommende Saugetier-Aminosauresequenz-Varianten davon; 

b) ein Polypeptid, umfassend die menschliche lnhibin-0 B -Ketten-Prodomanensequenz 

CTSCGGFRRPEELGRVDGDFLEAV ' 
(Aminosauren 7 - 30 aus Fig. 9) 

H ILSRLQMRGR PN I THAVPKAAMVTALRKLHAGKVREDGRVE I PHLDGHAS PGAD - 

GQERVSEIISFAETDGLASSRVRLYFFISNEGNQNLFWQASLWLYLKLLPYVLEKGS 
(Aminosauren 33- 145 aus Fig. 9) 

VRVKVYFQEQGHGDRWNMVEKRVDLKRSGWHTFPLTEAIQALFERGE 
(Aminosauren 149- 195 aus Fig. 9) 

LNLDVQCDSCQELAWPVFVDPGEESHRPFVWQARLGDSRHRI 
(Aminosauren 198 - 241 aus Fig. 9) 

Oder naturlich vorkommende Saugetier-Aminosauresequenz-Varianten davon; oder 

c) ein Polypeptid, das frei von der reifen a-Ketten-Aminosauresequenz ist, umfassend menschlichen 
Inhibin-a-Ketten-Prodomanensequenzen . 



54 



EP0 222 491 B1 



KVRAL F LD ALG ? P AVTREGGD PG V 
(Aminosauren 1 - 24 aus Fig. 6) 

HALGGFTHRGSEPEEEEDVSQAILFPATDASCEDKSAARGLAQEAEEGLFRYMFR- 

PSQHTRSRQVTSAQLWFHTGLDRQGTAASNSSEPLLGLLALSPGGPVAVPMSLGH- 

APPHWAVLHXJVTSALSLLTHPVL^LLRCPLCTCSARPEATPFLVAHTRTRPPSGGERA 
(Aminosauren 32 - 1 99 aus Fig. 6) 

oder naturlich vorkommende Saugetier-AminosSuresequenz-Varianten davon. 

41. Zellfreie Zusammensetzung, umfassend ein menschliches oder Schwein-lnhibin-^Ketten-Prodomanen- 
Polypeptid mit einer Sequenz, wie in Fig. 2B (Schweine-^-Ketten) und Fig. 8 und 9 (menschliche 0- 
Ketten) dargestellt, oder eine durch Einfugung, Loschung oder Substitution einer dieser ProdomSnen- 
Sequenzen erhaltene Aminosauresequenz-Variante, wobei die Variante im wesentlichen homolog zu 
einem Polypeptid mit einer der dargestellten Sequenzen und kreuzreaktiv mit gegen ein Polypeptid mit 
einer der dargestellten Sequenzen gebildeten Antikorpern ist. 

42. Zusammensetzung nach Anspruch 41, worin die /3-Kette die 0 A -Kette und die Zusammensetzung frei 
von reifer 0 A -Ketten- Sequenz ist. 

43. Zusammensetzung nach Anspruch 41, worin die £-Kette die fo-Kette und die Zusammensetzung frei 
von reifer jS B -Ketten-Sequenz ist. 

44. Zusammensetzung nach Anspruch 40, worin die Variante die entsprechende Schweine-Aminosaurese- 
quenz ist, wobei die Zusammensetzung enthalt: 

a) ein Polypeptid, umfassend die Schweine-lnhibin-jS A -Ketten-Prodomiinensequenz, die beschrieben 
wird durch die Aminosauren: 28 - 58; 61 - 87; 90 - 108; 111 - 179; 182 - 213; 216 - 258; oder 28 - 
87 aus Fig. 2B; oder 

b) ein Polypeptid, das frei von der reifen a-Ketten-Aminosauresequenz ist, umfassend die Schweine- 
Inhibin-a-Ketten-Prodomanen-Polypeptidsequenzen, die beschrieben werden durch die Aminosau- 
ren: 20 - 54 oder 70 - 228 aus Fig. 1 B. 

45. Zusammensetzung nach irgendeinem der Anspruche 39 - 44, worin das Polypeptid nicht von nativer 
Glykosylierung begleitet wird. 

46. Zusammensetzung nach irgendeinem der Anspruche 39 - 45, die steril ist, und worin das Polypeptid 
weiters ein immunogenes Polypeptid umfafit 

47. Verwendung einer Zusammensetzung nach irgendeinem der Anspruche 39 - 46 zur Herstellung eines 
Antikdrpers, der in der Lage ist, das Polypeptid zu binden. 

48. Zusammensetzung nach irgendeinem der AnsprOche 39 - 46, worin das Polypeptid an eine feststellba- 
re Gruppe konjugiert ist. 

49. Zusammensetzung nach Anspruch 48, worin die Gruppe ein Enzym, Fluorophor oder Radioisotop ist. 

50. Zusammensetzung nach irgendeinem der Anspruche 39 - 44, 48 oder 49, die durch nicht-kovalente 
Absorption oder kovalente Vernetzung an einen wasserunloslichen Trager unloslich gemacht ist. 

51. Zusammensetzung nach irgendeinem der AnsprUche 39 - 46, 48 oder 49, weiters umfassend eine 
physiologisch annehmbare, implantierbare Matrix zur gesteuerten Freisetzung des Polypeptids in die 
Gewebe eines Tieres. 



55 



EP 0 222 491 B1 



Patentanspruche fiir folgenden Vertragsstaat : GR 

1. Verfahren, umfassend das Kultivieren einer Wirtszelle, die mit einem Vektor transformiert wurde, der 
Nukleinsaure enthMIt, die fur eine menschliche oder Schweine-lnhibin-a-Kette und/oder eine menschli- 
che oder Schweine-lnhibin-£-Kette, deren Aminosauresequenzen wie in Fig. 1B (Schweine-a-Kette), 
Fig. 2B (Schweine-0-Ketten), Fig. 6A (menschliche a-Kette) und Fig. 8 und 9 (menschliche 0-Ketten) 
dargestellt sind, oder eine durch Einfugung, Loschung Oder Substitution einer der dargestellten 
Sequenzen erhaltene Aminosauresequenz- Variante kodiert, wobei die Variante im wesentlichen homo- 
log zu einem Polypeptid mit einer dargestellten Sequenz ist, und wobei die Variante 1) kreuzreaktiv mit 
gegen ein Polypeptid mit einer dargestellten Sequenz gebildeten Antikorpern ist; oder 2) kreuzreaktiv 
mit Zelloberflachen-Rezeptoren fiir ein Polypeptid mit einer dargestellten Sequenz ist; Oder 3) ahnliche 
hormonelle Aktivitat wie ein Polypeptid mit einer dargestellten Sequenz besitzt. 

2. Verfahren nach Anspruch 1 , worin die Nukleinsaure fur eine menschliche oder Schweine-lnhibin-a-Kette 
und/oder eine menschliche oder Schweine-j8-Kette kodiert, deren Aminosauresequenzen wie in Fig. 1B 
(Schweine-a-Kette), Fig. 2B (Schweine-0-Ketten), Fig. 6A (menschliche a-Kette) und Fig. 8 und 9 
(menschliche jS-Ketten) dargestellt sind. 

3. Verfahren nach Anspruch 1, worin der Vektor fur eine allelische Variante von menschlicher oder 
Schweine-lnhibin-a- und/oder -0-Kette kodiert. 

4. Verfahren nach irgendeinem der vorangegangenen Anspruche, worin die Nukleinsaure, die fiir die 
Inhibin-a-Kette und/oder -0-Kette kodiert, mit einem von der Wirtszelle erkannten Promotor operabel 
verbunden ist, und das den weiteren Schritt der Gewinnung der Inhibih-a-Kette, lnhibin-0-Kette, von 
Inhibin oder einem 0-Ketten-Dimer aus dem Kulturmedium umfaBt. 

5. Verfahren nach irgendeinem der vorangegangenen Anspruche, worin die Zelle ein Prokaryot ist. 

6. Verfahren nach irgendeinem der vorangegangenen Anspruche, worin der Vektor Nukleinsaure umfaBt, 
die fiir die Prapro-Form einer Inhibin-a-Kette oder einer Inhibin- /S-Kette kodiert. 

7. Verfahren nach Anspruch 6, worin der Vektor eine Nukleinsaure umfaflt, die sowohl fur die Prapro-Form 
einer Inhibin-a-Kette als auch einer lnhibin-/J-Kette kodiert. 

8. Verfahren nach irgendeinem der Anspruche 1 - 4, 6 oder 7, worin die Zelle eine Zelle aus einem 
vielzelligen Organismus ist und hormonell aktives Inhibin produziert wird. 

9. Verfahren nach Anspruch 4, worin der Promotor ein viraler Promotor ist. 

10. Verfahren nach Anspruch 9, worin der Promotor ein SV40-Promotor ist. 

11. Verfahren nach irgendeinem der vorangegangenen Anspruche, worin reifes Schweine- oder menschli- 
ches Inhibin gewonnen wird. 

12. Verfahren nach irgendeinem der Anspruche 1 - 5 oder 8-10, worin der Vektor Nukleinsaure umfaBt, 
die fur die Prapro-Form einer lnhibin-/S-Kette kodiert, und ein reifes jS-Ketten-Dimer frei von der a-Kette 
gewonnen wird. 

13. Verfahren nach Anspruch 8 oder 11, worin die £-Kette die £ A -Kette ist und das Inhibin in einer 
Konzentration von uber etwa 20 ng/ml im Kulturmedium vorliegt. 

14. Verfahren nach Anspruch 4, worin das Inhibin menschliches Oder Schweine-lnhibin ist, das aus einer a- 
und einer /S-Kette, wobei die Aminosauresequenzen der a- und /3-Ketten aus jenen in Fig. 1B 
(Schweine-a-Kette), Fig. 2B (Schweine-j8-Ketten), Fig. 6A (menschliche a-Kette) und Fig. 8 und 9 
(menschliche 0-Ketten) dargestellten ausgewahlt werden, und durch Einfugung, Loschung oder Substi- 
tution eines Polypeptids mit einer der dargestellten Sequenzen erhaltenen AminosSuresequenz-Varian- 
ten besteht, wobei die Varianten im wesentlichen homolog zu einem Polypeptid mit -einer dargestellten 
Sequenz und 1) kreuzreaktiv mit gegeh ein Polypeptid mit einer dargestellten Sequenz gebildeten 



56 



EP 0 222 491 B1 



Antikorpern; 2) kreuzreaktiv mit Zelloberflachen-Rezeptoren fur ein Polypeptid mit einer dargestellten 
Sequenz sind; Oder 3) ahnliche hormonelle Aktivitat wie ein Polypeptid mit einer dargestellten Sequenz 
besitzen; wobei die Zusammensetzung vollstandig frei von nicht-identifizierten menschlichen oder 
Schweine-Proteinen ist. 

6 

15. Verfahren nach Anspruch 14, worin das Inhibin menschliches oder Schweine-lnhibin ist, das aus einer 
a- und einer 0-Kette besteht, wobei die Sequenzen der a- und j9-Ketten aus jenen ausgewahlt werden, 
die in Fig. 1B (Schweine-a-Kette), Fig. 2B (Schweine-0-Ketten), Fig. 6A (menschliche a-Kette) und Fig. 
8 und 9 (menschliche 0-Ketten) dargestellt sind. 

70 

16. Verfahren nach Anspruch 4, worin die gewonnene Inhibin-Kette eine Prodomane von menschlichem 
oder Schweine-a- oder -£ B -lnhibin, wie in Fig. 1B (Schweine-a-Kette), Fig. 2B (Schweine-j3 B -Ketten), 
Fig. 6A (menschliche a-Kette) und Fig. 9 (menschliche /8 B -Kette) dargestellt, ist, die nicht mit nativer 
Glykosylierung assoziiert ist. 

75 

17. Verfahren nach Anspruch 4, worin ein 0-Ketten-Dimer gewonnen wird, das ein Homodimer von reifen 
menschlichen oder Schweine-lnhibin-j9 A - oder -/S B -Ketten, wobei die Ketten wie in Fig. 2B (Schweine-jS- 
Ketten) und Fig. 8 und 9 (menschliche ^-Ketten) dargestellt sind, oder eine durch Einfugung, Loschung 
oder Substitution eines Polypeptids mit einer der dargestellten Sequenzen erhaltene Aminosa'urese- 

20 quenz-Variante ist, wobei die Variante im wesentlichen homolog zu einem Polypeptid mit einer 
dargestellten Sequenz und 1) kreuzreaktiv mit gegen ein Polypeptid mit einer dargestellten Sequenz 
gebildeten Antikorpern; 2) kreuzreaktiv mit Zelloberflachen-Rezeptoren fOr ein Polypeptid mit einer 
dargestellten Sequenz ist; oder 3) ahnliche hormonelle Aktivitat wie ein Polypeptid mit einer dargestell- 
ten Sequenz besitzt; wobei das Dimer frei von der Inhibin-a-Kette ist. 

25 

18. Verfahren nach Anspruch 17, worin ein Homodimer von reifen menschlichen oder Schweine-0 B -Ketten 
oder eine der Aminosauresequenz-Varianten davon gewonnen wird. 

19. Verfahren nach Anspruch 4, worin ein 0- Ketten- Dimer gewonnen wird, das ein Heterodimer von reifem 
30 menschlichem oder Schweine-lnhibin-jS A mit reifem menschlichem oder Schweine-lnhibin-/S B , wobei die 

Ketten wie in Fig. 2B (Schweine-0- Ketten) und Fig. 8 und 9 (menschliche 0-Ketten) dargestellt sind, 
oder erne durch Einfugung, Loschung oder Substitution eines Polypeptids mit einer der dargestellten 
Sequenzen erhaltene Aminosauresequenz-Variante ist, wobei die Variante im wesentlichen homolog zu 
einem Polypeptid mit einer dargestellten Sequenz und 1) kreuzreaktiv mit gegen ein Polypeptid mit 
35 einer dargestellten Sequenz gebildeten Antikorpern; 2) kreuzreaktiv mit Zelloberflachen-Rezeptoren fur 
ein Polypeptid mit einer dargestellten Sequenz ist; Oder 3) ahnliche hormonelle Aktivitat wie ein 
Polypeptid mit einer dargestellten Sequenz besitzt; wobei das Dimer frei von der Inhibin-a-Kette ist. 

20. Nicht-chromosomale DNA, die fur eine menschliche oder Schweine-lnhibin-a- oder eine menschliche 
40 oder Schweine-lnhibin-/S-Kette, deren Aminosauresequenzen wie in Fig. 1B (Schweine-a-Kette), Fig. 2B 

(Schweine-0-Ketten), Fig. 6A (menschliche a-Kette) und Fig. 8 und 9 (menschliche j8-Ketten) dargestellt 
sind, oder fur eine durch Einfugung, Loschung oder Substitution eines Polypeptids mit einer der 
dargestellten Sequenzen erhaltene Aminosauresequenz-Variante kodiert, wobei die Variante im wesent- 
lichen homolog zu einem Polypeptid mit einer dargestellten Sequenz und 1 ) kreuzreaktiv mit gegen ein 
45 Polypeptid mit einer dargestellten Sequenz gebildeten Antikorpern; oder 2) kreuzreaktiv mit Zelloberfla- 
chen-Rezeptoren fur ein Polypeptid mit einer dargestellten Sequenz ist; Oder 3) ahnliche hormonelle 
Aktivitat wie ein Polypeptid mit einer dargestellten Sequenz besitzt. 



21. DNA nach Anspruch 20, die fur eine menschliche oder Schweine-lnhibin-a- oder eine menschliche oder 
so Schweine-lnhibin-jS-Kette kodiert, deren Aminosauresequenz wie in Fig. 1B (Schweine-a-Kette), Fig. 2B 

(Schweine-jS-Ketten), Fig. 6A (menschliche a-Kette) und Fig. 8 und 9 (menschliche £-Ketten) dargestellt 
ist. 

22. DNA nach Anspruch 20 oder 21 , die frei von intervenierenden untranslatierten Sequenzen ist. 

55 

23. DNA nach irgendeinem der Anspruche 20 - 22, die mit einer feststellbaren Gruppe markiert ist. 

24. Replizierbarer Vektor, umfassend eine DNA nach Anspruch 20 oder 21. 



57 



EP 0 222 491 B1 



25. Vektor nach Anspruch 24, umfassend einen viralen Promoter, der mit der fiir die lnhibin-a- oder 
Ketten kodierenden DNA operabel verbunden ist. 

26. Vektor nach Anspruch 24 oder 25, der DNA enthSIt, die sowohl ffjr eine lnhibin-a- als auch eine Inhibin- 
5 0-Kette kodiert. 

27. Vektor nach Anspruch 24 oder 25, der DNA enthalt, die fur eine lnhibin-/3-Kette, jedoch nicht fur eine 
eine Inhibin-a-Kette kodiert. 

io 28. Wirtszelle, die mit einem replizierbaren Vektor transformiert wurde, der DNA enthalt, die fur eine 
menschliche oder Schweine-lnhibin-a- und/oder eine -Inhibin-^S-Kette, deren Aminosauresequenzen wie 
in Fig. 1B (Schweine-a-Kette), Fig. 2B (Schweine-0-Ketten), Fig. 6A (menschliche o-Kette) und Fig. 8 
und 9 (menschliche 0-Ketten) dargestellt sind, oder fur eine durch EinfOgung, LSschung oder Substitu- 
tion eines Polypeptids mit einer der dargestellten Sequenzen erhaltene Aminosauresequenz-Variante 

75 kodiert, wobei die Variante im wesentlichen homolog zu einem Polypeptid mit einer dargestellten 
Sequenz und 1) kreuzreaktiv mit gegen ein Polypeptid mit einer dargestellten Sequenz gebildeten 
Antikorpern; oder 2) kreuzreaktiv mit Zelloberflachen-Rezeptoren fur ein Polypeptid mit einer darge- 
stellten Sequenz ist; oder 3) ahnliche hormonelle Aktivitat wie ein Polypeptid mit einer dargestellten 
Sequenz besitzt. 

20 

29. Wirtszelle nach Anspruch 28, worin die DNA fiir eine menschliche oder Schweine- Inhibin-a-Kette 
und/oder eine -lnhibin-0-Kette kodiert, deren Aminosauresequenzen wie in Fig. 1B (Schweine-a-Kette), 
Fig. 2B (Schweine-0-Ketten), Fig. 6A (menschliche a-Kette) und Fig. 8 und 9 (menschliche 0-Ketten) 
dargestellt sind. 

25 

30. Zelle nach Anspruch 28 oder 29, die eine eukaryotische Zelle ist. 

31. Verfahren nach Anspruch 4, worin die gewonnene Inhibin-Kette eine zellfreie Zusammensetzung ist, die 
frei von reifer a-Ketten-Sequenz ist, die eine menschliche oder Schweine-lnhibin-a-Ketten-Prodomanen- 

30 Polypeptidsequenz, wie in Fig. 1B (Schweine-a-Kette) oder Fig. 6A (menschliche a-Kette) dargestellt, 
oder eine durch Einfugung, Loschung oder Substitution einer dieser Prodomanen-Sequenzen erhaltene 
Aminosauresequenz-Variante enthalt, wobei die Variante im wesentlichen homolog zu einem Polypeptid 
mit einer der dargestellten Sequenzen und kreuzreaktiv mit gegen ein Polypeptid mit einer dargestell- 
ten Sequenz gebildeten Antikorpern ist. 

35 

32. Verfahren nach Anspruch 4, worin die gewonnene Inhibin-Kette eine zellfreie Zusammensetzung ist, 
umfassend 

a) ein Polypeptid, umfassend die menschliche lnhibin-/8 A -Ketten-Prodomanensequenz 

40 

HSAAPDCPSCALAALPKDVPNSQPEMVEAVKKHI LNMLHL 

(Aminosauren 1 - 40 aus Fig. 8) 

45 



50 



55 



58 



EP 0 222 491 B1 



PDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIG 
(Aminosauren 44 - 80 aus Fig. 8) 

AEMNELMEQTSEII7FAESGTARKTLHFEISKEGSDLSVVERAEVWLFLXVPKAN- 
RTRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDA 
(Aminosauren 83 - 185 aus Fig. 8) 

STWHVFPVSSSIQRLLDQGKSSLDVRIACEQCQESGASLVLLG 
(Aminosauren 188 - 230 aus Fig. 8) 

Oder naturlich vorkommende Saugetier-Aminosauresequenz-Varianten davon; 

b) ein Polypeptid, umfassend die menschliche lnhibin-0 B -Ketten-Prodomanensequenz 

CTSCGGFRRPEELGRVDGDFLEAV 

(Aminosauren 7 - 30 aus Fig. 9) 
H I LS R LQMRGRPNI TH A VPK AAM VT A LRKLH AGKVREDGRVE I PHLDGHASPG AD - 

GQERVSEIISFAETDGLASSRVRLYFFISNEGNQNLFVVQASLWLYLKLLPYV- 

LEKGS (Aminosauren 33 - 145 aus Fig. 9) 

VRVKVYFQEQGHDRWNMVEKRVDLKRSGWHTFPLTEAIQALFERGE 

(Aminosauren 1 49 - 1 95 aus Fig. 9) 

LNLDVQCDSCQELAVVPVFVDPGEESHRPFVVVQARLGDSRHRI 
(Aminosauren 198 - 241 aus Fig. 9) 

Oder naturlich vorkommende Saugetier-Aminosauresequenz-Varianten davon; Oder 

c) ein Polypeptid, das frei von der reifen a-Ketten-Aminosauresequenz ist, umfassend menschlichen 
Inhibin-a-Ketten-Prodomanensequenzen 

KVRALFLDALGPPAVTREGG PGV 
(Aminosauren 1 - 24 aus Fig. 6) 

HALGGFTHRGSEPEEEEDVSQAILFPATDASCEDKSAARGLAOEAEEGLFRYMFR- 
PSQHTRSRQVTSAQLWFHTGLDRQGTAASNSSEPLLGLLALSPGGPVAVPMSLGH- 
APPHWAVLHLATSALSLLTHPVLVLLLRCPLCTCSARPEATPFLVAHTRTRPPSG- GERA 

(Aminosauren 32- 199 aus Fig. 6) 

oder naturlich vorkommende Saugetier-Aminosauresequenz-Varianten davon. 

33. Verfahren nach Anspruch 4, worin die gewonnene Inhibin-Kette eine zellfreie Zusammensetzung ist, 
umfassend ein menschliches oder Schweine-lnhibin-j8-Ketten-Prodomanen-Polypeptid mit einer Se- 
quenz, wie in Fig. 2B (Schweine-£-Ketten) und Fig. 8 und 9 (menschliche j8-Ketten) dargestellt, oder 
eine durch Einfugung, Loschung oder Substitution einer dieser ProdomSnen-Polypeptide mit einer 
dieser Sequenzen erhaltene Aminosauresequenz-Variante ist, wobei die Variante im wesentlichen 
homolog zu einem Polypeptid mit einer der dargestellten Sequenzen und kreuzreaktiv mit gegen ein 



59 



EP 0 222 491 B1 



Polypeptid mit einer dargestellten Sequenz gebildeten Antikorpern ist. 

34. Verfahren nach Anspruch 33, worin die jS-Kette die 0 A -Kette und die Zusammensetzung frei von reifer 
^A-Ketten-Sequenz ist. 

5 

35. Verfahren nach Anspruch 33, worin die £-Kette die 0 B -Kette und die Zusammensetzung frei von reifer 
/3 B -Ketten- Sequenz ist. 

36. Verfahren nach Anspruch 32, worin die Variante die entsprechende Schweine-Aminosauresequenz ist, 
70 wobei die Zusammensetzung enthalt: 

a) ein Polypeptid, umfassend die Schweine-Inhibin-0 A -Ketten-Prodomanensequenz, die beschrieben 
wird durch die Aminosauren: 28 - 58; 61 - 87; 90 - 108; 111 - 179; 182 - 213; 216 - 258; Oder 28 - 
87 aus Fig. 2B; Oder 

b) ein Polypeptid, das frei von der reifen o-Ketten-Aminosauresequenz ist, umfassend die Schweine- 
75 Inhibin-a-Ketten-Prodomanen-Polypeptidsequenzen, die beschrieben werden durch die Aminosau- 
ren: 20 - 54 Oder 70 - 228 aus Fig. 1 B. 

37. Verfahren nach irgendeinem der Anspruche 31 - 36, worin das Polypeptid nicht von nativer Glykosylie- 
rung begleitet wird. 

20 

38. Verfahren nach irgendeinem der Anspruche 31 - 37, worin die Zusammensetzung steril ist, und weiters 
umfassend das Verbinden des Polypeptids mit einem immunogenen Polypeptid. 

39. Verwendung einer Zusammensetzung nach irgendeinem der Anspruche 31 - 38 zur HersteNung eines 
25 Antikorpers, der in der Lage ist, das Polypeptid zu binden. 

40. Verfahren nach irgendeinem der Anspruche 31 - 38, weiters umfassend das Binden des Polypeptids an 
eine feststellbare Gruppe. 

30 41. Verfahren nach Anspruch 40, worin die Gruppe ein Enzym, Fluorophor oder Radioisotop ist. 

42. Verfahren nach irgendeinem der Anspruche 31 - 38, 40 Oder 41 , worin die Zusammensetzung durch 
nicht-kovalente Absorption oder kovalente Vernetzung an einen wasserunloslichen Trager unloslich 
gemacht ist. 

35 

43. Verfahren nach irgendeinem der Anspruche 31 - 38, 40 oder 41, weiters umfassend das Mischen der 
Zusammensetzung mit einer physiologisch annehmbaren, implantierbaren Matrix zur gesteuerten Frei- 
setzung des Polypeptids in die Gewebe eines Tieres. 

40 Patentansprliche fur folgende Vertragsstaaten : AT, ES 

Verfahren, umfassend das Kultivieren einer Wirtszelle, die mit einem Vektor transformiert wurde, der 
NukleinsSure enthalt, die fur eine menschliche oder Schweine-lnhibin-o-Kette und/oder eine menschli- 
che oder Schweine-lnhibin-^-Kette, deren Aminosauresequenzen wie in Fig. 1B (Schweine-a-Kette), 
Fig. 2B (Schweine-0-Ketten), Fig. 6A (menschliche a-Kette) und Fig. 8 und 9 (menschliche 0-Ketten) 
dargestellt sind, Oder eine durch Einfugung, Loschung oder Substitution einer der dargestellten 
Sequenzen erhaltene Aminosauresequenz-Variante kodiert, wobei die Variante im wesentlichen homo- 
log zu einem Polypeptid mit einer dargestellten Sequenz ist, und wobei die Variante 1 ) kreuzreaktiv mit 
gegen ein Polypeptid mit einer dargestellten Sequenz gebildeten Antikorpern ist; oder 2) kreuzreaktiv 
mit Zelloberflachen-Rezeptoren fUr ein Polypeptid mit einer dargestellten Sequenz ist; Oder 3).ahnliche 
hormonelle Aktivitat wie ein Polypeptid mit einer dargestellten Sequenz besitzt. 

Verfahren nach Anspruch 1 , worin die Nukleinsaure fur eine menschliche Oder Schweine-lnhibin-o-Kette 
und/oder eine menschliche oder Schweine-lnhibin-0-Kette kodiert, deren Aminosauresequenzen wie in 
Fig. 1B (Schweine-a-Kette), Fig. 2B (Schweine-j3-Ketten), Fig. 6A (menschliche a-Kette) und Fig. 8 und 
9 (menschliche jfl-Ketten) dargestellt sind. 



60 



EP 0 222 491 B1 



3. Verfahren nach Anspruch 1, worin der Vektor fur eine allelische Variante von menschlicher oder 
Schweine-lnhibin-a- und/oder -0-Kette kodiert. 

4. Verfahren nach irgendeinem der vorangegangenen AnsprUche, worin die NukleinsSure, die fOr die 
5 Inhibin-a-Kette und/oder -£-Kette kodiert, mit einem von der Wirtszelle erkannten Promotor operabel 

verbunden ist, und das den weiteren Schritt der Gewinnung der Inhibin-a-Kette, Inhibin-jS-Kette, von 
Inhibin oder einem 0-Ketten-Dimer aus dem Kulturmedium umfaBt. 

5. Verfahren nach irgendeinem der vorangegangenen Anspruche, worin die Zelie ein Prokaryot ist. 

70 

6. Verfahren nach irgendeinem der vorangegangenen Anspruche, worin der Vektor Nukleinsaure umfaBt, 
die fur die Prapro-Form einer Inhibin-a-Kette oder einer lnhibin-0-Kette kodiert. 

7. Verfahren nach Anspruch 6, worin der Vektor eine Nukleinsaure umfaBt, die sowohl fur die Prapro-Form 
75 einer Inhibin-a-Kette als auch einer lnhibin-/8-Kette kodiert. 

8. Verfahren nach irgendeinem der Anspruche 1 - 4, 6 oder 7, worin die Zelle eine Zelle aus einem 
vietzelligen Organismus ist und hormonell aktives Inhibin produziert wird. 

20 9. Verfahren nach Anspruch 4, worin der Promotor ein viraler Promotor ist. 

10. Verfahren nach Anspruch 9, worin der Promotor ein SV40-Promotor ist. 

11. Verfahren nach irgendeinem der vorangegangenen Anspruche, worin reifes Schweine- oder menschli- 
25 ches Inhibin gewonnen wird. 

12. Verfahren nach irgendeinem der Anspruche 1 - 5 oder 8-10, worin der Vektor Nukleinsaure umfaBt, 
die fur die Prapro-Form einer Inhibin-jS-Kette kodiert, und ein reifes jS-Ketten-Dimer frei von der a-Kette 
gewonnen wird. 

30 

13. Verfahren nach Anspruch 8 oder 11, worin die /8-Kette die 0 A -Kette ist und das Inhibin in einer 
Konzentration von uber etwa 20 ng/ml im Kulturmedium vorliegt. 

14. Verfahren nach Anspruch 4, worin das Inhibin menschliches Oder Schweine-lnhibin ist, das aus einer a- 
35 und einer 0-Kette, wobei die Aminosauresequenzen der a- und /S-Kette aus jenen in Fig. 1B (Schweine- 

a-Kette), Fig. 2B (Schweine-0-Ketten), Fig. 6A (menschliche a-Kette) und Fig. 8 und 9 (menschliche 0- 
Ketten) dargestellten ausgewahlt werden, und durch EinfOgung, Loschung oder Substitution eines 
Polypeptids mit einer der dargestellten Sequenzen erhaltenen Aminosauresequenz-Varianten besteht, 
wobei die Varianten im wesentlichen homolog zu einem Polypeptid mit einer dargestellten Sequenz 
40 und 1) kreuzreaktiv mit gegen ein Polypeptid mit einer dargestellten Sequenz gebildeten Antikorpern; 
2) kreuzreaktiv mit ZelloberflSchen-Rezeptoren fur ein Polypeptid mit einer dargestellten Sequenz sind; 
oder 3) ahnliche hormonelle Aktivitat wie ein Polypeptid mit einer dargestellten Sequenz besitzen; 
wobei die Zusammensetzung vollstandig frei von nicht-identifizierten menschlichen oder Schweine- 
Proteinen ist. 

45 

15. Verfahren nach Anspruch 14, worin das Inhibin menschliches oder Schweine-lnhibin ist, das aus einer 
a- und einer 0-Kette besteht, wobei die Sequenzen der a- und /8-Ketten aus jenen ausgewahlt werden, 
die in Fig. 1B (Schweine-a-Kette), Fig. 2B (Schweine- /S-Ketten), Fig. 6A (menschliche a-Kette) und Fig. 
8 und 9 (menschliche j8-Ketten) dargestellt sind. 

so 

16. Verfahren nach Anspruch 4, worin die gewonnene Inhibin-Kette eine Prodomane von menschlichem 
oder Schweine-a- oder -fo-lnhibin, wie in Fig. 1B (Schweine-a-Kette), Fig. 2B (Schweine-j9 B -Ketten), 
Fig. 6A (menschliche a-Kette) und Fig. 9 (menschliche j8 B -Kette) dargestellt, ist, die mit der nativen 
Glykosylierung nicht assoziiert ist. 

55 

17. Verfahren nach Anspruch 4, worin ein 0-Ketten-Dimer gewonnen wird, das ein Homodimer von reifen 
menschlichen oder Schweine-Inhibin-0 A - oder -j9 B -Ketten, wobei die Ketten wie in Fig. 2B (Schweine-0- 
Ketten) und Fig. 8 und 9 (menschliche /3-Ketten) dargestellt sind, oder eine durch EinfOgung, Loschung 



61 



EP 0 222 491 B1 



Oder Substitution eines Polypeptids mit einer der dargestellten Sequenzen erhaltene Aminosaurese- 
quenz-Variante ist, wobei die Variante im wesentlichen homolog zu einem Polypeptid mit einer 
dargestellten Sequenz und 1) kreuzreaktiv mit gegen ein Polypeptid mit einer dargestellten Sequenz 
gebildeten Antikorpern; 2) kreuzreaktiv mit Zelloberflachen-Rezeptoren fOr ein Polypeptid mit einer 
s dargestellten Sequenz ist; Oder 3) ahnliche hormonelle Aktivitat wie ein Polypeptid mit einer dargestell- 
ten Sequenz besitzt; wobei das Dimer frei von der Inhibin-a-Kette ist. 

18. Verfahren nach Anspruch 17, worin ein Homodimer von reifen menschlichen Oder Schweine-0 B -Ketten 
oder eine der Aminosauresequenz-Varianten davon gewonnen wird. 

70 

19. Verfahren nach Anspruch 4, worin ein 0-Ketten-Dimer gewonnen wird, das ein Heterodimer von reifem 
menschlichem oder Schweine-Inhibin-0 A mit reifem menschlichem oder Schweine-lnhibin-fo, wobei die 
Ketten wie in Fig. 2B (Schweine-/S-Ketten) und Fig. 8 und 9 (menschliche 0-Ketten) dargestellt sind, 
oder eine durch Einfugung, Loschung oder Substitution eines Polypeptids mit einer der dargestellten 

75 Sequenzen erhaltene Aminosauresequenz-Variante ist, wobei die Variante im wesentlichen homolog zu 
einem Polypeptid mit einer dargestellten Sequenz und 1) kreuzreaktiv mit gegen ein Polypeptid mit 
einer dargestellten Sequenz gebildeten Antikorpern; 2) kreuzreaktiv mit Zelloberflachen-Rezeptoren fUr 
ein Polypeptid mit einer dargestellten Sequenz ist; oder 3) ahnliche hormonelle Aktivitat wie ein 
Polypeptid mit einer dargestellten Sequenz besitzt; wobei das Heterodimer frei von der Inhibin-a-Kette 

20 ist. 

20. Verfahren zur Herstellung von nicht-chromosomaler DNA, die fur eine menschliche oder Schweine- 
lnhibin-cr- oder eine menschliche oder Schweine-lnhibin-^-Kette, wie in Fig. 1B (Schweine-a-Kette), Fig. 
2B (Schweine-/9-Ketten), Fig. 6A (menschliche a-Kette) und Fig. 8 und 9 (menschliche /8-Ketten) 

26 dargestellt, oder fur eine durch Einfugung, Loschung oder Substitution eines Polypeptids mit einer der 
dargestellten Sequenzen erhaltene Aminosauresequenz-Variante kodiert, wobei die Variante im wesent- 
lichen homolog zu einem Polypeptid mit einer dargestellten Sequenz und 1) kreuzreaktiv mit gegen ein 
Polypeptid mit einer dargestellten Sequenz gebildeten AntikSrpern; oder 2) kreuzreaktiv mit Zelloberfla- 
chen-Rezeptoren fur ein Polypeptid mit einer dargestellten Sequenz ist; oder 3) ahnliche hormonelle 

30 Aktivitat wie ein Polypeptid mit einer dargestellten Sequenz besitzt; wobei das Verfahren chemische 
Synthese, Screenen von mRNA aus dem Eierstock oder Screenen der genomischen Sammlungen 
jeder Zelle unfaflt. 

21. Verfahren nach Anspruch 20, worin die DNA fur eine menschliche oder Schweine-lnhibin-a- oder eine 
35 menschliche Oder Schweine-lnhibin-0-Kette kodiert, deren Aminosauresequenz wie in Fig. 1B (Schwei- 
ne-a-Kette), Fig. 2B (Schweine-0-Ketten), Fig. 6A (menschliche a-Kette) und Fig. 8 und 9 (menschliche 
j8-Ketten) dargestellt ist. 

22. Verfahren nach Anspruch 20 oder 21, worin die nicht-chromosomale DNA frei von intervenierenden 
40 untranslatierten Sequenzen ist. 

23. Verfahren nach irgendeinem der Anspruche 20 - 22, weiters umfassend den Schritt des Markierens der 
nicht-chromosomalen DNA mit einer feststellbaren Gruppe. 

45 24. Verfahren zur Herstellung eines replizierbaren Vektors, wobei das Verfahren das Einfugen einer DNA 
nach Anspruch 20 Oder 21 in einen Klonungsvektor umfaBt. 

25. Verfahren nach Anspruch 24, worin die DNA in einen Vektor eingefUgt wird, der einen viralen Promotor 
besitzt, der mit der fur die lnhibin-a- und/oder -jS-Ketten kodierenden DNA operabel verbunden ist, um 

so dadurch einen Expressionsvektor zu produzieren. 

26. Verfahren nach Anspruch 24 oder 25, worin DNA, die sowohl fur eine lnhibin-a-als auch eine lnhibin-/8- 
Kette kodiert, in den Vektor eingefUgt wird. 

55 .27. Verfahren nach Anspruch 24 oder 25, worin DNA, die fur eine lnhibin-0-Kette, jedoch nicht fur eine eine 
Inhibin-a-Kette kodiert, in den Vektor eingefUgt wird. 



62 



EP 0 222 491 B1 



28. Wirtszelle, die mit einem replizierbaren Vektor transform iert wurde, der DNA enthalt, die fur eine 
menschliche Oder Schweine-lnhibin-a-Kette und/oder eine -lnhibin-0-Kette, deren Aminosauresequen- 
zen wie in Fig. 1B (Schweine-a-Kette), Fig. 2B (Schweine-0-Ketten), Fig. 6A (menschliche a-Kette) und 
Fig. 8 und 9 (menschliche 0-Ketten) dargestellt sind, oder fur eine durch Einfugung, Loschung Oder 
Substitution eines Polypeptids mit einer der dargestellten Sequenzen erhaltene Aminosauresequenz- 
Variante kodiert, wobei die Variante im wesentlichen homolog zu einem Polypeptid mit einer dargestell- 
ten Sequenz und 1) kreuzreaktiv mit gegen ein Polypeptid mit einer dargestellten Sequenz gebildeten 
Antikorpern; oder 2) kreuzreaktiv mit Zelloberflachen-Rezeptoren fur ein Polypeptid mit einer darge- 
stellten Sequenz ist; oder 3) ahnliche hormonelle Aktivitat wie ein Polypeptid mit einer dargestellten 
Sequenz besitzt. 

29. Wirtszelle nach Anspruch 28, worin die DNA fur eine menschliche oder Schweine-lnhibin-a-Kette 
und/oder eine -lnhibin-0-Kette kodiert, deren Aminosauresequenzen wie in Fig. 1B (Schweine-a-Kette), 
Fig. 2B (Schweine-0-Ketten), Fig. 6A (menschliche a-Kette) und Fig. 8 und 9 (menschliche 0-Ketten) 
dargestellt sind. 

30. Zelle nach Anspruch 28 oder 29, die eine eukaryotische Zelle ist. 

31. Verfahren nach Anspruch 4, worin die gewonnene Inhibin-Kette eine zellfreie Zusammensetzung ist, die 
frei von reifer a-Ketten-Sequeriz ist, die eine menschliche oder Schweine-lnhibin-a-Ketten-Prodomanen- 
Polypeptidsequenz, wie in Fig. 1B (Schweine-a-Kette) oder Fig. 6A (menschliche a-Kette) dargestellt, 
oder eine Aminosauresequenz-Variante einer dieser Prodomanen-Sequenzen enthalt, wobei die Varian- 
te im wesentlichen homolog zu einem Polypeptid mit einer der dargestellten Sequenzen und kreuzreak- 
tiv mit gegen ein Polypeptid mit einer dargestellten Sequenz gebildeten Antikorpern ist. 

32. Verfahren nach Anspruch 4, worin die gewonnene Inhibin-Kette eine zellfreie Zusammensetzung ist, 
umfassend 

a) ein Polypeptid, umfassend die menschliche lnhibin-/S A -Ketten-Prodomanensequenz 
HSAAPDCPSCALAALPKDVPNSQPEMVEAVKKHILNMLHL 

(Aminosauren 1 - 40 aus Fig. 8) 

PDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIG 
(Aminosauren 44 - 80 aus Fig. 8) 

AEMNELMEQTSEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKAN- 

TRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDA 
(Aminosauren 83- 185 aus Fig. 8) 

STWHVFPVSSSIQRLLDQGKSSLDVRIACEQCQESGASLVLLG 
(Aminosauren 188 - 230 aus Fig. 8) 

oder naturlich vorkommende Saugetier-Aminosauresequenz-Varianten davon; 



63 



EP 0 222 491 B1 



b) ein Polypeptid, umfassend die menschliche lnhibin-£ B -Ketten-Prodomanensequenz 

CTSCGGFRRPEELGRVDGDFLEAV 
(Aminosauren 7 - 30 aus Fig. 9) 

HILSRLQMRGRPNITHAVPKAAMVTALRKLHAGKVREDGRVEIPHLDGHASPGAD- 
GQERVSEI ISFAETDGLASSRVRLYFFISNEGNQNLFVVQASLWLYLKLLPYV- 
LEKGS (Aminosauren 33- 145 aus Fig. 9) 

VRVKVYFQEQGHDRWNMVEKRVDLKRSGWHTFPLTEAIQALFERGE 

(Aminosauren 149- 195 aus Fig. 9) 

LiNLDVQCDSCQE LA VVPVFVD PGEESH RPFVVVQARLGDSRHRI 
(Aminosauren 198 - 241 aus Fig. 9) 

Oder naturlich vorkommende Saugetier-Aminosauresequenz-Varianten davon; Oder 

c) ein Polypeptid, das frei von der reifen a-Ketten-Aminosauresequenz ist, umfassend menschlichen 
Inhibin-o-Ketten-Prodomanensequenzen 

(Aminosauren 1 - 24 aus Fig. 6) 

HALGGFTHRGSEPEEEEDVSQAILFPATDASCEDKSAARGLAQEAEEGLFRYMFR- 
PSQHTRSRQVTSAQLWFHTGLDRQGTAASNSSEPLLGLLALSPGGPVAVPMSLGH- 
APPHWAVLHLATSALSLLTHPVLVLLLRCPLCTCSARPEATPFLVAHTRTRPPSG- GERA 

(Aminosauren 32 - 1 99 aus Fig. 6) 

Oder naturlich vorkommende Saugetier-Aminosauresequenz-Varianten davon. 

33. Verfahren nach Anspruch 4, worin die gewonnene Inhibin-Kette eine zellfreie Zusammensetzung ist, 
umfassend ein menschliches Oder Schweine-lnhibin-j8-Ketten-Prodomanen-Polypeptid mit einer Se- 
quenz, wie in Fig. 2B (Schweine-j3-Ketten) und Fig. 8 und 9 (menschliche j8-Ketten) dargestellt, oder 
eine durch Einfugung, Loschung oder Substitution einer dieser Prodomanen-Polypeptide mit einer 
dieser Sequenzen erhaltene Aminosauresequenz- Variante ist, wobei die Variante im wesentlichen 
homolog zu einem Polypeptid mit einer der dargestellten Sequenzen und kreuzreaktiv mit gegen ein 
Polypeptid mit einer dargestellten Sequenz gebitdeten Antikorpern ist. 

34. Verfahren nach Anspruch 33, worin die jS-Kette die 0 A -Kette und die Zusammensetzung frei von reifer 
j8 A -Ketten-Sequenz ist. 

35. Verfahren nach Anspruch 33, worin die jS-Kette die 0 B -Kette und die Zusammensetzung frei von reifer 
j8 B -Ketten-Sequenz ist. 

36. Verfahren nach Anspruch 32, worin die Variante die entsprechende Schweine-Aminosauresequenz. ist, 
wobei die Zusammensetzung enthalt: 

a) ein Polypeptid, umfassend die Schweine-lnhibin-/8 A -Ketten-Prodomanensequenz, die beschrieben 
wird durch die Aminosauren: 28 - 58; 61 - 87; 90 - 108; 111 - 179; 182 - 213; 216 - 258; Oder 28 - 
87 aus Fig. 2B; oder 

b) ein Polypeptid, das frei von der reifen a-Ketten-Aminosauresequenz ist, umfassend die Schweine- 
Inhibin-a-Ketten-Prodomanen-Polypeptidsequenzen, die beschrieben werden durch die Aminosau- 
ren: 20 - 54 oder 70 - 228 aus Fig. 1B. 



64 



EP 0 222 491 B1 



37. Verfahren nach irgendeinem der Anspruche 31 - 36, worin das Polypeptid nicht von nativer Glykosylie- 
rung begleitet wird. 

38. Verfahren nach irgendeinem der Anspruche 31 - 37, worin die Zusammensetzung steril ist, und weiters 
6 umfassend das Verbinden des Polypeptids mit einem immunogenen Polypeptid. 

39. Verwendung eines Polypeptids nach irgendeinem der Anspruche 31 - 38 zur Herstellung eines 
Antikorpers, der in der Lage ist, das Polypeptid zu binden. 

10 40. Verfahren nach irgendeinem der Anspruche 31 - 38, weiters umfassend das Konjugieren des Polypep- 
tids an eine feststellbare Gruppe. 

41. Verfahren nach Anspruch 40, worin die Gruppe ein Enzym, Fluorophor Oder Radioisotop ist. 

75 42. Verfahren nach irgendeinem der Anspruche 31 - 38, 40 Oder 41, worin die Zusammensetzung durch 
nicht-kovatente Absorption oder kovalente Vernetzung an einen wasserunloslichen Trager unloslich 
gemacht ist. 

43. Verfahren nach irgendeinem der Anspruche 31 - 38, 40 oder 41 , weiters umfassend das Mischen der 
20 Zusammensetzung mit einer physiologisch annehmbaren, implantierbaren Matrix zur gesteuerten Frei- 
setzung des Polypeptids in die Gewebe eines Tieres. 

Revendications 

Revendications pour les Etats contractants suivants : BE, CH, DE, FR, GB, IT, LI, NL, SE 

25 

1. Procede comprenant la culture d'une cellule hote transform^ avec un vecteur qui renferme un acide 
nucleique codant pour une chatne a d'inhibine humaine ou porcine et/ou pour une chaTne 0 d'inhibine 
humaine ou porcine dont les sequences d'aminoacides sont representees sur la figure 1B (chaTne a 
porcine), la figure 2B (chaTnes 0 porcine), la figure 6A (chaTne a humaine) et les figures 8 et 9 (chaTnes 

30 £ humaines), ou un variant de sequence d'amino-acides forme par insertion, deletion ou substitution 
d'une des sequences representees, le variant etant fortement homologue avec un polypeptide d'une 
sequence representee mais excluant la chaTne a d'inhibine bovine et la chaTne 0 partielle d'inhibine 
bovine de sequence 

35 Gly-Leu-<31u-Cys-Asp- 
Gly-Lys-Val-Asn-Ile-Cys-CVs-Lys-Lys-Gln-Phe-Phe-Val-Ser-Phe- 
Lys-Asp-Ile-Gly-Trp-Asn-Asp-Trp-Ile-Ile-Ala-Pro-Ser*Gly-Tyr- 

4o His-Ala-Asn-Tyr-Cys-Glu-Gly-Glu-Cys-Pro-Ser-His-Ile-Ala-Gly- 
Thr-Ser-Gly-Ser-Ser-Leu-Ser-Phe-His-Ser-Thr-Val«Ile-Asn-His- 
Tyr-Arg-Met-Arg-Gly-His-Ser, 

45 et 

1) presentant une reactivate croisee avec des anticorps engendres contre un polypeptide d'une 
sequence representee ; ou 2) presentant une reactivite croisee avec des receptees de surface 
cellulaire pour un polypeptide d'une sequence representee, ou 3) presentant une activite hormonale 
similaire a celle d'un polypeptide d'une sequence representee. 

50 

2. Procede suivant la revendication 1, dans lequel I'acide nucieique code pour une chaTne a d'inhibine 
humaine ou porcine et/ou une chaTne jS d'inhibine ou porcine dont les sequences d'amino-acides sont 
representees sur la figure 1 B (chaTne a porcine), la figure 2B (chaTnes 0 porcines), la figure 6A (chaTne 
a humaine) et les figures 8 et 9 (chaTnes 0 humaines). 

55 

3. Procede suivant la revendication 1 , dans lequel le vecteur code pour une chaTne /S d'inhibine, ou un de 
ses variants de sequence d'amino-acides, autre que la chaTne £ A humaine ou porcine naturelle ou la 
chaTne & d'inhibine bovine partielle specifiee dans la revendication 1 . 



65 



EP 0 222 491 B1 



4. Proc£de suivant la revendication 1, dans tequel I'acide nucleique code pour un variant choisi entre : 
des derives d'inhibine porcine [Asn266—Gln]lnha ; [Cyss25 ou CyS324-*A]lnha ; [CyS3&i ou CyS36 3— 'AJ- 
Inha ; [Lys32i ou Lys322^A]lnh0 A ; [Lys322— His ou Ser]lnh0 A ; [LyS3i5~*Arg ; Valsu— Thr] lnh/8 A ; 
[Cyss88 ou CyS39o-*A]lnh/5 A ,[LyS4ii- ,, Gln]lnhj8A ; [Argsis-^Lys ; Val3H- , Thr]lnh0 B ; {Cys 3 is ou 

5 CyS32o- > A]lnh/9 B ; [Pro 3 8i Gly 3 s2— Pro Phe Gly]lnh0 B ; [Arg 39 s— Gln]lnhj8 B , dans lesquel Inh est une 
abr^viation d ? inhibine et les num^ros des residus pour la lnhj9 B sont ceux utilises pour le r^sidu de 
lnh£ A correspondent (voir figure 2B) ; les variants de chaTne £ A d'inhibine humaine poss^dant des 
variations qui consistent en une substitution ou une deletion au niveau d'un, ou une insertion aprfcs un, 
residu choisi entre les residus 293 a 297, 364 a 376 et 387 a 398 de la sequence representee sur la 

to figure 8 ; et des variants de la chatne a humaine qui pnSsentent une homologie de plus de 90 % avec 
la sequence representee sur la figure 6A. 

5. Procede suivant la revendication 1, dans lequel I'acide nucleique code pour un variant d'une chaTne 
d'inhibine 0 A humaine choisi entre Phe 3 o2^He ou Leu ; Gln2S7-*Asp ou Lys ; Trp 30 7-^Tyr ou Phe ; 

J5 . Trp3io—Tyr ou Phe ; lle 3 u— Phe ou Val ; Tyr 3 i7— Trp ou Thr ; Hissi8-*Lys ; Alasig-^Ser ; Asn32o-*Gln, 
Tyr ou His ; Tyr 3 2i— Thr ou Asp, Phes40-*Tyr ; His 353 — Asp ; His 3 53— Lys (a un hybride jS A /£ B ) ; 
Phe 3 56-*Tyr ; Vals64-^Phe ; Val3&4— Leu ; Tyr 3 75—Thr ; Tyr 3 7&-*Trp ; Asn389-*Gln, His ou Lys ; 
lle 39 i-*Leu ou Thr ; Metsso-^Leu ou Ser ; Val 392 ^Phe, Glu, Thr ou He ; ou une chaTne /S B humaine 
modifi6e de maniere comparable. 

20 

6. Procede suivant Tune quelconque des revendications precedentes, dans lequel I'acide nucleique 
codant pour la chaTne a et/ou la chaTne 0 d'inhibine est lie de maniere fonctionnelle a un promoteur 
reconnu par la cellule note et contenant Tetape supplemental de separation de I'inhibine ou d'un 
dimere de chaTne 0 du milieu de culture. 

25 

7. Procede suivant Tune quelconque des revendications precedentes, dans lequel la cellule est un 
procaryote. 

8. Procede suivant Tune quelconque des revendications pr^cedentes, dans lequel le vecteur comprend un 
30 acide nucieique codant pour la forme pre-pro d'une chaTne a d'inhibine ou d'une chaTne 0 d'inhibine. 

9. Procede suivant la revendication 8, dans lequel le vecteur comprend un acide nucleique codant pour la 
forme pre-pro de la chaTne a d'inhibine et de la chaTne /? d'inhibine. 

35 10. Procede suivant Tune quelconque des revendications 1 a 6, 8 et 9, dans lequel la cellule est une 
cellule provenant d'un organisme pluricellulaire et une inhibine douee d'activite hormonale est produite. 

11. Procede suivant la revendication 6, dans lequel le promoteur est un promoteur viral. 

40 12. Procede suivant la revendicatioon 11, dans lequel le promoteur est un promoteur de SV40. 

13. Procede suivant I'une quelconque des revendications precedentes, dans lequel de I'inhibine porcine ou 
humaine mature est recueillie. 

45 14. Procede suivant I'une quelconque des revendications 1 a 7 ou 10 a 12, dans lequel le vecteur 
comprend un acide nucieique codant pour la forme pre-pro d'une chaTne ■& d'inhibine et un dimere de 
chaTne & mature est recueilli en ('absence de chaTne a. 

15. Procede suivant la revendication 10 ou 13, dans lequel la chaTne 0 est la chaTne 0 A et I'inhibine 
so presente a une concentration superieure a environ 20 ng/ml de milieu de culture. 

16. Composition contenant de I'inhibine humaine ou porcine constituee d'une chaTne o et d'une chaTne 0, 
les sequences d'amino-acides desdites chaTnes a et £ eXanX choisies entre celles representees sur la 
figure 1 B (chaTne o porcine), la figure 2B (chaTnes & porcines), la figure 6A (chaTne a humaine) et les 

55 figures 8 et 9 (chaTnes 0 humaines), et des variants de sequence d'amino-acides engendres par 
insertion, deletion ou substitution d'un polypeptide d'une desdites sequences representees, variants qui 
sont fortement homologues avec un polypeptide d'une sequence representee, mais a I'exclusion de la 
chaTne a d'inhibine bovine, et 1) qui pr^sentent une reactivite croisee avec des antlcorps engendres 



66 



EP 0 222 491 B1 



contre un polypeptide cTune sequence representee ; 2) qui presentent une reactivity croisee avec les 
r^cepteurs de surface cellulaire pour un polypeptide d'une sequence representee ; ou 3) qui possectent 
une activity hormonale similaire a celle d'un polypeptide d'une sequence representee ; composition qui 
est totalement depourvue de prolines humaines ou pdrcines non identifiees. 

5 

17. Composition suivant la revendication 16, dans laquelle les sequences des chaTnes a et £ sont choisies 
entre celles representees sur la figure 1 B (chaTne a porcine), la figure 2B (chatne 0 porcine), la figure 
6A (chaTne a humaine) et les figures 8 et 9 (chaTnes 0 humaines). 

70 18. Composition suivant la revendication 16, dans laquelle les variants sont choisis entre : des derives 
d'inhibine porcine (Asn2&6— ln]Glnha ; [Cys325 ou Cys324— A]lnho ; [CyS36i ou CyS363— A]lnha ; 
[Lys 3 2i ou Lys 32 2— A]lnh/S A ; [Lys 3 22- f His ou Ser]lnhj8 A ; Lys 3 i5^Arg ; Vabu-Thr] lnh/9 A ; [Cys 38 8 ou 
Cys39o-A]lnhj8 A , [Lys*i i-"Gln]lnh£ A ; [Arg 3 i 5 ->Lys ; Val 3 i6-Thr]lnh/S B ; [Cys 3 i9 ou Cys 3 2o— A]lnh/8 B - 
[Prossi 0^382-* Pro Phe Gly]lnh£ B ; [Arg 395 — Gln]lnh£ B , dans lesquels Inh repr^sente une abr^viation 

75 d'inhibine et les num^ros des r^sidus pour lnhj8 B sont ceux utilises pour le r^sidu lnh£ A correspondant 
(voir figure 2B) ; des variants de chaTne 0 A d'inhibine humaine j9 A possedant des variations qui 
consistent en une substitution ou une deletion au niveau d'un, ou une insertion apres un, residu choisi 
entre les residus 293 a 297, 364 a 376 et 387 a 398 de la sequence representee sur la figure 8 ; et des 
variants de chaTne a humaine qui presentent une homologie de plus de 90 % avec la sequence 

20 representee sur la figure 6A. 

19. Composition suivant la revendication 16, comprenant un variant d'une chaTne d'inhibine $ A humaine 
choisi entre Phe302— Me ou Leu ; G1n297-*Asp ou Lys ; Trp 30 7-*Tyr ou Phe ; Trp3io—Tyr ou Phe ; 
He3i 1— Phe ou Val ; Tyr 3 i7-*Trp ou Thr ; His 3 is-»Lys ; Ala3i9— Ser ; Asn 3 2o- H *Gln, Tyr ou His ; 
25 Tyr 3 2i-»Thr ou Asp, Phe3«o— Tyr ; His 35 3-»Asp ; His35 3-*Lys ; (un hybride 0 A /0 B ) ; Phe 3 s&-*Tyr ; 
Val36A^Phe ; Val 3 &4— Leu ; Tyr 3 75— Thr ; Tyr 3 76— Trp ; Asn 38 9— Gin, His ou Lys ; lle 3 9i-*Leu ou Thr ; 
Met39o—Leu ou Ser ; Val 3 92-*Phe, Glu, Thr ou lie ; ou une chatne 0 B humaine modifiee de maniere 
comparable. 

30 20. Composition comprenant un prodomaine d'inhibine a ou 0 B humaine ou porcine representee sur la 
figure 1B (chaTne o porcine), la figure 2B (chaTne j8 B porcine), la figure 6A (chaTne a humaine) et la 
figure 9 (chaTne £ B humaine) non associe" a une glycosylation naturelle. 

21. Composition comprenant un homodimere de chaTnes 0 A ou 0 B d'inhibine humaine ou porcine mature, 
35 lesdites chaTnes etant representees sur la figure 2B (chaTnes 0 porcines) et les figures 8 et 9 (chaTnes 

j9 humaines), ou d'un variant de sequence d'amino-acides engendre par insertion, deletion ou 
substitution d'un polypeptide d'une des sequences representees, variant qui presente une forte 
homologie avec un polypeptide d'une sequence representee et 1) qui presente une reactivite croisee 
avec des anticorps engendres contre un polypeptide d'une sequence representee ; 2) qui presente une 
40 reactivite croisee avec des recepteurs de surface cellulaire pour un polypeptide d'une sequence 
representee ; ou 3) qui Presente une activite hormonale similaire a celle d'un polypeptide d'une 
sequence representee ; composition qui est depourvue de la chaTne a d'inhibine. 

22. Composition suivant la revendication 21, qui est un homodimerelde chaTnes /3 humaine ou porcine 
45 mature ou d'un de ses variants de sequence d'amino-acides. 

23. Composition comprenant un heterodimere d'inhibine 0 A humaine ou porcine mature avec I'inhibine 0 B 
humaine ou porcine mature, lesdites chaTnes etant representees sur la figure 2B (chaTnes jS porcines) 
et les figures 8 et 9 (chaTnes 0 humaines), ou d'un variant de sequence d'aminoacides engendre par 

so insertion, deletion ou substitution d'une des sequences representees, le variant presentant une forte 
homologie avec un polypeptide d'une sequence representee et 1) presentant une reactivite croisee 
avec des anticorps engendres contre un polypeptide d'une sequence representee ; 2) presentant une 
reactivite croisee avec des recepteurs de surface cellulaire pour un polypeptide d'une sequence 
representee ; ou 3) presentant une activite hormonale similaire a celle d'un polypeptide d'une 

55 sequence representee ; composition qui est depourvue de la chaTne a d'inhibine. 

24. ADN non chromosomique codant pour une chaTne a d'inhibine humaine ou porcine ou d'une chaTne 0 
d'inhibine humaine ou porcine dont les sequences d'amino-acides sont decrites sur les figures 1B 



67 



EP 0 222 491 B1 



(chaTne q porcine), 2B (chaTnes & porcines), 6A (chaTne a humaine) et 8 et 9 (chatnes & humaines) ou 
pour un variant de sequence d'amino-actdes engendre par insertion, deletion ou substitution d'un 
polypeptide d'une desdites sequences representees, ie variant presentant une forte homologie avec un 
polypeptide d'une sequence representee, a I'exclusion de la chaTne a d'inhibine bovine et de la chaTne 
5 $ d'inhibine bovine partielle de sequence 

Gly-Leu-Glu-Cys-Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys- 
Lys-Gln-Phe-Phe-Val-Ser-Phe-Lys-Asp-Ile-Gly-Trp-Asn-Asp-Trp- 
"> He-Ile-Ala-Pro-Ser-Gly-Tyr-His-Ala-Asn-Tyr-Cys-Glu»Gly-Glu- 
Cys-Pro-Ser-His-Ile-Ala-Gly-Thr-Ser-Gly-Ser-Ser-Leu»Ser-Phe- 
His-Ser-Thr-Val-Ile-Asn-His-Tyr-Arg-Met-Arg-Gly-His-Ser, 

75 

et 

1) presentant une reactivite croisee avec des anticorps engendres contre un polypeptide d'une 
sequence representee ; ou 2) presentant une reactivite croisee avec des recepteurs de surface 
cellulaire pour un polypeptide d'une sequence representee, ou 3) presentant une activite hormonale 
20 similaire a celle d'un polypeptide d'une sequence representee. 

25. ADN suivant la revendication 24, codant pour une chaTne a d'inhibine humaine ou porcine ou une 
chaTne 0 d'inhibine humaine ou porcine dont la sequence d'amino-acides est representee sur la figure 
1 B (chaTne a porcine), la figure 2B (chaTnes p porcines), la figure 6A (chaTne a humaine) et les figures 

25 8 et 9 (chaTnes @ humaines). 

26. ADN suivant la revendication 24, qui code pour un variant choisi entre : des derives d'inhibine porcine 
[Asn26&-*Gln]lnha ; [Cyss25 ou Cys324-*A]lnha ; [CyS36i ou CyS363-*A]lnha ; [Lys32i ou Lys322-*AJ- 
lnhj8 A ; [Lys 3 22^His ou Ser]lnh0 A ; [Lyssu^Arg ; Vafets-Thr] lnhj8 A ; [Cys 3 88 ou Cys 390 -*A]lnh A , 

30 [Lys*n— Gln]lnh0 A ; [Arg3i 5 —Lys ; Valsie— Thr]lnh/8 B ; [Cyssis ou Cys 3 2o— A]lnh/9 B ; [Prossi 
Gly 3 82- ,, Pro Phe-Gly]lnhj8 B ; [Arg3g 5 —Gln]lnhj8 B , dans lesquel Inh est une abreviation d'inhibine et les 
numeros des residus pour la lnh0 B sont ceux utilises pour le residu de lnh£ A correspondents (voir 
figure 2B) ; les variants de chaTne jS A d'inhibine humaine possedant des variations qui consistent en 
une substitution ou une deletion au niveau d'un, ou une insertion apres un, residu choisi entre les 

35 residus 293 a 297, 364 a 376 et 387 a 398 de la sequence representee sur la figure 8 ; et des variants 
de la chaTne a humaine qui presentent une homologie de plus de 90 % avec la sequence representee 
sur la figure 6A. 

27. ADN suivant la revendication 24, qui code pour un variant d'une chaTne j3 d'inhibine humaine choisi 
40 entre Phe 3 o2-*He ou Leu ; Gln2S7—Asp ou Lys ; Trp3 0 7-*Tyr ou Phe ; Trp3io-*Tyr ou Phe ; lle3ii-*Phe 

ou Val ; Tyr 3 i7-»Trp ou Thr ; Hissis— Lys ; Ala3is— Ser ; Asn 32 o-*Gln ( Tyr ou His ; Tyr 3 2i- k Thr ou Asp, 
Phe34o-*Tyr ; His 35 3— Asp ; Hiss53— Lys (a un hybride 0 A /0 B ) ; Phe 35 6-*Tyr ; Vab64 — Phe ; Val3S4->Leu 
; Tyr37s-»Thr ; Tyr 37 6-*Trp ; Asnsas-^GIn, His ou Lys ; lle39i-*Leu ou Thr ; Met39o—Leu ou Ser ; 
Val392- |, Phe, Glu, Thr ou He ; ou une chaTne jS B humaine modifiee de maniere comparable. 

45 

28. ADN suivant Tune quelconque des revendications 24 a 27, qui est depourvu de sequences non 
traduites intermedial res. 

29. ADN suivant Tune quelconque des revendication 24 a 28, qui est marque avec un groupement 
so detectable. 

30. Vecteur replicable comprenant un ADN suivant I'une quelconque des revendications 21 a 27. 

31. Vecteur suivant la revendication 30, comprenant un promoteur viral lie de mani§re fonctionnelle a I'AON 
65 codant pour les chaTnes a et/ou & d'inhibine. 

32. Vecteur suivant la revendication 30 ou 31, qui contient un ADN codant a la fois pour une chaTne a 
d'inhibine et une chaTne fi d'inhibine. 



€8 



EP 0 222 491 B1 



33. Vecteur suivant ta revendication 30 ou 31, qui contient un ADN codant pour une chatne 0 d'inhibine, 
mais pour la chaTne a d'inhibine. 

34. Cellule note transformation avec un vecteur replicable comprenant I'ADN codant pour la chaTne a et/du 
5 la chaTne 0 d'inhibine humaine ou porcine dont les sequences d'amino-acides sont representees sur la 

figure 1B (chaTne a porcine), la figure 2B (chaTnes 0 porcines), la figure 6A (chaTne a humaine) et les 
figures 8 et 9 (chaTnes 0 humaines) ou un variant de sequence d'amino-acides engendre par insertion, 
deletion ou substitution d'un polypeptide d'une telle sequence representee, le variant etant pratique- 
ment homologue avec un polypeptide d'une sequence representee, a I'exclusion de la chaTne a 
70 d'inhibine bovine et de la chaTne £ d'inhibine bovine partielle de sequence 

Gly-Leu-Glu-Cys- 

Asp-Gly-Lys-Val-Asn-Ile-Cys-Cys-Lys-Lys-Gln-Phe-Phe-Val-Ser- 
75 Phe-Lys-Asp-Ile-Gly-Trp-Asn-Asp-Trp-Ile-Ile-Ala-Pro-Ser-Gly- 
Tyr-His-Ala-Asn-Tyr-Cys-Glu-Gly-Glu-Cys-Pro-Ser-His-Ile-Ala- 
Gly-Thr-Ser-Gly-Ser-Ser-Leu-Ser-Phe-His-Ser-Thr-Val-Ile-Asn- 
2Q His-Tyr-Arg-Met-Arg-Gly-His-Ser, 

et 

1) presentant une reactivite croisee avec des anticorps engendres contre un polypeptide d'une 
sequence representee ; ou 2) presentant une reactivite croisee avec des recepteurs de surface 
25 cellulaire pour un polypeptide d'une sequence representee, ou 3) presentant une activite hormonale 
similaire a celle d'un polypeptide d'une sequence representee. 

35. Cellule hote suivant la revendication 34, dans laquelle i'ADN code pour une chaTne a et/ou une chaTne 
i9 d'inhibine humaine porcine, dont les sequences d'amino-acides sont representees sur la figure 1B 

30 (chaTne a porcine), la figure 2B (chaTnes 0 porcines), la figure 6A (chaTne a humaine) et les figures 8 et 
9 (chaTnes 0 humaines). 

36. Cellule hote suivant la revendication 34, dans laquelle I'ADN code pour un variant choisi entre : des 
derives d'ihibine porcine [Asn 2 66—Gln]lnha ; [Cys 3 25 ou Cys 3 24—A]lnha ; [Cys 3 ei ou CyS363— A]lnha ; 

35 [Lys32i ou Lys 3 22-*A]lnh/8 A ; [Lys322^His ou Ser]lnh/8 A ; [LySais^Arg ; Va^u—Thr] lnhj8 A ; [CyS388 
ou Cys 39o -'A]lnh0 A ,[LyS4ii-»Gln]lnhj8 A ; [Arg 3 i 5 — Lys ; Val 3 i6-Thr]lnh)3B ; [Cys 3 i 9 ou Cys 32 o-*A]- 
Inh^B ; [Prossi Gly382-»Pro Phe Gly]lnh/9 B ; [Argss^—Glnllnh^B, dans lesquel Inh est une abreviation 
d'inhibine et les numeros des residus pour la lnhjS B sont ceux utilises pour le residu de lnh0 A 
correspondant (voir figure 2B) ; les variants de, chaTne 0 A d'inhibine humaine possedant des variations 

40 qui consistent en une substitution ou une deletion au niveau d'un, ou une insertion apres un, residu 
choisi entre les residus 293-297, 364-376 et 387-398 de la sequence representee sur ta figure 8 ; et 
des variants de la chaTne a humaine qui presentent une homologie de plus de 90 % avec ta sequence 
representee sur la figure 6A. 

45 37. Cellule hote suivant ia revendication 34, dans laquelle I'ADN code pour un variant d'une chaTne £ A 
d'inhibine humaine choisie entre Phe302-*He ou Leu ; Gln297-*Asp!ou Lys ; Trp 3 o7—Tyr ou Phe ; 
Trpsio-^Tyr ou Phe ; Ile3n—Phe ou Val ; Tyrs^—Trp ou Thr ; Hissis— Lys ; Ala3i 9 — Ser ; Asns2o— Gin, 
Tyr ou His ; Tyr32i— Thr ou Asp, Phe 3 4o—Tyr ; His 3 53— Asp ; His 3 53— Lys (a un hybride 0 A /0 B ) ; 
Phe 3 56-*Tyr ; Valse*— Phe ; Val 3 64-»Leu ; Tyr 37 5— Thr ; Tyr 3 76—Trp ; Asn 38 9-> Gin, His ou Lys ; 

so lle 3 9i— Leu ou Thr ; Met3go-*Leu ou Ser ; Val 3 92— Phe, Glu, Thr ou lie ; ou une chaTne j8 B humaine 
modifiee de maniere comparable. 

38. Cellule suivant Tune quelconque des revendications 34 a 37, qui est une cellule eucaryotique. 

55 39. Composition acellulaire qui est depourvue d'un polypeptide de chaTne a mature, contenant une 
sequence polypeptidique de prodomaine de chaTne a d'inhibine humaine ou porcine representee sur la 
figure 1 B (chaTne o porcine) ou la figure 6A (chaTne o humaine) ou un variant de sequence d'amino- 
acides engendre par une insertion, deletion ou substitution d'une telle sequence de prodomaine, le 



69 



EP 0 222 491 B1 



variant presentant une forte homologie avec un polypeptide d'une desdites sequences pr^sentant une 
reactivity crois^e avec des anticorps engendr^s contre un polypeptide d'une sequence representee. 

40. Composition acellutaire contenant 

a) un polypeptide comprenant la sequence de prodomaine de chatne £ d'inhibine humaine 

HSAAPDCPSGALAALPKDVPNSQPEMVEAVKKHILNMLHL (aminoacides 1 a 40 

de la figure 8) , t PDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIG 
(aminoacides 44 & 80 de la figure 8), 

AEMNELMEQTSEIITFAESGTARKTLHFEISKEGSDLSWERAEVWLFLKVPKAN- 
RTRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDA ( aminoacides 
83 a 185 de la figure 8), STWHVFPVSSSIQRLLDQGKSSLDVRIACEQ- 
CQESGASLVLLG (amino-acides 188 a 230 de la figure 8), 

ou un de ses variants de sequence d'amino-acides naturels de mammifere ; 

b) un polypeptide comprenant la sequence de prodomaine de chatne 0b d'inhibine humaine 

CTSCGGFRRPEELGRV- 
DGDFLEAV ( amino -acides) 7-30 de la figure 9, 
HILSRLQMRGRPNITHAVPKAAMVTALRKLHAGKVREDGRVEIPHLDGHASPGAD- 

GQERVSEIISFAETDGLASSRVRLYFFISNEGNQNLFWQASLWLYLKLLPYVLEKGS 
(amino-acides 33 a 5 de la figure 9) , 

WVKVYFQEQGHGDRWNMVEKRVDLKRSGWHTFPLTEAIQALFERGE (amino-acides 
149 a 195 de la figure 9) , 

LNLDVQCDSCQELAWPVFVDPGEESHRPFVWQARLGDSRHRI ( amino- acides 
198 a 241 de la figure 9) , 

ou un de leurs variants de sequence d'amino-acides naturels de mammifere ; ou 

c) un polypeptide d£pourvu de la sequence d'amino-acides de chaTnes a mature comprenant les 
sequences de prodomaine de chatne a d'inhibine humaine 

KVRALFLDALGPPAVTREGGDPGV (aminoacides 1 3 24 de la figure 6), 
HALGGFTHRGSEPEEEEDVSQAILFPATDASCEDKSAARGLAQEAEE<3LFRYMFR- 
PSQHTRSRQVTSAQLWFHTGLDRQGTAASNSSEPLLGLLALSPGGPVAVPMSLGH- 

APPHWAVLHIATSALSLLTHP\njVLLL^ 
(amino-acides 32 a 199 de la figure 6) , 

ou un de leurs variants de sequence d'amino-acides naturels de mammifere. 

41. Composition acellulaire contenant un polypeptide de prodomaine de chatne 0 d'inhibine humaine ou 
porcine d'une sequence representee sur la figure 2B (chatnes 0 porcines) et les figures 8 et 9 (chatnes 
0 humaines) ou un variant de sequence d'amino-acides engendre* par insertion, de* lotion ou substitution 
d'une desdites sequences de prodomaine, variant qui prgsente une forte homologie avec un polypepti- 



70 



EP 0 222 491 B1 

de d'une desdites sequences et qui presente une r^activite croisee avec des anticorps engendr^s 
contre un polypeptide d'une sequence representee. 

42, Composition suivant la revendication 41, dans laquelle la chaTne /3 et la chaTne 0 A et la composition est 
5 d£pourvue de sequence de chaTne /9 A mature. 

43. Composition suivant la revendication 41 , dans laquelle la chaTne 0 est la chaTne j3 B et la composition 
est depourvue de sequence de chaTne /8 B mature. 

10 44. Composition suivant la revendication 40, dans laquelle le variant est la sequence d'amino-acides 
porcine correspondante, composition contenant : 

a) un polypeptide comprenant la sequence de prodomaine de chaTne & A d'inhibine porcine 
representee par : les amino-acides 28 a 58 ; les amino-acides 61 a 87 ; les amino-acides 90 a 108 ; 
les amino-acides 111 a 179 ; les amino-acides 182 a 213 ; les amino-acides 216 a 258 ; ou les 

75 amino-acides 28 a 87 de la figure 2B ; ou 

b) un polypeptide depourvu de la sequence d'amino-acides de chaTne a mature comprenant les 
sequences de polypeptides de prodomaine de chaTne a d'inhibine porcine representees par : les 
amino-acides 20 a 54 ; ou les amino-acides 70 a 228 de la figure 1 B. 

20 45. Composition suivant I'une quelconque des revendications 39 a 44, dans laquelle le polypeptide est non 
accompagne par une glycosylation naturelle. 

46. Composition suivant Tune quelconque des revendications 39 a 45, qui est sterile et dans laquelle le 
polypeptide comprend en outre un polypeptide immunogene. 

25 

47. Utilisation d'une composition suivant Tune quelconque des revendications 39 a 46 dans la preparation 
d'un anticorps capable de se lier audit polypeptide. 

48. Composition suivant I'une quelconque des revendications 39 a 46, dans laquelle le polypeptide est 
30 conjugue a un groupe detectable. 

49. Composition suivant la revendication 48, dans laquelle le groupe est un enzyme, un fluorophore ou un 
radioisotope. 

35 50. Composition suivant I'une quelconque des revendications 39 a 44, 48 et 49, qui est insolubilisee par 
une absorption non covalente ou une reticulation covalente a un support insoluble dans I'eau. 

51. Composition suivant I'une quelconque des revendications 39 a 46, 48 et 49, comprenant en outre une 
matrice implantable physiologiquement acceptable pour la liberation controiee du polypeptide dans les 
40 tissus d'un animal. 

Revendications pour I'Etat contractant suivant : LU 

1. Procede comprenant la culture d'une cellule note transformee avec un vecteur qui renferme un acide 
45 nucieique codant pour une chaTne a d'inhibine humaine ou porcine et/ou pour une chaTne 0 d'inhibine 

humaine ou porcine dont les sequences d'amino-acides sont representees sur la figure 1 B (chaTne a 
porcine), la figure 2B (chaTnes /3 porcines), la figure 6A (chaTne a humaine) et les figures 8 et 9 
(chaTnes £ humaines), ou un variant de sequence d'amino-acides forme par insertion, deletion ou 
substitution d'une des sequences representees, le variant etant fortement homologue avec un poly pep- 
so tide d'une sequence representee mais excluant la chaTne a d'inhibine bovine et 1) qui presente une 
reactivity croisee avec des anticorps engendres contre un polypeptide d'une sequence representee ; 
ou 2) qui presente une reactivite croisee avec des recepteurs de surface cellulaire pour un polypeptide 
d'une sequence representee ; ou 3) qui presente une activity hormonale similaire a celle d'un 
polypeptide d'une sequence representee. 

55 

2. Procede suivant la revendication 1, dans lequel I'acide nucieique code pour une chaTne a d'inhibine 
humaine pu porcine et/ou une chaTne 0 d'inhibine ou porcine dont les sequences d'amino-acides sont 
representees sur la figure 1B (chaTne a porcine), la figure 2B (chaTnes 0 porcines), la figure 6A (chaTne 



71 



EP 0 222 491 B1 



a humaine) et les figures 8 et 9 (chaTnes 0 humaines). 

3. Procede* suivant ta revendication 1 , dans lequel le vecteur code pour une chatne £ d'inhibine, ou un de 
ses variants de sequence d'amino-acides, autre que la /S A humaine ou porcine naturelle. 

5 

4. Procede suivant la revendication 1, dans lequel I'acide nucleique code pour un variant choisi entre : 
des derives d'inhibine porcine [Asn 2&6 -*Gln]lnha ; [Cys 325 ou Cys324— A]lnha ; {Cysssi ou Cys 3 &3*-*A]- 
Inha ; [Lys32i ou Lys322—A]lnh0 A ; [Lys322-»His ou Ser]lnhj9 A ; [Lys 3 is— Arg ; Val 3 i 6 -*Thr] lnh/3 A ; 
[Cys 3 88 ou Cys 3 9o—A]lnh/3 A ,[LyS4ii— Gln]lnhi9 A ; [Arg 3 i 5 — Lys ; Vabu^ThrJInhfo ; [Cys3i9 ou 

io Cys 32 o— A]lnhj3 B ; [Pro 3 si Gly 3 82^Pro Phe Gly]lnhj9 B ; [Arg 39 5->Gln]lnh/3 B , dans lesquel Inn est une 
abreviation d'inhibine et les num^ros des r^sidus pour la lnh£ B sont ceux utilises pour le r^sidu de 
lnh0 A correspondent (voir figure 2B) ; les variants de chatne j9 A d'inhibine humaine possedant des 
variations qui consistent en une substitution ou une deletion au niveau d'un, ou une insertion apres un, 
residu choisi entre les residus 293 a 297, 364 a 376 et 387 a 398 de la sequence representee sur la 

is figure 8 ; et des'variants de la chatne a humaine qui pr^sentent une homologie de plus de 90 % avec 
la sequence representee sur la figure 6A. 

5. Procede suivant la revendication 1, dans lequel I'acide nucleique code pour un variant d'une chatne 
d'inhibine 0 A humaine choisi entre Phe302— He ou Leu ; Gl^gy—Asp ou Lys ; Trp 3 o7- ,, Tyr ou Phe ; 

20 Trp 3 io-*Tyr ou Phe ; lle 3 n-*Phe ou Val ; Tyr 3 i 7 ^Trp ou Thr ; His 3 i8-*Lys ; Ala 3 i 9 -*Ser ; Asn 320 -*Gln, 
Tyr ou His ; Tyr 32 i—Thr ou Asp, Phe 3 4o—Tyr ; His 353 -»Asp ; HiS353-*Lys (a un hybride j8 A /0 B ) ; 
Phe 3 5G~»Tyr ; Val 3 &«— Phe ; Vabe^Leu ; Tyr 37 5-^Thr ; Tyr 3 77—Trp ; Asnsss—GIn, His ou Lys ; 
Ile39i— Leu ou Thr ; Met3 9 o— -Leu ou Ser ; Val392— Phe, Glu, Thr ou He ; ou une chatne 0 B humaine 
modified de maniere comparable. 

25 

6. Procede suivant Tune quelconque des revendications precedentes, dans lequel I'acide nucleique 
codant pour la chatne a et/ou la chatne 0 d'inhibine est lie de maniere fonctionnelle a un promoteur 
reconnu par la cellule note et contenant I'etape supplemental de separation de I'inhibine ou d'un 
dime re de chatne jS du milieu de culture. 

30 

7. Proced§ suivant Tune quelconque des revendications precedentes, dans lequel la cellule est un 
procaryote. 

8. Procede suivant Tune quelconque des revendications precedentes, dans lequel le vecteur comprend un 
as acide nucleique codant pour la forme pre-pro d'une chatne a d'inhibine d'une chatne /8 d'inhibine. 

9. Procede suivant la revendication 8, dans lequel le vecteur comprend un acide nucleique codant pour la 
forme de pre-pro de la chatne a d'inhibine et de la chatne 0 d'inhibine. 

40 10. Procede suivant Tune quelconque des revendications 1 a 6, 8 et 9, dans lequel la cellule est une 
cellule provenant d'un organisme pluricellulaire et une inhibine douee d'activite hormonale est produite. 

11. Procede suivant la revendication 6, dans lequel le promoteur est un promoteur viral. 

45 12. Procede suivant la revendicatioon 11, dans lequel le promoteur est un promoteur de SV40. 

13. Procede suivant Tune quelconque des revendications precedentes, dans lequel de I'inhibine porcine ou 
humaine mature est recueillie. 

50 14. Procede suivant I'une quelconque des revendications 1 a 7 ou 10 a 12, dans lequel le vecteur 
comprend un acide nucleique codant pour la forme pre-pro d'une chatne 0 d'inhibine et un dimere de 
chatne j9 mature est recueilli en I'absence de chatne a. 

15. Procede suivant la revendication 10 ou 13, dans lequel la chatne 0 est la chatne 0 A et I'inhibine 
55 presente a une concentration supeVieure a environ 20 ng/ml de milieu de culture. 

16. Composition contenant de I'inhibine humaine ou porcine constituee d'une chatne a et d'une chatne 0, 
les sequences d'amino-acides desdites chatnes a et /S etant choisies entre celles representees sur la 



72 



EP 0 222 491 B1 



figure 1B (chatne o porcine), la figure 2B (chaTnes £ porcines), la figure 6A (chaTne a humaine) et les 
figures 8 et 9 (chaTnes & humaines), et des variants de sequence d'amino-acides engendres par 
insertion, deletion ou substitution d'un polypeptide d'une desdites sequences representees, variants qui 
sont fortement homologues avec un polypeptide d'une sequence representee, mais a I'exclusion de la 
5 chaTne a d'inhibine bovine, et 1) qui presentent une r^activite croisee avec des anticorps engendres 
contre un polypeptide d'une sequence representee ; 2) qui presentent une reactivite croisee avec les 
recepteurs de surface cellulaire pour un polypeptide d'une sequence representee ; ou 3) qui possedent 
une activite hormonale similaire a celle d'un polypeptide d'une sequence representee ; composition qui 
est totalement depourvue de proteines humaines ou porcines non identifies. 

70 

17. Composition suivant la revendication 16, dans laquelle les sequences des chaTnes a et & sont choisies 
entre celles representees sur la figure 1B (chaTne a porcine), la figure 2B (chaTne 0 porcine), la figure 
6A (chaTne a humaine) et les figures 8 et 9 (chaTnes 0 humaines). 

75 18. Composition suivant la revendication 16, dans laquelle les variants sont choisis entre : des derives 
d'inhibine porcine (Asn 2 66-*ln]Glnha ; [Cys 3 2s ou Cys 32 4— A]lnha ; [CyS3ei ou Cys 363 — AJInha ; 
[Lyss2i ou LyS322—A]lnh^ A ; [Lys 3 22—His ou Ser]lnhj8 A ; Lys 3 i5— Arg ; Vabu— Thr] lnh/8 A ; [Cysass ou 
Cys 3 9o-*A]lnh/5 A( [Lys^i 1— Gln]lnh0 A ; [Arg 315 ->Lys ; Val 3 i 6 — Thr]lnh/8 B ; [Cys 3 i 9 ou Cys 3 2o-*A]lnhjS B - 
[Prossi Gly 3 82-* Pro Phe G1y]lnh0 B ; [Arg39 5 ->G!n]lnh0 B , dans lesquels Inh est une abreviation 

20 d'inhibine et les numeros des residus pour lnhj8 B sont ceux utilises pour le residu lnh/9 A correspondant 
(voir figure 2B) ; des variants de chaTne /S A d'inhibine humaine possedant des variations qui consistent 
en une substitution ou une deletion au niveau d'un, ou une insertion apres un, residu choisi entre les 
residus 293 a 297, 364 a 376 et 387 a 398 de la sequence representee sur la figure 8 ; et des variants 
de chaTne a humaine qui presentent une homologie de plus de 90 % avec la sequence representee sur 

25 la figure 6A. 

19. Composition suivant la revendication 16, comprenant un variant d'une chaTne d'inhibine 0 A humaine 
choisi entre Phe 30 2-*lle ou Leu ; Gln297-*Asp ou Lys ; Trp307—Tyr ou Phe ; Trp 3 io-*Tyr ou Phe ; 
He3ii— Phe ou Val ; Tyr 3 i7—Trp ou Thr ; His 3 i8—Lys ; Ala^s— Ser ; Asn 32 o— Gin, Tyr ou His ; 
30 Tyr 32 i—Thr ou Asp, Phe 3 4o—Tyr ; His 3 53->Asp ; His 353 -^Lys ; (un hybride j8 A /j8 B ) ; Phe 3 56->Tyr ; 
Val3G4^Phe ; Val 3& 4— Leu ; Tyr 37 5-*Thr ; Tyr 37 6-»Trp ; Asn 38 s-* Gin, His ou Lys ; lle 3 9i-*Leu ou Thr ; 
Metsgi— Leu ou Ser ; Val 39 2-*Phe, Glu, Thr ou He ; ou une chaTne 0& humaine modifiee de maniere 
comparable. 

35 20. Composition comprenant un prodomaine d'inhibine a ou 0 B humaine ou porcine represente sur la figure 
1 B (chaTne a porcine), la figure 2B (chaTne /9 B porcine), la figure 6A (chaTne a humaine) et la figure 9 
(chaTne 0 B humaine) non associe a une glycosylation naturelle. 

21. Composition comprenant un homodimfcre de chaTnes /3 A ou & B d'inhibine humaine ou porcine mature, 
40 lesdites chaTnes etant representees sur la figure 2B (chaTnes jS porcines) et les figures 8 et 9 (chaTnes 

& humaines), ou d'un variant de sequence d'amino-acides engendre par insertion, deletion ou 
substitution d'un polypeptide d'une des sequences representees, variant qui presente une forte 
homologie avec un polypeptide d'une sequence representee et 1) qui presente une reactivite croisee 
avec des anticorps engendres contre un polypeptide d'une sequence representee ; 2) qui presente une 
45 reactivite croisee avec des recepteurs de surface cellulaire pour un polypeptide d'une sequence 
representee ; ou 3) qui presente une activite hormonale similaire a celle d'un polypeptide d'une 
sequence representee ; composition qui est depourvue de la chaTne a d'inhibine. 

22. Composition suivant la revendication 21, qui est un homodimere de chaTnes £ B humaine ou porcine 
so mature ou d'un de ses variants de sequence d'amino-acides. 

23. Composition comprenant un heterodim^re de chaTne 0 A d'inhibine humaine ou porcine mature avec 
une chaTne 0 B d'inhibine humaine ou porcine mature, lesdites chaTnes etant representees sur la figure 
2B (chaTnes & porcines) et les figures 8 et 9 (chaTnes j8 humaines), ou d'un variant de sequence 

55 d'amino-acides engendre par insertion, deletion ou substitution d'une desdites sequences representees, 
variant qui presente une forte homologie avec un polypeptide d'une sequence representee et 1) qui 
presente une reactivite croisee avec des anticorps engendres contre un polypeptide d'une sequence 
representee ; 2) qui presente une reactivite croisee avec des recepteurs de surface cellulaire pour un 



73 



EP 0 222 491 B1 



polypeptide d'une sequence representee ; ou 3) qui pr£sente une activite hormonale similaire a celle 
d'un polypeptide d'une sequence representee ; composition qui est depourvue de la chatne a 
d'inhibine. 

5 24. ADN non chromosomique codant pour une chaTne a d'inhibine humaine ou porcine ou d'une chatne /? 
d'inhibine humaine ou porcine dont les sequences d'amino-acides sont representees sur la figure 1B 
(chaTne a porcine), la figure 2B (chaTnes 0 porcines), la figure 6A (chaTne a humaine) et les figures 8 et 
9 (chaTnes /? humaines) ou un variant de sequence d'amino-acides engendre par insertion, deletion ou 
substitution d'un polypeptide d'une desdites sequences representees, variant qui presents une forte 

70 homologie avec un polypeptide d'une sequence representee a I'exclusion de la chaTne a d'inhibine 
bovine et 1) qui presente une reactivite croisee avec des anticorps engendres contre un polypeptide 
d'une sequence representee ; ou 2) qui presente une reactivite croisee avec des recepteurs de surface 
cellulaire pour un polypeptide d'une sequence representee ; ou 3) qui presente une activite hormonale 
similaire a celle d'un polypeptide d'une sequence representee. 

T5 

25. ADN suivant la revendication 24 codant pour une chaTne o d'inhibine humaine ou porcine ou une 
chaTne & d'inhibine humaine ou porcine dont la sequence d'aminoacides est representee sur la figure 
1B (chaTne a porcine), la figure 2B (chaTnes j3 porcines), la figure 6A (chaTne a humaine) et les figures 
8 et 9 (chaTnes £ humaines). 

20 

26. ADN suivant la revendication 24, qui code pour un variant choisi entre : des derives d'inhibine porcine 
[Asn 2 &6-*Gln]lnhar ; [CyS325 ou Cys324-*A]lnha ; [Cys3si ou CyS36 3—A]lnha ; [Lys 32 i ou Lys 3 22-*A]- 
lnh/S A ; [Lys 3 22— His ou Ser]lnh0 A ; [LVS315— Arg ; Val 3 t6—Thr] lnhjS A ; [Cys 38 s ou Cys 39 o-*A]lnhjS A , 
[LyS4ii^Gln]lnh0 A ; [Argsis-^Lys ; Val 3 i 6 -Thr]lnh0 B ; [Cys 3 i9 ou CyS3 2 o- , A]lnhi9B ; [Pro 38 i 

25 Gly38 2— Pro Phe Gly]lnh£ B ; [Arg 395 ^G!n]lnhi3B, dans lesquels Inh est une abreviation d'inhibine et les 
numeros des residus pour la lnh£ B sont ceux utilises pour le residu de lnhj8 A correspondant (voir figure 
2B) ; des variants de chaTne 0 A d'inhibine humaine possedant des variations qui consistent en une 
substitution ou une deletion au niveau d'un, ou une insertion apres un, residu choisi entre les residus 
293 a 297, 364 a 376 et 387 a 398 de la sequence representee sur la figure 8 ; et des variants de 

30 chaTne a humaine qui presenter-it une homologie de plus de 90 % avec la sequence representee sur la 
figure 6A. 

27. ADN suivant la revendication 24, qui code pour un variant d'une chaTne d'inhibine 0 humaine choisi 
entre ; Phe302-Hle ou Leu ; Gln297-*Asp ou Lys ; Trp 30 7—Tyr ou Phe ; Trp 3 io-*Tyr ou Phe ; 

35 Ile3u— Phe ou Val ; Tyr 3 i7^Trp ou Thr ; Hissia—Lys ; Alaaia— Ser ; Asn 3 2o-*Gln, Tyr ou His ; 
Tyr 3 2i^Thr ou Asp, Phe 3 4o— Tyr ; His^s—Asp ; His 3 s3— Lys (a un hybride j8 A /0 B ) ; Phe 3 56— Tyr ; 
Val 3 64— Phe ; Val 3 64-*Leu ; Tyr 3 75—Thr ; Tyr 37 6-*Trp ; Asnsgg-^GIn, His ou Lys ; lle 3 9i— Leu ou Thr ; 
Met39Q—Leu ou Ser ; Val 3 92-*Phe, Glu, Thr ou He ; ou une chaTne j8 B humaine modifiee de maniere 
comparable. 

40 

28. ADN suivant Tune quelconque des revendications 24 a 27, qui est depourvue de sequences non 
traduites intermediates. 

29. ADN suivant I'une quelconque des revendications 24 a 28, qui est marquee avec un groupement 
45 detectable. 

30. Vecteur replicable comprenant un ADN suivant I'une quelconque des revendications 21 a 27. 

31. Vecteur suivant la revendication 30, comprenant un promoteur viral lie de maniere fonctionnelle a I'ADN 
so codant pour les chaTnes a et/ou 0 d'inhibine. 

32. Vecteur suivant la revendication 30 ou 31, qui contient un ADN codant a la fois pour une chaTne a 
d'inhibine et une chaTne £ d'inhibine. 

55 33. Vecteur suivant la revendication 30 ou 31, qui contient un ADN codant pour une chaTne 0 d'inhibine 
mais non pour la chaTne a d'inhibine. 



74 



EP 0 222 491 B1 



34. Cellule note transformed avec un vecteur rgplicable comprenant un ADN codant pour la chaTne a 
cTinhibine et/ou la chaTne 0 d'inhibine humaine ou porcine dont les sequences d'amino-acides sont 
representees sur la figure 1 B (chaTne a porcine), la figure 2B (chaTnes 0 porcines), la figure 6A (chaTne 
a humaine) et les figures 8 et 9 (chaTnes 0 humaines), ou un variant de sequence d'amino-acides 
engendre par insertion, deletion ou substitution d'un polypeptide d'une desdites sequences represen- 
tees, variant qui presente une forte homologie avec un polypeptide d'une sequence representee, a 
I'exclusion de la chaTne a d'inhibine bovine, et 1) qui presente une reactivite croisee avec des anticorps 
engendr^s contre un polypeptide d'une sequence representee ; ou 2) qui presente Line reactivite 
croisee avec des recepteurs de surface cellulaire pour un polypeptide d'une sequence representee, ou 
3) qui presente une activtte hormonale similaire a celte d'un polypeptide d'une sequence representee. 

35. Cellule hote suivant la revendication 34, dans laquelle I'ADN code pour une chaTne a d'inhibine et/ou 
une chaTne £ d'inhibine humaine ou porcine, dont les sequences d'amino-acides sont representees sur 
la figure 1 B (chaTne a porcine), la figure 2B (chaTnes 0 porcines) la figure 6A (chaTne a humaine) et les 
figures 8 et 9 (chaTnes /S humaines). 

36. Cellule hdte suivant la revendication 34, dans laquelle I'ADN code pour un variant choisi entre : des 
derives d'inhibine porcine [Asn266— Gln]lnha ; [CVS325 ou Cys 3 2*— A]lnha ; [CyS36i ou CyS36 3~*A]lnha 
; [LVS321 ou LyS322—A]lnh/9 A ; [Lys 3 22—His ou Ser]lnh/8 A ; [Lyssis—Arg ; Val 3 i 6 — Thr] lnh/8 A ; [Cys 3 8B 
ou Cys 3 9o-*A]lnh)3A.[Lys4ii-^GIn]lnh)SA ; [Arg 3 i5-*Lys ; Val 3 i6— Thr]lnh0 B ; [Cyssis ou Cys 32 o-*A]- 
Inh^B ; [Pro38i Gly382-*Pro Phe Giy]lnh0 B ; [Arg 39 5- #, Gln]lnh)9B, dans lesquels Inh est une abreviation 
d'inhibine et les numeros des r^sidus pour Inhfo sont ceux utilises pour le residu de lnh£ A 
correspondant (voir figure 2B) ; des variants de chaTne 0 A d'inhibine humaine possedant des variations 
qui consistent en une substitution ou une deletion au niveau d'un, ou une insertion apres un, residu 
choisi entre les residus 293 a 297, 364 a 376 et 387 a 398 de la sequence representee sur la figure 8 ; 
et des variants de chaTne a humaine qui presentent une homologie de plus de 90 % avec la sequence 
representee sur la figure 6A. 

37. Cellule hote suivant la revendication 34, dans laquelle I'ADN code pour un variant d'une chaTne 
d'inhibine 0 A humaine choisi entre Phe 30 2— He ou Leu ; Gln 2 97-^Asp ou Lys ; Trp 3 o7—Tyr ou Phe ; 
Trp 3 i 0 -*Tyr ou Phe ; lle 3 n— Phe ou Val ; Tyr 3 i7-*Trp ou Thr ; His 3 i8-*Lys ; Ala 3 ig-*Ser ; Asn 32 o-*Gln, 
Tyr ou His ; Tyr 32 i-Thr ou Asp, Phe 3 4o— Tyr ; His 353 — Asp ; His 3 53— Lys (a un hybride 0 a /0b) ; 
PheasB-^Tyr ; Val364~*Phe ; Val 3 64—Leu ; Tyr 37 5-*Thr ; Tyr 3 76— Trp ; Asn 38 9— Gin, His ou Lys ; 
He 3 9i— Leu ou Thr ; Met 3 go—Leu ou Ser ; Val 39 2-*Phe, Glu, Thr ou He ; ou une chaTne 0 B humaine 
modifiee de maniere comparable. 

38. Cellule suivant Tune quelconque des revendications 34 a 37, qui est une cellule eucaryotique. 

39. Composition acellulaire qui est depourvue de polypeptide de chaTne a mature, contenant une sequence 
potypeptidique de prodomaine de chaTne a d'inhibine humaine ou porcine representee sur la figure 1 B 
(chaTne a porcine) ou la figure 6A (chaTne a humaine) ou un variant de sequence d'amino-acides 
engendre par une insertion, deletion ou substitution d'une desdites sequences de prodomaine, variant 
qui presente une forte homologie avec un polypeptide d'une desdites sequences et qui presente une 
reactivite croisee avec des anticorps engendres contre un polypeptide d'une sequence representee. 

40. Composition acellulaire contenant 



75 



EP 0 222 491 B1 

a) un polypeptide comprenant la sequence de prodomaine de chaTne /3 d'inhibine humaine 

HSAAPDCPSCALAALPKDVPNSQPE^4VEAVKKHILNMLHL ( amino -acides l a 40 
de la figure 8) , PDVTQPVPKAALLNAIRKLHVGKVGENGYVEIEDDIG 
(aminoacides 44 a 80 de la figure 8), 

AEMNELMEQTSEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKAN- 
RTRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKWDA ( amino- 
acides 83 a 185 de la figure 8), STWHVFP VS SSI QRLLDQGKS S LD VR - 
IACEQCQESGASLVLLG (amino-acides 188 a 230 de la figure 8), 

ou un de ses variants de sequence d'amino-acides naturels de mammifere ; 

b) un polypeptide comprenant la sequence de prodomaine de chaTne B 0 d'inhibine humaine 

CTSCGGFRRPEELGRVD- 

GDFLEAV (amino-acides) 7 a 30 de la figure 9, 
HILSRLQMRGRPNITHAVPKAAMVTALRKLHAGKVREDGRVEIPHLDGHASPGAD- 

GQERVSEI I SFAETDGLASSRVRLYFFISNEGNQNLFVVQASLWLYLKLLPYV- 
LEKGS 

(amino-acides 33 a 145 de la figure 9) , 

VRVKVYFQEQGHGDRWNMVEKRVDLKRSGWHTFPLTEAIQALFERGE ( aminoacides 
149 a 195 de la figure 9) , 

LNLDVQCDSCQELAVVPVFVDPGEESHRPFVVVQARIjGDSRHRI (amino-acides 
198 a 241 de la figure 9) ( 

ou un de leurs variants de sequence d'amino-acides naturels de mammifere ; ou 

c) un polypeptide depourvu de la sequence d'amino-acides de chaTne a mature comprenant les 
sequences de prodomaine de chaTne a d'inhibine humaine 

KVRALFLDALGPPAVTREGGDPGV (amino-acides 1 a 24 de la figure 
6), 

HALGGFTHRGSEPEEEEDVSQAILFPATDASCEDKSAARGLAQEA*EEGLFRYMFR- 

PSQHTRSRQVTSAQLWFHTGLDRQGTAASNSSEPLLGLLALSPGGPVAVPMSLGH- ' 

APPHWAVLHLATSALSLLTHPVLVLLLRCPLCTCSARPEATPFLVAHTRTRPPSG- 
GERA 

(amino-acides 32 a 199 de la figure 6), 

ou un de leurs variants de sequence d'aminoacides naturels de mammifere. 

41. Composition acellulaire contenant un polypeptide de prodomaine de chaTne 0 d'inhibine humaine ou 
porcine d'une sequence representee sur la figure 2B (chaTnes 0 porcines) et les figures 8 et 9 (chaTnes 
0 humaines) ou un variant de sequence d'amino-acides engendre* par insertion, delation ou substitution 
d'une desdites sequences de prodomaine, variant qui pr^sente une forte homologie avec un polypepti- 
de d'une desdites sequences et qui pr^sente une r£activite* crois£e avec des anticorps engendr^s 



76 



EP 0 222 491 B1 



contre un polypeptide d'une sequence representee. 

42. Composition suivant la revendication 41 , dans laquelle la chaTne & est la chatne 0 A et la composition 
est dgpourvue de sequence de chaTne 0 A mature. 

5 

43. Composition suivant la revendication 41, dans laquelle la chaTne 0 est la chaTne 0 B et la composition 
est depourvue de sequence de chaTne 0 B mature. 

44. Composition suivant la revendication 40, dans laquelle le variant est la sequence d'amino-acides 
70 porcine correspondante, composition contenant : 

a) un polypeptide comprenant la sequence de prodomaine de chaTne /3 A d'inhibine porcine 
representee par : les amino-acides 28 a 58 ; les amino-acides 61 a 87 ; les amino-acides 90 a 108 ; 
les amino-acides 111 a 179 ; les amino-acides 182 a 213 ; les amino-acides 216 a 258 ; ou les 
amino-acides 28 a 87 de la figure 2B ; ou 
75 b) un polypeptide depourvu de la sequence d'amino-acides de chaTne a mature comprenant les 

sequences polypeptidiques de prodomaine de chaTne a d'inhibine porcine representees par : les 
amino-acides 20 a 54 ; ou les amino-acides 70 a 228 de la figure 1B. 

45. Composition suivant Tune quelconque des revendications 39 a 44, dans laquelle le polypeptide est non 
20 accompagne par une glycosylation naturelle. 

46. Composition suivant Tune quelconque des revendications 39 a 45, qui est sterile et dans laquelle le 
polypeptide comprend en outre un polypeptide immunogene. 

25 47. Utilisation d'une composition suivant Tune quelconque des revendications 39 a 46 dans la preparation 
d'un anticorps capable de se Her audit polypeptide. 

48. Composition suivant Tune quelconque des revendications 39 a 46, dans laquelle le polypeptide est 
conjugue a un groupe detectable. 

30 

49. Composition suivant la revendication 48, dans laquelle le groupe est un enzyme, un fluorophore ou un 
radioisotope. 

50. Composition suivant Tune quelconque des revendications 39 a 44, 48 et 49, qui est insolubilisee par 
35 une absorption non covalente ou une reticulation covalente a un support insoluble dans Teau. 

51. Composition suivant I'une quelconque des revendications 39 a 46, 48 et 49, comprenant en outre une 
matrice implantable physiologiquement acceptable pour la liberation controlee du polypeptide dans les 
tissus d'un animal. 

40 

Revendications pour I'Etat contractant suivant : GR 

1. Procede comprenant la culture d'une cellule d'une cellule note transformee avec un vecteur qui 
comprend un acide nucieique codant pour une chaTne a d'inhibine humaine ou porcine et/ou une 
45 chaTne 0 d'inhibine ou porcine dont les sequences d'amino-acides sont representees sur la figure 1 B <- 
(chaTne a porcine), la figure 2B (chaTnes /9 porcines), la figure 6A (chaTne o humaine) et les figures 8 et 
9 (chaTnes £ humaines), ou un variant de sequence d'amino-acides forme par insertion, deletion ou 
substitution d'une des sequences representees, le variant etant fortement homologue avec un polypep- 
tide d'une sequence representee et 1) qui presente une reactivite croisee avec des anticorps 
so engendres contre un polypeptide d'une sequence representee ; ou 2) qui presente une reactivite 
croisee avec des recepteurs de surface cellulaire pour un polypeptide d'une sequence representee, ou 
3) qui presente une activity hormonale similaire a celle d'un polypeptide d'une sequence representee. 

Procede suivant la revendication 1, dans lequel I'acide nucieique code pour une chaTne a d'inhibine 
humaine ou porcine et/ou une chaTne & d'inhibine ou porcine dont les sequences d'amino-acides sont 
representees sur la figure 1B (chaTne a porcine), la figure 2B (chaTnes 0 porcines), la figure 6A (chaTne 
a humaine) et les figures 8 et 9 (chaTnes 0 humaines). 



77 



EP 0 222 491 B1 



3. Proceed suivant la revendication 1 , dans lequel le vecteur code pour un variant allelique d'une chaTne a 
et/ou d'une chaTne ft d'inhibine humaine ou porcine. 

4. Procede suivant I'une quelconque des revendications precedentes, dans lequel I'acide nucieique 
5 codant pour la chaTne a et/ou la chaTne ft d'inhibine est liee de maniere fonctionnelle a un promoteur 

reconnu par la cellule note et comprenant retape supplemental consistant a separer la chaTne a 
d'inhibine, la chaTne ft d'inhibine, I'inhibine ou un dimere de chaTne ft du milieu de culture. 

5. Procede suivant Tune quelconque des revendications precedentes, dans lequel la cellule est un 
io procaryote. 

6. Procede suivant I'une quelconque des revendications precedentes, dans lequel le vecteur comprend un 
acide nucieique codant pour la forme pre-pro d'une chaTne a d'inhibine ou d'une chaTne ft d'inhibine. 

75 7. Procede suivant la revendication 6, dans lequel le vecteur comprend un acide nucieique codant pour 
les formes pre-pro de la chaTne a d'inhibine et d'une chaTne /S d'inhibine. 

8. Procede suivant I'une quelconque des revendications 1 a 4, 6 et 7, dans lequel la cellule est une 
cellule provenant d'un organisme pluricellulaire et de I'inhibine dou§e d'activite* hormonale est produite. 

20 

9. Procede suivant la revendication 4, dans lequel le promoteur est un promoteur viral. 

10. Procede suivant la revendication 9, dans lequel le promoteur est un promoteur de SV40. 

25 11. Procede suivant I'une quelconque des revendications precedentes, dans lequel de I'inhibine porcine ou 
humaine mature est recueillie. 

12. Procede* suivant I'une quelconque des revendications 1 a 5 ou 8 a 10, dans lequel le vecteur comprend 
un acide nucieique codant pour la forme pre-pro d'une chaTne ft d'inhibine et un dimere de chaTne ft 

30 mature est recueilli en I'absence de chaTne a. 

13. Procede* suivant la revendication 8 ou 11, dans lequel la chaTne ft et la chaTne ft A I'inhibine est 
presente a une concentration superieure a environ 20 ng/ml dans le milieu de culture. 

35 14. Procede suivant la revendication 4, dans lequel I'inhibine est une inhibine humaine ou porcine 
constitute d'une chaTne a et d'une chaTne ft, les sequences d'amino-acides desdites chaTnes a et ft 
etant choisies entre celles representees sur la figure 1B (chaTne a porcine), la figure 2B (chaTnes ft 
porcines), la figure 6A (chaTne a humaine) et les figures 8 et 9 (chaTnes ft humaines), et des variants de 
sequence d'amino-acides engendr£s par insertion, deletion ou substitution d'un polypeptide d'une 

40 desdites sequences representees, variants qui sont fortement homologues avec un polypeptide d'une 
sequence representee, et 1) qui presentent une reactivite croisee avec des anticorps engendr.es contre 
un polypeptide d'une sequence representee ; 2) qui presentent une reactivite croisee avec les 
recepteurs de surface cellulaire pour un polypeptide d'une sequence representee ; ou 3) qui possedent 
une activite hormonale similaire a celle d'un polypeptide d'une sequence representee ; composition qui 

45 est totalement depourvue de proteines humaines ou porcines non identifiees. 

15. Procede suivant la revendication 14, dans lequel I'inhibine est une inhibine humaine ou porcine 
constituee d'une chaTne a et d'une chaTne ft, les sequences d'amino-acides desdites chaTnes a et ft 
etant choisies entre celles representees sur la figure 1B (chaTne a porcine), la figure 2B (chaTnes ft 

so porcines), la figure 6A (chaTne a humaine) et les figures 8 et 9 (chaTnes ft humaines). 

16. Procede suivant la revendication 4, dans lequel la chaTne d'inhibine recueillie est un prodomaine 
d'inhibine a ou j8b humaine ou porcine representee sur la figure 1B (chaTne a porcine), la figure 2B 
(chaTnes ft B porcines), la figure 6A (chaTne a humaine) et la figure 9 (chaTnes ft humaines), non associe 

65 a une glycosylation naturelle. 

17. Procede suivant la revendication 4, dans lequel est recueilli un dimere de chaTne ft qui est un 
homodimere de chaTnes ft ou ft B d'inhibine humaine ou porcine mature, lesdites chaTnes etant 



78 



EP 0 222 491 B1 

representees sur la figure 2B (chaTnes & porcines) et les figures 8 et 9 (chatnes /S humaines) ou un 
variant de sequence d'amino-acides engendre par insertion, deletion ou substitution d'un polypeptide 
d'une desdites sequences representees, variant qui presente une forte homologie avec un polypeptide 
d'une sequence representee et 1) qui presente une reactivite croisee avec des anticorps engendres 
5 contre un polypeptide d'une sequence representee ; 2) qui presente une reactivity croisee avec des 
recepteurs de surface cellulaire pour un polypeptide de sequence representee ; ou 3) qui presente une 
activite hormonale similaire a celle d'un polypeptide d'une sequence representee ; dimere qui est 
depourvu de la chain e a d'inhibine, 

70 18. Procede suivant la revendication 17, dans lequel un homodimere de chaTnes 0b humaines ou porcines 
matures ou d'un d'un de leurs variants de sequence d'amino-acides est recueilli. 

19. Procede suivant la revendication 4, dans lequel est recueilli un dimere de chaTne £ qui est un 
heterodimere de chaTne /S A d'inhibine humaine ou porcine mature avec une chaTne /S B d'inhibine ou 

75 porcine mature, lesdites chaTnes etant representees sur la figure 2B (chaTnes 0 porcines) et les figures 

8 et 9 (chaTnes £ humaines) ou un variant de sequence d'amino-acides engendre par insertion, deletion 
ou substitution d'un polypeptide d'une desdites sequences representees, variant qui presente une forte 
homologie avec un polypeptide d'une sequence representee et 1) qui presente une reactivite croisee 
avec des anticorps engendres contre un polypeptide d'une sequence representee ; 2) qui presente une 

20 reactivite croisee avec des recepteurs de surface cellulaire pour un polypeptide d'une sequence 
representee ; ou 3) qui presente une activite hormonale similaire a celle d'un polypeptide d'une 
sequence representee ; dimere qui est depourvu de la chaTne a d'inhibine. 

20. ADN non chromosomique codant pour une chaTne a d'inhibine humaine ou porcine ou une chaTne 0 
25 d'inhibine humaine ou porcine, dont les sequences d'amino-acides sont representees sur la figure 1B 

(chaTne q porcine, la figure 2B (chaTnes j8 porcines), la figure 6A (chaTne a humaine) et les figures 8 et 

9 (chaTnes 0 humaines) ou un variant de sequence d'amino-acides engendre par insertion, deletion ou 
substitution d'un polypeptide d'une desdites sequences representees, variant qui presente une forte 
homologie avec un polypeptide d'une sequence representee et 1) 

30 qui presente une reactivite croisee avec des anticorps engendres contre un polypeptide d'une 
sequence representee ; 2) qui presente une reactivite croisee avec des recepteurs de surface cellulaire 
pour un polypeptide d'une sequence representee ; ou 3) qui presente une activite hormonale similaire 
a celle d'un polypeptide d'une sequence representee. 

35 21. ADN suivant la revendication 20, codant pour une chaTne a d'inhibine humaine ou porcine ou une 
chaTne 0 d'inhibine humaine ou porcine, dont la sequence d'amino-acides est representee sur la figure 
1 B (chaTne a porcine), la figure 2B (chaTnes 0 porcines), la figure 6A (chaTne o humaine) et les figures 
8 et 9 (chaTnes j8 humaines). 

40 22. ADN suivant la revendication 20 ou 21 , qui est depourvue de sequences non traduites intermediates. 

23. ADN suivant I'une quelconque des revendications 20 a 22, qui est marque avec un groupement 
detectable. 

45 24. Vecteur replicable comprenant un ADN suivant la revendication 20 ou 21 . 

25. Vecteur suivant la revendication 24, comprenant un promoteur viral lie de maniere fonctionnelle a I'ADN 
codant pour les chaTnes a et/ou 0 d'inhibine. 

50 26. Vecteur suivant la revendication 24 ou 25, qui contient un ADN codant a la fois pour une chaTne a 
d'inhibine et une chaTne 0 d'inhibine. 

27. Vecteur suivant la revendication 24 ou 25, qui contient un ADN codant pour une chaTne j8 d'inhibine, 
mais non pour la chaTne a d'inhibine. 

55 

28. Cellule hote transformee avec un vecteur replicable comprenant un ADN codant pour la chaTne a 
d'inhibine humaine ou porcine et/ou une chaTne 0 d'inhibine humaine ou porcine, dont les sequences 
d'amino-acides sont representees sur la figure 1 B (chaTne a porcine), la figure 2B (chaTnes 0 porcines), 



79 



EP 0 222 491 B1 



la figure 6A (chatne a humaine) et les figures 8 et 9 (chaTnes /3 humaines) ou un variant de sequence 
d'amino-acides engendre par insertion, deletion ou substitution d'un polypeptide d'une desdites 
sequences representees, variant qui presente une forte homologie avec un polypeptide d'une sequence 
representee et 1) qui presente une reactivity croisee avec des anticorps engendre s contre un 
polypeptide d'une sequence representee ; ou 2) qui presente une reactivite croisee avec des 
recepteurs de surface celiulaire pour une sequence representee ; ou 3) qui presente une activity 
hormonale similaire a celle d'une sequence representee. 

29. Cellule note suivant la revendication 28, dans laquelle I'ADN code pour une chaTne o et/ou une chaTne 
/8 d'inhibine humaine ou porcine, dont les sequences d'amino-acides sont representees sur la figure 1 B 
(a porcine), la figure 2B (chaTnes £ porcines), la figure 6A (chaTne a humaine) et les figures 8 et 9 
(chaTnes & humaines). 

30. Cellule suivant la revendication 28 ou 29, qui est une cellule eucaryotique. 

31. Procede suivant la revendication 4, dans lequel la chaTne d'inhibine recueillie est une composition 
celiulaire, depourvue de sequence de chaTne a mature, contenant une sequence polypeptidique de 
prodomaine de chaTne a d'inhibine humaine ou porcine representee sur la figure 1 B (a porcine) ou la 
figure 6A (chaTne a humaine) ou un variant de sequence d'amino-acides engendre par insertion, 
deletion ou substitution d'une desdites sequences de prodomaine, variant qui presente une forte 
homologie avec un polypeptide d'une desdites sequences representees et qui presente une reactivite 
croisee avec des anticorps engendres contre un polypeptide d'une sequence representee. 

32. Procede suivant la revendication 4, dans lequel la chaTne d'inhibine recueillie est une composition 
acellulaire contenant 

a) un polypeptide comprenant la sequence de prodomaine de chaTne 0 A d'inhibine humaine 

HSAAPDCPSCALAALPKDVPNSQPEMVEAVKKHILNMLHL (aminoacides 1 a 40 
de la figure 8), PDVTQPVPKAALLNA I RKLHVGKVGENGYVE I EDDIG 
(aminoacides 44 a 80 de la figure 8) , 

AEMNELMEQTSEIITFAESGTARKTLHFEISKEGSDLSWERAEVWLFLKVPKAN- 
RTRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKVVDA ( aminoacides 
83 a 185 de la figure 8), STWHVFPVSSSI^RLLDQGKSSLDWIACEQ- 
CQESGASLVLLG ( amino -acides 188 a 230 de la figure 8) , 

ou un de ses variants de sequence d'amino-acides naturels de mammifere ; 

b) un polypeptide comprenant la sequence de prodomaine de chaTne j9 B d'inhibine humaine 

CTSCGGFRRPEEIiGRVDG- 
DFLEAV (amino- acides) 7 a 30 de la figure 9, 

HILSRLQMRGRPN ITHAVPKAAMVTALRKLHAGKVREDGRVE I PHLDGHASPGAD - 

GQERVSEIISFAETDGLASSRVRLYFFISNEGNQNLFWOASLWLYLKLLPYVLEKGS 
(amino-acides 33-145 de la figure 9), 

WVKVYFQE<^HGDRWNMVEKRVDLKRSGWHTFPLTEAI<3ALFERGE (amino-acides 
149 a 195 de la figure 9) , 

LNLDVQCDSCQELAWPVFVDPGEESHRPFVWQARLGDSRHRI ( amino- acides 
198 a 241 de la figure 9) , 

ou un de leurs variants de sequence d'amino-acides naturels de mammifere ; ou 



80 



EP 0 222 491 B1 



c) un polypeptide depourvu de la sequence d'amino-acides de chatnes a mature comprenant les 
sequences de prodomaine de chaTne a d'inhibine humaine 

KVRAL FLDALG P P A VTREGGD PGV (amino-acides 1 a 24 de la figure 
6) , 

HALGGFTHRGSEPEEEEDVSQAILFPATDASCEDKSAARGLAQEAEEGLFRYMFR- 
70 PSQHTRSRQVTSAQLWFHTGLDRQGTAASNSSEPLLGLLALSPGGPVAVPMSLGH- 
APPHWAVLHLATSALSLLTHPVLVLLLRCPLCTCSARPEATPFLVAHTRTRPPSGGERA 

75 

(amino-acides 32 a 199 de la figure 6), 
ou un de leurs variants de sequence d'amino-acides naturets de mammifere. 

20 

33. Proc^de* suivant la revendication 4, dans lequel la chaTne d'i'nhibine recueillie est une composition 
acellulaire contenant un polypeptide de prodomaine de chaTne jS d'inhibine humaine porcine d'une 
sequence representee sur la figure 2B (chaTnes /S porcines) et les figures 8 et 9 (chaTnes 0 humaines) 
ou un variant de sequence d'amino-acides engendre par insertion, deletion ou substitution d'une 

25 desdites sequences polypeptidiques de prodomaine, variant qui presente une forte homologie avec un 
polypeptide d'une desdites sequences et qui presente une r^activite* crois^e avec des anticorps 
engendres contre un polypeptide d'une sequence representee. 

34. Procede* suivant la revendication 33, dans lequel la chaTne 0 est la chaTne /? A et la composition est 
30 dSpourvue de sequence de chaTne 0 A mature. 

35. Proc^de* suivant la revendication 33, dans lequel la chaTne j8 est la chaTne & B et la composition est 
d§pourvue de sequence de chaTne /3 B mature. 

35 36. Proc£de suivant la revendication 32, dans lequel le variant est la sequence d'amino-acides porcine 
correspondante, la composition contenant : 

a) un polypeptide comprenant la sequence de prodomaine de chaTne 0 A d'inhibine porcine 
representee par : des amino-acides 28 a 58 ; les amino-acides 61 a 87 ; les amino-acides 90 a 108 ; 
les amino-acides 111 a 179 ; les amino-acides 182 a 213 ; les amino-acides 216 a 258 ou les 

40 amino-acides 28 a 87 de la figure 2B ; ou 

b) un polypeptide depourvu de la sequence d'amino-acides de chaTne a mature, comprenant les 
sequences polypeptidiques de prodomaine de chaTne a d'inhibine porcine representees par : les 
amino-acides 20 a 54 ; ou les amino-acides 70 a 228 de la figure 1B. 

45 37. Proc^de* suivant I'une quelconque des revendications 31 a 36, dans lequel le polypeptide est non 
accompagne* par une glycosylation naturelle. 

38. Proc^de* suivant Tune quelconque des revendications 31 a 37, dans lequel la composition est sterile, et 
comprend en outre le couplage du polypeptide a un polypeptide immunogene. 

50 

39. Utilisation d'une composition suivant Tune quelconque des revendications 31 a 38 dans la preparation 
d'un anticorps capable de se lier au polypeptide. 

40. Proc^de* suivant Tune quelconque des revendications 31 a 38, comprenant en outre la conjugaison du 
55 polypeptide a un groupe detectable. 

41. Procede* suivant la revendication 40, dans lequel le groupe est un enzyme, un fluorophore ou un 
radioisotope. 



81 



EP 0 222 491 B1 



42. Proceed suivant Tune quelconque des revendi cations 31 a 38, 40 et 41 , dans lequel la composition est 
insolubilis4e par une absorption non covalente ou une reticulation covalente a un support insoluble 
dans I'eau. 

5 43. Procede suivant Tune quelconque des revendications 31 a 38 ou 40 a 41, comprenant en outre le 
melange de la composition a une matrice implantable physiologiquement acceptable pour la liberation 
controiee du polypeptide dans les tissus d'un animal. 

Revendications pour les Etats contractants suivants : AT, ES 

10 

1. Procede comprenant la culture d'une cellule note transformee avec un vecteur qui renferme un acide 
nucieique codant pour une chaTne a d'inhibine humaine ou porcine et/ou pour une chaTne d'inhibine 
humaine ou porcine dont les sequences d'aminoacides sont representees sur la figure 1B (chaTne a 
porcine), la figure 2B (chaTnes porcines), la figure 6A (chaTne a humaine) et les figures 8 et 9 

75 (chaTnes £ humaines), ou un variant de sequence d'amino-acides forme par insertion, deletion ou 
substitution d'une des sequences representees, le variant etant fortement homologue avec un polypep- 
tide d'une sequence representee et 1 ) qui presente une activity croisee avec les anticorps engendres 
contre un polypeptide d'une sequence representee ; ou 2) qui presente une reactivite croisee avec des 
recepteurs de surface cellulaire pour un polypeptide d'une sequence representee ; ou 3) qui presente 

20 une activite hormonale similaire a celle d'un polypeptide d'une sequence representee. 

2. Procede suivant la revendication 1, dans lequel Tacide nucieique code pour une chaTne a d'inhibine 
humaine ou porcine et/ou une chaTne & d'inhibine humaine ou porcine dont les sequences d'aminor 
acides sont representees sur la figure 1 B (chaTne a porcine), la figure 2B (chaTnes 0 porcines), la figure 

?5 6A (chaTne a humaine) et les figures 8 et 9 (chaTnes & humaines). 

3. Procede suivant la revendication 1, dans lequel le vecteur code pour un variant allelique de chaTne a 
et/ou de chaTne p d'inhibine humaine ou porcine. 

30 4. Procede suivant Tune quelconque des revendications precedentes, dans lequel Tacide nucieique 
codant pour la chaTne a et/ou la chaTne j9 d'inhibine est lie de maniere fonctionnelle a un promoteur 
reconnu par la cellule note, et comprenant retape supplemental de separation de la chaTne a 
d'inhibine, de la chaTne p d'inhibine, de I'inhibine ou d'un dime re de chaTne & du milieu de culture. 

35 5. Procede suivant Tune quelconque des revendications precedentes, dans lequel la cellule est un 
procaryote. 

6. Procede suivant Tune quelconque des revendications precedentes, dans lequel le vecteur comprend un 
acide nucieique codant pour la forme pre-pro d'une chaTne a d'inhibine ou d'une chaTne 0 d'inhibine. 

40 

7. Procede suivant la revendication 6, dans lequel le vecteur comprend un acide nucieique codant pour 
les formes pre-pro de la chaTne a d'inhibine et d'une chaTne j8 d'inhibine. 

8. Procede suivant Tune quelconque des revendications 1 a 4, 6 et 7, dans lequel la cellule est une 
45 cellule provenant d'un organisme pluricellulaire et une inhibine dou£e d'activite hormonale est produite. 

9. Procede suivant la revendication 4, dans lequel le promoteur est un promoteur viral. 

10. Procede suivant la revendication 9, dans lequel le promoteur est un promoteur de SV40. 

so 

11. Procede suivant I'une quelconque des revendications precedentes, dans lequel une inhibine porcine 
humaine mature est recueillie. 

12. Procede suivant I'une quelconque des revendications 1 a 5 ou 8 a 10, dans lequel le vecteur comprend 
55 un acide nucieique codant pour la forme pre-pro d'une chaTne & d'inhibine et un dimere de chaTne & 

mature est recueilli en I 'absence de chaTne a. 



82 



EP 0 222 491 B1 



13. Procede suivant la revendication 8 ou 11, dans lequel la chaTne $ est la chaTne 0 A et I'inhibine est 
presente a une concentration superieure a environ 20 ng/ml dans le milieu de culture. 

14. Procede suivant la revendication 4, dans lequel I'inhibine est une inhibine humaine ou porcine 
5 constitute d'une chaTne a et d'une chaTne 0, les sequences d'amino-acides desdites chaTnes a et 0 

etant choisies entre celles representees sur la figure 1B (chaTne a porcine), la figure 2B (chaTnes /S 
porcines), la figure 6A (chaTne a humaine), et les figures 8 et 9 (chaTnes 0 humaines), et les variants de 
sequence d'amino-acides engendres par insertion, deletion ou substitution d'un polypeptide d'une 
desdites sequences representees, variants qui presentent une forte homologie avec un polypeptide 
70 d'une sequence representee et 1) qui presentent une reactivity croisee avec des anticorps engendres 
contre un polypeptide d'une sequence representee ; 2) qui presentent une reactivate croisee avec des 
recepteurs de surface cellulaire pour un polypeptide d'une sequence representee ; ou 3) qui presentent 
une activite hormonale similaire a celle d'un polypeptide d'une sequence representee ; composition qui 
est totalement depourvue de proteines humaines ou porcines identifiees. 

75 

15. Procede suivant la revendication 14, dans lequel I'inhibine est une inhibine humaine ou porcine 
constitute d'une chaTne o et d'une chaTne 0, les sequences d'aminoacides desdites chaTnes a et 0 
etant choisies entre celles representees sur la figure 1B (chaTne a porcine), la figure 2B (chaTnes 0 
porcines), la figure 6A (chaTne a humaine) et les figures 8 et 9 (chaTnes 0 humaines). 

20 

16. Procede suivant la revendication 4, dans lequel la chaTne d'inhibine recueillie est un prodomaine 
d'inhibine a ou & B humaine ou porcine representee sur la figure 1B (chaTne a porcine), la figure 2B 
(chaTne 0 B porcine), la figure 6A (chaTne o humaine) et la figure 9 (chaTne £ B humaine) non associe et 
a une glycosylation naturelle. 

25 .... 

17. Proctde suivant la revendication 4, dans lequel est recuelli un dim£re de chaTne 0 qui est un 
homodimere de chaTne 0a ou j8b d'inhibine humaine ou porcine mature, lesdites chaTnes etant 
representees sur la figure 2B (chaTnes £ porcines) et les figures 8 et 9 (chaTnes ^humaines) ou un 
variant de sequence d'amino-acides engendre par insertion, deletion ou substitution d'un polypeptide 

30 d'une desdites sequences representees, variant qui presente une forte homologie avec un polypeptide 
d'une sequence representee et 1) qui presente une reactivite croisee avec des anticorps engendres 
contre un polypeptide d'une sequence representee ; 2) qui presente une reactivite croisee avec des 
recepteurs de surface cellulaire pour un polypeptide de sequence represents ; ou 3) qui presente une 
activite hormonale similaire a celle d'un polypeptide d'une sequence representee ; dimere qui est 

35 depourvu de la chaTne a d'inhibine. 

18. Procede suivant la revendication 17, dans lequel un homodimere de chaTnes j9 B humaines ou porcines 
matures ou un de ses variants de sequence d'amino-acides est recueilli. 

40 19. Procede suivant la revendication 4, dans lequel est recueilli un dimere de chaTne 0 qui est un 
heterodimere de chaTne £ A d'inhibine humaine ou porcine mature avec la chaTne d'inhibine humaine 
ou porcine mature, lesdites chaTnes etant presentees sur la figure 2B (chaTnes 0 porcines) et les 
figures 8 et 9 (chaTnes 0 humaines) ou un variant de sequence d'aminoacides engendre par insertion, 
deletion ou substitution d'un polypeptide d'une desdites sequences representees, variant qui presente 

45 une forte homologie avec un polypeptide d'une sequence representee et 1) qui presente une reactivite 
croisee avec des anticorps- engendres contre un polypeptide d'une sequence representee ; 2) qui 
presente une reactivite croisee avec des recepteurs de surface cellulaire pour un polypeptide de 
sequence represente ; ou 3) qui presente une activite hormonale similaire a celle d'un polypeptide 
d'une sequence representee ; homodimere qui est depourvu de'la chaTne a d'inhibine. 

so 

20. Procede de production d'un ADN non chromosomique codant pour une chaTne a d'inhibine humaine ou 
porcine ou une chaTne 0 d'inhibine humaine ou porcine representee sur la figure 1 B (chaTne o porcine), 
la figure 2B (chaTnes 0 porcines), la figure 6A (chaTne a humaine) et les figures 8 et 9 (chaTnes & 
humaines) ou un variant de sequence d'amino-acides engendre par insertion, deletion ou substitution 
55 d'un polypeptide d'une desdites sequences representees, variant qui presente une forte homologie 
avec un polypeptide d'une sequence representee et 1) qui presente une reactivite croisee avec des 
anticorps engendres contre un polypeptide d'une sequence representee ; 2) qui presente une reactivite 
croisee avec des recepteurs de surface cellulaire pour un polypeptide de sequence representee ; ou 3) 



83 



EP 0 222 491 B1 



qui presente une activity hormonale similaire a celle d'un polypeptide d'une sequence representee, 
procede comprenant une synthese chimique, une selection d'ARNm d'ovaires ou une selection de 
banques genomiques de n'importe quelle cellule. 

5 21. Procede suivant la revendication 20, dans lequel I'ADN code pour une chaTne a d'inhibine humaine ou 
porcine ou une. chaTne & d'inhibine humaine ou porcine dont la sequence d'amino-acides est 
representee sur la figure 1B (chaTne a porcine), la figure 2B (chaTnes 0 porcine), la figure 6A (chaTne a 
humaine) ou la figure 8 ou 9 (chaTnes p humaines). 

w 22. Procede suivant la revendication 20 ou 21, dans lequel I'ADN non chromosomique est depourvu de 
sequences non traduites intermediates. 

23. Procede suivant Tune quelconque des revendications 1 a 22, comprenant en outre I'etape de marquage 
de I'ADN non chromosomique avec un groupement detectable. 

75 

24. Procede de production d'un vecteur replicable, procede comprenant insertion d'un ADN suivant la 
revendication 20 ou 21 dans un vecteur de clonage. 

25. Procede* suivant la revendication 24, dans lequel I'ADN est inseVe* dans un vecteur qui possede un 
so promoteur viral lie de maniere fonctionnelle a I'ADN codant pour la chaTne q et/ou la chaTne 0 

d'inhibine afin de produire ainsi un vecteur d'expression. 

26. Procede suivant la revendication 24 ou 25, dans lequel un ADN codant a la fois pour une chaTne a 
d'inhibine et une chaTne 0 d'inhibine est inseree dans le vecteur. 

25 

27. Procede* suivant la revendication 24 ou 25, dans lequel un ADN codant pour une chaTne j8 d'inhibine 
mais non pour la chaTne a d'inhibine est in sere dans le vecteur. 

28. Cellule hote transformee avec un vecteur replicable comprenant un ADN codant pour la chaTne a 
30 d'inhibine humaine ou porcine et/ou une chaTne 0 d'inhibine dont les sequences d'amino-acides sont 

representees sur la figure 1 B (chaTne a porcine), la figure 2B (chaTnes £ porcines), la figure 6A (chaTne 
a humaine) et les figures 8 et 9 (chaTnes 0 humaines) ou un variant de sequence d'amino-acides 
engendre par insertion, deletion ou substitution d'un polypeptide d'une desdites sequences represen- 
tees, variant qui presente une forte homologie avec un polypeptide d'une sequence representee et 1) 
35 qui presente une reactivite croisee avec des anticorps engendres contre un polypeptide d'une 
sequence representee ; 2) qui presente une reactivite croisee avec des recepteurs de surface cellulaire 
pour un polypeptide de sequence representee ; ou 3) qui presente une activite hormonale similaire a 
celle d'un polypeptide d'une sequence representee. 

40 29. Cellule hote suivant la revendication 28, dans laquelle I'ADN code pour une chaTne a d'inhibine et/ou 
une chaTne 0 d'inhibine humaine ou porcine, dont les sequences d'amino-acides sont representees sur 
la figure 1 B (chaTne q porcine), la figure 2B (chaTnes 0 porcines), la figure 6A (chaTne a humaine) et les 
figures 8 et 9 (chaTnes £ humaines). 

45 30. Cellule suivant la revendication 28 ou 29, qui est une cellule eucaryotique. 

31. Procede suivant la revendicatioin 4, dans lequel la chaTne inhibine recueillie est une composition 
acellulaire depourvue de sequences de chaTne a mature, contenant une sequence polypeptidique de 
prodomaine de chaTne o d'inhine humaine ou porcine representee sur la figure 1B (chaTne a porcine) 
so ou la figure 6A (chaTne a humaine) ou un varjant de sequence d'amino-acides d'une desdites 
sequences de prodomaine, variant qui presente une forte homologie avec un polypeptide d'une 
desdites sequences et qui presente une reactivite croisee avec des anticorps engendres contre un 
polypeptide d'une sequence representee. 

55 32. Procede suivant la revendicatioin 4, dans lequel la chaTne d'inhibine recueillie est une composition 
acellulaire contenant 



84 



EP 0 222 491 B1 



a) un polypeptide comprenant la sequence de prodomaine de chatne /9 A d'inhibine humaine 

HSAAPDCPSCALAALPKDVPNSQPEMVEAVKKHILNMLHL (amino-acides 1 a 40 
5 de la figure 8) , PDVTQ PVPKAALLNAI RKLHVGKVGENGYVE I EDD IG 
(aminoacides 44 a 80 de la figure 8) , 

AEMNELMEQTSEIITFAESGTARKTLHFEISKEGSDLSVVERAEVWLFLKVPKAN- 
w RTRTKVTIRLFQQQKHPQGSLDTGEEAEEVGLKGERSELLLSEKWDA (amino- 
acides 83 a 185 de la figure 8), STWHVFPVSSSIQRLLDQGKSSLDVR- 
IACEQCQESGASLVLLG (amino-acides 188 a 230 de la figure 8) , 

75 ou un de ses variants de sequence d'amino-acides naturels de mammifere ; 

b) un polypeptide comprenant la sequence de prodomaine de chatne /3 B d'inhibine humaine 

CTSCGGFRRPEELGRVD- 
20 GDFLEAV (aminoacides) 7 S 30 de la figure 9, 

HILSRLQMRGRPNITHAVPKAAMVTALRKLHAGKVREDGRVEIPHLDGHASPGAD- 

GOERVSEI ISFAETDGLASSRVRLYFFISNEGNQNLFVVQASLWLYLKLLPYV- 
LEKGS 

25 (amino-acides 33 a 145 de la figure 9) , 

WVKVYFQEQGHGDRWNMVEKRVDLKRSGWHTFPLTEAIQALFERGE ( amino- acides 

30 149 a 195 de la figure 9), 

LNLDVQCDSCQELAWPVFVDPGEESHRPFVWQARLGDSRHRI (amino-acides 
198 & 241 de la figure 9) , 

35 

ou un de leurs variants de sequence d'amino-acides naturels de mammifere ; ou 

c) un polypeptide d^pourvu de la sequence d'amino-acides de chaTnes a mature comprenant les 
sequences de prodomaine de chaTne q d'inhibine humaine 

40 KVRALFLDALGPPAVTREGGDPGV (amino-acides 1 sL 24 de la figure 
6) ^ HALGGFTHRGSEPEEEEDVSQAILFPATDASCEDKSAARGLAQEAEEGLFRYMFR- 

PSQHTRSRQVTSAQLWFHTGLDRQGTAASNSSEPLLGLLALSPGGPVAVPMSLGH- 

45 APPHWAVLHLATSALSLLTHPVLVLLLRCPLCTCSARPEATPFLVAHTRTRPPSG- 
GERA 

(amino-acides 32 a 199 de la figure 6), 

so ou un de leurs variants de sequence d'amino-acides naturels de mammifere. 

33. Proc^de* suivant la revendication 4, dans lequel la chaTne d'inhibine recueillie est une composition 
acellulaire contenant un polypeptide de prodomaine de chaTne /S d'inhibine humaine porcine d'une 
sequence representee sur la figure 2B (chaTnes & porcines) et les figures 8 et 9 (chaTnes 0 humaines) 
55 ou un variant de sequence d'amino-acides engendre* par insertion, deletion ou substitution d'une 
desdites sequences polypeptidiques de prodomaine, variant qui pr^sente une forte homoiogie avec un 
polypeptide d'une desdites sequences et qui presente une reactivity crois£e avec des anticorps 
engendr£s contre un polypeptide d'une sequence representee. 



85 



EP 0 222 491 B1 



34. Proc6d6 suivant la revendication 33, dans lequel la chaTne & est la chaTne 0 A et la composition est 
dSpourvue de sequence de chaTne q a mature. 

35. Precede* suivant la revendication 33, dans lequel la chaTne 0 est la chaTne jS B et la composition est 
d^pourvue de sequence de chaTne 0b mature. 

36. Procede suivant la revendication 32, dans lequel le variant est la sequence d'amino-actdes porcine 
correspondante, la composition contenant : 

a) un polypeptide comprenant la sequence de prodomaine de chaTne 0 A d'inhibine porcine 
representee par : les amino-acides 28 a 58 ; les amino-acides 61 a 87 ; les amino-acides 90 a 108 ; 
les amino-acides 111 a 179 ; les amino-acides 182 a 213 ; les amino-acides 216 a 258 ou les 
amino-acides 28 a 87 de la figure 2B ; ou 

b) un polypeptide d§pourvu de la sequence d'amino-acides de chaTne a mature, comprenant les 
sequences polypeptidiques de prodomaine de chaTne a d'inhibine porcine representees par : les 
amino-acides 20 a 54 ; ou les amino-acides 70 a 228 de la figure 1 B. 

37. Proc6de* suivant Tune quelconque des revendications 31 a 36, dans lequel le polypeptide est non 
accompagne par une glycosylation naturelle. 

38. Procede suivant Tune quelconque des revendications 31 a 37, dans lequel la composition est sterile, et 
comprenant en outre le couplage du polypeptide a un polypeptide immunogene. 

39. Utilisation d'une composition suivant Tune quelconque des revendications 31 a 38 dans la preparation 
d'un anticorps capable de se lier au polypeptide. 

40. Procede suivant Tune quelconque des revendications 31 a 38, comprenant en outre la conjugaison du 
polypeptide a un groupe detectable. 

41. Proc£de" suivant la revendication 40, dans lequel le groupe est un enzyme, un fluorophore ou un 
radioisotope. 

42. Proc6d£ suivant Tune quelconque des revendications 31 a 38, 40 et 41, dans lequel la composition est 
insoiubilisee par une absorption non covalente ou une reticulation covalente a un support insoluble 
dans Teau. 

4a Procede suivant Tune quelconque des revendications 31 a 38 ou 40 a 41, comprenant en outre le 
melange de la composition a une matrice implantable physiologiquement acceptable pour la liberation 
contrdl£e du polypeptide dans les tissus d'un animal. 



EP 0 222 491 B1 



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