LU87361A1 - New DNA sequence encoding transforming growth factor beta 2 - used for large scale expression in eucaryotic cells - Google Patents

Abstract

A nucleotide sequence (I) encoding the transforming growth factor (TGF)beta-2 precursor (A) is new. The specification includes the sequence (1326 bases) and the derived amino acid sequence. Also new are (1) a similar sequence in which the 346-432 base region has been replaced by AAT; (2) a sequence encoding TGFbeta2 itself (bases 991-1326 of the (A) sequence); (3) a sequence encoding a TGFbeta1/TGFbeta2 hybrid precursor (about 1825 bases); (4) proteins derived from all these sequences, and (5) eucaryotic cells contg. such sequences.

Description

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Λ T 7 C 4 GRAND-DUCHY OF LUXEMBOURG

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Mr. Minister of,, 1.0 ....., P.C'fcQbrS .19.3.8 of the Economy and the Middle Classes

Tjfrp ^ ρΐΐνγ-ρ Intellectual Property Service

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Patent Application ............................................ ............._ (!) I. Request ..... The ....... company ..... called: ........ ONCOGEN . 3005 First Avenue, SEATTLE, _______ (2) .Washington 98121, United States of America, represented by__________________

Mr. Jacques de Muyser, acting as agent ---------------------------------------- ---------------------------------------------------------- ------: ------------------------------------------- --------------------------------------- (3) submitting) this ..... ... ten ...... October 1900 ..... eighty-eight__________________________________________________ (4) at ____________________ hours, at the Ministry of the Economy and the Middle Classes, in Luxembourg: 1. this request for obtaining 'a patent for an invention concerning: "Nucleotide sequence coding for the precursor of __________ ^ 5 \ transforming growth factor j2, precursor of transforming growth factor β2, and ...... transforming growth factor £ 2." .. .................................................. .................................................. .................................................. ......................................

2. description in French _______ language ........................................... ..of the invention in triplicate; 3. ................? ............................... .... drawing boards, in triplicate; 4. the receipt of the taxes paid to the Luxembourg Registration Office on ..- LQ ........ October 1988.

5. the delegation of power, dated ......................................... .................................................. ....... the.......................................... ................................................; 6. the successor document (authorization); UV declared (s) assuming responsibility for this declaration, that the inventor (s) is (are): (6) | ..... not ...... mention________________________________________________________________________________________________________________________________________________________________________________________________________ • r-1 QJ Q)

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.................................................. .................................................. .................................................. .................................................. ........................................ — ......... .....................- C CD _ ......................... .................................................. .................................................. .................................................. .................................................. ........................... __ ........... _ o ro 3 + 31-1 co claims ( nt) for the above patent application the priority of one (or more) application (s) of (7) 8-S ο Έ ......... patents ........... .................................................. ................................................. filed (s) & i (8) in the United States of America "lëS I 1e (Q)" 6 - eotobre— l „e. 2.5. January 1988 and August 18, 19J8.8 N ° (10). · 75ΒNo. 148.267 ____________________________ No. 234.065 ________ ___________________ in the name of (11) s ..... inventors .______________________________________________________________________________________________________________________________________________________________________________ elect (elect) domicile for him / her and, if designated, for his / her representative, in Luxembourg_____________________________________________ ............. 3.5 .., boulevard Royal___________________________________________________________________________________________________________________________________ (12) requests (s) the grant of a patent for the invention for the subject described and represented in the abovementioned appendices, with postponement of this grant to .......... ................................ 6 ................. .................................................. ........................................month. (13)

JktftßpBtent / authorized representative: Ά ____________________________________________________________________________________________________________________________________________________________________________________________________________________________ (14) -verbal de Dépôt

The above; patent application has been filed with the Ministry of the Economy and the Middle Classes, Intellectual Property Service in Luxembourg, dated: October 1988

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J i \ / "J The head of the will be" already intellectual property,

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A 68007_ 'V'i_PiY _ ΡΧΡΓ .ΓΓΑΤίΠΝ.ς RFTATTVFS A11 FOR MT f Jf [I (1) if applicable "Request for certificate of addition aubrSVefflRnctpal, at main patent application No / .... ....... from ............ ”- (2) enter the last name, first name, profession.

Γ A A A / address of the applicant, when the applicant is an individual or company name, legal form, address of the head office, when the applicant is a legal person - (3) enter {. / "((V the surname, first name, address of the professional representative, industrial property attorney, with special powers, if applicable:" represented by ............ acting as agent "^ - (4) date of filing in full - (5) title of invention - (6) enter the names, first names, addresses of the inventors or the indication" (see) separate designation (will follow ) ”, When the designation is made or will be made in a separate document, or the indication" not to mention ", when the inventor or his own a non-mention document to be attached to a designation • 88 /B.52.784

CLAIM OF PRIORITY

of the patent application / • In the United States of America October 6, 1987 (No. 106.752), January 25, 1988 (No. 148.267) and .. August 18, 1988 (No. · 234.065) Brief Description filed in support of a request for

PATENT

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Luxembourg on behalf of: oncogen SEATTLE, Washington 98121 (United States of America) for: "Nucleotide sequence coding for the precursor of the transforming growth factor ß2 precursor of the transforming growth factor | 3 2, and transforming growth factor jS2." "Nucleotide sequence encoding the precursor of transforming growth factor ß2, precursor of transforming growth factor ß2, and transforming growth factor ß2." The present invention generally relates to the cloning and expression of the transforming human growth factor beta2.

In this technical field, the transforming growth factor beta (TGF-ß) is known to be a member of a recently described family of polypeptides which regulate cell differentiation and proliferation. Other members of this family include Muller's inhibitory substance (Cate et al, 1986, Cell 45: 685-698), inhibins (Mason et al, 1985, Nature 318: 659-663) and a protein predicted to from a transcript of the Drosophila decapentaplegic gene complex (Padgett et al, 1987, Nature 325: 81-84)

The transforming growth factor Beta 20 (TGF-ß) consists of two identical subunits linked by disulfide bridges having molecular weights of 13,000 (Assoian et al., 1983, J. Biol. Chem. 258: 7155-7160; Frolik et al, 1983, Proc. Natl. Acad. Sci. USA 80: 3676-3680; Frolik et al.

25 1984, J. Biol. Chem. 260: 10995-11000). It has been purified from several tissue sources including the placenta (Frolik et al., 1983, Nature 325: 81-84), blood platelets (Childs et al., 1982, Proc. Natl. Acad. Sci. USA 79: 5312-5316; Assoian et al., 1983; J. Biol. Chem. 258: 7155- 7160), kidney (Roberts et al., 1983, Biochemistry 22: 5692-5698), and bone demineralized (Seyedin et 2 al., 1985, Proc. Natl. Acad._ Sci. USA 82: 119-123). In the presence of 10% serum and epidermal growth factor, TGF-β promotes growth independent of the anchoring of fibroblasts from kidneys of normal rats (Roberts et al., 1981, Proc Natl. Acad. Sci. USA 78 : 5339-5343; Roberts et al., 1982, Nature 295: 417-419; Twardzik et al., 1985, J.

Cell. Biochem. 28: 289-297); in the presence of 10% serum alone, it is capable of inducing the formation of 10 colonies of AKRr-2B fibroblasts (Tucker et al.

1983, Cancer Res. 43: 1518-1586). TGF-ß has also been shown to differentiate mesenchyme cells from rat fetus muscles to produce cartilage-specific macromolecules (Seyedin et al., 1986, J. Biol. Chem. 261: 5693-5695 ).

In contrast to its effect on cell proliferation, TGF-ß purified from human platelets as well as a functionally related protein isolated from African callitrich cells (BSC-1) has been shown to inhibit the growth of certain cells in culture (Tucker et al., 1984, Science 226: 705-707).

‘TGF-ß has also been shown to inhibit the growth of several human cancer cell lines (Roberts et al., 1985, Proc. Natl. Acad. Sci. USA 82: 119-123). This inhibiting / stimulating effect of TGF-ß may depend on several factors including cell type and physiological state of cells (for a review see Sporn et al., 1986, Science 233: 532-534).

CDNA clones encoding human TGF-β (Derynck et al., 1985, Nature 316: 701-705), murine (Derinck et al., 1986, J. Biol. Chem. 261: 4377-4379) 4 3 and simien (Sharples et al., 1987, DNA 6: 239-244) have been isolated. Analysis of the DNA sequences of these clones indicates that TGF-ß is synthesized as a large precursor polypeptide, the carboxy terminus of which is cleaved to form the mature monomer of TGF-ß. Strong sequence homology was found throughout the TGF-ß precursor protein from all of the above sources.

Very recently, a protein isolated from demineralized bovine bone has been identified as being related to TGF-ß (Seyedin et al., 1987, J. Biol. Chem. 262: 1946-1949). The protein has also been isolated from pig platelets (Cheifetz 15 et al., 1987, Cell 48: 409-415), from a human prostate adenocarcinoma cell line, PC-3 (Ikeda et al. ., 1987, Biochemistry 26: 2406-2410), and a human glioblastoma cell line (Wrann et'al., 1987, EMBO 6: 1633-1636) '. A partial amino acid sequence of this protein indicated that it was homologous to TGF-ß and was designated TGF-B2. Human TGF-ß (Derynck et al., '1985, Nature 316: 701-705), murine (Derynck et al., 1986, J. Biol. Chem. 261: 4377-4379) and simian 25 (Sharples et al ., 1987, DNA 6: 239-244) described above was called TGF-ß1.

The present invention relates to the production of large quantities of TGF-B2 by eukaryotic host cells transfected with recombinant DNA vectors containing a sequence coding for TGF-B2 controlled by expression regulating elements. In a specific embodiment, cDNA clones encoding the human TGF-B2 precursor were obtained from a library of cDNA constituted from a cell line of human prostate adenocarcinoma treated with tamoxifen, PC-3. The cDNA sequence of such a clone predicts that TGF-B2 5 is synthesized in the form of a polypeptide precursor of 442 amino acids from which the mature TGF-B2 subunit of 112 amino acids is derived by proteolytic cleavage . This TGF-B2 precursor, designated TGF-B2-442, shares 41% homology with the precursor of-TGF-β1. In another embodiment, cDNA clones encoding the simian TGF-B2 precursor were obtained from a cDNA library constructed from a cheat calli-15 kidney cell line African, BCS-40. The cDNA sequence of such a clone predicts that TGF-ß2 is also synthesized as a precursor ion of a 414 amino acid polypeptide from which the mature 112 amino acid TGF-ß2 subunit i is derived by cleavage. proteolytic. This TGFrß2 precursor, named TGF-B2-414, has an amino acid sequence of 414 amino acid residues and is identical to the amino acid sequence of TGF-ß2-442, except that it contains a single asparagine residue instead of the 29 amino acid sequence of the residues

Nos. 116 to 135 of the sequence of human TGF-ß2-442.

Clones from the BSC-40 cDNA library that encode a precursor of TGF-B2-442 simien as well as clones from the human PC-3 cDNA library that encode a TGF precursor -B2-414 human have also been identified. The precursors of human and simian TGF-B2-442 appear to be perfectly homologous at the amino acid level, as are the precursors of human and simian TGF-ß2-414.

The mature monomers of 112 amino acids of TGF-ß1 and TGF-ß2 show 71% homology.

Expression vectors containing the mature sequence coding for TGF-B2 in phase joined to the signal and precursor sequences of TGF-β1 (US patent application pending and in co-ownership No. 189,984) were constructed and used to transfect Chinese hamster ovary cells (CHO cells). The resulting CHO transfectants produce and secrete a TGF-B2

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* mature, biologically active.

The following terms as used herein in the singular or plural have the meanings indicated.

TGF-ß2: a transforming growth factor ß2 of human or simian origin comprising the amino acid sequence substantially as shown in Figure la from about the remainder of amino acid No. 331 to about the remainder amino acid 442.

TGF-B2 precursor: A family of transforming growth factor molecules B2 of human or simian origin comprising an amino acid sequence substantially as shown in FIG. 1 a from approximately i 6 the amino acid residue No. 1 to about amino acid residue No. 442 in which the amino acid sequence from amino acid residue No. 116 to amino acid residue No. 144 is deleted and replaced by a single residue of asparagine.

The term means a TGF-1332 precursor designated TGF-32-442 or TGF-B2-414 of human or simian origin.

TGF-ß1 / 15 TGF-B2 hybrid precursor: New growth factor precursor molecule transforming ß comprising the amino acid sequence substantially as shown in Figure 1b from approximately amino acid residue No 1 to about amino acid residue No. 390.

"

TGF-ßl Precursor 25: Precursor and signal sequences of the simian transforming growth factor β1 substantially as shown in Figure 1b from about amino acid residue No.1 to about amino acid residue No. 278.

* 7

The characteristics and advantages of the invention will appear better in the detailed description which follows and which refers to the appended figures given by way of example, in which: - Figure la represents the nucleotide sequence of TGF-β2 cDNA -442 human and the deduced amino acid sequence; the bp 2597 insertion sequence of PC-21 was subcloned into 10 pEMBL (Dante et al., 1983, Nucleic Acids Res. 11: 1645-1654) and sequenced on both strands using the reaction of dideoxy termination. nucleotide (Sänger et al., 1977, Proc. Natl. Acad. -Sei. USA 74: 5463-5467); the coding sequence is shown and the deduced amino acid sequence is shown directly above; the mature TGF-B2 sequence is framed and the signal peptide is surmounted by a line; potential glycosylation sites are indicated by asterisks; the arrow indicates the presumed site of cleavage of the signal sequence; the nucleotide sequence of simian TGF-32-414 cDNA is identical to the cDNA sequence of human TGF-ß2-442 except that nucleotides 346 to 432 (in square brackets) are deleted and replaced by the sequence AAT , and s except that several silent nucleotide changes take place elsewhere in the structure (this is indicated by isolated letters directly below the modified nucleotide); the deduced amino acid sequence for the simian TGF-32-414 precursor is identical to the amino acid sequence for human TGF-ß2-442 precursor except that asparagine replaces the amino acid residues 116 to 144 in the structure of human TGF-B2-442 i 8; the nucleotide sequence of a human TGF-B2-414 cDNA has been sequenced in the underlined region in broken lines and has been found to be perfectly homologous to the human TGF-B2-442 cDNA sequence except that the nucleotides 346 to 432 are deleted and replaced by the sequence AAT; FIG. 1b represents the nucleotide sequence of hybrid precursor DNA of TGF-β1 / TGF-ß2 and the deduced amino acid sequence; the. coding sequence is shown and the deduced amino acid sequence is shown directly above; the mature TGF-B2 sequence is boxed -and the precursor signal peptide is surmounted by a line. The glycosylation sites are indicated by asterisks; the arrow indicates the presumed site of cleavage of the signal sequence. The mature coding sequence of TGF-B2 described is of human origin. The mature coding sequence of simian TGF-B2 is approximately identical to the human sequence: only three silent base changes take place and are indicated by letters ‘isolated directly above the modified nucleotide; details of cDNA cloning of TGF-B2 and construction of the hybrid * TGF-ß1 / TGF-ß2 gene are given in the text; - Figure le represents a schematic diagram of the precursor gene of hybrid TGF-ß1 / TGF-ß2; FIG. 1d represents the restriction endonuclease sites of pPC-14 (2.2 kb) and pPC-21 (2.3 kb); the boxed regions indicate the coding sequences for the monomer of TGF-B2 ; ATG represents the initiation methionine codon; “9 the distance between ATG and the KpnI site in pPC-21 (2.34 kb) is approximately 420 bp; the black area indicates the position of the 84 bp insertion in pPC-21 (2.3 kb); 5 - FIG. 1a represents a partial analysis of DNA sequences from pPC-14 (2.2 kb); a synthetic oligonucleotide 5'-AGGAGCGACGAAG-AGTACTA-3 'which' hybridized approximately 140 bp upstream of the Kpnl site within the insertion sequence in pPC-21 (2.3.kb) was used to, initiate DNA sequencing reactions; in this region, the sequence of pPC-14 (2.2 kb) (upper line) is identical to pPC-21 (2.3 kb.) 4 '. up to the nucleotides coding for Asn-116; The insertion of 84 bp inside the codon Asn-116 of pPC-14 (2.2 kb) which was found in pPC-21 (2.3 kb) is shown; the Kpnl site within the insertion sequence is shown; FIG. 2 represents homologies of 20 precursor sequences of TGF-β1 and human TGF-B2-442; a) primary sequence homology: identical residues are boxed; the asterisks • designate the potential glycosylation sites in TGF-B2; the potential cleavage site of the signal sequence and, the cleavage site of the mature polypeptide are indicated; b) comparison of point matrices using Gene Pro software; each point locates a point where 5 amino acids are identical among 10 amino acids; the diagonal lines indicate the regions of homology; FIG. 3 represents a Northern blot analysis of the polyadenylated RNA of BSC-40 and of PC-3; polyadenylated RNA was isolated from BSC-40 and PC-3 cells, fractionated on an agarose-formaldehyde gel, transferred to Hybond-N filters and hybridized with a specific labeled TGF-B2 probe at [32p1, with pPC-21 (part A) or a mixture of specific TGF-β1 and 5 TGF-132 probes labeled with [32P] (part B) (Sharples et al., 1987) as described in the part of materials and methods ·, band 1, polyadenylated RNA of BSC-4Q (5 micrograms) j band 2, polyadenylated RNA of PC-3 (5 micrograms); 10 - FIG. 4 represents an analysis at

Northern blot of polyadenylated RNA from different sources; polyadenylated RNA was isolated from MCF-7 (human breast carcinoma), SK-MEL 28 (human melanoma), KB 15 (nasopharyngeal carcinoma) and HBL-100 (human mammary epithelium) cells and analyzed by hybridization with the Northern blot with a specific probe of TGF-S2 (pPC-21) as described in the materials and methods section; each band contains S 20 micrograms of polyadenylated RNA originating from cells of SK-MEL 28 (band 1), of MCF-7 (band 2). HBL-100 (band 3) or KB (band 4); - Figure 5 corresponds to a test for bioactivity of recombinant TGF-B2; cells of 25 clone 36 of 1B9 "12.5, the cell line of which was deposited at the ATCC (American Type Culture Collection) under the access number CflL 9800. were cultivated until the confluence of the tanks of 100 mm tissue culture; the cells were raised three times with serum-free medium "and were incubated for 24 hours in 5 ml of serum-free medium} the medium was collected, dialyzed against 0" 2M acetic acid, and tested for inhibition of DNA synthesis in

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CCL64 cells as described (Gentry et al., 1307, Moi. Coll. Biol. 7 "3410"). Däne oo toefe, 3.3 μg of natural purified TOf-Ol from Otondard gave 60% inhibition; the specific activity of natural purified TGF-32 5 was calculated: it was approximately half that of TGF-β1 and - Figure 6 represents a Western blot analysis of recombinant proteins secreted by clone 36 of lß9, 12 , S whose cell line 10 has been deposited at the ATCC (American Type culture collection) under the numei-u u'cuxea crl 0600 j a conditioned medium devoid of acid dialyzed serum from cells' of clone 36 of 1ΑΘ, 12.5 ', was fractionated by SDS-polyacrylamide gel electrophoresis and analyzed by Western blot with an anti-serum prepared against the synthetic peptide NH2-YNTINPEASASPC-COOH as described (Gentry et al., 1987, Mol Cell Biol. 7} 3418).

T..3 the present invention relates to the production of a mature biologically active form of TGF-Ö2 from a coding sequence of the precursor gene of TGF-ß and its product # The mature biologically active TGF-B2 can be produced by cloning and expressing the entire length of the nucleotide coding sequence of the TGF-A2 precursor or its functional equivalent in a host cell which correctly transforms the precursor so that a mature TGF-ß2 having been produced a biological activity which virtually cannot be distinguished from that of authentic natural TGF-β2, the functional equivalents of the full length of the nucleotide coding sequence of the TGF-B2 precursor include any DNA sequence which, when 'it is expressed inside an appropriate host cell, is capable of directing the synthesis, transformation and export of mature TGF-B2. In this regard, hybrid precursor coding sequences including, for example, the precursor sequence of TGF-β1 in the context of reading the mature sequence of TGF-B2, can be constructed and used to produce a biologically active TGF-B2 . The method according to the invention can be divided into the following steps for the purpose of description only: (a) isolation or production of the coding sequence for a form of TGFe-β2 precursor; (b) constructing an expression vector which will direct the expression of a coding sequence for TGF-ß2; (c) transfection of appropriate host cells which are capable of replicating and expressing the gene and transforming the gene product to produce the mature, biologically active form of TGF-B2; (d) identification and purification of mature biologically active TGF-B2. The transfectant which expresses high levels of mature bioactive TGF-β2 being identified, the practice of the invention includes expansion of this clone and isolation of the expressed gene product.

The method of the invention is demonstrated herein by means of examples in which cDNAs from the coding region of the TGF-B2 precursor have been prepared, cloned, sequenced and used to construct expression vectors which direct the high level expression of TGF-B2 in CHO cells. In a specific embodiment, the complete amino acid sequence of the mature form of human TGF-B2 has been determined "13 and has an overall homology of 71% with TGF-β1. Using synthetic oligonucleotic probes we identified clones from a PC 3 cDNA library encoding 5 for TGF-B2. Analysis of the DNA sequences of one of these clones revealed that TGF-B2, like TGF-ß1, is synthesized as a larger precursor protein, the end of which carboxy is cleaved to produce the mature TGF-B2 monomer. While there is 71% homology between TGF-ßl and TGF-B2 throughout the mature portions of these molecules, there is only a maximum of 31% homology in the rest of the precursor, suggesting that the amine end regions of TGF-β1 and TGF-B2 may be functionally distinct.

In a specific embodiment of the invention, expression of a new hybrid gene for TGF-β1 / TGF-ß2 in CHO cells is used to produce large amounts of biologically active TGF-B2. The various aspects of the process of the invention are described in more detail in the following subsections and in the examples which follow.

The nucleotide coding sequence for TGF-B2 is shown in Figure la. In practicing the method of the invention, the nucleotide sequence shown here, or fragments or functional equivalents thereof, can be used to produce the recombinant molecules that will direct expression of the TGF-B2 product in appropriate host cells. In a specific embodiment, a hybrid gene of "14 TGF-ß1 / TGF-ß2 (Figure 1b) was constructed and used to transfect CHO cells. Transfectants producing as much as 500 µg of mature biologically active TGF-B2 per ml of culture medium have been isolated.

Due to the degeneracy of the nucleotide coding sequences, other DNA sequences which code for substantially the same amino acid sequences as those shown in Figure 1a and Figure 1b may be used in the practice of the present invention for cloning. and expression of TGF-ß2. Such modifications include deletions, additions or substitutions of different nucleotide residues resulting in a sequence which codes for the same gene product or for a functionally equivalent gene product. The gene product may contain deletions, additions or substitutions of amino acid residues in the sequence, which results in a silent modification thereby producing a bioactive product. Such amino acid substitutions can be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, bydrophilicity and / or the amphipathic nature of the residues involved. For example, negatively charged amino acids include aspartic acid and glutamic acid; the 30 positively charged amino acids include lysine and arginine; amino acids having uncharged polar head groups or non-polar head groups having similar values of hydrophilicity include the following: leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, tyrosine, the nucleotide coding sequence for TGF-B2 can be obtained from cellular sources which produce activity similar to TGF-B2. The coding sequence can be obtained by cloning by cDNA from RNA isolated and purified from such cellular sources or by genomic cloning. CDNA or genomic libraries. clones can be prepared from DNA fragments produced using methods well known in the art, including without being

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the use of restriction enzymes is limited thereto. The fragments which code for TGF-82 can be identified by screening such libraries with a nucleotide probe which is substantially complementary to any portion of the sequence shown in Figure 1a. Full length clones, i.e., those containing the full coding region for the TGF-J32 precursor can be chosen for expression.

In another embodiment of the invention, the coding sequence of Figure la can be synthesized in whole or in part, using chemical methods well known in the art. See, for example, Caruthers et al., 1980, Nue. Acids Res. Nice. Ser. 7: 215-223; Créa and Horn, 1980, Nue Acids. Res. 9 (10): 2331; 30 Matteucci and Carruthers, 1980, Tetrahedron Letters 21: 719 and Chow and Kempe, 1981, Nude. Acids. Res. 9 (12): 2807-2917. Alternatively, the protein could be produced using chemical methods to synthesize the amino acid sequence shown in Figure la in whole or in part. For example, peptides can be synthesized by solid phase methods on a Beckman 990 instrument, and cleaved from the resin as previously described (Gentry, LE, et al, 1983, J. Biol. Chem. 258: 11219 -11228; Gentry, LE and Lawton, A., 1986, Virology 152: 421-431). The purification can be carried out by high performance preparative liquid chromatography. The composition of the peptides was confirmed by analysis of. amino acids.

In a specific embodiment, described in the examples included herein, the F \ coding sequence of TGF-B2 was obtained by cloning by cDNA of coding sequences of human TGF-B2 precursor derived from isolated polyadenylated RNA from a human prostate adenocarcinoma cell line treated with tamoxifen, PC3, which it has previously been shown to produce TGF-B2.

The entire coding region of a cDNA clone was sequenced and compared to the published sequence of human TGF-β1 (see Figure 2).

DNA sequence analysis of TGF-B2 cDNA clones indicates that TGF-B2 as well as TGF-ß1 is synthesized as a large * precursor protein, the carboxy terminus of which is cleaved to produce the mature TGF-B2 monomer of 112 amino acids. TGF-B2 has been shown to have a molecular weight of 24,000 composed of two 13,000 dalton subunits linked by disulfide bridges (Ikeda et al., 1987, Biochemistry 26: 2406-2410; Cbeifetz et al., 1987, Cell 48: 409-415). Thus, the production of TGF-B2 requires appropriate proteolytic cleavage as well as the formation of intra- and intermolecular disulfide bridges. A hydrophobic leader sequence at the amine terminus (residue 3-19) is present in the precursor and may be responsible for directing the protein out of the cell. Mature TGF-B2 can still be associated with the remaining part of the precursor during this process.

TGF-B2 has 71% homology with TGF-β1 in the entire mature part of the precursor, which implies a functional similarity which is supported by experimental evidence (Seyedin et al., 1987, J. Biol. Chem. 262 : 1946-1949; Cheifetz et al., 1987, Cell 48: 409-415). The amine portion of the TGF-β1 precursor region from human, rodent or simian sources (Derynck et al., 1985, Nature 316: 701-705; Derynck et al., 1986, J. Biol Chem 261: 4377-4379;

Sharples et al., 1987, DNA 6: 239-244) is highly conserved * and suggests that this part of the molecule may have an important biological function. In contrast, there is no more than 31% homology between the N-terminal precursor * regions of TGF-ß1 and TGF-ß2. After cleavage of the supposed signal peptide, the TGF-B2 precursor would also contain more amino acids than the TGF-β1 precursor. Primary structural differences within the amine terminus region of the TGF-β1 / TGF-ß2 precursor proteins may reflect functional differences. However, significantly homologous regions within the precursors are found in isolated blocks suggesting "18 the conservation of important functional domains even within the N-terminal precursor region.

Northern blot analysis revealed two large size classes of the 4.1 and 6.5 kb TGF-B2 specific mRNA in BSC-40 cells. PC-3 cells treated with tamoxifen contain three transcribed sequences of TGF-B2 of 4.1 kb, 5.1 kb and 6.5 kb. These different sized messages could be the result of differential RNA splicing, polyadenylation, or both as has been described for other genes (Helfman et al1986, '• Mol Cell. Biol. 6: 3582 -3595; Sayre et al., 1987, 15 Proc. Natl. Acad, Sci. USA 84: 29841-2945). Preliminary analysis of another TGF-B2 cDNA clone shows that it contains a 3 'untranslated region approximately 1 kb larger than that of pPC-211 and pPC-14 and that it contains a different polyadenylation site suggesting that another polyadenylation is a factor responsible for the production of multiple TGF-B2 mRNAs seen on Northern blots.

BSC-40 cells contain comparable levels of specific transcribed sequences of TGF-ß'l and TGF-B2: PC-3 cells treated with tamoxifen contain more TGF-ßl mRNA than TGF-B2 (Figure 3B). This latter result is unexpected since these cells produce more TGF-B2 protein than TGF-ß1 (Ikeda et al, 1987, Biochemistry 26: 2406-2410) and suggests a level of regulatory post-transcription regarding the synthesis of this protein. growth modulator. Experiments to understand the transcriptional and translational control mechanisms that lead to the production of TGF-β1 and TGF-B2 are underway. The production of adequate amounts of TGF-B2 by recombinant DNA techniques, as has been done for TGF-β1, should further help in designing experiments to explore the different effects of this protein.

In another embodiment, the coding sequence of TGF-B2. was obtained by cDNA cloning of coding sequences of simian TGF-B2 precursor derived from polyadenylated RNA isolated from a cell line of African jDalli-cheat, BSC-40. The entire coding region of a cDNA clone was sequenced. The precursors of human and simian TGF-β2 appear to have identical amino acid sequences, and their nucleotide sequences are approximately identical. In order to express a mature, biologically active form of TGF-B2, an expression vector / host system should be chosen which not only provides high levels of transcription and translation, but also the correct production of the gene produced. . This is particularly important when using the entire coding sequence of a TGF-B2 precursor in expression vectors since the mature form of TGF-B2 appears to be derived from the precursor product via transformation phenomena. cellular. In addition, an expression / host cell system which provides for the secretion of the product can be chosen.

4:20

In particular, it appears that mature TGF-B2, a disulfide bonded homodimer of 112 amino acids per subunit can be formed by cellular transformation involving a proteolytic cleavage between the amino acids Arg-Ala of the precursor (residues numbers 330 and 331 in figure la). In addition, the TGF-B2 precursor contains three potential N-glycosylation sites not found in the mature form. Appropriate glycosylation of the precursor may be important for. cell synthesis and the release or secretion of the mature molecule. In addition, the mature form of TGF-B2 comprises a disulfide-linked dimer 1 involving 9 cysteine residues per 15 subunits. Some of these are involved in the intercatenarian disulfide bridges and others in the intracatenary disulfide bridges which modify the tertiary structure and configuration of the mature molecule and therefore its biological activity. Thus, the ability of a host cell used in the expression system to properly express and transform the TGF-B2 gene product is important for the production of a mature biologically active TGF-B2. A variety of animal host / expression vector systems (i.e., vectors which contain the elements necessary to direct replication, transcription and translation of the coding sequence of TGF-32 in a cell Suitable host) can be used by those skilled in the art as well. They include, but are not limited to, mammalian virus / host cell expression vector systems (eg, cytomegalovirus, vaccinia virus, 4-21 adenovirus, and the like); insect virus / insect cell expression vector systems (eg, baculovirus); or non-viral promoter expression systems derived from mammalian cell genomes (e.g., the mouse metallothionine promoter).

The expression elements of these vectors vary with regard to their strength and specificities. Depending on the host / vector system used, any one of a number of transcription and translation elements. can be used. For example, - when cloning into mammalian cellular systems, promoters isolated from the genome of mammalian cells, (eg the mouse metallothionine promoter) or from viruses which develop in these cells, (for example) the 7.5K vaccinia virus promoter) can be used. Promoters produced by recombinant DNA techniques or by synthetic techniques can also be used to provide for transcription of the inserted sequences.

Specific initiation signals are also required for sufficient translation of the inserted protein coding sequences. These signals include the ATG initiation codon and the adjacent sequences. In cases where the entire TGF-B2 gene including its own initiation codon and adjacent sequences is inserted into the appropriate expression vectors, additional translation control signals may not be required. However, in cases where only part of the coding sequence is inserted, exogenous translation control signals, including the ATG initiation codon; must be provided. In addition, the initiation codon must be in phase with the reading frame of the coding sequences of TGF-B2 to ensure translation of the entire insertion sequence. These exogenous translation control signals and the initiation codons can be of varied, natural or synthetic origin. Expression efficiency can be improved by including transcription attenuation sequences, enhancement elements, etc.

Any of the methods described above for inserting DNA fragments into a vector can be used to construct expression vectors containing the TGF-ß gene and the appropriate transcription / translation control signals. . These methods may include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombinations (genetic recombination).

In cases where an adenovirus is used as an expression vector, the coding sequence of TGF-B2 can be linked to an adenovirus transcription / translation control complex, for example the last promoter and the tripartite leader sequence. This chimeric gene can then be inserted into the genome of the adenovirus by in vitro or in vivo recombination. Insertion into a nonessential region of the viral genome (eg, the E1 or E3 region) will result in the formation of a recombinant virus which is viable and <23 capable of expressing TGF-B2 in affected hosts.

Similarly, the 7.5K vaccinia promoter can be used.

Another expression system which could be used to express TGF-B2 is an insect system. In such a system, the Autograph californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in the cells of Spodoptera frugiperda. The coding sequence of TGF-B2 can be cloned into nonessential regions (eg the polyhedrine gene) of the virus and placed under »control

AT

of an AcNPV promoter (for example the polyhedrine promoter). Successful insertion of the TGF-B2 coding sequence will result in the inactivation of the polyhedrine gene and the production of non-occluded recombinant virus (i.e., a virus lacking the protein coating for which the gene codes polyhedrine). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the -inserted gene is expressed.

In addition, a host cell strain can be chosen which modulates the expression of the inserted sequences, or which modifies and transforms the gene product in the specific manner desired. The expression of certain promoters can be increased in the presence of certain inducers (eg zinc and cadmium ions for metallothionine promoters). Thus, the expression of TGF-B2 obtained by genetic engineering can be controlled. This is important if the protein product of the cloned foreign gene is lethal to the host cells. In addition, modifications (eg glycosylation) and transformations (eg cleavage) of protein products are important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational transformation and modification of proteins. Appropriate cell lines or host systems can be chosen to ensure correct modification and transformation of the expressed foreign protein.

Host cells which contain the recombinant TGF-B2 coding sequence and which express the mature biologically active product can be identified by at least four general approaches: (a) DNA-DNA hybridization; (b) presence or absence of “marker” gene functions; (c) estimation of the level of transcription as measured by the expression of the transcription sequences of TGF-B2 mRNA in the host cell and (d) detection of the mature gene product as measured by immunoassay and, finally , by its biological activity.

In the first approach, the presence of the coding sequence, of TGF-B2 inserted into the expression vector * can be detected by DNA-DNA hybridization using probes comprising the nucleotide sequences which are homologous to the sequence encoding TGF-B2 substantially as shown in Figure la, or parts thereof or derivatives thereof.

In the second approach, the recombinant expression vector / host system can be identified and chosen on the basis of the presence or "absence of certain" marker "gene functions (eg, thymidine kinase activity, resistance antibiotics, methotrexate resistance, transformation phenotype, formation of an occlusion body in baculovirus, etc.). For example, if the coding sequence of TGF-1332 is inserted into a marker gene sequence of the vector, the recombinants containing the coding sequence of TGF-B2 can be identified by the absence of the marker gene function. Alternatively, a marker gene can be placed in tandem with the TGF-B2 sequence under the control of the same promoter or a different promoter used to control expression of the TGF-B2 coding sequence. Expression of the marker in response to induction or selection indicates expression of the TGF-B2 coding sequence.

In the third approach7 the transcription activity for the coding region of TGF-B2 can be assessed by hybridization assays. For example, polyadenylated RNA can be isolated and analyzed by Northern blotting using a probe homologous to the coding sequence of TGF-D2 or to specific parts thereof. Alternatively, the total nucleic acids of the host cell can be extracted and tested for hybridization with such probes.

In the fourth approach, the expression of the mature protein produced can be assessed immunologically, for example by Western blots, immunoassays such as radioimmunoprecipitation, enzyme linked immunoassays and the like. However, the ultimate test for the success of the expression system includes detection of the biologically active TGF-B2 gene product. When the host cell secretes the gene product, the cell-free medium obtained from the cultured transfecting host cell can be tested for TGF-B2 activity. When the gene product is not secreted, cell lysates can be tested for such activity. In each case, bioassays such as the growth inhibition test described herein or la. stimulation of growth independent of anchoring in target cells (Twardzik and Sherwin, 1985, J. Cell. Biochem. 28: 289-297; '· Delarco and Todaro, 1978, Proc. Natl. Acad. Sci.

15 U.S.A. 75: 4001-4005) or the like may be used.

A clone producing high levels of mature biologically active TGF-B2 being identified, the clone can be expanded and TGF-B2 'can be purified using methods well known in the art. Such methods include immunoaffinity purification, chromatographic methods including high performance liquid chromatography, and the like.

The following examples describe the cloning by cDNA of TGF-B2 precursor coding sequences from the human prostate adenocarcinoma cell line, PC-3, from which TGF-B2 was isolated previously.

The following procedures, illustrating materials and methods, were used to clone cDNAs encoding the human TGF-B2 precursor.

4 27

The human prostate adenocarcinoma cell line, PC-3, was cultivated in a medium supplemented with tamoxifen as described (Ikeda et al., 1987, Biochemistry 26: 2406-2410). MCF-7 cells were grown in Dulbecco-modified Eagle medium containing 10% fetal calf serum and 6 units / ml insulin. All other cell lines were grown in the same medium without insulin. Polyadenylated RNA was isolated by chromatography on oligo [[dT] -cellulose as described (Purcbio and Fareed, 1979, J. Virol. 29: 763-769).

Double stranded cDNA was synthesized from polyadenylated RNA isolated from PC-3 cells treated with tamoxifen for 24 hours as described (Maniatis et al., 1982, in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York). CDNA fractions greater than 1000 base pairs were cloned into lambda gt10 as described (Webb et al., 1987, DNA 6: 71-79). The library was first screened in duplicate with a 24 times qp • degenerate probe labeled with [_ Pj complementary amino acids WKWIHEP coding for DNA (probe 1) which are stored between TGF-β1 and TGF- B2: l · 4 28 [5'-GGTTCGTGTATCCATTTCCA-3 ']

C A G C A

Positive clones were then screened with a second degenerate probe 128 times complementary to amino acids CFRNVQD encoding DNA (probe 2); five of these seven amino acids are specific for TGF-B2: [5'TCTTGAACGTTTCTGAAGCA-3 ']

10 C C A C A A

G

\ T

Hybridization was performed at 42 ° C in 6X SSC, in 5X Denhart solution, in 0.15 mM pyrophosphate, in 100 micrograms / ml denatured calf hymus DNA, -in 100 micrograms / ml of yeast tRNA and ImM of EDTA (Maniatis et al., 1982, in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold 20 -Spring Harbor, New York). The filters were washed at 42 ° C in 2XSSC, 0.1% NaDodSO 4, four times for 30 minutes. Several cDNA clones were isolated which hybridized to the two probes and which were subcloned into pEMBL (Dante et al., 1983, Nucleic Acids Res. 11: 1645-1654). A clone (pPC-21) containing an insertion sequence of 2.6 kb was sequenced on both strands by the dideoxy nucleotide termination reaction (Sänger et al., 1977, Proc. Natl. Acad. Sci. USA 74 : 5463-5467) using different restriction and deletion fragments of exonuclease III combined with priming "29 of specific oligonucleotide (Henikoff, 1984, Gene 28: 351-359). Another clone (pPC-14) containing an insertion sequence of 2.2 kb has been partially sequenced. The dot matrix analysis was performed on an IBM PCAT using Gene Pro software from Riverside Scientific Enterprises (Seattle, WA).

The polyadenylated RNA was fractionated on a 1% agaroseformaldehyde gel (Lehrach et al., 1977, Biochemistry 16: 4743-4751), transferred to a.

nylon membrane (Hybond, Amersham), and hybridized with an L pl labeled probe · Hybridization was carried out at 42 ° C in 50% formamide containing 0.9 M NaCL, 50 mM phosphate 15 sodium (pH 7), 5mM EDTA, 0.1% NaDodSO4, 4X Denhardt's solution, 0.4 mg / ml yeast tRNA, and 0.25 mg / ml calf thymus DNA denatured. Filters were washed at 65 ° C in 0.25X SSC, 0.1% NaDodSO 4, dried and exposed to Cronex-4 X-ray film (DuPont) using intensifying screens Lightening Plus (DuPont).

A cDNA library was constructed using polyadenylated RNA isolated from PC-3 cells treated with tamoxifen. Previous observations indicated that treatment with tamoxifen caused a 2-5 fold increase in secretion of TGF-B2 (Ikeda et al., 1987, Biochemistry 26-2406-2410). The library was screened with probes 1 and 2 as described above. Five clones were obtained which hybridized to the two probes. A clone, pPC-21, containing an insertion sequence of 2.6 kb, was chosen for sequencing. Another clone, pPC-14 4 30 containing an insertion sequence of 2.2 kb, was partially sequenced. The DNA and deduced amino acid sequences are shown in Figure 1.

pPC-21 contains a single open reading frame encoding a polypeptide derived from 442 amino acids; the 112 amino acids of the carboxy terminus comprise the mature TGF-β2 monomer (boxed in Figure la). The first methionine encoded by the open reading frame is immediately followed by an expanse of hydrophobic and uncharged amino acids (topped by a line in Figure la) characteristic of a signal peptide. Neither the nucleotide sequence coding for '-this methionine nor those coding for the following two methionines present in the open reading frame are homologous to the consensus sequence for the initiating methionine sequence (kozak, 1986, Cell 44: 283-292). Since translation usually begins with the first methionine in an open reading frame and because homologous regions of TGF-β1, as discussed below, occur upstream of the second methionine, an attempt has been made to attribute at the first methionine the site of the start of translation. It then appears that TGF-ß2, like TGF-ß1, is expressed as part of a much larger secreted precursor. The pPC-21 clone contains 467 bp upstream of the supposed priming methionine and a 3 'untranslated region of approximately 800 bp including a poly [A] termination, fifteen bases upstream of which is located a sequence polyadenylation signal (Proudfoot and Brownlee * 1976,

Nature 263: 211-214).

4 31 The homology of nucleotide sequences within the coding regions of the cDNA clone pPC-21 of TGF-β1 and TGF-B2 was determined to be 53%. The regions encoding the 5 mature proteins have 57% homology while the upstream precursor regions have 48% homology. After optimal alignment of the two sequences, several nucleotide insertions were noted in the precursor region of TGF-ß2, one of which spans. 75 nucleotides. It is unknown whether these insertions are due to the presence of additional exons in TGF-B2. No significant homology was detected between the DNA sequences in the non-coding regions of the two clones. In fact, while TGF-ß1 had extensive non-coding regions rich in G-C, TGF-ß2 had extensive non-coding regions rich in A-T. Both cDNA clones contain repeating structural units in the 3 'non-coding region, the repeating units in TGF-β1 consisting of CCCC (purine) (Sharples et al., 1987, DNA 6: 239-244) and in TGF-B2 in ATG • or A (pyrimidine) (purine).

The localization of the restriction sites of many clones revealed that one clone, pPC-14, lacked a Kpn1 restriction site located in the amine part of the coding sequence of TGF-B2. The restriction sites of pPC-14 and pPC-21 are shown in Figure 1d. pPC-14 was sequenced over a range of approximately 100 nucleotides corresponding to this region of the molecule by specific priming with a 20-mer oligonucleotide complementary to nucleotides 277 to 296 in FIG. The results show that the 432 pPC-14 clone contains a deletion of 87 nucleotides (nucleotide positions 346 to 432 in FIG. 1a; see also FIG. 1a) which explains the missing Kpnl site and which is replaced by the sequence AAT, the codon for asparagine.

The results suggest that the clone pPC-14 codes for a shorter TGF-B2 precursor of 414 amino acids different from the sequence for which code pPC-21 only in that the 10 amino acid residues 116 to 144 are deleted and . replaced by a single residue of asparagine.

Although the entire coding region of pPC-14 has not been determined, it is probably in complete agreement with the coding sequence of pPC-21 since, with the exception of the KpnI site, the restriction sites of the two clones overlap perfectly (Figure 1d). In addition, a simian clone encoding a TGF-β precursor with 414 amino acids corresponding to the same deletion of 29 amino acids and the same replacement was identified as described in section 7 as an example below. This simian clone has a coding sequence which is roughly identical to that of the human pPC-21 clone in the 5 'and 3' regions with respect to the deletion.

Figure 2A shows the deduced protein sequence of human TGF-β1 (Derynck et al., 1985, Nature 316: 701-705) compared to that of human TGF-B2-442. TGF-B2 has been determined to be 71% homologous to human TGF-β1 throughout the mature portion of the molecule as previously described (Marquardt et al. 1987, J. Biol. Chem., In press). The amine part of the precursor upstream of. the mature molecule shows 31% homology between TGF-ß133 and TGF-ß2-442. The comparison of homology by dot matrix shown in FIG. 2B reveals that a significant homology exists in several specific areas of the proteins. Comparison of the N-terminal amino acid sequences in the supposed signal peptide region reveals no significant homology.

In TGF-B2, the signal sequence cleavage site is predicted to be located after amino acid 20 (serine) and after amino acid 29 (glycine) in TGF-ß1 (Von Heijne, 1983, Eur. J Biochem. 133: 17-21). This cleavage site directly precedes the first block of homology between \ TGF-β1 and TGF-B2 which spans 34 amino acids downstream. After elimination of the signal sequences, the precursors of TGF-ß1 and TGF-ß2 would share identical N termini on the first four amino acids, including cysteine at position 4. Fourteen amino acids downstream of this supposed N terminus, 19 amino acids among the following 21 amino acids are conserved between TGF-ß1 and TGF-B2, a larger block of homology than 'all those already seen even in the C-terminal region containing the mature TGF-ß protein. Several other blocks of strong homology exist within the region upstream of the mature protein as shown in Figures 2A and 2B. These domains are separated by long stretches of non-homologous amino acids.

The TGF-β2 precursor has three potential N-glycosylation sites (located at residues 72, 168 and 269 in Figure la). Only the first site is conserved in TGF-β1, and is located within a larger block of conserved residues 434, suggesting that this potential glycosylation site has important structural and / or functional characteristics.

After elimination of the signal sequence, the precursor of TGF-B2 would contain 31 or 59 amino acids more than its counterpart of TGF-β1. An additional cysteine residue in TGF-62 is located just upstream of a large non-homologous amino acid region which precedes the mature sequence. As with TGF-ß1, the cleavage site of the mature TGF-ß2 protein is located just after a region of 4-5 basic amino acids as shown in Figure 2A. The mature region contains nine cysteines. The conservation of 15 7 of the 9 cysteines is characteristic of the different members of the TGF-ß family. Hydropathy analyzes of TGF-ß1 and TGF-ß2 reveal similar patterns in the precursor and in mature regions, the two proteins generally being hydrophilic in nature (data not shown).

Figure 3A shows Northern blot analysis using pPC-21 to test polyadenylated RNA from BSC-40 (a kidney, African callitrich cell line) and pPC-3 cells treated with tamoxifen. The PC-3 cells contain three major species of TGF-B2 specific mRNAs having a size of 4.1, 5.1 and 6.5 kb (Figure 3A, lane 2); BSC-40 cells predominantly contain transcription sequences of 4.1 and 6.5 kb and lesser amounts of RNA of 5.1 kb (Figure 3A, lane 1). It should be noted that the pPC-21 probe does not detect the species of 2.5 kb TGF-β1 specific mRNA 4 present in this cell line under the hybridization conditions used here. These results and previous observations (Sharples et al., 1987, DNA 6: 239-244) 5 suggest that BSC-40 cells contain specific mRNAs for TGF-ß1 and TGF-ß2. In order to demonstrate this more clearly, Northern blots were hybridized to a mixture containing equal amounts of TGF-β1 and TGF-ß2 probes radiolabelled with the same specific activity. Lane 1 in Figure 3B shows that the BSC-40 cells contain the 2.5 kb TGF-ß1 specific mRNA as well as the 4 ^ 4.1 and 6.5 kb TGF-ß2 mRNA species : lane 2 in FIG. 3B shows that the PC-3 cells treated with tamoxifen also contain the 2.5 mb TGF-β1 specific mRNA. Figure 3B also demonstrates that PC-3 cells treated with tamoxifen contain more of a specific TGF-β1 message than a specific message of TGF-B2.

The identification of the TGF-B2 specific cDNA clones enabled us to screen for the TGF-β2 mRNA in different cell lines. The Northern blot shown in Figure 4, shows that specific transcription sequences for TGF-ß * 2 could be detected in HBL100 (a normal epithelial cell line derived from human milk, obtained from Dr. Greg Schultz), MCF-7 (a human mammary carcinoma cell line), SK-MEL 28 (a melanoma cell line) and KB cells (a nasopharyngeal carcinoma cell line) containing very low levels of TGF-.B2 mRNA.

"36

The following examples describe the cloning by cDNA of TGF-B2 coding sequences from the African callitrichian kidney cell line, BSC-40, which has been shown to contain mRNAs specific for TGF-B2 (section 6 below). above). The results indicate that simian TGF-B2, like human TGF-B2, is synthesized as one of at least two longer precursors from which the mature TGF-B2 molecule is derived by proteolytic cleavage.

The following procedures were used to clone the cDNAs encoding the simian TGF-B2 precursor.

BSC-40 cells were grown in Eagle medium modified by Dulbecco containing 10% fetal calf serum. Polyadenylated RNA was isolated by chromatography on oligoLdT] -cellulose as described (Purchio and Fareed, 1079, J Virol. 29: 763-769 ^.

A double stranded cDNA was synthesized from polyadenylated RNA from BSC-40 as described (Maniatis et al., 1982, Molecular Cloning: A • Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 371-372 ) and, after treatment with EcoRI methylase, was linked to oligonucleotide binders containing an EcoRI restriction enzyme recognition site (EcoRI binders). The cDNA was digested with EcoRI and was fractionated by chromatography on Sephacryl S-1000. CDNA fractions greater than 750 base pairs were pooled and linked in lambda gt10 which had been cut with EcoRI (Davis et al., 1980, A Manual for Genetic Engineering: Advanced Bacterial Genetics; "37

Cold Spring Harbor Laboratory, Cold Sring Harbor, NY), packaged (Grosveld et al., 1981, Gene 13: 227-237) and plated on E. coli 0ΟΛΛ rK_mK + hfl.

................- OR

The library was screened by plate hybridization (Bentonet et al., 1977, Science 196: 180-182) to p P-21 and p P-14 labeled probes. The clone pBSC-40-16, which hybridized the pPC-21 probe, and the clone pBSC-40-1, which hybridized the pPC-14 probe, were isolated and subcloned into 10 pEMBL. The coding sequence, from TGF-B2 to pBSC-40-1. was determined by sequencing the two strands using the dideoxy nucleotide termination reaction (Sänger et al., 1977, Proc. NatJ .. Acad.

é \ Sei. USA 74: 5463-5467). pBSC-40-16 has been partially sequenced.

Two clones were obtained from a cDNA library of BSC-40 which hybridized in turn with probes constructed from the coding sequences of precursor of TGF-B2-442 and TGF-B2- 414 human.

The pBSC-40-16 clone, which hybridized to the TGF-B2-442 probe, was sequenced over a range of 150 nucleotides (nucleotides 300 to 450 in FIG. 1 a) supposed to contain the coding sequence for the segment of, 29 amino acids from position 346 to position 432 in Figure la. The results show that, in this region, pBSC-40-16 codes for an amino acid sequence which is identical to the corresponding sequence of the human TGF-B2-442 cDNA clone, pPC-21, and suggests that pBSC-40-16 codes for a TGF-B2 precursor of 442 amino acids.

4 38

The clone pBSC-40-1, which hybridized to the TGF-B2-414 probe, was sequenced over the entire coding region. The results show that this clone codes for a TGF-B2 precursor of 414 amino acids which is identical to the precursor of human TGF-B2-442 except that the amino acid residues 116 to 144 of human TGF-B2-442 are deleted and replaced with a single residue of asparagine. At the nucleotide level, pBSC-40-1 differs from human TGF-B2-442 in the deletion region:.

nucleotides 346 to 432 in FIG. 1a are deleted and replaced by the codon for asparagine, AAT. With the exception of 13 base-silent changes, the two structures are otherwise perfectly homologous to the rest of the coding sequence.

The following examples describe the expression of biologically active mature TGF-B2 in Chinese Hamster ovary cells (CHO cells) transfected with a recombinant plasmid containing the coding sequence for mature human TGF-B2 linked downstream and framed with the sequence encoding the precursor of TGF-β1 simien, under the regulatory control of the SV40 promoter sequences. The construction sequence directed synthesis and secretion of mature biologically active TGF-B2 to a level of approximately 0.5 mg / L.

Chinese Hamster ovary (CHO) cells deficient in dihydrofolate reductase (dhfr) (Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci.

U.S.A. 77: 4216) were propagated in Ham's F-12 medium (Gibco Laboratories, NY) supplemented with "39 10% fetal bovine serum (FBS) and 150 μl £ / πι1 of L-proline. Penicillin and streptomycin were incorporated at 100 U / ml and 100 jag / ml, respectively. The CHO 5 transfectants were cultivated in an Eagle medium modified by Dulbecco containing the same supplements as those listed above. CHO cells and their derivatives were routinely separated from their supports by trypsinization at a separation ratio of 1: 5.

Methotrexate (Sigma, MO) was prepared at a reserve concentration of 10 mg / ml in water. Diluted NaOH (0.2 M) was added to dissolve the drug (final pH 6). The reserve was sterilized by filtration and stored at -20 ° C. Stock solutions of methotrexate in medium (100 µm) were kept at 4 ° C for no more than a month.

Deb restriction enzymes, T4 DNA ligase, calf intestinal phosphatase, Klenow fragment of DNA polymerase I and other DNA reagents were purchased from Bethesda • Research Laboratories, MD. Standard DNA manipulations were performed as described in Maniatis, T., et al., 1982, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

Plasmid pSV2 (B1-TGF-dhfr) which contains the TGF-β1 cDNA and the tandem mouse dhfr gene as well as intervening sequences of SV40, was constructed as described (Gentry et al., 1987, Mol. Cell. Biol. 7: 3418) '.

"40

The plasmid pSV2 / ß1-ß2 / dhfr was constructed as described below on pages 37, 38 and 39 first paragraph.

Approximately 24 hours after seeding 5 × 10 10 dhfr-deficient CHO cells in 100 mm dishes, the cultures were transfected with 20 μg of plasmid pSV2- (ß1-TGF-dhfr) linearized by Ndel in the form of a precipitate. calcium phosphate (Wigler, M., et al., 1979, Proc. Natl.

10 Acad. Sei. U.S.A. 76: 1373-1376). Briefly, 20 µg of linearized DNA was added to 1 ml of sterile 250 mM CaClg. A 1 ml portion of 2X HEPES solution (280 mM NaCl, 50 mM HEPES, 1.4 mM ·, sodium phosphate, pH 7.1) was then added dropwise, and allowed to stand. mix on ice for 30 minutes. The precipitate was then dispersed dropwise on the cells containing 10 ml of the F12 medium. After incubation at 37 ° C for 4 hours, the medium was removed and replaced with 10 ml of F12 medium containing 25% glycerol for 90 seconds at room temperature. The cells were rinsed once. with 20 ml of F12 medium and · were incubated in the non-selective F12 medium (20 ml) for a further 48 hours. Selection of the dhfr-expressing transfectants was accomplished by replacing the medium with DMEM supplemented with 10% dialyzed FBS (Gibco, N.Y.) and 150 µg / ml L-proline. Colonies were observed after culturing the cells for 10-14 days in the selection medium. Ten colonies were aspirated using a pasteur pipette and spread.

"41

Cells amplified with dihydrofolate reductase (dhfr) were derived from the primary transfectants essentially as described (Gasser, C.S. and Schimke, R.T., 1986, J. Biol. Chem.

5 261: 6938-6946). After spreading, 105 cells were seeded in 100 mm tanks and adapted to increasing concentrations of methotrexate.

The plate containing colonies visible at the highest methotrexate concentration was trypsinized and matched to this concentration of methotrexate during at least two further 1: 5 cell separations. Cells (10) were then seeded in tanks of 100 ^ mm to 5 des ·. times the concentration of methotrexate. Tray 15 containing visible colonies was again trypsinized and adapted to the medium containing methotrexate. The cells were again frozen at different stages of amplification in media containing 40% FBSL, 10% dimethyl sulfoxide and 50% DMEM. Methotrexate was not added to the freezing medium.

Mink lung epithelial cells, Mv 1 Lu (serial number CCL-64, American Type Culture Collection), which are extremely sensitive to TGF-β1, were used for the growth inhibition assay. The test was performed at -1 ΛΕ using the thymidine analog 5'-C 1J-iodo-2 'desoxyuridine (IdU) to estimate DNA synthesis. One unit of activity was defined as the amount required to inhibit 50% 125 of IdU incorporation compared to untreated CCL-64 cells.

"42

To test the transfected cells for secretion of active TGF-B2, the serum-free supernatants were collected from a 24-hour harvest from the confluent cell cultures and widely dyalised against acetic acid 0.2 M. The acetic acid was removed by lyophilization and the sample was redissolved in the sterile complete culture medium for the tests.

A hybrid TGF-ß precursor gene consisting of a coding for a simian TGF-ß1 precursor and in 5 'untranslated sequences joined by framing with the mature coding of human TGF-432 λ v and with 3' untranslated sequences was constructed as illustrated in Figure le.

pPC-21 was first digested with EcoRI, filled with the Klenow enzyme, the 2.3 kb fragment linked to pEMBL digested with HincII, and used to transform E; coli. Two clones, pPC-21 / HincII + and pPC-21 / HincII ~, having insertion sequences in opposite orientations, were used to produce digestion fragments of ExoIII overlapping by digestion with SstI and BamHI then by digestion. by ExoIII, Klenow repair, DNA binding, and E. coli transformation. Two clones, Exo 5.9 and Exo 25c were found to contain different lengths of the 5 'and 3' sequences respectively, and were subcloned into pEMBL to produce pEMBL 5.9 and pEMBL 25C. pEMBL 5.9 was digested with HinduI, cut with blunt edges by the enzyme Klenow, digested with Kpnl, and the 0.6 kb fragment (fragment 1) was isolated. Exo 25C was digested with EcoRI and Kpnl and the fragment <43 1.1 kb (fragment 2) was isolated. pGS62 was digested with BamHI, filled with the Klenow enzyme, digested with EcoRI and linked to fragments 1 and 2 (pGS62 was derived from pGS20 (Mackett et al, 5 1984, J. Virol. 49: 857) by deletion of a single site

EcoRI). The mixture was used to transform E. coli and pGS62 / CIFB was isolated.

pGS62 / CIFB was digested with PstI and EcoRI and the 1600 bp fragment was isolated and further digested with XhoII. The 400 bp XhoII-EcoRI fragment was isolated (fragment 3). pSV2-beta-TGF (Gentry et al., 1987, Mol. Cell.

Biol. 7: 3418) was digested with Apa1 and 4 * EcoRI and the large 3000 bp fragment was isolated (fragment 4).

Two complementary strands of DNA with the sequences shown below were synthesized, phosphorylated, fused and linked to the "3" and "4" fragments above. _

20 '5 CAA CAT CTG CAA AGC TCC CGG CAC CGC CGA GCT TTG

GAT GCG GCC TAT TGC TTT AGA AAT GTG CAG GAT AAI

TGC TGC CTA CGT CCA CTT TAC · ATT GAT TTC AAG AGG 3 '

5 'GATC CCT CTT GAA ATC AAT GTA AAG TGG ACG TAG GCA

GCA ATT ATC CTG CAC ATT TCT AAA GCA ATA GGC CGC

25 ATC CAA AGC TCG GCG GTG CCG GGA GCT TTG CAG ATG

TTG GGCC 3 '

The binding mixture was used to transform Ε ^ _ coli and pßl / B2 was isolated.

Plasmid pßl / ß2 was digested with EcoRI, filled with the Klenow fragment of DNA polymerase I, cut with HindIII and the fragment of i 44 1600bp was isolated: pSV2, ßl / ß2 was constructed in inserting this fragment into pSV2, neo which had previously been digested with HindIII and Hpal to eliminate the neo gene.

5 pSV2, ß1 / ß2 was digested with Pvul and

EooRI, filled with the Klenow enzyme, digested with Ndel and the 2.6 kb Ndel-EcoRI fragment (approximately) was isolated and linked to pSV2, dhfr which had been digested with Ndel and Pvull. The binding mixture was used to transform E. coli and pSV2 / ß1 / ß2 / dhfr was isolated.

PSV / ß1-ß2 / dhfr was used to transfect dhfr-deficient CHO cells and dhfr-amplified cells were derived from the primary transfectants described in the above materials and methods.

Positive clones were identified by bioassay (inhibition of mink lung epithelial cells (CCL-64) as described (Gentry et al. 1987, Mol. Cell, Biol. 7: 3418). Recombinant proteins were also detected by performing a Western blot using anti-sera - anti-peptides prepared against the NH2-YNTINPEASASPC-C00H sequence (Gentry et al., 1987, Mol.

25 Cell, Biol. 7: 3418) which is present in mature TGF-B2. *

One band, 1B9, 12.5, was found to secrete 240 ng / ml of TGF-B2 (Figure 5). This strip was then cloned by limiting the dilution in 96 30 well plates. One clone, 1B9, 12.5, cl 36, produced approximately 500 ng / ml (Figure 5).

The analysis of the protein secreted by this clone using a Western blot using an anti-peptide antiserum is presented in FIG. 456, showing the presence of the mature 24 kd TGF-B2 dimer as well as the larger precursor form (approximately 96 kd).

The following microorganisms have been deposited with the Agricultural Research Culture Collection, Northern Regional Research Center (NRRL) and have been assigned the following deposit numbers.

10 Plasmid microorganism Deposit number

Escherichia coli HB101 pPC-21 _ B-18256% Escherichia coli HB101 pPC-14 B-18333

Escherichia coli HB101 pBSC-40-1 B-18335

Escherichia coli HB101 pBSC-40-16 B-18334 15 Chinese hamster ovary pSV / ßl-ß2 / dhfr

J

4

Claims (25)

46 1. Nucleotide sequence coding for the precursor of transforming growth factor 62, characterized in that it comprises the coding sequence of nucleotides substantially as represented in FIG. 1a from approximately the residue of nucleotide number 1 to approximately the residue of nucleotide number 1326. 2. Nucleotide sequence coding for the precursor of transforming growth factor 62, characterized in that it comprises the coding sequence of nucleotides substantially such as 4 \ shown in FIG. La from about the nucleotide residue number 1 up to approximately the nucleotide residue * number 1326 in which the nucleotide sequence from nucleotide residue number 346 to nucleotide residue number 432 is deleted and replaced by the nucleotide sequence "AAT". 3. Nucleotide sequence coding for mature transforming growth factor 62, characterized in that it comprises the sequence of ^ nucleotides substantially as shown in FIG. 1a from approximately the nucleotide residue number 991 to approximately nucleotide residue number 1326. 4. Transforming growth factor 62 precursor characterized in that it comprises the amino acid sequence substantially as shown in Figure la from about the amino acid residue number 1 to about the acid residue amino number 442. 4 47 5. A precursor of the transforming growth factor ß2, characterized in that it comprises the amino acid sequence substantially as represented in FIG. 1a from approximately the amino acid residue number 1 to approximately the residue of amino acid number 442 in which the amino acid sequence from residue number 116 to residue number 144 is deleted and replaced by a single residue of asparagine. 6. A precursor of the transforming growth factor B2, characterized in that it comprises the amino acid sequence substantially as shown in FIG. 1 a from approximately J The residue Λ of amino acid number 20 to approximately the residue 15 amino acid number 442. 7. A precursor of the transforming growth factor B2, characterized in that it comprises the amino acid sequence substantially as shown in FIG. 1a from approximately the amino acid residue number 20 to approximately the residue d amino acid number 442 in which the amino acid sequence from residue number 116 to residue number 144 is deleted and replaced by a single asparagine residue. 8. Transforming growth factor ß2, characterized in that it comprises the amino acid sequence substantially as shown in FIG. 1a from approximately the amino acid residue number 331 to approximately the amino acid residue number 442. 9. Nucleotide sequence coding for the precursor of transforming growth factor β1 / transforming growth factor, hybrid B2, characterized in that it comprises the sequence “48 coding for nucleotides substantially as represented in FIG. 1b from approximately the residue of nucleotide number -70 to approximately the residue of nucleotide number 1755. 10. Precursor of the transforming growth factor β1 / transforming growth factor B2 hybrid, characterized in that it comprises the amino acid sequence substantially as represented in FIG. 1b from approximately amino acid residue number 1 up to 'to about amino acid residue number 390. 11. Precursor of transforming growth factor ß1 / transforming growth factor x hybrid ß2 characterized in that it comprises the amino acid sequence substantially as shown in FIG. 1b from approximately amino acid residue number 30 up to 'to about amino acid residue number 390. 12. A method for producing the transforming growth factor B2, characterized in that it comprises: (a) culturing a eukaryotic cell - containing a nucleotide sequence coding for the transforming growth factor B2 under the control of a second nucleotide sequence which regulates the expression of the gene so that a peptide or a protein having a growth factor activity transforming ß2 is produced by the eukaryotic cell; and (b) obtaining the transforming growth factor ß2 from the culture. 13. The method of claim 12, characterized in that the nucleotide sequence coding for the transforming growth factor i 49 ß2 comprises the nucleotide sequence substantially as shown in Figure la from nucleotide number 1 to nucleotide number 1339. 14. Method according to claim 12, characterized in that the nucleotide sequence coding for the transforming growth factor B2 comprises the nucleotide sequence substantially as shown in FIG. 1b from nucleotide number -70 to nucleotide number 1755. 15. The method of claim 12, characterized in that the eukaryotic cell ^ comprises a Chinese ovary hamster cell. 16. The method according to claim 12, characterized in that the second nucleotide sequence which controls the expression of the gene comprises an SV40 promoter. 171 Method according to claim 12, characterized in that the second nucleotide sequence comprises a promoter and a sequence coding for a marker which can be chosen for which the eukaryotic cell is deficient, so that the eukaryotic cell containing the coding sequence for transforming growth factor ß2 can * be identified. 18. The method of claim 17, characterized in that the marker capable of being chosen comprises dihydrofolate reductase. 19. The method of claim 18, characterized in that it further comprises exposing the eukaryotic cell to methotrexate, so that resistant colonies which contain amplified levels of the "50 coding sequences for dihydrofolate reductase and transforming growth factor ß2, are selected. 20. The method of claim 19, 5 characterized in that the eukaryotic cell comprises a Chinese hamster ovary cell deficient in dihydrofolate reductase. 21. A method for producing the transforming growth factor β2, characterized in that it comprises (a) the culture of the transfectant 1B9, 12.5, clone 36 as deposited with ATCC; and (b) obtaining the growth factor A s transforming ß2 from the culture. 22. The method of claim 21, characterized in that the transfectant is cultured in the presence of methotrexate. 23. Eukaryotic cell, characterized in that it contains a nucleotide sequence coding for the transforming growth factor β2 under the control of a second nucleotide sequence which regulates the expression of the gene so that the. eukaryotic cell produces the active transforming growth factor ß2. 24. Eukaryotic cell according to claim 23, characterized in that the nucleotide sequence coding for transforming growth factor B2 comprises the nucleotide sequence substantially as shown in FIG. 1b from nucleotide number -70 to nucleotide number 1755 . 25. Eukaryotic cell according to claim 23, characterized in that it is a Chinese hamster ovary cell. 4 51 26. A eukaryotic cell according to claim 23, characterized in that the second nucleotide sequence which controls the expression of the gene comprises an SV40 promoter. 27, A eukaryotic cell according to claim 23, characterized in that the second nucleotide sequence comprises a promoter and a sequence coding for a selectable marker for which the eukaryotic cell is deficient, so that the eukaryotic cell containing the sequence coding for the 82 simian transforming oroissance factor can be identified. 28. Eukaryotic cell according to claim 27, characterized in that the marker which can be chosen comprises dihydrofolate reduotase. PC. gall him e Atjcaryctô according to claim 28, characterized in that it is a Chinese hamster ovary cell deficient in dihydrofolate reduotase. 20 30, Cell line, characterized in that it is line 189, 12.5, olone 36 as deposited with the ATCC "see the access number CRL 9800,