NL8802442A - HUMANE TRANSFORMATION GROWTH FACTOR-BETA2 (TGF-BETA). - Google Patents

Description

a

VO 1272

Title: Human transformation growth factor-beta2 (TGF-β).

I. Introduction

The present invention relates to the cloning and expression of human transformation growth factor beta2.

2. Background of the invention

Growth factor beta (TGF-β) transformation belongs to a recently described family of polypeptides that regulate cell differentiation and proliferation. This family also includes Mullerian inhibitory substance (Cate et al, 1986, 10 Cell 45, 685-698), the inhibins (Mason et al, 1985, Nature 318, 659-663) and a protein predicted from a transcript of the Drosophila decapentaplegic gene complex (Padgett et al, 1987, Nature 325, 81-84).

Growth Factor Beta (TGF-β) transformation consists of two identical, disulfide-linked subunits with molecular weights of 13000 (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, 1984, J. Biol. Chem. 260, 10995-11000). It has been purified from various types of tissues, including placenta (Frolik et al, 1983, Nature 325, 81-84), platelets (Childs et al, 1982, Proc. Natl. Acad. Sci. USA 79,5312-5316; Associan et al, 1983, J. Biol. Chem. 258, 7155-7160), kidney (Roberts et al, 1983, Biochemistry 22, 5692-5698) and demineralized bone (Seyedin et al, 1985, 25 Proc. Natl. Acad. Sci. USA 82, 119-123).

In the presence of 10% serum and epidermal growth factor, TGF-β promotes anchorage-independent growth of normal rat renal fibroblasts (Roberts et al, 1981, Proc. Natl. Acad. Sci. USA 78, 5339-5343; Roberts 30 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 colony formation of AKR-2B

8802442

a

to induce fibroblasts (Tucker et al., 1983, Cancer Res.

43, 1518-1586). TGF-β has also been shown to induce fetal rat muscle mesenchyme cells to differentiate and form cartilage-specific macromolecules (Seyedin et al, 1986, 5 J. Biol. Chem. 261, 5693-5695).

In contrast to this effect on cell proliferation, both growth of TGF-β and a functionally related protein (BSC-1) isolated from African green monkey cells have been demonstrated in both human platelets and the growth of certain cells in culture. inhibits (Tucker et al, 1984,

Science 226, 705-707). TGF-3 has also been shown to inhibit growth of various human cancer cell lines (Roberts et al, 1985, Proc. Natl. Acad. Sci. USA 82, 119-123). This inhibitory / stimulatory action of TGF-β may depend on various factors, including the cell type and physiological state of the cells (for review, see Sporn et al, 1986, Science 233, 532-534).

CDNA clones encoding human TGF-β (Derynck et al, 1985, Nature 316, 701-705), mouse (Derynck et al, 1986, J. Biol. Chem. 261, 4377-) have been isolated. 4379) and the monkey (Sharpies et al, 1987, DNA 6, 239-244). Analysis of the DNA sequence of these clones indicates that TGF-β is synthesized as a large precursor polypeptide, from which the carboxy terminus is cleaved to obtain the mature TGF-β monomer. A large sequence homology has been found across the entire TGF-β precursor protein from all of the above sources.

Most recently, a protein isolated from demineralized bovine bone has been identified as related to TGF-β (Seyedin et al, 1987, J. Biol. Chem. 262, 1946-1949). The protein has also been isolated from porcine platelets (Cheifetz et al, 1987, Cell 48, 409-415), a human prostatic adenocarcinoma cell line, PC-3 (Ikeda et al, 1987, Biochemistry 26, 2406-2410), and human glioblastoma cell-35 line (Wrann et al, 1987, EMBO 6, 1633-1636). The amino acid 8802442-3 ft sequence of a portion of this protein indicated that it was homologous to TGF-β and was termed TGF-β2. The previously described TGF-β from human (Derynck et al, 1985, Nature 316, 701-705), mouse (Derynck et al, 1986, J. Biol.

Chem. 261, 4377-4379) and the monkey (Sharpies et al, 1987, DNA 6, 239-244) has been designated TGF-β1.

3. Summary of the invention.

The present invention relates to the preparation of large amounts of TGF-β 2 by. eukaryotic host cells transfected with recombinant DNA vectors containing a TGF-β2 coding sequence under control of expression regulation elements. In a specific embodiment, human TGF-β2 precursor coding cDNA clones were obtained from a cDNA library made from a tamoxifen-treated human prostate adenocarcinoma cell line, PC-3. The cDNA sequence of one such clone predicts that TGF-β2 is synthesized as a 442 amino acid polypeptide precursor from which the mature 112 amino acid TGF-β2 subunit is derived by proteolytic cleavage. This TGF-β2 precursor, designated TGF-0: 2-442, has a homology of 41% in common with the precursor of TGF-βΙ. In another embodiment, monkey TGF-β2 coding precursor cDNA clones were obtained from a cDNA library made from an African green monkey kidney cell line, BCS-40.

The cDNA sequence of one such clone predicts that TGF-β2 is also synthesized as a 414 amino acid polypeptide precursor from which the mature 112 amino acid TGF-β2 subunit is derived by proteolytic cleavage. This TGF-B2 precursor designated TGF-B2-414 has an amino acid sequence of 414 amino acid residues and is identical to the amino acid sequence of TGF-B2-442, except that it contains a single Asparagine residue e 8802442.

* - 4 - in place of the 29 amino acid sequence with numbers 116-135 of the human TGF-62-442 sequence.

Also clones from the BSC-40 cDNA library encoding a TGF-62-442 precursor of the monkey and 5 clones from the human PC-3 cDNA library containing a human TGF-β2-414 precursor encoding identified.

The TGF-62-442 human and monkey precursors appear to be perfectly homologous at the amino acid level, as do the TGF-62-414 human and monkey precursors.

The 112 amino acid mature monomers of TGF-61 and TGF-62 show a homology of 71%.

Expression vectors containing the mature TGF-62 coding sequence, in phase with the TGF-61 signal and precursor sequences (copending U.S. Patent Application No. 15, 189,984), were constructed and used to transfect Chinese Hamster Ovary (CHO) cells ). The resulting CHO transfectants produce mature, biologically active TGF-62 which is also secreted.

20 3.1 Definitions

The following terms used in this description, whether used in the singular or in the plural, will have the meanings indicated here. .

TGF-62: A transformation growth factor-62 derived from the human or the monkey comprising substantially the amino acid sequence depicted in Figure 1a from about amino acid residue No. 331 to about amino acid residue No. 442.

TGF-62 A family of transformation growth factor-62 mole-30 precursor: human- or monkey-derived cells and an amino acid sequence substantially from approximately amino acid residue No. 1 to approximately amino acid residue No. 442, as shown in Figure 1a, or from about 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 has been removed and replaced by a single Asparagine residue.

The term will refer to a TGF-32 precursor, designated TGF-32-442 or TGF-62-414, regardless of whether it comes from humans or the monkey.

Hybrid A novel transformation growth factor-6 precursor TGF-β1 / sor molecule comprising substantially the amino acid sequence depicted in Figure 1b TGF-β2 from about precursor: amino acid residue No. 1 to about amino acid residue No. 390.

TGF-31 Transformation Growth Factor-31 Precursor of the Precursor: Monkey and Signal Sequences, substantially as depicted in Figure 1b from about amino acid residue No. 1 to about amino acid residue No. 278.

4. Brief description of the figures.

FIG. 1a shows the nucleotide sequence of human TGF8-2-442 cDNA and the amino acid sequence derived therefrom. The 2597 base pair long insert of PC-21 was subcloned into pEMBL (Dante et al, 1983, Nucleic Acids Res. 11: 1645-1654 and sequenced on both strands using the dideoxy chain termination method (Sanger et al, 1977, Proc. Natl. Acad. Sci. USA 74: 5463-5467) The coding sequence is shown and the amino acid sequence derived therefrom is shown directly above. The sequence of the mature TGF-32 is boxed and the signal peptide is indicated by a line above it Potential glycosylation sites are indicated by asterisks The arrow points to the putative splice site of the signal sequence The monkey cS602 442 - 6 nucleotide sequence of TGF-32-414 cDNA is identical with the human TGF-β2-442 cDNA sequence, except that the nucleotides 346-432 (placed in brackets) are removed and replaced by the sequence AAT, 5 and further apart from the fact that different silent nucleotides The changes occur elsewhere in the structure (which is indicated directly below the altered single letter nucleotide). The deduced amino acid sequence for monkey TGF-β2-414 precursor is identical to the Τ0Ρ-β2-442 precursor human amino acid sequence, except that Asparagine replaces amino acid residues 116-144 in the human TGF-B2-442 structure. The nucleotide sequence of a human TGF ^ 2-414 cDNA was determined by the region indicated by a dashed underline and was found to be perfectly homologous to the human TGF ^ 2-442 cDNA sequence, except that the nucleotides are 346-432 have been removed and replaced by the sequence AAT.

Fig. 1b shows the nucleotide sequence of hybrid TGF-B1 / TGF-B2 precursor DNA and the amino acid sequence derived therefrom. The coding sequence is shown and directly above the amino acid sequence derived therefrom is indicated. The sequence of the mature TGF ^ 2 is boxed and the precursor signal peptide is indicated by a dashed line above it. The glyco-25 sites are indicated with asterisks. The arrow points to the putative splice site of the signal sequence. The coding sequence of the mature TGF-β2 depicted is of human origin. The monkey coding sequence of mature TGF-β2 is almost identical to the human sequence. Only three silent base changes occur and are indicated directly below the altered single letter nucleotide. Details on the cDNA cloning of TGF-β2 and the construction of the hybrid TGF-βΙ / ΤΟΡ-β 2 gene are given in the text.

FIG. lc gives a schematic diagram of the hybrid TGF-β1 / TGF-B2 precursor gene.

> 8802442 - 7 -

Fig. 1d shows restriction endonuclease maps of pPC-14 (2.2 chicken ') and pPC-21 (2.3 kb). The boxed regions indicate coding sequences for TGF-82 monomer. ATG denotes the initiating methionine codon. The distance between the MUG site and the Kpnl site in pPC-21 (2.34 kb) is approximately 420 bp. The dark area indicates the position of the 84-bp insert in pPC-21 (2.3 kb).

Fig. Ie shows the partial DNA sequence analysis of 10 pPC-14 (2.2 kb). A synthetic oligonucleotide 5'-AGGAGCGACGAAGAGTACTA-3 'that hybridized approximately 140 bp upstream of the Kpnl site within the insert into pPC-21 (2.3 kb) was used as a primer for the DNA sequencing reactions. In this region, the sequence of pPC-14 (2.2 kb) (the top line) is identical to pPC-21 (2.3 kb) up to the nucleotides encoding Asn-116. The 84-bp insertion within the Asn-116 codon of pPC-14 (2.2 kb) found in PPC-21 (2.3 kb) is shown. The Kpnl site located within the insert is indicated. FIG. 2 shows the homologies of the human TGF-βΙ and TGF-β2-442 precursor sequences. a) Primary sequence homology: identical residues are boxed. Asterisks indicate potential glycosylation sites in TGF-32. The potential splice site of the signal sequence and the splice site of the mature polypeptide are indicated, b) Dot matrix comparison using Gene Pro software. Each dot locates a point where five out of ten amino acids are identical. Diagonal lines indicate areas of homology.

FIG. 3 shows Northern blot analysis of BSC-40 and PC-3 polyadenylated RNA. Polyadenylated RNA was isolated from BSC-40 and PC-3 cells, fractionated on an agarose-formaldehyde gel, transferred to Hybond-N filters 32 and hybridized with a [Pj-labeled TGF-82 specific 32 probe, pPC-21 (Panel A) or a mixture of [P] -labeled TGF-B2 and TGF-βΙ (Sharpies et al, 1987) specific probes O & 802442-8 - (Panel B) as described in Materials and Methods; lane 1, BSC-40 polydenylated RNA (5 micrograms); lane 2, PC-3 polydenylated RNA (5 micrograms).

Fig. 4 shows the Northern blot analysis of polyade-5 methylated RNA from different sources. Polyadenylated RNA was isolated from MCF-7 (human mammary carcinoma), SK-MEL 28 (human melanoma), KB (nasopharangeal carcinoma) and HBL-100 (human mammary epithelial cells) and analyzed by Northern blot hybridization with a TGF-g2 speci Specific probe (pPC-21) as described in Materials and

Methods. Each lane contained 5 micrograms of polyadenylated RNA from (SK-MEL 28 (lane 1), MCF-7 (lane 2), HBL-100 (lane 3) or KB (lane 4) cells.

Fig. 5 shows the bioactivity analysis of recombinant TGF-82. In 12.5 mm tissue culture dishes, 189 12.5, clone 36 cells were grown to confluency. The cells were washed three times with serum-free media and incubated in 5 mm serum-free media for 24 hours. Media were collected, dialyzed against 0.2 M acetic acid, and analyzed for inhibition of DNA synthesis in CCL64 cells as described by Gentry et al, 1987, Mol. Cell. Biol. 7:34 18. In this analysis, 3.3 µg of purified natural TGF-81 standard gave 50% inhibition; the specific activity of purified natural TGF-82 was calculated to be about half that of TGF-81.

Fig. 6 shows Western blot analysis of recombinant proteins secreted by 189, 12.5, clone 36. Acid dialyzed serum-free conditioned media from 189, 12.5, clone 36 cells were fractionated by SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting using antiserum made against the synthetic peptide NH2-YNTINPEASASPC-COOH as described by Gentry et al, 1987, Mol. Cell. Biol. 7: 3418.

* β & 02442.

- 9 - 5. Description of the invention.

The present invention relates to the preparation of a biologically active, mature form of TGF-32 from the coding sequence of a TGF-3 precursor 5 gene and its product. The mature biologically active TGF-32 can be prepared by cloning and expressing the entire coding nucleotide sequence of the TGF-32 precursor or a functional equivalent thereof in a host cell that correctly processes the precursor so that a mature TGF-32 is obtained with a biological activity that is virtually indistinguishable from that of authentic natural TGF-32. Functional equivalents of the complete coding nucleotide sequence of the TGF-32 precursor include any DNA sequence capable of controlling the synthesis, processing, and output of mature TGF-32 within a suitable host cell. In this regard, hybrid precursor coding sequences, including, for example, the TGF-31 precursor sequence in phase linked to the mature TGF-32 sequence, can be constructed and used to produce biologically active TGF-32.

The method of the invention, for reasons of clear description later, may be divided into the following stages: (a) isolation or formation of the coding sequence for a precursor form of TGF-32; (b) construction of an expression vector that will control the expression of a TGF-32 coding sequence; (c) transfection of suitable host cells capable of replicating and expressing the gene and processing the gene product into the mature, biologically active form of TGF-32; and (d) identification and purification of the mature, biologically active TGF-32.

. 8802 442.

- 10 -

Once a transfectant expressing large amounts of bioactive mature TGF-B2 has been identified, the practice of the invention includes expanding that clone and isolating the expressed gene product.

The method of the invention is illustrated herein by examples in which cDNAs from the TGF-β2 precursor coding region were prepared, cloned, sequenced, and used to construct expression vectors that express high level TGF- & 2 in Control CHO cells. In a specific embodiment, the complete amino sequence of the mature form of human TGF-β2 has been determined; it shows a total homology of 71% with TGF-βΙ. Clones from a PC-3 cDNA library encoding TGF-32 have been identified using synthetic oligo-15 nucleotide probes. DNA sequencing from one of these clones showed that TGF-32, like TGF-βΙ, is synthesized as a larger precursor protein, from which the carboxy terminus is cleaved to obtain the mature TGF-β2 monomer. Although there is a 71% homology between TGF-β1 and TGF-β2 over the mature parts of these molecules, only a maximum of 31% exists in the rest of the precursor, suggesting that the amino terminal regions of TGF-βΙ and TGF-β2 may be functionally different.

In a specific embodiment of the invention, expression of a new TGF-β1 / TGF ^ 2 hybrid gene in CHO cells is used to produce large amounts of biologically active TGF-B2.

The various aspects of the method according to the invention are described in more detail in the subsections below and in the following examples.

<8802442 - 11 - 5.1 Isolation or formation of the TGF-β 2 coding region.

The coding nucleotide sequence for TGF-β2 is depicted in Figure 1a. In the practice of the method of the invention, the nucleotide sequence or fragments or functional equivalents depicted therein can be used to generate the recombinant molecules that will control expression of the TGF & 2 product in suitable host cells. In a specific embodiment, a TGF-1 / TGF-2 hybrid gene (Fig. 1b) was constructed and used for transvection of CHO cells. Transvectants producing mature, biologically active TGF-β2 in amounts of up to 500 µg per ml of culture medium were isolated.

Due to the degeneration of the coding nucleotides sequences, other DNA sequences depicted essentially for the same amino acid sequences as shown in Figures 1a and 1b can encode in the practice of the present invention and to expressing TGF-β2 can be used. Such changes involve deletions, additions or replacements of different nucleotide residues that result in a sequence encoding the same or a functionally equivalent gene product. The gene product may contain deletions, additions or replacements of amino acid residues within the sequence which result in a silent change to yield a bioactive product. Such amino acid substitutions can be performed based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or the amphipathic nature of the residues involved. For example, aspartic acid and glutamic acid are among the negatively charged amino acids; lysine and arginine to the positively charged amino acids; leucine, isoleucine, valine; glycine, alanine; asparagine, glutamine; serine, threonine; phenylalanine, tyrosine to the amino acids with 35 uncharged polar groups in the head or non-polar groups in the head, which have comparable hydrophilicity values.

The coding nucleotide sequence for TGF-32 can be obtained from cell sources that produce a TGF-32-like activity. The coding sequence can be obtained by cDNA cloning of RNA isolated and purified from such cellular sources or by genomic cloning.

From the DNA fragments generated using well-known techniques including, but not limited to, the use of restriction enzymes, cDNA or genomic libraries of samples can be prepared. The fragments encoding TGF-32 can be identified by searching such libraries with a nucleotide probe substantially complementary to any part of the sequence depicted in Figure 1a. Full length clones, i.e. clones containing the entire coding region for the TGF-32 precursor, can be selected for expression.

In an alternative embodiment of the invention, the coding sequence of Figure 1a could be fully or partially synthesized using known chemical methods. See, for example, Caruthers et al, 1980, Nuc. Acids Res. Symp. Ser. 7: 215-223; Crea and Horn, 1980, Nuc. Acids. Res. 9 (10); 25 2331; Matteucci and Carruthers, 1980, Tetrahedron Letters 21: 719 and Chow and Kempe, 1981, Nuc. Acids. Res. 9 (12): 2807-2817. Also, the protein could be produced by chemical methods to fully or partially synthesize the '30 amino acid sequence depicted in Figure 1a. For example, peptides can be synthesized by solid phase techniques on a Beekman 990 instrument, and cleaved from the resin in a manner previously described (Gentry, LE> et al, 1983, J. Biol. Chem. 258: 11219-11228; Gentry, LE and Lawton, A., 1986, 35 Virology 152: 421-431 Purification can be performed by .0802442-13 preparative high performance liquid chromatography The composition of the peptides is confirmed by amino acid analysis.

In a specific embodiment, described in the examples, the TGF-32 coding sequence was obtained by cDNA cloning of human TGF-32 precursor coding sequences derived from polyadenylated RNA isolated from the tamoxifen-treated human prostate adenocarcinoma cell line PC-3, previously shown to produce TGF-β2. The entire coding region of one cDNA clone was sequenced and compared to the published sequence of human TGF-β1 (see Figure 2).

DNA sequence analysis of TGF-β2 cDNA clones indicates that TGF-2, like TGF-β1, is synthesized as a major precursor protein, the carboxy terminus of which is cleaved to obtain the mature 112 amino acid TGF-β2 monomer. TGF-β2 has been shown to. it has a molecular weight of 24,000, and consists of two 20 disulfide-bonded subunits of 13,000 daltons (Ikeda et al, 1987, Biochemistry 26: 2406-2410; Cheifetz et al, 1987, Cell 48: 409-415. mature TGF-B2 therefore requires proper proteolytic cleavage and formation of intra- and inter-molecular disulfide bonds 25. The precursor contains an amino terminal hydrophobic leader sequence (residues 3-19) which may be responsible for the release of the cell sending the protein The mature TGF-β2 may still be linked to the remainder of the precursor during this process.

TGF-β2 shows a 71% homology to TGF-βΙ throughout the mature portion of the precursor, implying functional similarity supported by experimental evidence (Seyedin et al, 1987, J. Biol. Chem. 262: 1946 1949; Cheifetz et al, 1987, Cell 48: 409-415 The amino portion of the precursor region of TGF-βΙ, derived from humans, rodents and the monkey (Derynck et al, 1985, 8802442: -14-

Nature 316: 701-705; Derynck et al, 1986, J. Biol. Chem. 261: 4377-4379; Sharpies et al, 2987, DNA 6: 239-244 is highly conserved and suggests that this part of the molecule may have an important biological function. In contrast, no more than 31% homology exists between the N-terminal precursor regions of TGF-β1 and TGF-β2. After cleavage of the putative signal peptide, the TGF-β2 precursor would also contain more amino acids than TGF-β1 precursor. The primary structural differences within the amino terminal region of the TGF-β1 and TGF-32 precursor proteins may indicate functional differences. In the precursors, however, significant homologous regions are found in isolated blocks suggesting the conservation of important functional domains even in the N-terminal precursor region.

Northern blot analysis revealed two major classes of TGF-32 specific 4.1 and 6.5 kb mRNA in BSC-40 cells. Tamoxifen-treated PC-3 cells contained three TGF-32 transcripts of 4.1 kb, 5.1 kb and 6.5 kb. These different size messages could be the result of differences in RNA splicing, polyadenylation, or both as described for other genes (Helfman et al, 1986, Mol. Cell.

Biol. 6: 3582-3595; Sayre et al., 1987, Proc. Natl. Acad.

25 Sci. USA 84: 2941-2945). An initial analysis of another TGF-32 cDNA clone shows that it contains a 3'-untranslated region about 1 kb larger than that of pPC-21 and pPC-14 and contains a different polyadenylation site suggesting that a different polyadenylation factor 30 is responsible for the formation of multiple TGF-β2 mRNAs observed on Northern blots.

BSC-40 cells contain similar amounts of TGF-βΙ and TGF-β2-specific transcripts: PC-3 cells treated with tamoxifen contain more TGF-βΙ mRNA than 35 TGF-β2 (Fig. 3B). The latter result is unexpected, because these cells produce more TGF-32 protein than TGF-31 (Ikeda et al, 1987, Biochemistry 26: 2406-2410) and suggests regarding the synthesis of this growth. modulator an arrangement at the post-transcriptional level.

Experiments aimed at understanding the transcriptional and translational control mechanisms leading to the formation of TGF-31 and TGF-32 are underway. Production of adequate amounts of TGF-32 by recombinant DNA techniques, such as that performed for TGF-31, would further contribute to designing experiments to explore the different effects of this protein.

In another embodiment, the TGF-32 coding sequence was obtained by cDNA cloning of monkey TGF-3 2 precursor coding sequences derived from polyadenylated RNA isolated from an African green monkey cell line BSC-40. The entire coding region was sequenced from one cDNA clone.

The TGF-32 human and monkey precursors appear to have identical amino acid sequences, and their nucleotide sequences are nearly identical.

5.2 Construction of expression vectors containing the TGF-32 coding sequence.

To express a biologically active, mature form of TGF-32, an expression vector / host system should be chosen that not only ensures high levels of transcription and translation, but also correct processing of the gene product. This is especially important when the full coding sequence of a TGF-32 precursor is used in the expression constructs because the mature form of TGF-32 appears to be derived from the precursor product via cellular processing. In addition, an expression / host cell system can be selected which ensures secretion of the product.

In particular, it appears that the mature TGF-32, a .8802441-16 disulfide-linked homodimer of 112 amino acids per subunit, can be formed by cellular processing involving a proteolytic cleavage between the Arg-Ala amino acids of the precursor (the amino acid residue Nos. 330 and 5,331 in Fig. 1a). In addition, the TGF-6 2 pre-cursor contains three potential N-glycosylation sites not found in the mature form; the correct glycosylation of the precursor can be important for cellular synthesis and the release or secretion of the mature molecule. In addition, the mature form of TGF-62 includes a disulfide-bonded dimer with nine cysteine residues per subunit. Some of these are involved in interchain disulfide bonds and others in disulfide bonds in the chain, these disulfide bonds affecting the tertiary structure and configuration of the mature molecule and thus its biological activity. Therefore, the ability of a host cell used in the expression system to properly express and process the TGF-β2 gene product is important for the production of a biologically active, mature TGF-62.

The skilled artisan may as well use various animal host / expression vector systems (i.e., vectors that are the necessary elements for controlling the replication, transcription and translation of the TGF-62 coding sequence in a suitable host cell). These include, but are not limited to, virus expression vector / mammalian host cell systems (e.g., cytomegalovirus, vaccinia virus, adenovirus, and the like); insect virus expression vector / insect cell systems (e.g., baculo-30 virus); or nonviral promoter expression systems derived from the genomes of mammalian cells (e.g., the mouse metallothionine promoter).

The expression elements of these vectors vary in strength and specificity. Depending on the host / vector system used, different. 8802442.

- suitable transcription and translation elements are used. For example, in cloning in mammalian cell systems, promoters isolated from the genome of mammalian cells (e.g., the mouse metallothionine promoter) or from viruses growing in these cells (e.g., vaccinia virus 7.5 K promoter) may be used. Promoters produced by recombinant DNA or synthetic techniques can also be used for transcription of the inserted sequences.

Specific initiation signals are also required for sufficient translation of inserted sequences encoding protein. These signals include the ATG initiation codon and adjacent sequences. In cases where the entire TGF-32 gene, including its own initiation codon and adjacent sequences, has been inserted into the appropriate expression vectors, additional signals for translation translation may not be necessary.

However, in cases where only part of the coding sequence has been inserted, exogenous translation control signals, including the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the TGF-32 coding sequences to ensure translation of the entire insert. These exogenous translation control signals and initiation codons can be of different origins, both natural and synthetic origins. The efficiency of the expression can be increased by including transcription attenuation sequences, enhancer elements, etc.

Any of the previously described methods for inserting DNA fragments into a vector can be used to construct expression vectors that contain the TGF-32 gene with appropriate transcription / translation control signals. These methods can include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombinations (genetic recombination).

, 8102442 * - 18 -

In cases where an adenovirus is used as an expression vector, the TGF-32 coding sequence may be linked to an adenovirus transcription / translation control complex, for example, the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (eg region E1 or E3) will result in a recombinant virus that is viable and capable of expressing TGF-B2 in infected hosts. Likewise, the vaccinia 7.5 K promoter can be used.

Another expression system that could be used to express TGF-B2 is an insect system. In one such system, Autographa 15 californica nuclear polyhedrcsis virus (AcNPV) is used as a vector for expressing foreign genes. The virus grows in Spodoptera frugiperda cells. The TGF-B2 coding sequence can be cloned into non-essential regions (eg, the polyhedrin gene) of the virus and placed under the control of an AcNPV promoter (eg, the polyhedrine promoter). Successful insertion of the TGF-B2 coding sequence will result in inactivation of the polyhedrin gene and production of non-included recombinant virus (ie, virus lacking the protein coat encoding the polyhedrin gene).

These recombinant viruses are then used to infect Spodoptera frugiperda cells expressing the inserted gene.

In addition, a host cell strain can be selected that modulates the expression of the unauthorized sequences or modifies and processes the gene product in the specific desired manner. Expression can be enhanced from certain promoters in the presence of certain inducing agents (eg zinc and cadmium ions for metallo-thionine promoters). Expression of the genetically engineered TGF-B2 can therefore be regulated. This is important when the protein product of the cloned foreign gene for host cells is lethal. Furthermore, modifications (eg glycosylation) and processing (eg cleavage) of protein products are important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines or host systems can be selected to ensure the correct modification and processing of the expressed foreign protein.

5.3 Identification of transfectants or transformants expressing the TGF-32 gene product.

The host cells containing the recombinant TGF-32 coding sequence and expressing the biologically mature mature product can be identified according to at least four general approaches: (a) DNA-DNA hybridization; (b) the presence or absence of "marker" gene functions; (c) determination of the transcription level as measured by the expression of TGF-32 mRNA transcripts in the host cell; and (d) detection of the mature gene product as measured by immunoassay and ultimately its biological activity.

In the first approach, the presence of the TGF-32 coding sequence inserted into the expression vector can be detected by DNA-DNA hybridization using probes comprising nucleotide sequences homologous to the TGF-32 coding sequence essentially as in Fig. 1a, or 30 parts or derivatives thereof.

In the second approach, the recombinant expression vector / host system can be identified and selected based on the presence or absence of certain "marker" gene functions (eg, thymidine. && 02442 - 20 - kinase activity, antibiotic resistance, methotrexate resistance, transformation phenotype. , inclusion body formation in baculovirus, etc.). For example, when the TGF-g2 coding sequence is inserted into a marker 5 gene sequence of the vector, recombinants containing the TGF-$ 2 coding sequence can be identified by the absence of the function of the marker gene. Also, a marker gene in tandem with the TGF-β2 sequence can be placed under the control of the same or a different promoter, used to control the expression of the TGF-f5 2 coding sequence. Expression of the marker in response to induction or selection indicates expression of the TGF-B2 coding sequence.

In the third approach, transcription activity for the TGF & 2 coding region can be determined by hybridization analyzes. For example, polyadylated RNA can be isolated and analyzed by Northern blotting using a probe homologous to the TGF-β2 coding sequence or certain parts thereof.

Alternatively, the total nucleic acid material of the host cell can be extracted and analyzed for hybridization with such probes.

In the fourth approach, expression of the mature protein product can be determined immunologically, for example, by Western blots, immunoassays such as radioimmunoprecipitation, enzyme-linked immunoassays and the like. However, the final test for the success of the expression system involves the detection of the biologically active TGF-β2 gene product. When the host cell secretes the gene product, the cell-free media obtained from the cultured transfectant host cell can be analyzed for TGF-β 2 activity. When the gene product is not secreted, cell lysates can be analyzed for such activity. In both cases, biological assays can be used such as the growth inhibition assay described herein, or the stimulation of anchorage-independent growth in target cells (Twardzik and Sherwin, 1985, J. Cell. Biochem. 28: 289-297; Delarco and Todaro, 1978, Proc Natl Acad Sci USA 75: 4001-4005) and the like.

Once a clone producing large amounts of biologically active, mature TGF-32 has been identified, the clone can be expanded and the TGF-32 purified using techniques known per se. Such methods include immunoaffinity purification, chromatographic methods including high performance liquid chromatography and the like.

6. Example: cDNA cloning of TGF-32 precursor from PC-3 cells.

The following examples describe the cDNA

cloning of TGF-32 precursor coding sequences from the human prostatic adenocarcinoma cell line PC-3, from which TGF-32 had previously been isolated.

6.1 Materials and Methods.

The following procedures were used to clone cDNAs encoding the human TGF-32 precursor.

6.1.1 Cell culture and RNA extraction

The human prostate adenocarcinoma cell line PC-3 was cultured in tamoxifen supplemented medium, as described by Ikeda et al, 1987, Biochemistry 26: 2406-2410.

MCF-7 cells were grown in Dulbecco's modified Eagle's 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 oligo CdT1 cellulose chromatography as described by Purchio and Fareed, 1979, J. Virol. 29: 763-769. .

c 8802442 - 22 - 6.1.2 Constructing and searching the cDNA library

Double stranded cDNA was synthesized from polyadenylated RNA isolated from PC-3 cells treated with tamoxifen for 24 hours, as described by 5 Maniatis et al, 1982 in Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbar, New York. In the manner described by Webb et al, 1987, DNA 6: 71-79, cDNA fractions larger than 1000 base pairs were cloned into lambda gt10. The library was first searched in duplicate with a 24-fold degenerate E32?] -Labeled probe complementary to DNA encoding the amino acids WKWIHEP (probe 1) conserved between TGF-81 and TGF-É52: [ 5 '-GGTTCGTGTATCCATTTCCA- 3']

15 C A G C

a

Positive clones were then searched with a second 128-fold degenerate probe complementary to DNA encoding the amino acids CFRNVQD (probe 2); Five of these seven amino acids are specific for TGF-82: [5'TCTTGAACGTTTCTGAAGCA-3 ']

C C A C A A G T

Hybridization was performed at 42 ° C in 6XSSC, 5X

Denhart's solution, 0.15 mM pyrophosphate, 100 micrograms / ml denatured calf thymus DNA, 100 micrograms / ml yeast tRNA and 1 mM EDTA (Maniatis et al, 1982, in Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring 30 Harbor, New York). Filters were washed at 42 ° C in 2XSSC, 0.1% NaDodSO 4, four times for 30 minutes. Several cDNA clones were isolated that hybridized to both probes and subcloned into pEMBL (Dante et al, 1983, Nucleic Acids Res. 11: 1645-1654).

One clone (pPC-21) containing a 2.6 kb insert, .8802442-23 - was sequenced on both strands using the dideoxy chain termination method (Sanger et al, 1977, Proc. Natl. Acad. Sci USA 74: 5463-5467) using different restriction and exonuclease III deletion fragments in combination with specific oligonucleotide primers (Henikoff, 1984, Gene 28: 351-359). Another clone (pPC-14) containing a 2.2 kb insert was partially sequenced. Dot matrix analysis was performed on an IBM ATPC using Gene Pro software 10 from Riverside Scientific Enterprises (Seattle, WA).

6.1.3 Other blot analysis.

Polyadenylated RNA was fractionated on a 1% agarose formaldehyde gel (Lehrach et al, 1977, Biochemistry 16: 4743-4751), transferred to a nylon membrane, (Hybond, Amersham), and hybridized with a D ^ P] labeled probe. Hybridization was performed at 42 ° C in 50% formamide containing 0.9 M NaCl, 50 mM sodium phosphate (pH 7.0), 5 mM EDTA, 0.1% NaDodSO 4, 4X Denhardt's solution, 0.4 mg / ml yeast tRNA, and 0.25 mg / ml denatured calf thymus DNA.

Filters were washed at 65 ° C in 0.25X SSC, 0.1% NaDodSO 2, dried and exposed to Cronex-4 X-ray film (DuPont) using Lightening Plus reinforcement screens (DuPont).

6.2 Results

A cDNA library was constructed using polyadenylated RNA isolated from tamoxifen-treated PC-3 cells. Previous observations indicated that tamoxifen treatment resulted in a 2- to 5-fold increase in TGF-82 secretion (Ikeda et al, 1987, Biochemistry 26: 2406-2410). The library was searched with probes 1 and 2 as described above. Five clones were obtained which hybridized with both probes: one clone, pPC-21, containing a 2.6 kb insert was chosen to sequence.

.8802442 * - 24 -

Another clone, pPC-14, which contained a 2.2 kb insert, was partially sequenced. The DNA and deduced amino acid sequences are shown in Figure 1.

The clone pPC-21 contains a single open reading frame encoding a derived polypeptide of 442 amino acids; the 112 carboxy terminal amino acids comprise the mature TGF-32 monomer (boxed in Figure 1a). The first methionine encoded by the open reading frame is immediately followed by a series of hydrophobic and uncharged amino-10 acids (indicated in Figure 1a by a line above), which is characteristic of a signal peptide. Neither the nucleotide sequence encoding this methionine, nor the nucleotide sequences encoding the next two methionines present in the open reading frame, are homologous with the consensus sequence for the initiating methionine sequence (Kozak, 1986, Cell 44: 283-292). Because translation is usually initiated in an open reading frame at the first methionine, and because with TGF-31 homologous regions, as discussed below, occur upstream of the second methionine, the first methionine has been tentatively referred to as the translation initiation site. It then appears that TGF-32, as well as TGF-βΙ, is expressed as part of a much larger secreted precursor. The pPC-21 clone contains 467 bp upstream of the putative initiating methionine and a 3 'untranslated region of approximately 800 bp including a poly [A] trace, fifteen bases upstream of which is a polyadenylation signal sequence (Proudfoot and Brownlee, 1976, Nature 263: 211-214).

The nucleotide sequence homology within the coding regions of the TGF-β1 and TGF-32 pPC-21 cDNA clone was determined to be 53%. The regions encoding the mature proteins have a homology of 57% while the upstream precursor regions have a homology of 48%. After optimal alignment of the two sequences, different nucleotide insertions in the TGF-32 precursor region were noted, one of which spanned 75 nucleotides. Whether these insertions are due to the presence of extra exons in TGF-32 is unknown. No significant homology was detected between the DNA sequences in the non-coding regions of the two clones. While TGF-3I has long G-C rich non-coding regions, TGF-32 actually has long A-T rich non-coding regions.

Both cDNA clones contain recurring structural motifs 10 in the 3 'non-coding region, the repeats in TGF-31 consisting of (purine) CCCC (Sharpies et al, 1987, DNA 6: 239-244) and in TGF-32 from ATG or A (pyrimidine) (purine).

Restriction maps from many clones revealed that one clone, pPC-14, lacked a Kpnl restriction site located in the amino part of the TGF-32 coding sequence. Restriction maps of pPC-14 and pPC-21 are shown in Figure 1d. PPC-14 was sequenced over a length of about 100 nucleotides corresponding to this region of the molecule, using as a primer specifically a 20 base oligonucleotide which was oligonucleotide complementary to nucleotides 277-296 in Fig. 1a. . The results show that the pPC-14 clone contains a deletion of 87 nucleotides (the nucleotide positions 346-432 in Fig. La; see also Fig. Ie) responsible for the missing 25 Kpnl site and replaced by the sequence AAT, the codon for Asparagine. The results suggest that the pPC-14 clone encodes a shorter 414 amino acid TGF-82 precursor, which differs from the sequence encoded by pPC-21 only in that the amino acid residues are deleted 116-144 and replaced by a single

Asparagine residue.

Although not the entire coding region of pPC-14 was determined, it probably matches the pPC-21 coding sequence perfectly because the restriction maps of the two clones, apart from the Kpnl site, perfectly overlap .8862442-26 pen (Fig. Id). Furthermore, a monkey-derived clone encoding a 414 amino acid long TGF-3 precursor containing the same 29 amino acid deletion and replacement has been identified, as described in the example of paragraph 7, see below. This monkey-derived clone has a coding sequence substantially identical to that of the human pPC-21 clone in the regions 5 'and 3' to the deletion.

Fig. 2A shows the derived protein sequence of human TGF-31 (Derynck et al, 1985, Nature 316: 701-705) compared to that of human TGF-32-442. TGF-32 was found to be 71% homologous to human TGF-31 over the entire mature portion of the molecule as previously described (Marquardt et al, 1987, J. Biol. Chem., In press). The amino 15 portion of the precursor, upstream of the mature molecule, shows a 31% homology between TGF-31 and TGF-32-442.

The dot matrix homology equation shown in Figure 2b shows that there is significant homology in several specific regions of the proteins. Comparison of the N-terminal amino acid sequences in the putative signal peptide region shows no significant homology.

In TGF-32, the cleavage site for the signal sequence according to the prediction is behind amino acid 20 (serine) 25 and behind amino acid 29 (glycine) in TGF-31 (Von Heijne, 1983, Eur. J. Biochem. 133: 17- 21). This cleavage site directly precedes the first block of homology between TGF-31 and TGF-β2 that extends 34 amino acids downstream. Upon removal of the signal sequences, the TGF-31 and TGF-32 precursors would share identical N-termini across the first four amino acids including the cysteine at position 4. Fourteen amino acids downstream of this putative N-terminus are 19 of the following 21 amino acids conserved between TGF-31 and TGF-32, a homology block larger than any other, even in the C-terminal .8802442-27 region containing the mature TGF-32 protein. Still several other blocks of strong homology exist in the region upstream of the mature protein as can be seen in Figures 2a and 2b. These domains are separated by 5 long stretches of non-homologous amino acids.

The TGF-β2 precursor has three potential N-glycosylation sites (located on residues 72, 168 and 269 in Figure 1a). Only the first site is conserved in TGF-β1, 10 and lies within a larger block of conserved residues, suggesting that this potential glycosylation site has important structural and / or functional properties.

After removing the signal sequence, the 15 TGF- $ 2 precursor would contain 31 or 59 more amino acids than its TGF-βΙ counterpart. An additional cysteine residue in TGF-β2 is located just downstream of a large region of non-homologous amino acids preceding the mature sequence. As with TGF-βΙ, the cleavage site of the mature TGF-β2 protein is just behind a region of 4-5 basic amino acids as shown in Figure 2A. The mature area contains nine cysteines. Conservation of 7 of the 9 cysteines is characteristic of the different members of the TGF-3 family. Hydropathic analyzes of TGF-β1 and TGF-B2 show similar patterns in both the precursor and the mature regions, while both proteins are generally hydrophilic in nature (data not shown).

Fig. 3A shows a Northern blot analysis using pPC-21 as a probe for polyadenylated RNA from BSC-40 (an African green monkey kidney cell line) and tamoxifen-treated PC-3 cells. PC-3 cells contain three predominant TGF-B2 specific mRNA species sized 4.1, 5.1 and 6.5 kb (Fig. 3A, lane 2); BSC-40 cells predominantly contain the 4.1 and 6.5 kb transcripts and 35 aliquots of the 5.1 kb RNA (Fig. 3A, lane 1). Note that the pPC-21 probe does not detect the 2.5 kb 8802442-28 TGF-β1-specific mRNA species present in these cells under the hybridization conditions used here. These results and previous observations (Sharpies et al, 1987, DNA 6: 239-244) suggest that BSC-40 cells contain TGF-βΙ and 5 TGF-g2 specific mRNAs. To demonstrate this more clearly, Northern blots were hybridized with a mixture containing equal amounts of the same specific activity radiolabeled TGF-βΙ and TGF-32 probes. Lane 1 of Figure 3B shows that BSC-40 cells contain both the 2.5 kb TGF-βΐ-specific mRNA and the 4.1 and 6.5 kb TGF-β2 mRNA species: Lane 2 of Figure 3B shows that with termoxifen-treated PC-3 cells also contain the 2.5 kb TGF-βΐ-specific mRNA. Fig. 3B also shows that termoxifen-treated PC-3 cells contain more TGF-61 specific than TGF-f52 specific messages.

The identification of TGF-62 specific cDNA clones has made it possible to search for TGF-β2 mRNA in different cell lines. The Northern blot shown in Figure 4 shows that TGF-32 specific transcripts could be detected in HBL 100 (a normal human milk epithelial cell line, obtained from Dr. Greg Schultz), MCF-7 (a human mammary carcinoma cell line ), SK-MEL 28 (a melanoma cell line), and that KB cells (a nasopharyngeal carcinoma cell line) contain very small amounts of TGF-32 mRNA.

7. Example: cDNA cloning of TGF-β2 precursor from BSC-40 cells.

The following examples describe the cDNA cloning of TGF- $ 2 coding sequences from the African green monkey kidney cell line BSC-40, which was shown to contain TGF-β2-30 specific mRNAs (see section 6 above). The results indicate that monkey TGF-32, as well as human TGF-32, is synthesized as one of at least two longer precursors from which the mature TGF-β2 molecule is derived by proteolytic cleavage.

8802442 - 29 - 7.1 Materials and Methods

The following procedures were used to clone cDNAs encoding monkey TGF-β2 precursor.

7.1.1 Cell culture and RNA extraction BSC-40 cells were grown in Dulbecco's modified Eagle medium containing 10% fetal calf serum. Polyadenylated RNA was isolated by oligoEdT] cellulose chromatography as described by Purchio and Fareed, 1979, 10 J. Virol. 29: 763-769.

7.1.2 Constructing and searching the cDNA library

Double-stranded cDNA was synthesized from BSC-40 polyadenylated RNA as described by Maniatis et al, 1982, Molecular Cloning: A Laboratory Manual, Cold Spring 15 Harbor Laboratory, Cold Spring Harbor, NY, 371-372 and after treatment with EcoRI, methylase was linked with oligonucleotide linkers containing an EcoRI restriction enzyme recognition site (EcoRI linkers). The cDNA was treated with EcoRI and fractionated by chromatography on Sephacryl 20 S-1000. cDNA fractions greater than 750 base pairs were collected and ligated into lambda gt10 cut with EcoRI (Davis et al, 1980, A Manual for Genetic Engineering: Advanced Bacterial Genetics; Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), packaged (Grosveld et al, 1981, Gene 13: 227-237) and plated on E. coli Cggg rK ~ mK + hfl. The library was searched by plaque hybridization (Bentonet et al, 1977, Science 196: 180-182) with [P] labeled pPC-21 and pPC-14 probes. Clone pBSC-40-16, which hybridized to the pPC-21 probe, and clone pBSC-40-1, which hybridized to the pPC-14 probe, were isolated and subcloned into pEMBL. The TGF-32 coding sequence of pBSC-40-1 was determined by sequencing .8802442 - 30 - from both strands using the dideoxy chain termination method (Sanger et al, 1977, Proc. Natl. Acad. Sci. USA 74 : 5463-5467). The sequence was partially sequenced for pBSC-40-16.

7.2 Results

Two clones were obtained from a BSC-40 cDNA

library each hybridizing with a different probe constructed from the coding sequences of the human TGF-82-442 and TGF-82-414 precursor.

Clone pBSC-40-16, which dissected with the TGF-82-442 probe hybrid-10, was sequenced over a length of 150 nucleotides (nucleotides 300-450 in Figure 1a) expected to encode the coding sequence for the 29 amino acid segment from positions 346-432 in Figure 1a.

The results show that over this region pBSC-40-16 encodes an amino acid sequence identical to the corresponding sequence in the human TGF-82-442 cDNA clone, pPC-21, and suggests that pBSC-40-16 encodes a 442 amino acid long TGF-82 precursor.

Clone pBSC-40-1, which hybridized with the TGF-82-414 probe hybrid, was sequenced over the entire coding region. Results show that this clone encodes a 414 amino acid long TGF-82 precursor that is identical to the human TGF-82-442 precursor, except that the amino acid residues 116-144 have been removed from human TGF-82-442 and replaced by a single Asparagine residue. At the nucleotide level, pBSC-40-1 differs from human TGF-82-442 in the region of the deletion: nucleotides 346-432 in Figure 1a have been deleted and replaced with the codon for Asparagine, AAT. Apart from 13 silent base changes, the two structures for the remainder are perfectly homologous to the rest of the coding sequence.

.8802442 - 31 - 8. Example; Expression of TGF-32

The following examples describe the expression of mature, biologically active TGF-β 2 in Chinese hamster ovary (CHO) cells transfected with a recombinant binant plasmid containing the coding sequence for mature human TGF-β2 downstream and in phase linked to the coding sequence for the monkey TGF-βΙ precursor, under regulatory control of the SV40 promoter sequences. The construct controlled the synthesis and secretion of mature, biologically active TGF-β 2 at a level of approximately 0.5 mg / l.

8.1 Materials and Methods 8.1.1 Cell Culture

Dihydrofolate reductase (dhfr) -deficient Chinese Hamster Ovary (CHO) cells (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 10% fetal bovine serum (FBS) and 150 µg / ml L-proline. Penicillin and streptomycin were included in amounts of 100 U / ml and 100 µg / ml, respectively. CHO transvectants were grown in Dulbecco's modified Eagle's medium containing the same supplements as listed above. CHO cells and derivatives thereof routinely underwent trypsinization at a 1: 5 split ratio. Methotrexate (Sigma, MO) was prepared in a 10 mg / ml aqueous solution of the stock solution. Diluted NaOH (0.2 M) was added to dissolve the drug (final pH 6). The stock solution was sterilized through a filter and stored at -20 ° C. Stock solutions of methotrexate in media (100 µM) were kept at 4 ° C for no longer than 1 month.

8802442 - 32 - 8.1.2 DNA manipulations and plasmid constructions

Restriction enzymes, T4 DNA ligase, calf intestinal phosphatase, the Klenow fragment of DNA polymerase I and other DNA reagents were purchased from Bethesda Research Labora-tories, MD. Standard DNA manipulations were performed according to the protocols given in Maniatis, T., et al, 1982, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

Plasmid pSV2 (81-TGF-dhfr), which contained the monkey TGF-β1 cDNA 10 and mouse dhfr gene in tandem as well as intermediate SV40 sequences, was constructed as described by Gentry et al, 1987, Mol. Cell. Biol. 7: 3418).

Plasmid pSV2 / 8l-B2 / dhfr was constructed in the manner described here in section 8.2.

8.1.3 DNA transfections g

Approximately 24 hours after seeding 10 dhfr-deficient CHO cells on 100 mm dishes, the cultures were transfected with 20 µg of Ndel-linearized pSV2 (31-TGF-20 dhfr) plasmid as a calcium phosphate precipitate (Wigler, M., et al, 1979, Proc Natl Acad Sci USA 76: 1373-1376). Briefly, 20 µg of linearized DNA was added to 1 ml 250 mM sterile CaCl 3. A 1 ml portion of 2X HEPES solution (280 mM NaCl, 50 mM HEPES, 1.5 mM sodium phosphate, pH 7.1) was then added dropwise and the mixture was left on ice for 30 minutes. The precipitate was then distributed dropwise to the cells containing 10 ml of the F12 media. After 4 hours of incubation at 37 ° C, the media was removed and replaced with 10 ml of F12 media containing 25% glycerol for 90 seconds at room temperature. The cells were rinsed once with 20 ml of F12 media and incubated in 20 ml of the non-selective F12 media for an additional 48 hours. Selection for dhfr-expressing .8802442-33 transfectants was made by replacing the media with DMEM supplemented with 10% dialyzed FBS (Gibco, N.Y.) and 150 µg / ml L-proline. Colonies were observed after culturing the cells in the selection media for 10-14 days. Ten colonies were aspirated and expanded with a Pasteur pipette.

8.1.4 Selection of methotrexate resistant cells

Dihydrofolate reductase (dhfr) enhanced cells were prepared essentially as described by Gasser, C.S. and Schimke, R.T., 1986, J. Biol. Chem. 261: 6938-6946. Derived from the primary transfectants after extension, 10 cells were seeded on 100 mm dishes and used to increasing concentrations of methotrexate. The plate containing visible colonies at the highest methotrexate concentration was trypsinized and used to that concentration of methotrexate 5 for at least two additional 1: 5 cell passages. Then 10 cells were seeded on 100 mm dishes at 5 times the concentration of methotrexate. The dish containing visible colonies was retrypsinized and used in the methotrexate containing medium. Cells were frozen back at various stages of amplification in media containing 40% FBS, 10% dimethyl sulfoxide and 50% DMEM. Methotrexate was not included in the freezing media.

8.1.5 Growth inhibition analysis For the growth inhibition analysis, mink lung epithelial cells, Mv 1 Lu (accession number CCL-64, American

Type Culture Collection), which are extremely sensitive to TGF- $ 1. The analysis was performed using 125 of the thymidine analog 5 '- [13-iodo-2' deoxyuridine 125 (Idü) to determine DNA synthesis. A unit of activity was defined as the amount needed to prevent 50% uptake of IdU compared to untreated CCL-64 cells.

»8802442 - 34 -

To analyze transfused cells for secretion of active TGF-32, serum-free supernatants from one 24-hour pool were collected on confluent cell cultures and dialyzed extensively against 0.2 M acetic acid. Acetic acid was removed by lyophilization and the sample was returned to sterile complete culture medium solved for analyzes.

8.2 Construction of TGF-3I / TGF-32 hybrid precursor gene for TGF-32 expression

A hybrid TGF-32 precursor gene, consisting of 10 monkey TGF-3I precursor coding sequences and 5 'untranslated sequences, phase-linked to mature human TGF-32 coding sequences and 3' untranslated sequences, was presented in the Figure 1c shown shown.

First, pPC-21 was digested with EcoRI, filled with Klenow enzyme, then the 2.3 kb fragment was ligated into pEMBL digested with Hindi and used to transform E. coli. Two clones, (pPC-21 / HincII + and pPC-21 / HincII, containing inserts in opposite orientations, were used to generate overlapping ExoIII digestion fragments by cutting them both with SstI and BamHI followed by cutting with ExoIII, Klenow repair , DNA Religion, and Transformation of E. coli Two clones, Exo 5.9 and Exo 25C were found to contain different lengths of 5 'and 3' sequences, respectively, and were subcloned into pEMBL to form pEMBL 5.9 and pEMBL 25C obtained.

The plasmid pEMBL 5.9 was digested with HindIII, blunted with Klenow enzyme, digested with Kpnl, and the 0.6 Kb fragment (fragment I) was isolated. Exo 25C was cut with EcoRI and Kpnl and the 1.1 Kb fragment (fragment 2) was isolated. The plasmid pGS62 was digested with BamHI, filled with Klenow enzyme, digested with EcoRI and ligated to fragments 1 and 2 (pGS62 was derived from pGS20 (Mackett et al, 1984, J. Virol. 49: 857) by deletion of a c SB02442 - 35 - single EcoRI site). The mixture was used to transform E. coli and pGS62 / CIFB was isolated.

pGS62 / CIFB was cut with PstI and EcoRI and the 1600 bp fragment was isolated and further cut with 5 XhoII. The resulting 400 bp XhoII-EcoRI fragment was isolated (fragment 3). The plasmid pSV2-3-TGF (Gentry et al, 1987, Mol. Cell. Biol. 7: 3418) was digested with Apal and EcoRI and the 3000 bp size fragment was isolated (fragment 4).

Two complementary strands of DNA with the sequences shown below were synthesized, phosphorilated, annealed and ligated with fragments 131 and '41 described above.

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

15 HOLE GCG GCC TAT TGC TTT AGA AT GTGG CAG HOLE AT

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 CAG ATT TCT AAA GCA ATA GGC CGC

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

20 TTG GGCC 3 "

The ligation mixture was used to transform E. coli and p βΐ / 32 was isolated.

The plasmid p 31/32 was digested with EcoRI, filled with the Klenow fragment of DNA polymerase I, digested with HindIII and the 1600 bp fragment was isolated: pSV2, 31/32 was constructed by inserting this fragment into pSV2, neo previously cut with HindlII and Hpal to remove the neo gene.

The plasmid pSV2, 31/32 was cut with Pvul and EcoRI, filled with Klenow enzyme, cut with Ndel and the Ndel-EcoRI fragment of '2.6 kb (approximate) was isolated and ligated with pSV2, dhfr that was cut with Ndel and PvuII. The ligation mixture was used to transform E. coli and pSV2 / 31/32 / dhfr isolated.

c8802442 - 36 - 8.3 Expression of TGF-82 in CHO cells

The plasmid pSV / 8l / 82 / dhfr was used for transfection of dhfr-deficient CHO cells and dhfr-enhanced cells were derived from the primary transfectants as described above under Materials and Methods.

Positive clones were identified by a bioassay (inhibition of minklong epithelial cells (CCL-64) as described by Gentry et al, 1987, Mol. Cell, Biol.

10 7: 34 18. Recombinant proteins were also detected with

Western blotting using an antipeptide antiserum made against the sequence NH2-YNTINPEASASPC-COOH (Gentry et al, 1987, Mol. Cell, Biol. 7: 3418) present in mature TGF-82.

One cell line, 189, 12.5, was found to secrete 240 ng / ml TGF-82 (Fig. 5). This cell line was then cloned by limiting dilution in 96-well plates. One clone, 189, 12.5, cl 36, produced about 500 ng / ml (Fig. 5).

Analysis of the protein secreted by this clone by Western blotting using anti-peptide antiserum is shown in Figure 6, disclosing the presence of the mature 24kd TGF-82 dimer and of the larger (about 90kd) precursor form .

25 9. Deposits of microorganisms

The following microorganisms have been deposited with the Agricultural Research Culture Collection, Northern Regional Research Center (NRRL) with the following access numbers: 30 Microorganism Plasmid Access 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 35 Chinese Hamster Ovary pSV / 81-32 / dhfr, 8902442

Claims (30)

Nucleotide sequence encoding transformation-growth factor-β2 precursor comprising the encoding nucleotide sequence substantially as shown in Fig. 1a from about nucleotide residue No. 1 to about nucleotide residue No. 1326. Nucleotide sequence, encoding transformation-growth factor-β2 precursor, comprising the encoding nucleotide sequence substantially as shown in Fig. 1a from about nucleotide residue No. 1 to about nucleotide residue No. 1326, wherein the nucleotide sequence of nucleotide residue is 346-432 removed and replaced with the nucleotide sequence AAT. 3. Nucleotide sequence, encoding mature transformation growth factor-β2, comprising the nucleotide sequence substantially as shown in Figure 1a from about nucleotide residue No. 991 to about nucleotide residue No. 1326. A transformation growth factor-62 precursor, comprising the amino acid sequence substantially as shown in Figure 1a from about amino acid residue No. 1 to about amino acid residue No. 442. 5. Transformation growth factor β2 precursor, comprising the amino acid sequence substantially as depicted in Figure 1a from about amino acid residue No. 1 to about amino acid residue No. 442, wherein the amino acid sequence of amino acid residue No. 116-144 has been removed and replaced by a single Asparagine residue. 6. Transformation growth factor-β2 precursor, comprising the amino acid sequence substantially as shown in Figure 1a from about amino acid residue No. 20 to about amino acid residue No. 442. 7. Transformation growth factor-β2 precursor, comprising the amino acid sequence substantially as shown in Figure 1a from about amino acid residue No. 20 to about amino acid .8802442 »(, - 38 - residue No. 442 wherein the amino acid sequence of residues Nos. 116-144 has been removed and replaced with a single Asparagine residue. 8. Transformation growth factor-82, comprising the amino acid sequence substantially as shown in Figure 1a, from about amino acid residue No. 331 to about amino acid residue No. 442. Nucleotide sequence, encoding a hybrid transformation growth factor-51 / transformation growth factor-6 2 10 precursor, comprising the encoding nucleotide sequence substantially as shown in Figure 1b from about nucleotide residue No. -70 to about nucleotide residue No. 1755. 10. Hybrid transformation growth factor-31 / transformation growth factor-32 precursor comprising the amino acid sequence 15 substantially as shown in Fig. 1b from about amino acid residue No. 1 to about amino acid residue No. 390. 11. Hybrid transformation growth factor-31 / transformation greeting factor-32 precursor, comprising the amino acid sequence substantially as shown in Fig. 1b from about amino acid residue No. 30 to about amino acid residue No. 390. A method of producing transformation growth factor-32 comprising: (a) culturing a eucaryotic cell containing a nucleotide sequence encoding transformation growth factor-3.2 under control of a second nucleotide sequence that regulates gene expression such that a peptide or protein with transform growth factor-32 activity is produced by the eucaryotic cell; and 30 (b) recovering the transformation growth factor-32 from the culture. The method of claim 12, wherein the nucleotide sequence encoding transform growth factor-32 comprises the nucleotide sequence substantially as shown in Fig. 1a from nucleotide No. 1 to 1339. "8802442 J - 39 - The method of claim 12, wherein the nucleotide sequence encoding transform growth factor-6 comprises the nucleotide sequence substantially as shown in Fig. 1b from nucleotide No. -70 to 1755. The method of claim 12, wherein the eucaryotic cell comprises a Chinese hamster ovary cell. The method of claim 12, wherein the second nucleotide sequence controlling gene expression comprises an SV40 promoter. The method of claim 12, wherein the second nucleotide sequence comprises a promoter and a sequence encoding a selectable marker for which the eucaryotic cell is deficient so that the eucaryotic cell containing the transformation growth factor-62 coding sequence can be identified. The method of claim 17, wherein the selectable marker comprises dihydrofolate reductase. 19. The method of claim 18, further comprising exposing the eucaryotic cell to methotrexate, so that resistant colonies are selected that contain enhanced levels of the coding sequences for dihydrofolate reductase and transformation growth factor-62. 20. The method of claim 19, wherein the eucaryotic cell comprises a dihydrofolate reductase deficient Chinese hamster Ovary cell. A method of producing transform growth factor-β2 comprising (a) culturing transfectant 169, 12.5, clone 36 as deposited with the ATCC; and (b) recovering the transformation growth factor-62 from the culture. The method of claim 21, wherein the transfectant is grown in the presence of methotrexate. 23. Eucaryotic cell containing a nucleotide sequence 35 encoding transformation growth factor-6 2 under control of a second nucleotide sequence that regulates gene expression such that the eucaryotic cell actively produces transformation growth factor-62. The eucaryotic cell of claim 23, wherein the nucleotide sequence encoding the transformation growth factor-8 comprises the nucleotide sequence substantially as shown in Fig. 1b from nucleotide No. -70 to 1755. The eucaryotic cell of claim 23, which comprises a Chinese hamster ovary cell. The eucaryotic cell of claim 23, wherein the second nucleotide sequence controlling gene expression comprises an SV40 promoter. The eucaryotic cell of claim 23, wherein the second nucleotide sequence comprises a promoter and a sequence encoding a selectable marker for which the eucaryotic cell is deficient, so that eucaryotic cells containing the monkey transformation growth factor-62 coding sequence can be identified. 28. The eucaryotic cell of claim 27, wherein the selectable marker comprises dihydrofolate reductase. The eucaryotic cell of claim 28, comprising a dihydrofolate reductase deficient Chinese hamster ovary cell. 30. Cell line, comprising 169, 12.5, clone 36 as deposited with the ATCC. .8862442.