DD296691A5 - PROCESS FOR THE PREPARATION OF ANTIBODIES AGAINST AN EPITOPE OF AMPHIREGULIN - Google Patents

Abstract

The invention relates to a process for the production of antibodies against an epitope of amphiregulin for the qualitative and quantitative detection of mature AR and its precursor and its subcomponents. These can be used in the affinity purification of AR proteins and in the detection of AR biosynthesis, its metabolism and function. Antibodies to AR can also be used in diagnostic and therapeutic agents. {Method; manufacture; amphiregulin; fragments; peptides; proteins; nucleotide sequence; modified cell; plasmid; Antibody; pharmaceutical agent; Medicine; neoplasia; Wound care; bone resorption; Immune response}

Description

1986, Science 234: 161-166; Pardee, 1987, Cancer Res. 47: 1488-1491; Sachs, 1986, Sei. Amer. 254: 40-47; Marshall 1987, Cell 50: 5-6; Melcher and Anderson, 1987, Cell 30: 715-720; Clemens and McNurlan, 1985, Biochem. J. 226: 345-360; Nathan, 1987, J. Clin. Invest. 79: 319-326; Sporn and Roberts, 1986, J. Clin. Invest. 78: 329-332; Old, 1987, Nature, 326: 330-331; Beutler and Cerami, 1987, New Eng. J. Med. 316: 379-385; Weinstein, J. Cell. Biochem., 33: 213-224; Zarling et al., 1987, Proc. Natl. Acad. Be. USA 83: 9739-9744; Sporn and Todaro, 1985, N. Eng. J. Med. 303: 878-880; Sporn and Roberts, 1985, Nature 313; 745-747). Bioactive phorbol esters such as ^ -O-tetradecanoylphorboMS acetate (TPA) are in vivo effective tumor promoters and triggers and modulators for a wide variety of biological and biochemical reactions both in vivo and in vitro (Blumberg, 1981, Crit. Rev. Toxicol. 9: 153-197; Slaga, 1983, Cancer Surv. 2: 595-612). It has been known for some time that TPA inhibits the growth of the human breast adenocarcinoma cell line MCF-7. In addition, TPA alters the morphology of MCF-7 cells in that TPA-treated cells exhibit morphological features of secretory cells (Osborne et al., 1981, J. Clin Invest 67: 943-951, Valette et al., 1987 , Cancer Res. 47: 1615-1620).

Object of the invention

The aim of the invention is the provision of antibodies against amphiregulin, a growth-regulating glycoprotein.

Explanation of the essence of the invention

The invention has for its object to provide a method for the production of antibodies against amphiregulin.

3. Summary of the invention

The present invention relates to a method for the production of antibodies against amphiregulin. Amphiregulin is a growth regulating factor that has unusual cell growth regulating activity. Amphiregulin inhibits the growth of neoplastic cells and enhances the growth of certain normal cells. Amphiregulin is an extremely hydrophilic glycoprotein with an average molecular weight of 22,500 daltons and is found in two distinct, but functionally equivalent forms, a truncated and a larger. With the exception of the additional six N-terminal residues found in the larger form, the two forms are absolutely homologous at the amino acid level (Figure 12). The present invention is based in part on the observation that TPA-treated MCF-7 cells release a glycoprotein which inhibits the growth of the A431 and other tumor cell epidermal cancer cell lines but promotes the growth of normal human fibroblasts and some other cells. The present invention will be described by way of example in which amphiregulin is detected, purified to homogeneity and characterized structurally and functionally accurately. In other examples, the isolation and sequencing of both cDNA and genomic clones encoding the amphiregulin precursor is described. These clones are used to produce expression vectors capable of directing a high rate of synthesis of biologically active amphiregulin in transformed bacteria and transfected eucaryotic host cells. A wide variety of applications for this factor is shown in the description.

4. Short description of the figures

Fig. 1: preparative reversed-phase HPLC of flow and wash fraction Fig. 2: semi-preparative reversed phase HPLC of the pooled fractions 47 to 62 from Fig. 1 Fig. ЗA: analytical reversed-phase HPLC of pool I from the previous run 3 B: analytical reversed-phase HPLC of pool II from the previous run,

FIG. 4: Gel permeation chromatography of fractions from FIGS. 6A and 3B with Bio-Sil TSK 250 columns. The chromatography was carried out as described in Section 6.3.2.

(A) HPLC of the concentrated fraction 35 (Fig.

(B) HPLC of Concentrated Fraction 36 (Fig.

(C) HPLC of Concentrated Fraction 37 (Fig.

(D) HPLC of concentrated fractions 41 and 42 together (Fig. 3B);

Figure 5: Analysis of purified AR and S-pyridylethylated-AR (SPE-AR) by gel permeation HPLC on Bio-Sil TSK 250 columns. Chromatography was performed as described in Section 6.3.2. described, performed. The molecular weight standard used was ovalbumin, 43kD, chymotrypsinogen A, 25kD; Ribonuklease14kD; undlnsulin6kD;

(A) AR

(B) SPE-AR;

Figure 6: Analysis of AR and SPE-AR on a Reversed-Phase Ultrapore RPSC C3 column (4.6 x 75 mm). A gradient was used between 0.1% TFA as the first solvent and acetonitrile with 0.1% TFA as the second solvent at a flow rate of 1 ml / min, at room temperature. 1 ml fractions were collected.

(A) AR

(B) SPE-AR

Figure 7: SDS-PAGE analysis of AR proteins

(A) A 15% SDS-PAGE gel (0.75 mm χ 18 cm χ 15 cm, Bio-Rad) in a discontinuous buffer system was developed at a constant current of 30 mA. The molecular weight standards used were: phosphorylase B, 92.5 kD; BSA, 66.2kD; Ovalbumin, 43kD; Carbonic anhydrase 31 kD; Chymotrypsinogen A, 25.7 kD; Soyabohnentrypsin inhibitor, 21,5kD; Lactoglobulin, 18.4kD; Lysozyme, 14.4kD; Aprotonin, 6.2kD; and insulin subunit, 3kD

 Lane 1: AR, Lane 2: NG-AR

Lane 3: SPE-AR

Lane 4: NG-SPE-AR (B) 20% STS-PA-GE minigel (0.75mm χ 10cm x 7cm) developed at a constant voltage of 200V.

The same molecular weight standards were used.

Lane 1: AR

Lane 2: NG-AR

Lane 3: Phospholipase treated with NG-AR separated on rp HPLC

Fig. 8: IEF analysis of ¹²⁶ L-labeled AR. The following standards with pI values between 4.65 and 9.6 were used: phycocyanin, 4.65; β-lactoglobulin B, 5.1; Bovine carboanhydrase, 6.1; Human carbonic anhydrase, 6.5; Equine myglobin, 7.0; Wal-Carboanhydrase6,5; a-chymotrypsin, 8.8; and cytochrome C, 9,6. Figure 9: (A) Dose-response curve of AR on inhibition of ¹²⁵ I-deoxyuridine incorporation into DNA of A431 cells;

(B) Influence of AR on stimulation of ¹²⁶ L deoxyuridine incorporation into human foreskin fibroblast DNA (Sadamoto).

Fig. 10: Influence of ¹²⁶ l-EGF binding on fixed A431 cells or A-431 plasma membranes by mouse EGF and AR. The binding assays were performed as described in Section 6.1.5. described carried out. ЮО-μΙ- or 50-цІ-РгоЬеп in wells were used for tests with fixed cells or membranes.

symbol Type of recipe competitor • Fixed cells EGF ▲ Fixed cells AR O membrane EGF Δ membrane AR

Fig. 11: Hydropathicity analysis of AR and human EGF (Kyte and Doolittle)

(A) AR (residues 1-84)

(B) AR (residues 44-84)

(C) EGF (residues 1-53)

(D) AR precursor (residues 1-252) and EGF (residues 1-53)

(The arrangement follows, as shown in FIG. 13, such that cysteine, residue 46 of mature AR is cysteine residue no

matured EGF); Figure 12: Amino acid sequence of mature AR and truncated AR. The standard single-letter code for amino acids was used:

Alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), histidine (H), isoleucine (I), Leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y) and valine (V).

Fig. 13: Comparison of the amino acid sequences of amphiregulin (AR) and members of the EGF superfamily. Fig. 14: (A) ¹²⁵ I-AR binding to cellulose (OO), denatured DNA-cellulose (GG) and native DNA-cellulose ( Δ-Δ)

(B) ^12S I-AR binding to cellulose (OO) and to DNA-cellulose (DG) is compared to ¹²⁵ I-EGF binding

Cellulose (Δ-Δ) and DNA-cellulose (V-V) Fig. 15: Synthetic amphiregulin peptides produced by solid phase technique. Five peptides corresponding to residues 166-184 (# 259), 108-130 (# 264), 31-50 (# 279) 71-90 (# 280) and 221-240 (# 281)

are synthesized by solid-phase synthesis and then purified by reverse-phase HPLC. Figure 16: Nucleotide sequence and deduced amino acid sequence of cDNA clone pARI encoding human amphiregulin; Figure 17: Genomic human amphiregulin sequence Figure 18: Schematic diagram of the exon structure and protein domains of the AR molecule relative to the genomic sequence

Fig. 19: nucleotide sequence of the pDCHBARI expression vector Fig. 20: nucleotide sequence for pTacAPARI; Fig. 21: Nucleotide sequence of pTacAPHILE.

5. Detailed description of the invention

The present invention relates to amphiregulin (AR), nucleotide sequences encoding AR and the AR precursor, and the production of AR using conventional or recombinant DNA techniques.

AR, a novel cell growth regulating factor, is expressed and secreted by TPA-treated MCF-7 cells in two distinct but functionally equivalent forms. The two forms are structurally identical except for six additional amino-terminal residues in the larger form. AR has a very potent inhibitory activity on DNA synthesis in neoplastic cells and therefore can be used as an effective antitumor compound. Of particular interest is the ability of AR to promote the growth of fibroblasts and other normal cells. AR may therefore be of use in the treatment of such conditions requiring acceleration of cell growth (eg, burns and wounds).

AR, its fragments or derivatives have widespread potential therapeutic utility in the treatment and monitoring of neoplasia, as well as in wound treatment. AR can also be used to modulate bone resorption and immune response as well as to stimulate the arachidonic acid cascade. The amphiregulin gene and the gene products, processes for their preparation and their use are described in detail in the following subsections and in the examples.

5.1 Preparation and purification of amphiregulin

Amphiregulin is secreted by the human breast carcinoma cell line MCF-7 upon treatment with the tumor-stimulating compound 12-O-tetradecanoyl-phorbol-13-acetate (TPA). Amphiregulin can be isolated from the conditioned media of such cultured TPA-treated MCF-7 cells and subsequently purified to obtain high specific activity. Amphiregulin may also be isolated from other cell lines, with or without induction, by treatment with TPA or other tumor-stimulating compounds. On the other hand, amphiregulin can be produced by recombinant DNA techniques or by solid phase peptide synthesis.

Amphiregulin may be purified using various methods and techniques known in the art, e.g. Chromatographic (e.g., reversed phase, liquid permeation, ion exchange, size exclusion, and affinity chromatography), centrifugation, electrophoretic methods, differential solubility, or other standard techniques for purifying a protein ,

According to a specific embodiment of the present invention, MCF-7 cells are treated with TPA for 48h and grown in fresh serum-free medium for 4 days. The resulting conditioned medium is separated and used as described in Section 6.2 to prepare crude AR. A combination of reversed-phase liquid and gel permeation chromatography can be used to purify AR to homogeneity as described in Section 6.3. Purified AR is obtained which is 1842 times to 2 270 times enriched in comparison to the starting material. The method is reproducible and gives AR preparations with specific activities between 2.7 and 3.4 × 10 ⁶ units / mg protein.

5.2 Amphirae training

5.2.1 Isolation and cloning of amphiregulin gene

According to the invention, the coding nucleotide sequence for human amphiregulin or a functional equivalent thereof can be used to generate recombinant molecules which direct the expression of the human amphiregulin product. The coding nucleotide sequence for AR can be isolated from cells that produce AR-like activity. For example, according to a specific embodiment, the human breast carcinoma cell line MCF-7 is used as the source of the AR-encoding nucleotide sequence. The coding sequence may be obtained by cDNA cloning of the RNA recovered from such cells, or by genomic cloning. One can either make a cDNA or genomic library of clones from the generated DNA fragments by techniques known in the art using, for example, restriction enzymes.

Fragments containing the AR gene can be identified in a variety of ways using known methods. For example, part of the amino acid sequence of AR can be used to derive the corresponding DNA sequence, and then the DNA sequence is chemically synthesized, radioactively labeled and used as a hybridization probe.

Other methods for isolating the AR gene include, for. B. the chemical synthesis of the gene sequence itself based on a known sequence z. B. derived from the amino acid sequence of AR. On the other hand, in vitro translation of selected mRNA followed by functional or immunological assays with the translated products can be used. The identified and isolated gene can then be inserted into a suitable cloning vector. A large number of vector host systems are known and applicable in the art. Possible vectors include e.g. As plasmids or modified viruses, wherein the vector system and the host cell are compatible. Such vectors are z. B. bacteriophages, such as λ derivatives or plasmids, such as pBR322 and pUC plasmid derivatives. Recombinant molecules can be introduced into the host cell via transformation, transfection, infection, electroporation, etc. In a particular embodiment, the AR gene expressed by MCF-7 cells is cloned by selecting mRNA which is produced in response to treatment of MCF-7 cells with TPA and constructing a cDNA library in bacteriophage Xgt10. Since untreated MCF-7 cells apparently do not synthesize and secrete AR protein, it has been thought that the induction of AR by TPA is also noticeable at the transcriptional level and that such a cDNA library contains elevated levels of AR-containing sequences Contains dimensions. Screening of the cDNA library with degenerate and best guess oligonucleotides based on the use of human codons (Lathe, 1985, J. Mol. Biol. 183: 1-12 and Section 8.1., Supra) resulted in isolation These cDNA clones were then used to characterize the AR mRNA and the AR gene, and the nucleotide sequence of the AR cDNA (Section 8.2, above and Fig. 16) can be used to derive the primary AR cDNA clones. Due to the inherent degeneracy of the coding nucleotide sequence, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may also be used to carry out the methods of the present invention Such changes in the AR nucleotide sequence include deletions, additions, or Substitutions of different nucleotides leading to a sequence coding for the same or a functionally equivalent gene The gene product may contain deletions, additions or substitutions of amino acid residues within the sequence which result in silent changes to produce a bioactive product. Such amino acid substitutions may be on the basis of similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphatic nature of the residues involved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; Amino acids with uncharged polar head groups or non-polar head groups with similar hydrophilicity values include the following compounds; Leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, tyrosine.

The AR cDNA can be used as a probe to detect AR mRNA in TPA-induced MCF-7 cells. The AR mRNA has a length of about 1400bp.

As described in Section 9, supra, the AR cDNA can be used to characterize the AR gene. The assignment of the chromosome for the human AR gene was determined by in situ hybridization of ³ H-labeled AR cDNA with normal metaphase chromosomes. As a result, the human AR gene was found on chromosome 4 in the region 4q13-21, which was thought to play some role in the differentiation of lymphocytes (see Section 9.2. Below). The cDNA was also used to isolate the genomic DNA comprising the entire AR gene, including

the 5'-regulatory region. It has been found that the AR gene is 1Okb in length and divided into 6 exons. The 5 'regulatory region was incorporated into an expression vector containing a promoter-free chloramphenicol acetyltransferase (CAT) gene. This construction was capable of stimulating transcription of the CAT gene after transient introduction into MCF-7 cells. Addition of TPA stimulated 6- to 7-fold activity (see section 9.4 below). According to particular embodiments of the present invention, this 5'-regulatory region can be used in expression vectors to control the transcription of the AR gene or other structural genes. The chimeric AR-CAT construction can also be used to search for factors that regulate the expression of AR (see Section 9 and following).

2.5.2 Construction of the Expression Vector Containing the Amphiregulin Coding Sequence In order to express biologically active AR in maturer form, such a vector / host system should be chosen, not for high transcription and translation rates, but also for correct processing of the gene product provides. This is particularly important if the entire coding sequence of the amphiregulin precursor is used in the expression construction, as the mature form of amphiregulin is likely to be derived from a precursor product via cellular processing events. For example, a mammalian host cell system may be selected for its ability to correctly process and secrete amphiregulin into the extracellular environment. In the present case, in particular, two forms of mature amphiregulin appear to be synthesized as the middle portion of a common 252-amino acid precursor, from which the two mature forms are released by differential proteolytic processing. In addition, amphiregulin is glycosylated and may additionally be subjected to tyrosine sulfation, once again underscoring the importance of selecting the expression system capable of performing these post-translational modifications, if desired in the final product. A variety of animal / host expression vector systems (i.e., vectors containing the necessary elements to direct replication, transcription, and translation of the coding AR sequence in a suitable host cell) are known to those skilled in the art. These include, for example, 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., baculovirus); or non-viral promoter expression systems derived from the genomes of mammalian cells (e.g., the mouse metallothionein promoter). The expression elements of these vectors differ in their strength and specificity. Depending on the host / vector system used, any of a number of suitable transcriptional and translational elements may be used. For example, when cloning in a mammalian cell system, promoters can be used which are isolated from the genome of mammalian cells (e.g., mouse metallothionein promoter) or from viruses that proliferate in these cells (e.g., vacinavirus-7 , 5K promoter or the "Moloney murine sarcoma virus long terminal repeat.") Promoters made by recombinant DNA technology or by synthetic methods can also be used to transcribe the inserted sequence.

In addition, specific initiation signals are required for adequate translation of the inserted protein coding sequence. These signals include the ATG initiation codon and adjacent sequences. In the event that the entire AR gene, including its own initiation codon and adjacent sequences, were inserted into an appropriate expression vector, no additional translation control signals are required. However, if only part of the coding sequence is inserted, exogenous translational control signals including the ATG initiation codon must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the AR coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of different, both natural and synthetic origin. The effectiveness of the expression can be increased by incorporation of transcriptional attenuation sequences, enhancer elements, etc.

Any method already described for inserting DNA fragments into a vector may be used to construct expression vectors containing the AR gene and appropriate transcriptional / translational control signals. These methods may include in vitro recombinant DNA techniques, synthetic techniques, and in vivo recombinations (genetic recombination).

For example, if an adenovirus is used as the expression vector, the AR coding sequence can be linked to the adenovirus transcriptional / translational control complex, e.g. B. the "late promoter" and "tripartite Ieader" sequence are ligated. These chimeric genes 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 (e.g., region E1 or E3) results in an intact recombinant virus capable of expressing AR in the infected host. Similarly, the Vaczina 7.5K promoter can be used. An alternative expression system for the expression of AR is an insect system. In such a system, nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Spodoptera frugiperda cells. The AR coding sequence can be cloned into non-essential regions (e.g., the polyhedrin gene) of the virus under the control of an AcNPV promoter (e.g., the polyhedrin promoter). Successful insertion of the AR coding sequence results in inactivation of the polyhydric ring and production of non-occluded recombinant viruses (i.e., viruses without the protein envelope encoded by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed.

Retroviral vectors provided in amphotropic packaging cell lines enable very effective expression in numerous cell types. This method allows the determination of a cell-type-specific processing, regulation or function of the inserted protein-coding sequence.

In addition, a host cell strain can be selected which modulates the expression of the inserted sequence or modifies and processes the gene product as desired. Promoter-dependent expression may be increased in the presence of certain inducers (eg, zinc or cadmium ions for metallothionine promoters). This allows the expression of genetically engineered AR to be controlled. This is important in the event that the protein product of the cloned foreign gene acts lethal on host cells. Furthermore, modifications (eg, glycosylation) and processing (eg, cleavage) of the protein product are important to the function of the protein. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Suitable cell lines of the

Host systems can be selected to ensure the correct modification and processing of the expressed foreign protein.

Examples of expression vectors used according to the invention are:

Plasmid pSV2Neo

Plasmid pSV2Neo is described in Southern et al., (1982) J. Mol. Applied Genetics 1,327-341.

Plasmid pSV2dhfr

The plasmid pSV2dhfr (Subramani et al., 1981, Mol. Cell. Biol. 1,854-864) contains the mouse dedyloid reductase gene (dhfr) under the control of the SV40 promoter.

Plasmid рНЗМ

The plasmid рНЗМ (Aruffo et al., 1987, Proc Natl. Acad. Sci., USA, 84, 3665-3369) contains a chimeric promoter composed of the human cytomegalovirus (CMV) AD169 immediate early enhancer linked to HIV LTR. Positions -67 to +80. LTR is followed by a polylinker with the 5 'to 3' junctions: HindIII, XbaI, Xhol, BstXI, Xhol, PstI and XbaI. The small t antigen splice / polyadenylation signals of pSV2 and an SV40 origin of replication are located downstream of the polyclinker (in the З 'direction), which allows amplification in cells carrying the SV40 large T antigen "(the COS and WOP cells). A suppressor tRNA gene favors growth in pVX-related vectors and bacteria bearing the P3 plasmid (such as MC1061 / P3).

Plasmid рНЗМ / ЬОпсМ

Plasmid рНЗМ / ЬОпсМ was provided by Najma Malik, Oncogen. It contains the simian TGF-β-5 'untranslated sequence and the leader sequence linked to the Oncostatin M coding sequence and inserted in рНЗМ and at the HindIII / XhoI (filled) interface. The TGF-β sequence consists of the 85pb 5 'untranslated region beginning at a HindIII site (starting from the pSP65 polylinker) and an adjacent PstI site of the TGF-β cDNA, followed by 87 bp, which is responsible for the 29 amino acid long signal sequence, which is normally cleaved between a Glycinleucindipeptidsequenz.

Plasmid pH3ARP

The plasmid pH3ARP is a рНЗМ-derived vector designed to transiently express the AR precursor in COS cells. The plasmid contains 13bp of the AR 5 'untranslated region and the entire coding sequence as well as З' untranslated sequences. This plasmid was prepared by ligating the 4kb fragment of the рНЗМ vector after digestion with HindIII and XbaI, with the oligonucleotides EVSAL3 and EVSAL4 and the gel-purified 1.1 kb Hgal / XbaI cDNA fragments from pARI. EVSAL3 and EVSAL4 are complementary to the 5'-HindIII, EcoRV, Sail, and Hgal interfaces. The construction also contains the EcoRI to Xbal polylinker sites of pEMBL18 following the З'-untranslated region.

5 ' EcoRV Hgal AGCTTGATATCGTCGA 3 ' Hind Sail ACTATAGCAGCTGACGC EVSAL3 EVSAL4

Plasmid pSVDR / bOM

The plasmid pSVDR / bOM was provided by Jeff Kallestad, Oncogen. It represents a fusion between pSV2dhfr and pH3M / bOM and is a vector with a cDNA driven by the CMV / HIV promoter from рНЗМ and flanked by the TGF-β signal sequence and the SV40 polyadenylation signals and additionally the mouse -high gene controlled by the SV40 early promoter. The plasmid was prepared by ligation of BamHI / Nrul (filled) digested pH3M / bOM with BamHI digested pSV2dhfr.

Plasmid pHras

The plasmid pHras is a mammalian expression vector and has been described by Stan McKnight, Univ. of Washington, Dept. developed by Pharmacology. The piasmidistein pML-related vector containing a 754 bp EcoRI to Tthllll fragment from Harvey Ras LTR, a polyclinker with Smal, BamHI, Sail, PstI, HindIII and ClaI sites followed by the mouse DHFR cDNA and the hepatitis B virus З contains untranslated / polyadenylation signal. The distance from BamHI in the polylinker to the ATG of DHFR is 54bp.

Plasmid pHLARGE

The plasmid pHLARGE represents a mammalian expression construct containing the 10kb AR genomic sequences (SmaI to HindIII) driven by Harvey ras LTR and followed by a non-functional, promoter-free DHFR gene. It was prepared by triplicating the following fragments: 4.6kb pHras SmaI / HindIII fragment; 6.0kb SmaI / HindIII Ar genomic fragment of pARH12; and 6.4 kb HindIII AR genomic fragment of pARH6.

Plasmid pHLARGIpcD

The plasmid pHLARGIpcD is a polycistronic mammalian expression construct. It contains the AR precursor cDNA sequence followed by the mouse dhfr gene under the control of a single Harvey ras LTR sequence. The primary transcript represents a 74 bp polycistronic information between the stop codon of AR and dhfr's ATG start codon, thereby reinitiating the ribosome and continuing translation. pH3ARP was digested with EcoRV and a 700bp fragment was isolated. This fragment contained the 13 bp AR-5 'untranslated region, the complete AR precursor coding region and

21 bp of the 5 'untranslated sequence. This fragment was ligated with the BamHI cut, filled and phosphatase treated vector pHras.

Plasmid pLOSNL

The plasmid pLOSNL was provided by Dusty Miller, Fred Hutchison Cancer Research Center, Seattle, WA. This retroviral expression vector was designed to generate high titers of amphotropic helper-free viral material capable of infection but not only replication in a wide variety of hosts. The vector contains: a mutant Moloney murine leukemia virus LTR with deleted packaging signals; psi ± sequences; the ornithine transcarbamylase (OCT) gene with two Xhol sites for insertion of a cDNA and subsequent inactivation of the OTC gene; SV2Neo, a selectable marker; the 3 'LTR; and a pBR322 amp-resistance scaffold.

Plasmid pLARSNL

The plasmid pLARSNL is an amphotropic retroviral expression construct containing the AR precursor encoding cDNA region lacking a poly (A) tail and controlled by viral LTR. SV2Neo allows the selection of expressing transfectants. pLARSNL was generated by ligation of the 800bp Sall fragment from pHLARGIpcD with the 7.7kb fragment of the Xhol digested and phosphatase treated pLOSNL vector fragment.

5.2.3 Identification of transfectants or transformants expressing the amphiregulin gene.

The host cells containing the recombinant AR coding sequence and capable of expressing the biologically active mature product can be identified by at least four general methods:

(a) DNA-DNA, DNA-RNA or RNA antisense (reverse RNA) hybridization

(b) presence or absence of "marker" gene functions;

(c) determining the transcription rate based on the expression of the AR mRNA transcript in the host cell; and

(D) Detection of the mature gene product by immunoassay and finally by its biological activity.

In the first method, the human AR coding sequence inserted in the expression vector is detected by DNA-DNA hybridization using probes. These probes include nucleotide sequences that are homologous to the coding sequence of human AR.

In the second method, the recombinant expression vector and host system can be identified and selected based on the presence or absence of particular marker gene functions. Examples of marker gene functions are thymidine kinase activity, antibiotic resistance, methotrexate resistance, transformation phenotype, formation of occlusion body in baculovirus, etc. If e.g. the AR coding sequence is inserted within a marker gene sequence of a vector, recombinants containing the AR coding sequence can be identified by the lack of marker gene function. Alternatively, a marker gene may be placed in tandem with the AR sequence under the control of the same or a further promoter to control the expression of the AR coding sequence. The expression of the marker due to induction or selection indicates the expression of the AR coding sequence.

In the third method, the transcriptional activity for the AR coding region can be determined by hybridization assays. For example, polyadenylated RNA can be isolated and analyzed by Northern blotting using a probe homologous to the AR coding sequence or portions thereof. In addition, the entire nucleic acid of the host cell can be extracted and assayed for hybridization with such probes. In the fourth method, expression of the mature protein product by immunological means, e.g. by Western blotting, immunoassays such as radioimmuno-precipitation, enzyme-linked immunoassays, etc. In a final assay to determine the function of the expression system, the biologically active AR gene product is detected. When the gene product is secreted by the host cell, the cell-free medium of the transfected host cell culture can be separated and assayed for AR activity. If the gene product is not excreted, cell lysates can be tested for activity. In both cases, biological assays such as the growth inhibition and stimulation assays described herein or the like may be used.

5.3 Structure of Amphiregulin

Amino acid sequencing of purified AR revealed that the preparation consisted of an unequal mixture of two nearly identical forms of AR (see Figure 12). The larger of the two forms comprises about 16% of the preparation. The second form, the truncated AR, comprises the vast majority of the preparation and differs from the longer form in the absence of the amino-terminal hexapeptide SVRVEQ. Otherwise, the two forms are absolutely homologous at the amino acid level.

AR is an extremely hydrophilic glycoprotein with an average molecular weight of 22,500 daltons. Upon N-glycanase treatment to remove N-linked carbohydrates, AR migrates as a single band having a relative molecular weight of 14,000. This roughly matches the molecular weight determinations for AR from gel permeation chromatography data. Under non-reducing conditions AR migrates in a similar way. AR is therefore a single-stranded glycoprotein.

The primary structure of AR was compared to a variety of protein sequences. These comparisons show that AR is a novel protein that shows no significant structural homology with any of the compared proteins. However, the primary structure of AR is related to the structure of several other growth factors, most of which belong to the EGF superfamily. It therefore makes sense to classify AR as a member of this group of proteins since several structural features of this protein run parallel to highly conserved structural features found within the EGF protein family. For example, the N-terminal AR sequence is similar to the N-terminal sequences of TGFa, VGF and MGF. These regions are rich in proline, serine and threonine residues. AR also has sites for N-linked glycosylation similar to the N-terminal precursor regions of TGFs and VGF. AR also shows sequence homology with other EGF-like proteins by conserving 6 cysteine residues that bind to

3 Disulfide bonds are involved and define the secondary structure of the mature forms of these growth factors. An analysis of the hydropathic properties, however, reveals significant differences between homologous AR and EGF sequences. For further comparisons between AR and other members of the EGF superfamily, see Table I, Figure 13 and Sections 9.7 and 9.8.

Table I

Protein Species Region # 1 Region # 2

EGF humanely CLHDGVCMYIE CNCVVGYIGERC TGF-a humanely CFH GTCRFLV CFCHSGYVGARC VGF vaccinia CLH GDCIHAR CRCSHGYTGIRC SGF Shope CLNNGTCFTIA CVCRINYEGSRC Factor IX humanely CLNXGSCKDDI CWCPFGFEGKNC Factor X humanely CQNXGKCKDGL CTCLEGFEGKNC Factor XII humanely CLHXGRCLEVE CHCPVGYTGPFC Protein C humanely CCGXGTCIDGI CDCRSGWEGRFC AR humanely CIH GECKYIE CKCQQEYFGERC , combined Homology Scores "for regions # 1 and # 2 COAG. Score AR score EGF score

Growth Factor Family> 20 <20 <40

Coagulation factors <25> 25 <40

Amphiregulin subfamily <20 <20> 40

The amphiregulin-amino acid sequences given in Figure 12, as well as functional equivalents, are the subject of this invention. For example, the amphiregulin product may include deletions, additions or substitutions of amino acid residues within the sequence that result in silent changes and produce a bioactive product. Such amino acid substitutions may be made on the basis of similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathic nature of the corresponding residues. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; Amino acids with uncharged polar head groups of similar hydrophobicity include the following compounds: leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine and tyrosine.

5.4 Properties of Amphiregulin

Amphiregulin is a single-stranded glycoprotein with an average molecular weight of 22,500 daltons and exhibits bifunctional growth-modulating activities in a variety of cell cultures. AR is structurally related to the EGF family of growth factors and may additionally be functionally similar to other members of this family. One indication of this is the pronounced competitive behavior towards EGF in receptor binding. AR represents a monomeric protein that requires intramolecular disulfide bond activity to show activity. Glycosylation is not required for its biological activity. The protein is stable to mild acid and base treatment; also at 30 minute heat treatment at 56 ⁰ C. The biological activity of AR is completely lost after reduction, After a 5 minute heat treatment at 100 "C, and by digestion with trypsin, endopeptidase Lys-C, endopeptidase Arg and GIu endopeptidase.

By proteolytic cleavage with phospholipase D, AR appears to be converted to an active fragment or active fragments having a relative molecular weight of about 5600 (as determined by SDS-PAGE analysis). Active fragments of AR are the subject of the present invention and are discussed in more detail in Section 5.5, below. AR shows in vitro pronounced anti-proliferative effects on several human cancer cell lines of epithelial origin. For example, AR is used to synthesize DNA from epidermal carcinomas of vulvar A431 cells, breast adenocarcinoma HTB 132 cells, cervical epidermoid carcinoma CRL1550 cells, ovarian papillary adenoma HTB 75 cells, ovarian teratocarcinoma НТВ 1572 cells and breast adenocarcinoma -HTB-26 cells are effectively inhibited. One observes a 50% inhibition of DNA synthesis in A431 cells after treatment with 0.13 nM AR.

Normal Rat Kidneys NRK-SA6 cells usually do not form colonies in soft agar. However, in combination with exogenous EGF and TGF-β in the culture medium of these cells, anchorage-independent growth is induced, indicating a transformed phenotype. A combination of exogenous AR and TGF-β also induces anchorage-independent growth of NRK-SA6 cells, but to a lesser extent (see Table V). This provides a direct indication of the functional relationship of AR and EKF.

The synergistic effect of AR and TGF-ß implicates AR as an important factor in the regulation of cell growth. For the first time, the present invention provides auxiliaries for investigating the complex relationships of normal cell growth control and the characteristic loss of growth control in neoplastic cells. AR also induces proliferation of human foreskin fibroblasts (Table IV). There is a two-fold increase in DNA synthesis in these cells at an AR concentration of about 5OpM. A maximum, about sixfold stimulation is observed at about 5nM.

The mechanism of influencing cell proliferation or growth inhibition by AR is unknown. Preliminary studies to characterize AR receptor binding indicate that AR may have a specific receptor. AR shows a competitive behavior with EGF in binding to the EGF receptor and possibly also in binding to other common receptors, possibly including the AR-specific receptor itself. It is known that the binding of a ligand to the EGF receptor produces a growth-stimulating signal, in part by activation of tyrosine kinase activity. The binding of AR to the EGF receptor may similarly have a proliferative response

and / or, conversely, block the initiation of a proliferative signal by limiting the number of available EGF receptors for binding EGF or other signal-producing ligand. On the other hand, another mechanism by which AR inhibits cell growth is conceivable and is suggested by the data of Table I. Four clones of the A431 cell line with variable EGF binding sites per cell were tested for their sensitivity to AR inhibitory activity. The observed lack of correlation between AR sensitivity and the number of EGF binding sites per cell indicates that the AR-generated growth inhibition signal is initiated by a receptor binding that does not involve the EGF receptor. Such a receptor binding may possibly antagonize growth-proliferative signals which are dependent on other growth factors, such as e.g. B. TGF-a were triggered. AR can thus exert its antiproliferative effect by specifically blocking the cell's mitogenic response to TGF-α or other autocrine growth factors.

AR is an extremely hydrophilic protein whose amino acid sequence produces a distinctive hydropathicity profile that shows only a slight similarity to the profiles of other proteins of the EGF family (Figure 11). AR is a basic protein with a pI of 7.6 to 8.0.

5.4.1 Characterization of AR induction by TPA

TPA induces a plauotropic cell response including changes in cell morphology, cell proliferation and differentiation in membrane transport, in receptor-ligand interactions, in protein phosphorylation, and in phospholipid metabolism. Protein kinase C (PKC) is a Ca ²⁺ and phospholipid-dependent protein kinase and acts as a receptor for TPA. The binding of TPA to PKC results in the phosphorylation of a variety of substrates, such as growth factor receptors, cytoskeletal proteins, and trans-activating regulatory proteins.

c-AMP represents another regulatory molecule that exerts various effects on cells predominantly via the c-AMP-dependent protein kinase A. The intracellular c-AMP content is regulated by a combination of receptor mediated activation and adenylate cyclase inhibition. Some studies indicate that TPA and c-AMP-induced gene expression converge to a common pathway. To investigate the regulation of AR gene expression, the influence of various activators or inhibitors of these two regulatory pathways on the production of AR-specific RNA in MCF-7 cells was determined.

Forskolin is an active ingredient that activates adenylate cyclase and increases the level of intracellular cAMP. When administered to MCF-7 cells at a concentration of 25μΜ over a period of 4h, a marked stimulation of the AR mRNA is observed. The results indicate that cAMP also plays a role in the regulation of AR expression.

5.5 Derivatives derived from ampiregulin, analogues and peptides

The production and use of derivatives, analogs and peptides derived from AR are also provided by the present invention. Such derivatives, analogs and peptides showing growth modulating activity can be used in the diagnosis, prognosis and treatment of a variety of neoplasias. Such derivatives, analogs or peptides may have enhanced or decreased biological activity compared to native AR and may expand or decrease the range of AR GIA susceptible cells. Similarly, for the production and use of derivatives, analogs and peptides derived from AR, useful applications, e.g. As in the treatment of wounds and burns revealed. These AR-derived proteins may show increased or decreased growth-stimulating activity and / or show greater or lesser range of action on cells that respond to the growth-stimulating activity of AR. AR derivatives, analogs and peptides of the present invention may be prepared using a variety of prior art adjuvants. Methods and manipulations on the genetic level as well as on the protein level are likewise provided by the invention. At the protein level, a variety of chemical modifications can be made to produce AR-like derivatives, analogs, or peptides using prior art methods. Examples include specific cleavage by cyanogen bromide, endopeptidases (trypsin, chymotrypsin, V8 protease and the like) or exopeptidases or acetylation, formylation, oxidation, etc.

5.6 Anti-amphiregulin antibody production

The present invention also relates to the production of polyclonal and monoclonal antibodies which recognize amphiregulin or related proteins.

Various methods known in the art may be used to produce polyclonal antibodies to AR epitopes. For the production of antibodies, various host animals, such. As rabbits, mice, rats, etc. are immunized by injection of the AR protein or a synthetic AR peptide. For this purpose, various adjuvants can be administered to enhance the immune response depending on the nature of the host, such as. Freund's (complete or incomplete) adjuvant, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhollyme-thymocyanines, dinitrophenol, or possibly suitable human adjuvants such as BCG (Bacillus Calmette -Guinin) and Corynebacterium parvum. A monoclonal antibody for an AR epitope can be prepared using any technique that allows the production of antibody molecules by continuous cell line cultures. These include, for example, the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256: 455-497), and the more recent human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72) and EBV- Hybridoma technique (CoIe et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96).

Antibody fragments containing the idiotype of the molecule can be prepared by known techniques. For example, such fragments include the F (ab ') ₂ fragment which can be produced by pepsin digestion of the antibody molecule; the Fab 'fragments generated by reduction of the disulfide bridges of the F (ab') ₂ fragment and the two Fab 'or Fab fragments that can be generated by treating the antibody with papain and a reducing agent.

Antibodies against AR can be used for the qualitative and quantitative detection of mature AR and its precursor and its subcomponents, in the affinity purification of AR proteins and in the detection of AR biosynthesis, its metabolism and function. Antibodies to AR can also be used in diagnostic and therapeutic agents.

5.7 Uses of Amphiregulin

The bifunctional nature of AR allows a wide variety of in vitro and in vivo applications. Any type of compounds containing AR or fragments or derivatives thereof which exhibit growth inhibition and / or growth-stimulating activity alone or in conjunction with other biologically active growth factors, inhibitors or immunomodulators can be employed in the applications and methods of the invention. Localization of the AR gene in a region involved in lymphocyte differentiation indicates that AR may be involved in hematopoietic cell development, activation, or immunosuppression. This finds support by the homology between the AR-3 untranslated region and similar regions of other cytokines. The present compounds can be used to modulate angiogenesis, bone resorption, immune response, and synaptic and neuronal effector functions. AR can also be used to modulate the arachidonic acid cascade. The enzymatic oxidation of arachidonic acid leads to a variety of important products, such as prostaglandins, thromboxanes, prostacyclins and leukotrienes. These products are extremely effective, ubiquitous agents with a variety of physiological effects, such as. Muscle contraction, platelet aggregation, leukocyte migration and gastric secretion. AR, AR-related molecules and mixtures thereof can be used particularly in the treatment of injuries as well as for the diagnosis and treatment of cancer.

5.7.1 Treatment of wounds

AR can be used in a procedure for treating injuries such as cutaneous injury, corneal injury, various other epithelial and stromal injuries, such as a chronic ulcer, burns, surgical incisions, traumatic wounds, and injuries from hollow, epithelial-limited organs, such as the esophagus , the stomach, the large and small intestine, the mouth, the genital area and the urinary tract. The method is based on the topical application of the treatment agent containing AR in a physiologically acceptable carrier. The compositions of this invention can be used to treat a variety of injuries, including essentially all cutaneous injuries, corneal injuries, and injuries to epithelium-restricted hollow organs of the body. Wounds suitable for treatment include injuries caused by trauma, such as burns, abrasions, cuts, etc., as well as surgery-induced injuries such as surgical incisions and skin grafts. Other conditions suitable for treatment with the agents of the invention further include chronic conditions such as chronic ulcer, diabetic ulcer, and other non-healing (trophic) conditions.

AR may be included in physiologically acceptable carriers for application to the affected area. The type of carrier is variable and depends on the respective application site. For an application on the skin, a cream or ointment is used as a base; suitable bases include lanolin, silvaden (Marion) (especially for the treatment of burns), Aquaphor (Duke Laboratories, South Norwalk, Connecticut), etc. If desired, it is possible to incorporate AR-containing agents into bandages and other dressings around the wound continuously suspend the peptide. Aerosol applications are also possible. There is no critical concentration for AR in the agent used for treatment; however, it should be sufficient to induce epithelial cell proliferation. The agents may be applied topically to the respective area, typically using eyedrops for the eyes or creams, ointments or lotions for the skin. In the case of the eyes, repeated treatment is desirable, usually at intervals of 4 hours or less. In the case of the skin, it is desirable to continuously maintain the treatment agent on the respective surface during the healing process, the treatment agent being applied two to four times a day or more frequently. The amount of the polypeptide of the invention used depends on the mode of administration, the use of other active compounds, etc., and is generally in the range of 1 \ ig to 100 .mu.g. The polypeptide of the invention may be used with a physiologically acceptable carrier such as saline, phosphate buffered saline, etc. The amount of compound used is determined empirically based on the response of the cells observed in vitro and based on the response of experimental animals to the polypeptides of the invention or to formulations containing the polypeptides of the invention.

AR compounds may be used alone or in combination with other growth factors, inhibitors or immunomodulators.

5.7.2 Diagnosis and treatment of neoplasia

The agents of the invention can be used for the diagnosis, prognosis and treatment of a wide variety of neoplasias.

There are a variety of diagnostic applications of AR and related molecules.

According to the present invention, the polypeptides of the present invention may be labeled such as a radioisotope, an enzyme, a fluorescent marker, a chemiluminescent marker, an enzyme fragment, a particle, etc.

get connected. Such compounds can be used to determine the number of AR receptors on a cell. The identification of AR receptors serves as an indication of the potential responsiveness of the cell to biological effects of AR and related molecules. AR, AR-related molecules and / or antibodies thereto can be used in competitive assays for the detection of AR in media, especially in physiological media. A variety of diagnostic tests known in the art may be used.

The presence and content of AR in body fluids and tissues may be proportional or inversely proportional to the presence and spread of certain cancers. Assays for the determination and / or quantification of AR may find application in cancer diagnosis and prognosis (see section 5.6, above).

The present invention further relates to the detection of AR mRNA. Tests which use nucleic acid probes to detect sequences which wholly or partly contain the known gene sequence are known and applicable in the art. The content of AR mRNA may indicate the presence or persistence of neoplasia. Therefore, assays to quantify AR mRNA levels are useful diagnostic tools.

In addition, malignant cells expressing the AR receptor can be detected using labeled AR or AR-related molecules by a receptor binding assay or by using antibodies to the AR receptor. One can distinguish cells due to the presence and density of the AR receptors, predicting the susceptibility of such cells to biological activities of AR. AR can be used as an anti-neoplastic compound. For in vivo use, the agent of the invention may be administered in a variety of ways. Examples include injection, infusion, as well as topical and parenteral administration, etc. Administration can be carried out in any physiologically acceptable carrier, such as e.g. As phosphate-buffered saline, saline, sterilized water, etc. take place. AR and related molecules may also be encapsulated in liposomes as well as conjugated to antibodies that recognize and bind to the tumor or cell-specific antigens, thereby enabling targeting of the agents of the invention.

AR may be useful in vivo for inducing terminal differentiation of tumor cells. Such cells have abandoned the normal pathway of cell differentiation characteristic of normal cells and are capable of uninterrupted proliferation. In contrast, normal cells differentiate into cells which in most circumstances are incapable of further cell division. Therefore, the ability of AR to reactivate normal cell differentiation in tumor cells, and ultimately disrupt tumor growth, may find valuable use in tumor therapy. AR and related derivatives, analogues and peptides may be used alone or in combination with at least one other antiproliferative compound such as interferon, TGF-β1, TGF-β2, tumor necrosis factor-β, tumor necrosis factor-a, etc. in the treatment of hormone responsive carcinomas, which may affect a variety of organs, such as the lungs, breast, prostate, large intestine, etc. may be used. Hormone-responsive carcinomas can also be treated by induction of AR production in the carcinoma cells. Examples of inducers are estrogens, such as 17-B-estradiol, compounds with antiestrogen activity, such as tamoxifen. Any combinations of these compounds can be used as inducers.

The compounds of the invention can be used in vitro to inhibit cell growth of cell lines which, unlike non-sensitive cells, exhibit AR sensitivity. In this way, heterogeneous mixtures or cell lines can be freed of unwanted cells, the undesired cells reacting to the growth-inhibiting activity of AR. For example, the compounds of the present invention can be used in vitro to eliminate malignant cells from bone marrow or autologous medulla transplants, as well as to eliminate or inhibit the proliferation of malignant cells in blood prior to reinfusion.

The most effective AR concentration for inhibiting the proliferation of a given cell can be determined by adding different concentrations of AR to the respective tumor cells and by recording the inhibitory potency of cell proliferation. The most effective concentration of a single inducer and / or a combination of inducers can be determined by recording AR production in carcinoma cells.

embodiments

Example 6 Production, Purification and Characterization of Human Amphiregulin The following subsections describe the purification and characterization of amphiregulin from TPA-treated MCF-7 cells.

6.1 Material and Methods

The following procedures were used to induce AR synthesis in MCF-7 cells and to prepare and characterize substantially pure AR.

6.1.1 SDS polyacrylamide gel electrophoresis

Proteins were analyzed on SDS-PAGE Slab gels (normal or mini-Bio-Rad system) as described (Laemmli, 1970, Nature 227: 680-685) and detected by silver staining (Merril et al., 1981, Science 211: 1437-1439).

6.1.2 Isoelectric focusing

The isoelectric focusing was performed essentially based on the description of the Isolab Technical Data Sheet for Resolve IEF-GeI. The samples were loaded onto preformed agarose gels (85 x 100 mm, pH 3-10) and focused on a Resolve FR-2500 isoelectric focusing unit using a Bio-Rad-3000 Xi computer-controlled electrophoretic susceptor. IEF standards were focused next to the sample, stained, and the dry gel was placed on a Kodak X-Omat AR-FiIm in a DuPont Cronex illumination and intensification screen.

6.1.3 Proteiniodination

Proteins were labeled with ¹²⁶ I using the chloramine-T method (Barridge, 1978, Methods Enzymol. 50: 54-65).

6.1.4 Protein determination

The protein concentrations were determined by absorption at different wavelengths (210 to 280 nm). The methods of Lowry (Lowry et al., 1951, J. Biol. Chem. 193: 265-275) and Bradford (1976, Anal. Biochem. 72: 248-252) were performed using bovine serum albumin as a standard.

6.1.5 ¹²⁵ I-EGF binding assay

Binding assays were performed in either 48-well tissue culture dishes when freshly grown and / or formalin-fixed A431 cells were used as described (Carpenter et al., 1979, J. Biol. Chem. 254, 4884-4891, Shoyab et al. , 1979, Nature 279: 387-391; DeLarco et al., 1980, J. Biol. Chem. 225: 3685-3690). The determination was carried out in 96-well polyvinyl chloride plates by immobilization of the plasma membrane (Kimball and Warren, 1984, Biochem Biophys Acta771: 82-88).

6.2 Production of Amphiregulin

6.2.1 Separation of conditioned media from TPA-treated MCF-7 cells

MCF-7 cells were cultured in T150 Corning tissue culture bottles in a total volume of 25 ml of 50% IMDM (Iscove's Modified Dulbecco's Media) + 15% DMEM with 0.6pg / ml insulin and 15% heat-inactivated fetal bovine serum (complete MCF-7 -Medium). Approximately 1x 10 ⁶ cells are inoculated per bottle and incubated at 37 ° C with 5% CO2. On the 6th day, the entire medium is removed and 20ml of fresh complete MCF-7 medium containing 100ng / ml of TPA is added to each flask. 48 hours later, the medium is removed and each flask is washed with 15 ml of 50% IDMM + 50% DMEM (serum-free medium) and incubated after addition of 25 ml of fresh serum-free medium to each flask at 37 ° C in 5% CO ₂ . 4 days later, the conditioned serum-free medium is isolated and stored after centrifuging cell components at -20 ⁰ C. The bottles are recharged with 25 ml of serum-free medium and conditioned serum-free medium is isolated every 3 or 4 days. Part of the conditioning medium of each individual isolate is tested for growth inhibitory activity (GIA) on A431 human epidermal carcinoma cells. Usually, three to four times conditioned medium is separated from each series of TPA-treated MCF-7 cells.

6.2.2 Isolation of crude AR

About 4500ml of conditioned medium is thawed and 15min. centrifuged at 4 ° C and 3500 rpm. The supernatant is used in a 2-liter concentrator of Fa. Amicon YM using a lO-membrane (Amicon) at 4 ⁰ C and concentrated. If the volume of the retentate is about 200 ml, add 1000 ml of cold Mili-EQ water and concentrate the mixture again to 200 ml.

The concentrate is removed and transferred to a precooled 250 ml Corning centrifuge container. Concentrated acetic acid is added slowly with stirring to a final concentration of 1 M acetic acid. The mixture is stored for 1 h at 4 ° C and centrifuged for 20 min. At 40,000 x g at 4 ° C in a Sorval RC-5 B centrifuge. The supernatant is separated and stored at 4 ° C. The pellet is suspended in 30 ml of 1M acetic acid and recentrifuged as described above. The supernatant is carefully separated again and combined with the first supernatant and then dialyzed against 17 μl of 0.1 M acetic acid in a Spectroplater dialysis tube No. 3 (molecular weight exclusion about 3000). The dialysis buffer is changed 3 times within 2 days. The retentate is lyophilized. The dry product is isolated, pooled, weighed and stored at -20 ° C until further use. This product is called raw powder.

6.3 Purification of amphiregulin

6.3.1 Reversed-phase liquid chromatography

950 mg of the raw powder (from about 91 serum-free conditioned medium) is suspended in 300 ml of 0.1% TFA (trifluoroacetic acid) and centrifuged for 20 min. At 7000 x g. The supernatant is carefully separated and applied to a C 18 preparative column (2.54 cm x 27 cm, 55-105 microns, water equilibrated in water with 0.1% TFA). Then the chromatographic support was suspended in acetonitrile (MeCN) with 0.1% TFA, the slurry was poured into a 2.54 cm diameter column and washed with 400 ml MeCN in 0.1% TFA and then with 600 ml 0.1 % TFA equilibrated in water. The flow rate is 4 ml / min and the chromatography is carried out at room temperature. The column is washed with 650 ml in 0.1% TFA in water. The run and wash fraction are collected together. Then elute as follows:

(1) 650ml 20% MeCN / H ₂ O with 0.1% TFA

(2) 650 ml of 40% MeCN / HjO with 0.1% TFA

(3) 650 ml of 60% MeCN / H ₂ O with 0.1% TFA

(4) 750ml 100% MeCN with 0.1% TFA.

A sample of each fraction is tested for GIA. Approximately 77% of total GIA activity is found in the run and wash fractions.

Run and wash fractions are isocratically applied to a preparative Partisil 10 ODS-3 column (10 microns, 2.2 cm χ 50 cm, Whatman) attached to a high-resolution liquid chromatography (HPLC) system (Waters).

The flow rate is 4 ml / min. After applying the sample to the column, the column is washed with 250 ml of 0.1% TFA in water.

A linear gradient is generated between 0.1% TFA in water as the first solvent and acetonitrile with 0.1% TFA as the second solvent. The conditions for the gradient are as follows: Obis 15% in 10 minutes, 15 to 15% in 30 minutes, 15 to 25% in 150 minutes, 25 to 65% in 100 minutes, and 65 to 100% in 10 min. HPLC grade solvents were used.

14 ml fractions are collected and samples from each fraction are assayed for GIA. Two broad activity peaks are found (Figure 1). The first eluted peak (eluted between 20-23% acetonitrile) is further purified and characterized.

The fractions 47 to 62 are combined. Add 224 ml of 0.1% TFA in water to the pooled fractions. The mixture is isocratically applied to a semipreparative μ-Bondapak C18 column (7.8 x 300 mm, Waters) at a flow rate of 2 ml / min, at room temperature. The parameters of the linear gradient are: Obis 17% in 10 minutes, 17 to 17% in 30 minutes, 17 to 25% in 320 minutes and 25 to 100% in 40 minutes. The flow rate is 1 ml / min. During the application of the gradient, and fractions with a volume of 4 ml are collected. Samples of these are tested for GIA. Two main activity maxima are obtained, which are eluted at an acetonitrile concentration of about 20% and 21%, respectively (FIG. 2).

Fractions 49 to 53 are combined and 20 ml of 0.1% TFA are added. The mixture is isocratically applied to a μ-Bondapak C 18 column (3.9 x 300 mm, Waters) at a flow rate of 1 ml / min, at room temperature. The

Gradient parameters are: 0-18% in 10 min, 18-18% in 30 min, 18-25% in 280 min, and 25-100% in 20 min. The flow rate is 0.4 ml / min, and the Fraction volume 2 ml. The majority of the activity is eluted from the column at an acetonitrile concentration of about 21.5% (Figure ЗA). Fractions 54-59 (Figure 2) are pooled and chromatographed under identical conditions as indicated for Figure 3A. Most of the activity is eluted from the column at an acetonitrile concentration of about 22.2% (Figure 3B).

6.3.2 Gel permeation chromatography

The fractions 35 to 38 (FIG. 6A) are each concentrated to 70 μΙ using a Speed-Vac concentrator (Savant) and admixed with the same volume of acetonitrile with 0.1% TFA. These 140-цІ-РгоЬеп are applied in tandem on two Bio-sil TSK-250 columns (each 7.5 mm χ 300 mm, Βίο-wheel). Elution is isocratic with a mobile phase of 50% acetonitrile / H ₂ O with 0.1% TFA at room temperature. The flow rate is 0.4 ml / min, and a recorder speed of 0.25 cm / min .; 0.4 ml fractions are collected and samples thereof tested for GIA. The elution profiles of fractions 35, 36 and 37 (Fig. A) are shown in Figs. 4A, 4B and 4C, respectively. Fractions 41 and 42 (Fig. 3B) are pooled, concentrated to 70 μΙ and subjected to gel permeation chromatography as described above. The elution profile is shown in Fig.4D.

6.4 cell growth tests

6.4.1 Cell Growth Modulation Assays Using ¹²⁵ L Deoxyuridine Incorporation in DNA

The tests are carried out on 96-well plates (Falcon 3072). Human epidermal powder carcinoma cells (A431) are used as growth inhibitory activity (GIA) and human foreskin fibroblast (Sadamoto) test cells as indicator cells for determining growth-stimulating activity (GSA). 3.5 × 10 ⁴ cells in 50 μM DMEM with 5% heat-inactivated fetal bovine serum (FBS), penicillin / streptomycin (PS) and glutamine (test medium) are placed in all wells except the peripheral wells. In the peripheral wells transferred 50 μΙ PBS. 3 hours later, ιτ> 3η50μΙ of the sample to be tested in test medium is added to each well, while only 50 μΙ of test medium is added to the control wells. 3 wells are used per concentration of the sample to be tested. The plates at 37 ° C 2 to

Incubated for 3 days. Then you give 100μ | a solution of ¹²⁵ I-iodo-2'- ¹²⁵ I-deoxyuridine (4 Ci / mg-0.5mCi / ml, 2 μM / ml in assay medium) and incubate the plates at 37 ° C. After 4 to 6 days, the medium is aspirated from the wells and washed 1 χ with 200 μΙ PBS. 200 μl of methanol are then added to each well, the plates are incubated for 10 minutes at room temperature and the methanol is filtered off with suction. Add 200 μL of sodium hydroxide to each well and incubate the plates for 30 min at 37 ° C. Sodium hydroxide is removed by means of "Titertek plugs" (Flow Labs), the plugs are transferred to 12 mm χ 75 mm plastic tubes and measured in a gamma counter to quantitate the ¹²⁶ l IUdR incorporation.

6.4.2 Softagar Colon Test

A 0.5 ml base layer of O, 5% agar (Agar Noble, Difco Laboratories, Detroit, Michigan) in DMEM with 10% heat-inactivated FBS is added to Costar 24-well tissue culture plates. 0.5 ml of 0.3% agar with the same medium-FBS mixture, 5 to 10 x 10 ³ test cells and factors to be tested at various concentrations are used to cover the first agar layer. Incubate the plates at 37 ⁰ C in a humidified atmosphere of 5% CO ₂ in air and feeding again after 7 days by addition of 0.5 ml of 0.3% agar containing the same medium and the same concentrations of the compound to be tested contains. The colonies are counted unfixed and unstained and the number of colonies with more than 20 cells are determined between the 7th and the 14th day.

6.4.3 Cell growth inhibition test

A431 cells are incubated with 2.1 χ 10 ⁴ cells per well in 0.3 ml medium (DMEM with 10% FBS, P / S glutamine) in Costar plated (24-well about 2 cm ² per well), and 2 h at 37 ⁰ C incubated. Subsequently, the test compounds are added at different concentrations each 2x in 0.3 ml of medium in each well. The control wells contain medium without sample. The plates are incubated at 37 ° C for 72h. The medium is then aspirated, the cells washed with 1 ml PBS / well, detached with 0.5 ml trypsin (0.5% EDTA, 0.5 mM) and counted using a Coulter counter.

6.5 Analysis of the AR primary structure

6.5.1 Reduction and S-pyridylethylation

AR (20-30μ9) is dried in a 1.5 ml microcentrifuge polypropylene tube and suspended in ΙΟΟμΙ 3M urea in 0.05M TRIS-HCl, pH 7.5.

4 μM 2-mercaptoethanol are added to the mixture, mixed, purged with nitrogen and incubated at 25 ° C. After 2.5 h, add 4.5 μΙ of freshly distilled 4-vinylpyridine to the mixture, rinse the tube again with nitrogen and incubate for 2 h at 25 ° C. The reaction mixture is acidified to pH 2.0 with 10% TFA. The S-pyridylethylated AR is purified by rp HPLC using a μ-BondapakCie column (3.9 x 300 mm, Waters). The acetonitrile concentration is increased linearly (1% / min) within 55 min. At a flow rate of 1 ml / min. The first solvent used is 0.1% TFA. S-pyridylethylated AR (SPE-AR) is eluted at about 26.5% acetonitrile, an about 4% higher acetonitrile concentration than for untreated AR.

6.5.2 N-glycanase treatment

AR or S-pyridylethylated AR (10 to 20 μg) is dried in 1.5 ml polypropylene microcentrifuge tubes and suspended in 80 μl 0.1 M phosphate buffer, pH 5.5. Incubate the mixture at 37 ° C for 16h, add 0.9ml of 0.2TFA to the reaction mixture and inject a sample into an Ultrapore RPSC C3 rp HPLC column (4.6mm x 75mm, Altex) at a flow rate of 1 ml / min, following equilibration of the column with 0.1% TFA as the first solvent. Acetonitrile with 0.1% TFA is used as the second solvent. The acetonitrile concentration is increased linearly (0.4% / min.) For 85 min. At room temperature. N-deglycosylated AR and N-deglycosylated S-pyridylethylated AR are eluted at an acetonitrile concentration of approximately 16.5 and 20.5%, respectively.

6.5.3 Enzymatic cleavage of N-glycanase-treated S-pyridylethylated AR

Cleavage with endopeptidase Lys-C is carried out in 60 μM 0.1 M TRIS-acetic acid buffer, pH 8.0 within 16 h at 25 ° C. The enzyme / substrate ratio is 1 to 5 (w / w).

Endopeptidase Arg and S.Aureus V8 protease digestions are carried out in 80 μM 0.05 M TRIS-HCl, pH 8.0 and 0.1 M ammonium bicarbonate within 16 h at 37 ° C. The enzyme / substrate ratio is again 1 to 5th

6.5.4 Chemical cleavage

For CNBr cleavage on the methionine residue, 5 μl of N-glycanase-treated S-pyridylethylated AR are dried in 1.5 ml polypropylene microcentrifuge tubes, suspended in 75 μl of 70% formic acid and then mixed with 25 μl of a CNBr solution (25 mg / ml in 70 % HCOOH). Mix and rinse the tube with nitrogen. The reaction is carried out for 22 h at 25 ° C in the dark. The reaction mixture is diluted to 1.1 ml with water and dried in a Speed Vac concentrator. The dried sample suspended in 20μΙ 2-aminoethanol, stored for 10 min. At 22 ° C and then vacuum-dried. The mixture is suspended in 0.9 ml of 0.2% TFA.

6.5.5 Peptide isolation

The peptides are separated on a reverse-phase HPLC C8 column (4.6 mm χ 100 mm, Whatman) which is connected to a Waters (HPLC) system. The acidified sample (pH 2.0) is applied at a flow rate of 1 ml / min to a column equilibrated with 0.1% TFA (first solvent). Subsequently, the column is washed with 15 ml of 0.1% TFA. Linear gradients are used between the first solvent and the second solvent acetonitrile with 1% TFA. The gradient parameters are: 0 to 50% in 125 min. At a flow rate of 0.5 ml / min.

6.5.6 Amino acid analysis

Dried samples with constant boiling HCl (5.7 M Pierce) containing 1% (v / v) phenol under reduced pressure in a Teflon-sealed Glashydrolyseampulle (Pierce) was hydrolyzed at 105 ⁰ C during 16 h. The hydrolysates are dried in a Speed Vac concentrator (Savant Instruments) and derivatized with phenylisothiocyanate for 20 min. At room temperature. The phenylthiocarbamyl amino acid derivatives are analyzed by rp HPLC on an octadecyl column (4.5mm χ 250mm, IB). There is a linear gradient between the first solvent (0.15 M sodium acetate, pH 6.4, 0.05% triethylamine, titrated to pH 6.4 with acetic acid) and the second solvent (60% acetonitrile) at a flow rate of 1 ml / min, used at 38 ⁰ C.

6.5.7 Determination of the amino acid sequence

Peptide sequences are determined on an Applied Biosystem Model 475 gas phase sequencer as described (Hewick et al., 1981, J. Biol. Chem. 256: 7990-7997). Three pre-cycles of Edman degradation are performed prior to loading the sample before each run. 25% TFA is used to convert the triazoline derivatives to the phenylthiohydantoin amino acids. Identification of the phenylthiohydantoin amino acids is performed on-line on a PTH Analyzer Model 120A (Applied Biosystem) as described (Hunkapiller and Hood, 1983, Science 219: 650-659).

6.5.8 Different treatments

50 to 100 units of either semi-purified (semi-preparative rp C 18-step) or purified AR are used for these experiments (Table 1). N-glycanase and O-glycanase are purchased from Genzyme Corp., Boston; all other enzymes are purchased from Boehringer Mannheim Biochemicals or Worthington Biochemicals. The treatments are carried out in 50 to 200 μΙ of a suitable buffer. The enzymes are used in excess, usually 5 to 20% (w / w) of the substrate concentration. After the treatments, samples are tested for GIA after removal of interfering compound by various analytical means. In most cases, the pH of the samples is adjusted to about 2 with 10% TFA. The samples are loaded on a reverse-phase Ultra-pore RPSC C3 column (4.6 mm x 75 mm, Altex) equilibrated with 0.1% TFA. The column is washed with the first solvent 0.1% TFA. Subsequently, the elution is carried out using acetonitrile with 0.1% TFA as the second solvent. The gradient parameters are 0 to 50% 0.2 min and 50 to 50%, 19.8 min. The flow rate is 0.5 ml / min and fractions of 2 ml volume are collected. Samples of these are tested for GIA. The AR activity is usually eluted with fraction 4.

6.6 DNA binding studies

The binding of AR to native calf thymus DNA cellulose, denatured calf thymus DNA, and cellulose is performed as described (Herick and Alberts, 1971, Methods Enzymology 21: 198). The celluloses (10 mg) are rehydrated in 500 μM loading buffer (20 mM Tris pH 7.4.10% glycerol, 1 mM EDTA, 1 mM β-mercaptoethanol, 100 mg / □ BSA and 50 mM NaCl). The reactions are carried out twice in 1.5 ml Eppendorf containers with 300μΙ hydrated (packed volume) cellulose samples, which are washed 5x with loading buffer. Next, add 0.2 ng of ¹²⁵ I-AR (specific activity 425pCi ^ g) or other labeled proteins in 250μl of loading buffer to each tube. The samples are mixed and incubated for 20 min. At 4 ° C with periodic shaking and then washed 5x with 200μΙ loading buffer. Elution was gradual with 0.1M, 0.15M, 0.25M, 0.6M, 1M and 2M NaCl in loading buffers (5x at 200μΙ at each concentration). As the specifically bound material, one which is eluted at a NaCl concentration of 0.25 M or greater is considered. The material eluted at a concentration of 0.15M NaCl or less is considered nonspecifically bound.

6.7 results

6.7.1 Production of AR by TPA-treated MCF-7 cells

MCF-7 cells grown on a plastic substrate show typical ephitelial properties: the cells are small and polygonal in shape. TPA induces marked morphological dose-dependent changes. Following TPA treatment, MCF-7 cells lose their defined morphology, become round and broad

out. TPA results in a dose-dependent reduction in cell number and an increase in cell volume compared to untreated cells.

Serum-free conditioned medium of MCF-7 cells does not contain any detectable growth-inhibiting activity for A431 cells. However, serum-free supernatant, which is isolated after 2 to 3 days treatment of MCF-7 cells with TPA, inhibits the proliferation of A431 epidermal carcinoma cells. Optimal induction of inhibitory activity is observed at 25 to 100 ng / ml TPA. Even after removal of TPA, TPA-treated MCF-7 cells secrete this activity into the serum-free medium for at least another 8 days. Although a decrease in growth-inhibiting activity is observed within this period after TPA removal, these results indicate amphiregulin, the expression of which has been implicated or enhanced in TPA-treated MCF-7 cells.

6.7.2 Initial characterization

Table II summarizes the influence of various physical, chemical and enzymatic treatments on the antiproliferative activity of amphiregulin. Growth inhibitory activity (GIA) remains despite treatments with 1 M acetic acid, 1 M ammonium hydroxide, 6 M urea, 0.01 M sodium metaperiodate, 56 ° C for 30 minutes, and treatments with neuraminidase, N-glycanase, O-glycanase, lipase, phospholipase A2 , C or D received. However, the activity is responsive to 10-minute heat treatment at 100 ⁰ C, to reduction, to reduction and 4-Vinylpyridinbehandlung and to digestion with proteinases such as trypsin, endopeptidase Lys-C, endopeptidase Arg and Glu endopeptidase (V8). These results indicate that amphiregulin is a protein containing cysteine in disulfide bridges, which are of essential importance for biological activity. The protein may contain oligosaccharide and / or lipid moieties which are not obligatory for biological activity.

Table II Influences of various treatments on the GIA of amphiregulin

Treatment percent

the control

1M acetic acid, 2h4 ° C 102

1MNH ₄ OH for 2 hours at 4 ⁰ C 97

56 ° C at 30 in 96

100 ⁰ C at 10 in 5

6 M urea (2 h at 25Ό 82

0.2 M 2-mercaptoethanol (2.5 h at 25 ° C) 6 2-mercaptoethanol + 4-vinylpyridine

(2.5h at 25 ° C) + (2h at 25 ° C) 1

0.01 M sodium metaperiodate (6 h at 4 ° C in the dark) 86

TPCK trypsin (6 h at 37 ° C) 3

TPCK trypsin + soybean inhibitor (6h at 37 ° C) 82

Soybean trypsin inhibitor (6h at 37 ° C) 109

Endopeptidase Lys-C (20 h at 25 ° C) 5

Endopeptidase Arg (16h at 37 ° C) 4

Endopeptidase Glu (16h at 37 ° C) 6

Neuraminidase (16 h at 37 ° C) 103

N-glycanase (16h, 37 "C) 90

O-glycanase (16h at 37 ° C) 102

Neuraminidase + N-glycanase (16 h at 37 ° C) 94

Neuraminidase + O-glycanase (16 h at 37 ° C) 105

PhospholipaseA2 (6h at 37 ° C) 82

Phospholipase C (6 h at 37 ° C) 95

Phospholipase D (6 h at 37 ° C) 80

Lipase (6 h at 37 ^° C.) 111

6.7.3 Purification of amphiregulin

A summary of amphiregulin purification is included in Table III. A 1 842-fold enrichment with 5.1% yield for the TSK 250 l fraction I and a 2 270 fold enrichment with 1.7% yield for the TSK fraction II is achieved. The process is reproducible. Amphiregulin was purified 4x with similar results. The specific activity of purified amphiregulin ranges from 2.7 to 3.4x10 ⁶ units / mg protein. Purified amphiregulin from the TSK 250 L column has a specific activity of 3.0x10 ⁶ and was used for structure determination and other biochemical studies.

Table III Summary of Amphiregulin (GIA) Purification

fraction Volume (mL) Protein (Mg) GIA * (Units x KT ³ ) Specific activity Units x 10 " ³ per mg Yield% Cleaning (-fold) Crude 300 916.600 1363 1:49 100 1 Prep. C18 pass u. wash fraction 950 532000 1052 1.98 77.2 1.3 Prep.rp.ODS 225 2,281 286 125.43 21.0 84.2 Semiprep. rp.C18-l -Il 20 20 339 235 97 92 286.14 391.49 7.1 6.7 192.0 262.7 Analrp. C18-1 -Il 6 6 242 173 50 46 206.61 265.89 3.7 3.4 138.7 178.4 TSK 250-1 I-A I-B I-C 3.6 1.6 1.2 0.8 25.5 6.6 11.4 7.5 70 15 33 22 2,745.10 2,272.73 2,894.74 2,933.33 5.1 1.1 2.4 1.6 1,842.3 1,525.3 1,942.8 1,968.7 TSK 250-11 1.2 6.8 23 3,382.35 1.7 2,270.0

* The details are given in section 6.1.3 and the following subsections. One unit of GEA corresponds to the amount of factor required to inhibit ⁵ 'deoxyuridine uptake in A431 cells by 50%.

6.7.4 Analysis of amphiregulin (AR) and S-pyridylethylated amphiregulin (SPE-AR) by HPLC

Gel permeation chromatography of AR and SPE-AR over a TSK-250 column (7.5 mm x 600 mm, Bio-Rad) is shown in Figures 5A and 5B, respectively. The activity was eluted along with the protein peak (5 Å). The molecular weight of AR and SPE-AR was determined from the data in Figure 5 and is approximately 14,000 and 17,000, respectively. The calculated molecular weights for the structures of truncated AR and AR in Figure 12 are approximately 8500 and 9100 daltons, respectively.

AR and SPE-AR were eluted as symmetrical single peaks from an Ultrapore RPSC C3 rp HPLC column (4.6 mm χ 75 mm, Altex) (Figure 6 A and b). The GIA was eluted along with the protein peak (Fig. 6A). SPE-AR was eluted at an acetonitrile concentration of about 21%, and thus at about 4% higher acetonitrile concentration compared to untreated protein. These results indicate that AR is rich in cysteine residues that are modified by 4-vinylpyridine, rendering AR more hydrophobic and thus eluting at a higher acetonitrile concentration.

6.7.5 SDS-PAGE analysis of AR

Figure 7A shows an analysis of AR, N-glycanase-treated AR (NG-AR), SPE-AR and N-glycanase-treated AR (NG-SPE-AR) in 15% acrylamide under reducing conditions (Fig A). AR and SPE-AR migrate the gel as broad diffuse single band with an average molecular weight of 22,500 within a range of 20,000 to 25,000 (lanes 1 and 3). The treatment of AR and SPE-AR with N-glycanase leads to a higher migration rate of these proteins. NG-AR and NG-SPE-AR migrate in the form of a single lane with an average molecular weight of 14000 to 14500 Datton. Similar results are obtained when the proteins are analyzed in a 15% gel under nonreducing conditions (data not shown). Treatment of AR with neuraminidase, O-glycanase or neuraminidase and O-glycanase together does not result in alteration of electrophoretic mobility in a gel under reducing or nonreducing conditions. AR is thus a single-stranded glycoprotein and contains one or more N-linked oligosaccharide chains that are not required in vitro for the GIA of AR.

Treatment of NG-AR or AR with phospholipase D (PLD) (cabbage) results in GIA reduction to 80% of the control value. PLD-treated NG-AR is applied to a СЗ-rp HPLC column and the purified active peaks are analyzed on a 20% SDS-PAGE (Figure 7B). A single band with Mr 5 600 can be observed. These results indicate that PLD or one or more protease contaminants in the PLD preparation converts AR into one or more active fragments.

6.7.6 Analysis of AR by Isoelectric Focusing (IEF)

An IEF analysis of ^12S I-labeled AR is performed according to the instructions in Section 6.1.1.2, above. AR is focused as a single broad band with a p _r value between 7.6 and 8 (Figure 8).

NG-AR is fixed as a single band with a pI value of 8.05. AR thus represents a basic single N-bridged glycoprotein.

6.7.7 Biological properties

The inhibition of ¹²⁵ I-deoxyuridine uptake into DNA of A431 cells by various concentrations of purified AR is shown in Figure 9A. A 50% inhibition of DNA synthesis is observed at approximately 0.2 ng / well. A 50% inhibition of DNA synthesis in A431 human epidermal carcinoma cells is therefore observed at a pure protein concentration of about 0.13 nM. It should be noted that the growth-inhibiting activity of AR is dependent on experimental conditions such as the number of cells per well (cell density), the time of factor application, the duration of treatment, the serum concentration and other variables.

When A431 cells are cultured in the presence or absence of various concentrations of AR, and cell growth is recorded by counting the cells, AR is found to inhibit A431 proliferation dose-dependently (data not shown). The extent of ¹²⁶ l deoxyuridine incorporation into the DNA thus represents a good measure of cell growth.

The stimulation of ²⁵ I-deoxyuridine uptake into DNA of human foreskin fibroblasts (Sadamoto) as a function of various concentrations of purified AR is shown in Figure 9B. There is a twofold stimulation of ¹²⁶ L deoxyuridine incorporation at about 0.7 ng / ml or about 0.05 nM AR. The maximal response is a six-fold stimulation of ^12S I deoxyuridine incorporation into these fibroblasts. AR thus acts as a growth factor for human fibroblasts even in the presence of 5% FBS. The effect of AR on the incorporation of ¹²⁵ I-deoxyuridine into the DNA of various tumor cell lines and non-Tumorhumanzellinien as well as some non-human cell lines was investigated. The data are summarized in Table IV. AR inhibits the growth of various clones of A431 cells, human mammary carcinoma cells HTP 132, human ovarian teratocarcinoma cells HTP 1575, human cervical epidermal carcinoma cells CRL 155, human ovarian papillary adenoma cells НТВ 75 and human breast adenocarcinoma cells НТВ 26 The factor shows no significant effect on a variety of cells (Table III). AR stimulates the uptake of ^126- I-deoxyuridine into several human fibroblast cell lines, into human pituitary tumor cells CRL 7386, into human ovarian adenocarcinoma cells NT 77, in the liver cells of African green monkey BSC 1 and rat kidney cells SA 6. AR does not show significant influence the proliferation of T, B or endothelial cells. AR does not suppress human proliferative or cytotoxic T cell responses in mixed leukocyte culture reactions (MLC).

AR represents a monomeric protein which requires disulfide bonds within the protein chain for its activity. Glycosylation is not required for the activity. AR is stable under weakly acidic and basic conditions, and at 30-minute heat treatment at 56 ⁰ C. The biological activity of AR is completely lost by reduction, by 5 minute heat treatment at 100 ⁰ C, by digestion with trypsin, endopeptidase Lys-C, endopeptidase Arg or Endopeptidase GLU.

Table IV

Effect of amphiregulin on the proliferation of cells ^a

indicator cells

Human vulvar epidermal carcinoma A431

Human breast adenocarcinoma HTB132

Human cervix epidermal carcinoma CRL1550

Human ovarian papillary aortic HTB75

Human ovarian teratocarcinoma НТВ 1572

Human breast adenocarcinoma HTB26

Human melanoma A375

Human breast adenocarcinoma ZR7530

Human breast adenocarcinoma MCF-7

Human lung adenocarcinoma A549

Human prostate adenocarcinoma PC3

Human colon carcinoma H 3347

Human lymphoblastoid (T cells) CEM

Human EBV-transformed B cells

Human laryngeal epidermal carcinomaHep2

Human cervical carcinoma CRL1594

Human ovaria! AdenocarcinomaHTB77

Human foreskin fibroblasts (Sadamoto)

Human foreskin fibroblasts (Goodwin)

Bovine fetal heart endothelial cells

Murin Balb / 3T3

Simian African green monkey kidney cells BSC-1 kidney SA6

growth growth inhibitor stimulator activity activity GIA (Units) " (Units) GSA 125 240 28 19 55 5 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 25 225 70 N.D. N.D. N.D. N.D.

65 45

a) 125 Units (GIA on A 431 cells) of AR are suspended in 250μΙ assay medium, serially diluted 5-fold with assay medium. Six dilutions are used for each cell. The fractions are tested for growth modulating activity and the GIA and GSA units are calculated

b) A GIA unit is defined as the amount of factor needed to inhibit ¹²⁵ I deoxyuridine uptake in cells by 50%

c) a GSA unit is defined as the amount of factor required to increase 100% of ¹²⁵ l deoxyuridine uptake into the cells

N.D. = undetectable

The influence of AR and EGF on NRK-SAe cell colony formation in soft agar in the presence or absence of TGF-β was tested. The results are summarized in Table V. In the presence of TGF-ß, EgF induces a dose-dependent and anchorage-independent growth of SA6 cells. AR is a very weak inducer for SA-G colony formation in Softagar.

Table V

Effect of AR and EGF on NRK-SA6 cell colony formation in soft agar

encore (1 ng) Number of colonies (per ml) (2 ng) per 6 fields none (4ng) 0 TRGF-p (2ng) + TGF-ß (1ng) 0 AR (4ng) + TGF-ß (1ng) 0 AR (2 ng) 0 AR (4 ng) 7 AR (2ng) + TGF-ß (1ng) 8th EGF (4ng) + TGF-ß (1ng) 0 EGF 6 EGF 30 EGF 114

The tests were as described in section.

6.7.8 Influence of AR on EGF Binding to its Specific Receptors AR is found to inhibit the binding of ¹²⁶ I-EGF to A431 cells as well as to A431 plasma membranes (Figure 10). A 50% inhibition of ¹²⁵ I-EGF binding to fixed cells and membranes is observed at approximately 1.1 nM and 1.8 nM EGF, respectively, while a 50% reduction in EGF binding to cells and membranes at approximately 1 , 8nM and 5.7nM AR, respectively. Unlabeled EGF inhibits the ¹²⁶ l-EGF receptor interaction at higher concentrations in both systems. The competition maxima with AR were 75% and 50% for binding to cells and membranes, respectively. The competition curves for AR are not parallel with those of EGF. These results indicate that AR exhibits less activity on EGF receptors than EGF itself. In addition, the AR-specific receptor could be closely related to the EGF receptor.

From several selected clones of the A431 cell line with different sensitivity to AR inhibition, a lack of correlation between AR sensitivity and the number of EGF receptors per cell or Kp value is observed.

Table VI

Lack of correlation between amphiregulin sensitivity of different A431 clones and the co-value of the EGF receptor

EGF binding parameters K ₀ (NM) relative Binding site per cell 1.80 sensitivity A 431 clones 3.13 (GIA) to AR A-3 4,6x10 ⁶ 1.38 34 F-8 9,0X10 ⁶ 3.18 20 A-2 5.1 x 10 ⁶ 11 A-8 5,3x10 ⁵ 1

Different A431 clones were selected by growth in soft agar. EGF binding was performed according to section 6.6.1.5 above. K _d and the number of binding sites were derived from Scatchards plots (Scatchard et al., 1949, Ann. NY Acad., 51: 660-672).

6.7.9 Chemical structure of amphiregulin

The amino acid sequence of AR was derived from the microsequence analysis of S-pyridylethylated protein (SPE-AR) and the fragments produced by endoproteinase Lys-C, Staphylococcus aureus Proptease V8 and endopeptidase Arg. The sequences of the truncated protein as well as the protein with six additional amino acids are the following:

Shortened AR

1 10 20

VVKPPQNKTE SENTSDKPKR KKKGGKNGK

30 40 50

N RRNRKKKNPC NAEFQNFCIH GECKYI

60 70 78

EHLE AVTCKCQQEY FGERCGEK

Bigger AR

1 10 20

SVRVEQVVKP PQNKTESENT SDKPKRKKK

30 40 50

G GKNGKNRRNR KKKNPCNAEF QNFCI

60 70 80 84

HGECK YIEHLEAVTC KCQQEYFGER CGEK

In the sequences given above, the amino acids are named with the usual one-letter code for an amino acid. Amino acids whose identity is not fully protected are given in parentheses. The letter "X" denotes amino acids of unknown identity The amount of the larger AR is about 16% of the truncated AR The protein sequence of AR was identified with all proteins in the National Biomedical Research Foundation database (PIR 13) and in the Genetic Sequence Database (Bolt Beranek and Newman, Inc., Los Alamos National Laboratory DNA Sequence Library (Release γ # 12).) This research has revealed that AR is a novel protein belonging to the EGF superfamily of growth factors and includes EGF (mouse, human , Rat, bovine, etc.), TGF-a, viral growth factors (Vaczina, Myso, Shopefibroma), human plasminogen activator, bovine factor IX, human factor X, LDL receptor, bovine protein C, human proteoglycan core protein, product of Drosophila Notch gene, product of C. elegans lin 12 gene, and the product of the "cell lineage" specific sea urchin S. purpuratus gene A comparison of the AR structure with the structures of proteins of the EGF ob Erfamilie shows that AR, like other members of the EGF superfamily containing characteristic six essential cysteine residues, maintains the preservation of the gaps between the cysteine residues and contains a portion of the characteristic strongly conserved amino acids. It appears that AR is to be classified between EGF and TGF-like members and MGF and SFGF-like members of the growth factor family, in particular with regard to the occurrence of asparagine. For example, at position 2 (first cysteine = 1) all TGFs and EGFs have a proline residue, VGF shows a glycine residue and MGF, SFGF and AR have an asparagine residue. In the same loop at position 7, EGF's and VGF's have a glycine residue, TGF's a glutamine residue and MGF, SFGF and AR an asparagine residue. However, in the third loop, AR does not have the conserved beta at position 34 (either asparagine in MGF and SFGF and one glycine residue in all the other members), but has a glutamic acid residue (low beta potential) at this point. AR has a different structure for the highly conserved third loop, which may affect receptor binding. The AR also lacks the asparagine residues, which may be used in the MGF and SFGF as glycosylation sites within the growth factor structure.

The N-terminal sequence of AR shows some analogy with the N-terminal sequences of TGFs, VGF and MGF in that it is rich in prolines, serines and threonines and how TGFs and VGF are potential N-linked glycosylation sites (in reality a site ) and also the possibility for O-bridged glycosylation in this area, since this region is rich in serines, threonines and prolines. This region is highly conserved between the N-terminus of the TGF precursor and the N-terminus of VGF and appears to be a highly glycosylated region.

AR is an extremely hydrophilic protein, especially in the region of amino acids 8 to 45 (Figure 2). The hydropathicity profile of AR shows little similarity to the profiles of other members of the EGF family. The hydrophilicity profile of the C-terminal region of AR differs from other members of the EGF family in structural similarity to other members of this family (Figures 11 B and C).

6.7.10 Specific binding of AR to DNA

¹²⁶ I-AR is tested for its ability to bind to cellulose, denatured DNA-cellulose and native DNA-cellulose according to the test conditions in section 6.6, above. The results in Figure 14A indicate that AR specifically binds to both denatured and native DNA cellulose but not cellulose itself. The binding to native DNA was 3 to 4 times greater than the binding of denatured DNA.

The ability of AR to bind specifically to DNA distinguishes AR from all other members of the EGF superfamily. For example, EGF is unable to bind to DNA cellulose (Figure 14B). The results strongly suggest that the hydrophilic 45 amino acid N-terminal domain of AR, which is found neither in EGF nor in other members of the EGF family, is a nuclear localization sequence involved in the "targeting" process from AR to the nucleus, where then the factor interacts with the DNA.

Example 7: Antibodies to Amphalic Gulin

The production of antibodies to AR, synthetic AR and AR precursor peptides is described in the following subsections.

7.1 Material and Methods

7.1.1 Solid Phase Peptide Synthesis

Amphiregulin is prepared and purified in accordance with Section 6. Five peptides containing residues 31-50 (# 279), 71-90 (# 280), 108-130 (# 264), 166-184 (# 259) and 221-240 (# 281) corresponding to the AR primary structure (Figure 15) are synthesized by solid phase technique on an automated peptide synthesizer made by Applied Biosystems Inc. as described (Merrifield, 1963, J. Amer. Chem. Soc. 85: 2149). The synthesized peptides are cleaved from the carrier resin by means of HF (Tarn et al., 1988, J. Amer. Chem. Soc. 105: 6442). The synthetic peptides are purified by reverse phase HPLC.

7.1.2 Antibody production

Polygonal antisera to mature AR and synthetic AR peptides chemically conjugated to keyhole limpet hemocyanin (KLH) are produced in young adult New Zealand white rabbits. The synthetic peptides are conjugated with KLH as described (Ishikawa et al., 1983, Immunoassay 4: 235). Matured AR was not paired. AR or the peptide conjugates are emulated with the Ribi adjuvant system (Ribi Immunochem Research, Inc., Hamilton, MT). A total dose of 1 ml is administered to each rabbit: 0.8 ml intramuscularly at two sites in each hind leg and 0.2 ml subcutaneously at one site. For the generation of antisera to mature AR 30pg mature AR is used for the first inoculation and 15k for the following "booster injections. To generate antisera to synthetic AR peptides, 100 μg of the conjugated peptide are used for the initial injection and 50 mg for the subsequent booster injections. The booster injections are made every three to four weeks, and the rabbit is bled 10 to 14 days after the booster injection.

7.1.3 Immunoprecipitation

AR is radiolabeled with ¹²⁵ I using the chloramine-T method (Barridge, 1978, Methods Enzymol. 50: 54-65). 10μl polyclonal serum (or diluted in PBS) is combined with 10βl ¹²⁶ I-AR (50ng / ml, specific activity 425βCi / Mg) and 30μΙ PBS and assayed essentially as described (LeBier et al., 1982, J. Immunol : 2287-2292).

7.1.4 Radioimmunoassay

Polygonal sera are diluted 1:50 in PBS. ¹²⁶ I-AR (1 μg / ml, specific activity 425pCi / pg) is assayed 1: 200 with TNEN / 0.1% BSA (20 mM Tris pH 7.4, 5 mM EDTA, 15 mM NaCl, 0.05% NP-40 , 0.1% BASA). 10 μΙ unlabelled AR (1 mg / ml), 10 μΙ diluted polyclonal serum and 30 μΙ diluted ¹²⁶ I-AR are combined and incubated at room temperature for 45 min. A protein A-Sepharose solution is prepared as follows: 200mg of dry protein A-Sepharose (Pharmacia) is rehydrated with 1.4ml of PBS , 0.5% Triton X-100, 2 mM PMSF, 1% aprotinin and O, 5 mg / ml Levpeptin diluted. 20 μΙ protein A solution is then added to the mixture and incubated for a further 30 min. The Sepharose is then washed 2x with 500μΙ TNEN / 0.1% BSA, suspended in 50μΙ SB (80mM Tris pH 6.8.3% SDS, 15% glycine, 0.01% bromophenol blue, 5% β-mercaptoethanol) and heated to 100 ⁰ C for 5 min. The samples are centrifuged and the supernatants are checked for radioactivity in a gamma counter. With this procedure, 100pg of AR can still be detected.

7.1.5 Enzyme-linked immunosorbent assay

The solid phase micro ELISA method has been modified from U.S. Patent 740,124. Terasaki microtrays are added to each well by adding 5 μΙ of the synthetic AR peptide diluted in PBS to various concentrations. Then the solution is allowed to dry overnight at room temperature. 5% Blotto in PBS is added to the plates and allowed to stand at room temperature for 1 hour. In the next step, 10 μΙ dilutions of the polyclonal antibody solution (diluted in PBS / 1% Blotto / 0.05% Triton X-100) are added to each well, incubated for 1 h at room temperature and then washed 4x with PBS. 5 μΙ of peroxidase-conjugated goat anti-rabbit IgG (Cappel) is added to each well at a dilution of 1: 1000 in PBS / 1% Blotto / 0.05% Triton X-100, incubated for 1 h at room temperature and then Washed 6 times with PBS. 10μΙ of the MicroEIA Chromogan solution (Genetic Systems Corp.) are added in a 1:20 dilution. Subsequently, the absorbance at OD _{492 is determined} after 20 min. And 40 min. According to the Genetic Systems Micro EIA instructions.

7.1.6 Western blotting

The samples are electrophoresed on a 15% polyacrylamide gel or a 16% tricine polyacrylamide gel in a BioRad minigel apparatus. The gel is then placed in the cassette with a nitrocellulose sheet so that the nitrocellulose is placed on the cathode side. The protein transfer is carried out at a current of 40 oMA and constant power within 30 min. The nitrocellulose is then removed and immersed in 2.5% Blotto / PBS / 0.2% NP-40 overnight at 4 ° C.

The nitrocellulose filter is then treated with antiserum diluted in 2.5% Blotto / PBS / 0.2% NP-40 reaction buffer, 90 min. At room temperature on a shaker, followed by 4 washes (5 min. Each) in 2, 5 Blotto / PBS / 0.2% NP-40.

Alkaline phosphatase-conjugated Protein A (Cappel) diluted 1: 500 in 2.5% Blotto / PBS / 0.25% NP-40 is then added to the nitrocellulose sheet and incubated for 60 min. At room temperature in a shaker. The filter is then washed 4x with PBS / 0.2% NP-40 (3 min each), followed by incubation in APS buffer (100mM Tris pH 9.5, 10mM NaCl, 5mM MgCl ₂ ) for 5 min. The filters are prepared in a color reagent (40 ml of APS buffer, 12 mg of б-Вгот ^ -сЫог-З-indoylphosphat (Sigma), 6.8 mg Nitroblautetrazolium (Sigma) prepared immediately before use and filtered through a 0.2 μιη filter) The reaction is quenched with H ₂ O and then air dried.

7.2 Characterization of AR Antibodies

Polyclonal antibodies to mature AR and synthetic AR peptides are characterized by radioimmunoprecipitation, radioimmunoassay, ELISA, and Western blotting. The results are summarized in Table VII.

Table VII

Characterization of antibodies to amphiregulin and synthetic AR peptide

polyclonal Test Method ELISA WB Serum against RIP RIA ND 1: 250 Matured AR1: 256 ^a 1: 250 Ong) (.100pb) ^b ND ND Peptide 259 1: 256 ND 1: 100 1: 100 Peptide 264 1: 256 1: 250 (5 ng) (0.1 ng) (.100 pb) 1: 500 (1 ng) 1: 100 (5 ng) ND Peptide 279 ND ND 1: 100 (5 ng) ND Peptide 280 ND ND 1: 100 (5 ng) ND Peptide 281 ND ND

a) dilution of the antisera required

b) The values in parentheses indicate the sensitivity of the method

RIP = radioimmunoprecipitation, RIA = radioimmunoassay, ELISA = enzyme-linked immunosorbent assay WB = western blotting, ND = not determined

8. Example: cDNA cloning of the amphiregulin precursor

The following example describes cloning and analysis of cDNAS encoding the amphiregulin precursor from TPA-treated MCF-7 human breast carcinoma cells.

8.1. material and methods

8.1.1 RNA preparation

The RNA is isolated from subconfluent cells grown in T150 tissue culture flasks or from freshly thawed tissue cultures using the guanidinium method described by Chirgwin (1979, Biochemistry, 19, 5294). Cells from four T150 bottles are washed in PBS, trypsinized and washed again in PBS. The pellet is suspended in 8 ml of 4 M guanidinium isothiocyanate solution. The DNA is reduced during the passage of the solution through a "18 gauge" needle. With the sample 2.4 ml of a 5.7 M CsCl solution are overlaid in a Beckman SW41Ti beaker and centrifuged at 32000 Upm20hbei 20 ⁰ C. The pellets are resuspended in 2.5 M CsCl and precipitated at room temperature with 2 volumes of EtOH, the pellets are resuspended in 300 μM H ₂ O, and after addition of 35 μM of 3% sodium acetate (pH 5.1) and 800 μM EtOH, RNA becomes -7O precipitated ⁰ C. the precipitation is repeated and the RNA dried briefly. the RNA is then resuspended in 100μΙ H ₂ O ₂ M quantified and stored at -7O ⁰ C at OD.

8.1.2 Random PrimecT Marking with ³² P-TTP

Specific fragments for the AR coding region are excised from "low gel temperature" agarose (BioRad) and probed with ³² P-TTP (NEN, ЗOOSC / mmol) using the random primed method of Feinberg (1983, Anal. Biochem., 137, 266). Unincorporated deoxyribonucleosides are removed by chromatography on a 1.5 ml Sephadex 50 column. Specific activities are typically in the range of 0.5-2.5 χ 10 ⁹

8.1.3 RNA Gene and Northern Blot Analysis

10 or 20 μg of total RNA are electrophoresed on 1.2% agarose / formaldehyde gels for Northern blot analysis. The agarose / formaldehyde gels are prepared in 40 mM N-morpholinopropanesulfonic acid (MOPS), pH 7.0 / 1 mM EDTA / 10 mM sodium acetate / 2 M deionized formaldehyde (pH 5.6) in an IBI VCV chilled-plate gel apparatus. RNA is denatured in the same buffer with 50% formamide at 65 ⁰ C and separated in 1 x MOPS at 20 to 3OmA for 3 to 5h. The gel will be ЗОМіп. rinsed in 10x SSC and transferred to Hybond-N membrane (Amersham), prehybridized UV-crosslinked (1,200μϋουΙβ3) and 5x SSPE (1 χ SSPE is 0.18M NaCl / 10 mM sodium phosphate, pH7.7.0.1mM EDTA ), 5x Denhardt's, 0.5% SDS, and 20 μg / ml denatured salmon sperm DNA hybridized as a carrier. Hybridizations are performed at 42 ° C for 16h with 2x10 ⁶ cpm / ml ³² P-ARBPI. The blots are washed several times in 2x SSC with 0.1% SDS at room temperature and at 1 χ SSC / 0.1% at 65 ° C and on a Kodak X-OMAT-FiIm with two Dupont-Lightning Plus intensifying screens at - 70 ⁰ C launched. The bands are classified as (+) if they are visible overnight, as (+/-) if they are visible after 3 days and as (++) if they are detectable after 6 hours.

8.1.4 cDNA cloning

RNA is isolated from MCF-7 cells after a 24, 40, and 72 h continuous treatment with 100 mg / L-tetradecanoyl-phorboMS acetate (TPA). Poly (A) ⁺ RNA is isolated from individual pools of these samples and used as template for double-stranded cDNA synthesis as described (Gubler and Hoffman, 1983, Gene 25: 263). G-extended cDNA is ligated into the Eco RI site of \ gt 10 using the BRI oligonucleotide adapters (Rose et al., 1986, Proc Natl Acad Sci., USA, 83: 1261-1265). Specifically, 5 μg of poly (A) ⁺ RNA are heat denatured from TPA-treated MCF-7 cells and treated with 5 μg of oligo (dT) using 100 U reverse transcriptase in 45 μM reaction volume. The synthesis of the second strand is performed using 4U RNaseH and 115U E. coli DNA pol I. 10μg T4DNA polymerase is used to remove the З'-protruding ends to produce blunt ends. The entire 6 ^ g of the double-stranded cDNA are separated by size on a Sephadex G 50 column. It selects cDNAs greater than 500bp. Subsequently, 150ng cDNA is extended by dG tailing using the terminal deoxynucleotidyltransferase. The dG tailed cDNA is ligated to the EcoRI site of Agt 10 using the BRI adapter (AATTCCCCCCCCCCCC). The ligated DNA is packaged in vitro (Grosveld et al., 1981, Gene 13: 227-237) and plated on E. coli C 600 rK ~ mK hf 1 with an efficiency of 10 ⁶ recombinant gcDNA. Double nitrocellulose filterprints of 2.5χ 10 ⁵ recombinants are made and the filters are screened with [ ³² P] -labeled "best guess" and degenerate oligonucleotide probes (Table VII, below). Derived from the automated Edman degradation of AR to N-reduction and S-pyrridyl-ethylated AR (Section 6, supra).

Table VIII

Probe sequence length

degeneration

Degenerate

CK C QQ E (Y)

ARD41 3 'ACA TTT ACA GTT GTT CTT AT5' 20

GC GCCC (64-fold)

ECKYI E (H)

ARD 58 3 'CTT ACA TTT ATA TAA CTT GT 5' 20

CGCG C (32x)

Best Guess

KNPCNAEFQNFC ARK31 3 'TTC TTG GGT ACG TTA CGA CTC AAG GTC TTG AAG ACG 53

I H G E C (K)

DAY GTA CCG CTC ACG TT 5 '

(Y) I EHLEAVTCKC

ARK41 3 'AG TAA CTC GTA GAC CTC CGA CAC TGG ACG TTC ACG 67

QQE YFGE RCG (E)

GTC GTC CTC ATG AAT CCG CTC GCC ACA CCG CT 5 '

VVKPPODKTESE ARNT 3 'CAC CAC TTC GGG GGG GTC CTG TTC TGT CTC AGG GTC 59

NT SDK PK (R) TTG TGG AGA CTC TTC GGG TTG TG

Restriction analysis reveals a small number of cleavage sites in the pAR 1 cDNA inserts, with an interface of Bsml, EcoRV, Hgal, Nael, Pvull, SmaI and SstI, and two Sspl sites. No digestion within the insert occurs with BamHI, CIaI, EcoRI, Hind III, KpnI, PstI, Pvul, Sphl, Stul, and XbaI. A 170bp BspI-PvUII fragment is isolated and used as a probe for a second cDNA library of 100,000 recombinants prepared as described above. One identifies thirteen positive clones with inserts in a larger range from 300 bp to 1.3kb, with six inserts larger than 1kb and five inserts having a single SstI interface known to be within a 100 bp Fragment at the 5 'end of pAR1. Four of the five inserts (pAR3, pAR5, pAR9, pAR13) are subcloned for further restriction and sequence analysis. All of these clones have identical restriction patterns based on Bsml, EcoRV, Pvull, Sstl, and SmaI, except for pAR9, which is shortened by 100bp at the 3 'end, and which was later found to be an As sequence. which may be suitable for priming with oligo (dT).

8.2 Nucleotide sequence analysis of AR cDNA clones

Exact oligonucleotide primers are used to sequencing both strands of the 1,230 bp pAR 1 clone using the dideoxy chain termination method (Sanger et al., 1977, Proc. Natl., Acad. Sci. USA, 74: 5463-5467 ). The entire nucleotide sequence and the deduced amino acid sequence of the clone pAR 1 is shown in FIG. 16. An open reading frame of 965 bp starts at nucleotide # 1. The first AUG is at position 210, but does not match the optimal Kozak consensus for translation start regions (Kozak, 1984, Nucleic Acids Research 12: 857-872, Kozak, 1986 , Cell 44: 283-292) because a purine is missing at position -3. However, such AUG triplets may serve as the initiator codon (as Kozak has found for about 3% of the information studied, possibly also important is the presence of adenosine at position -1 in all of these "less preferred" AUGs and in the mRNA of Although a second methionine is present at nucleotide 378 and is consistent with the initiator consensus sequence, it is believed that the first AUG is the translation start point proper since it is followed by a predicted 19 amino acid sequence consisting predominantly of hydrophobic residues. which is interrupted by 3 prolines, which is typical of a signal peptide sequence (Heijne, 1983, J. Biochem., 133: 17-21).

The longest open reading frame, starting with a methionine, encodes a 252 amino acid polypeptide and receives the 19-residue signal peptide. The coding sequence follows a 5'-untranslated sequence of 209 nucleotides. The coding sequence is followed by the translation stop signal (TAA) and the 262 nucleotide 3'-untranslated sequence. A potential poly (A) addition signal sequence (AATAA) is found 64 nucleotides upstream (in the 5 'direction) of a series of 15 adenylate residues, which is likely to represent the poly (A) -TaU. The З'-untranslated region of AR contains four copies of the sequence ATTTTA, which is also found in certain lymphokines, cytokines and proto-oncogenes. In GM-CSF, this sequence mediates the de-stabilization and degradation of RNA (Shaw, 1986, Cell 46: 659-667). Conservation of this moiety in functionally similar molecules indicates that AR is also related to this class of lymphokines. The cDNA sequence confirms the mature AR peptide sequence (Section 6, supra) with the exception of amino acid 113 (Figure 15), which is determined by protein sequencing as aspartic acid (D). An asparagine (AAT = N) is derived from the cDNA clones for this purpose. Of five cDNAs examined, all 5 'ends were within the first 25 bp of the pAR1 sequence.

A comparison of the cDNA sequence with the best guess probes revealed a total homology of 74% (ARK41) and 77% (ARK31) .No of the samples matched at more than 8 consecutive base pairs, but 50 out of 67 nucleotides from ARK41 revealed a detectable signal under conditions of very low stringence The codon usage in the AR mRNA sequence differs considerably from the frequency of use according to the data from Lathe (Lathe, 1985, J. Mol., Biol., 183: 1-12) why the degenerate oligonucleotides gave stronger signals in this case: From the cDNA and protein sequences, two proteins with a molecular weight of 9772 and 9173, respectively, for the two forms of mature AR on the basis of the cDNA sequence (Figure 16) and the protein sequence (Section 6.7.9) The hydropathic profile of the AR precursor is given in Figure 11D.

The AR precursor has three potential N-glycosylation sites, one in the N-terminal domain (position 30 in Figure 16) and two in the hydrophilic region of the mature AR (positions 113 and 119). It is known that glycosylation contributes 10 to 12 kd to the molecular weight of mature AR and is probably caused by carbohydrate addition to one or both of these positions in the hydrophilic domain.

The N-terminal serine-rich domain of the AR precursor (Figure 15) contains three potential tyrosine sulfation sites at Y ⁸¹ , Y ⁸³ , Y ^8? due to the presence of acid residues, turn-inducing amino acids and the absence of residues that could contribute to steric hindrance (Huttner, 1987, TIBS 12: 301-303). Most typrosinsulfated proteins are secretory proteins. It is believed that modifications by tyrosine sulfation are involved in the activation, transport, or proteolytic processing of the precursor proteins. The serine rich domain of AR may also contain O-bridged carbohydrate chains due to the large number of serine / threonine residues (23 out of 81) in this region of the molecule, which are bridging sites for this. In this regard, it should be noted that O-glycosylated forms of another member of the TGF family, TGF-a, have been detected (Ignotz et al., 1986, PNAS, 83, 6307-6311). None of the serine residues in the AR precursor are consistent with the consensus sequence for the cAMP-dependent kinase phosphorylation sites (Grima et al., 1985, Proc Natl Acad., See, USA, 82: 617-621). In addition, none of the typrosine residues have flanking sequences similar to those known as phosphotyrosine residues.

The hydrophilic domain of the mature AR protein consists of a variety of positively charged amino acids (16 of 37 residues are lysine or arginine residues) including two consecutive regions of 4 to 5 basic charged residues. The SV40 large T antigen core targeting signal is similar to this AR region and contains a characteristic KKKRK sequence, with small amino acids (glycine, alanine, proline) possibly precedent for the formation of an helical structure (Burglinetal., 1987, EMBO 6: 2617-2625, Dingwall et al., 1987, EMBO 6: 69-74) Mutational analysis identifies four consecutive basic residues as the dominant feature of the SV40 core localization sequence (Lanford , 1984, Cell, 37, 801-813.) Other nuclear proteins with similar portions of basic amino acids that may be involved in nuclear targeting include nucleoplasmin, polyomavirus large T, histones, and m-myc. A fusion of several nuclear signal sequences to the genes for chicken pyruvate kinease, albumin, immunoglobulin, ferritin and β-galactosidase lead to a localization of these otherwise cytoplasmic proteins on the nucleus (Moreland, 198 7, Mol. Cell Biol. 7: 4048-4057). Whether the hydrophilic sequences in AR participate in nuclear targeting of this growth modulator has not been verified. However, the ability of AR to specifically bind to DNA suggests a functional role for AR at the core. Here, AR can regulate DNA synthesis, the cell cycle, or a variety of other nuclear processes.

The TGF-α precursor contains a variety of cysteines in the C-terminal cytoplasmic domain, with some of these residues covalently attached to plamitate (Bringman, 1987, Cell, 48, 429-440). In contrast, the AR precursor lacks any cysteine residues in the cytoplasmic domain, and it is believed that the precursor contains no palmitate residues. Although the biological function of palmitate attachment is unknown, this is another difference between AR and other members of the EGF superfamily.

8.3 Cellular sources of amphiregulin synthesis

Northern blot analysis (Thomas, 1980, PNAS, 77, 5201) using a ³² P-labeled AR cDNA fragments shows hybridization of a single 1.4 Kb RNA species in a variety of normal Human tissue cell lines and tumor cell lines. A single band is found in Northern blots when either AREB1 (a 480 bp BstEII-Pvu II fragment of pAR 1 containing the entire coding region of mature AR and part of the N-terminal precursor and the transmembrane domains) or AR 170 (a 170 bp Bsml Pvull fragment of pAR 1 containing only the C-terminal half of mature AR) as labeled probes. The results in Table IX below show that low expression (+) of AR RNA is found in normal adult lung tissue and pancreatic tissue. A lower, just detectable expression (+/-) can be detected in normal kidney, liver and brain tissue.

Table IX

normal

Human tissue ARRNA

lung

liver

pancreas

kidney

spleen

brain

placenta

Intestine, fetal

Since AR is originally isolated from TPA-treated MCF-7 cells, the determination of the time course of TPA induction of AR RNA in these cells is of interest. MCF-7 cells are treated with TPA for 0,1,3,6,18,24 and 48h, the total RNA is isolated and 10 цd per lane are separated on a 1.2% formaldehyde agarose gel, transferred to nylon membranes and screened with an ARBPI probe that is complementary to the entire coding region of mature AR. An impressive increase in the 1.4 kg AR-RNA species can be observed as early as 1 h after TPA treatment, reaching maximum values between 18 and 24. Subsequent Northern blots using a ³² P-ARBPI probe demonstrate TPA stimulation of AR-RNA synthesis in other breast cancer cell lines, however, peak values never reach the high levels of TPA-induced MCF-7 cells.

A number of tumor cell lines are examined for their AR RNA content both before and 24 h after TPA induction. The results are summarized in Table X. With few exceptions, TPA-treated human breast cancer cell lines are the only sources of detectable levels of AR RNA. One such exception is Caki-1, a human clear cell renal carcinoma cell line that expresses high levels of AR RNA, which are comparable to the levels detected in the TPA-induced MCF-7 cells. All reviewed TPA-treated breast carcinoma cell lines show AR-specific hybridization.

AR apparently has a functional role in the adult lung, pancreas, kidney, liver, and brain, and may be involved in a variety of processes, such as wound healing, tissue repair, and maintenance of neuronal cells.

Table X

Human cell line origin AR RNA content

non-induced TPA-induced

MCF-7 (HTB22) breast adenocarcinoma + + + + +

HBL-IOO (DHB 124) chest, normal - +

BT-474 (HTB20) Breast ductal carcinoma - +

MDA-MB-157IHTB24) Breast medullary carcinoma + + +

MDA-MB-36KHTB27) Breast adenocarcinoma + + +

SK-BR-3 (HTB30) brain metastasis - +

BT-476 breast adenocarcinoma - +

JEG-3 (HTB36) breast carcinoma - +/-

Caki-1 (HTB46) choriocarcinoma +++ · + + +

SK-HEP-KHTB 52) Kidney Clear Cell Carcinoma

G-401 (CRL 1441) Skin Metastasis

HEPM (CRL 1486) Liver adenocarcinoma - +/-

A-431 (CRL 1555) Embyrinal palatal mesenchyme -

HBL-299 (CCL 137) epidermoid carcinoma

HUF foreskin fibroblast - +/-

Example 9: Genomic cloning and analysis of the amphiregulin gene

The following example describes the genomic cloning of the human amphiregulin gene, its structure and intron / exon organization. Functional and evolutionary links to the EGF superfamily of growth factors are also discussed.

9.1 Material and Methods

9.1.1 Southem blot hybridizations

Total genomic DNA is isolated from subconfluent cells in T150 tissue culture flasks (Maniats, 1982, In Molecular Cloning: A Laboratory Manual). 20mM of DNA are digested with the indicated restriction enzymes, electrophoresed on a 0.8% agarose gel, and hybridized on Hybond-N (Amersham) with 20x SCC bottom buffer (Southern, 1975, J. Mol. Bio., 98, 503-517). blotted. The DNA is bound under the action of short-wave UV (1200pJoules). The filters are hybridized at 65 ° C overnight in the hybridization buffer containing 2x10 ⁶ cpm of the ^3Z P-labeled AR-specific fragment per ml (6x SSC, 5x Denhardt's solution, 0.5% SDS and 20 μg / ml sheared denatured salmon sperm DNA). The probes are labeled to a specific activity of 5 to 25 x 10 ⁸ срт / цд "random prime." The filters are extensively washed at 65 ° C in 1x SSC and autoradiographed overnight at -70 ° C.

9.1.2 Construction of genomic library

The bacteriophage lambda L41,7 is selected as a cloning vector and the phosphatase-treated arms are prepared after digestion with BamHI, EcoRI and HindIII. High molecular weight DNA from MCF-7 cells is digested with HindIII, electrophoresed on 0.8% low gel temperature agarose (BioRad) and fractionated by size, the agarose fraction maximally enriched with the desired restriction fragment being at 65 ° C The DNA is then melted and the DNA extracted therefrom with phenol and NaOAc, followed by ethanol precipitation and resuspension to 25-10 (g / ml. Genbank constructions consist of a ligation (overnight at 14X) of 100ng Lambda L47.1 Arms and 40 ng extracted and size-fractionated DNA in a reaction volume of 5 μΙ in the presence of T4 DNA ligase (Biolabs) Recombinant phages are amplified in vitro with extracts from the E. coli strains BHB2688 N205 recA "(lambda imm ^434- clts Ь2 red3 Eam4 Sam 7) and BHB2690 N202 recA "(lambda imm ⁴³⁴ clts b2 red3 Dam 15 Sam7) (Grosveld, 1981, Gene, 13, 227-223) Genes are determined on E. coli LE392 Genomic clones are identified with the AR -spezifis probes were screened by in situ plaque hybridization (Benton, 1987, Science 196, 180-182) and the DNA isolated from the plaque-purified positive clones (Huynh, 1985, In DNA cloning techniques: a practical approach, D.G. , Ed. [Oxford: IRL Press], p.49-78). Cut out the Hind III inserts and subclone them in pEMBL18 for restriction analysis and propagation.

9.1.3 Primer Extension Test

Isolate RNA from MCF-7 cells as described in Section 8. Synthetic oligonucleotides which are complementary to nucleotides 40 to 60 (ARAP) and 76 to 97 (ARCP) in the AR cDNA sequence are amplified by T4 polynucleotide kinase to a specific activity of 2 to 5 χ 10 ⁸ cpm ³² g P-end-labeled. 1 million cpm of the labeled oligonucleotide are used as primers for 1-strand cDNA synthesis with 50 μg MCF-7 RNA as described in Section 8.1.4. The product is treated with RNase A, extracted with phenol and chloroform, ethanol precipitated, denatured in 80% formamide at 100 ^° C. for 5 min and analyzed by electrophoresis on a standard 8% polyacrylamide M-urea sequencing gel.

9.1.4 CAT test

MCF-7 cells are cultured as described in Section 6.2.1. For each test, 1-2 x 10 ⁶ cells are plated in a 100 mm dish in 10 ml of medium, and added 24h later with 20 cc of calcium phosphate-precipitated supercoiled plasmid DNA (Southern and Berg, 1982) rinsed 4h after transfection and shock treated with 25% glycerol for 90 sec, rinsed again and overlaid with 20 ml of fresh medium containing 0 or 100 ng / ml TPA, the cells are washed and harvested 40 hours after glycerol shock and lysed by sonication in 100 μL of 0.25 M Tris-HCl (pH 7.8). CAT activity is determined essentially as described by Gorman et al., (1982) The cell extract (3-50 μΙ) is assayed 2.5mCi ^u C-chloramphenicol (NEN) in a reaction volume of 150μΙ 0.5M Tris (pH 7.8) and 0.5mM AcetylCoA The reaction mixture is incubated for 2 h at 37 ° C, extracted with 1 ml of ethyl acetate and on a Silica gel TLC plate with CHCl ₃ : 1-butanol (95: 5) The TLC plates are dried and autoradiographed. The acetylated and the non-acetylated C-chloramphenicol is quantified by counting the cut bands in Optifluor The units for CAT enzyme activity refer to those μg chloramphenicol which are acetylated per hour and per μg protein in the cell extract.

9.1.5 In situ chromosomal hybridization

Plasmid pAR9 is used randomly with ³ H-TTP and for hybridization with normal human metaphase cells prepared from phytohemagglutinin-stimulated peripheral blood lymphocytes (500 to 800 band stages). This probe corresponds to an AR cDNA lacking much of the З'-untranslated region and inserted into the EcoRI site of pEMPL18.

Hybridization is performed with 2,4,20 and 40ng of the probe per ml of the hybridization mixture as described (Le Beau, 1985, PNAS, 82, 6692-6696). The autoradiograms are prepared using a Kodak NTP-2 nuclear track emulsion and the films are applied for 7 to 60 days. Chromosome bands are visualized by staining with "quinicrine mustard".

9.2 Chromosomal location of the AR gene

The chromosomal attachment of the human AR gene is determined by in situ hybridization of the ³ H-labeled AR cDNA with normal metaphase chromosomes. Silver grains are distributed on 50 metaphase smears, with 30% not randomized to bands 4q13-4q21. This result is confirmed by the polymerase chain reaction (PCR) with hamster / human somatic cell hybrid DNA containing only human chromosome 4. Oligonucleotide primers derived from AR exon 3 and the flanking intron generate a 220bp PCR fragment only in human DNA and in the chromosome 4-containing hybrid DN A, while the hamster DNA gives a negative result.

The chromosomal region 4q 13-4q 21 also contains the genes for major or melanoma growth-stimulating activity (Anisowicz, A. et al., 1987, Proc. Natl. Acad. Sei., USA, 84, 7188-7192; Richmond et al., 1988, J. EMBO, 2025-2033), for the c-kit receptor (Yarden et al., 1987, J. EMBO 6,3341-3351), platelet factor 4 (PF4) (Griffin et al., 1987, Cytogenetic Cell Genet., 45, 43-73), the interferon-gamma inducible factor IP-10 (Luster, AD et al., 1987, Proc. Natl. Acad. Sei, USA, 84, 1868-1871 ), for the vitamin D binding protein (group specific component) (NE Cook et al., 1986, Human Genetics 73,225-229, JL McCombs et al., 1986, Cytogenet Cell Genet, 42, 62-64), and for statherin , a calcium-regulating salivary protein (Sabatini LM et al., 1987, Am. J. Hum. Genet., 41, 1048-1060). The gene for EGF is located further away at 4q25. Gro, IP-10 and PF4 belong to a class of structurally related peptides that may form a growth factor family clustered on chromosome 4. c kit encodes a cell surface receptor structurally and functionally related to multiple growth factor receptors having tyrosine-specific kinase activity, such as. EGF receptor, the neu oncogene, the PDGF receptor and the insulin receptor. Genes for ligands and the associated receptors are generally located on different chromosomes, but some are also common in a particular chromosomal region (Groffin, 1983, Nucl. Acids Res. 11: 6331-6339; Pettenati, 1987, Proc. Natl. Acad., P. USA 84: 2970-2974). The ligand for c-kit has not yet been identified, so AR should be tested for such activity.

Specific translocations are found in a variety of malignant changes. The most commonly observed cytogenetic aberration in congenital acute lymphoblastic leukemia (ALL), t (4; 11) (q21; q23), relates to the region in which AR has been localized (S. Heim et al., 1987, Leukemia, 1 , 26-23). ALL are now classified by morphology, as defined by the French-American-British Cooperative Group (FAB), with B and T cell markers and by chromosome analysis. These classifications serve as the basis for diagnosis, prognosis and treatment of ALL. One third of all cases of ALL involve specific translocations and t (4; 11) corresponds to a prognostic grouping that is commonly found in children less than 16 months of age with a median life of less than 1 year (Kocova et al., 1985, Cancer Genetics and Cytogenetics 16, 21-32).

Translocations involving region 4q 21 are also found in T lymphomas (EG Levine et al., 1986, Cancer Res. 466, 581-6488) and in a case of acute myeloblastic leukemia (AML) (A. Selypes, 1987; Human Genet., 76, 106-108). This region also contains the genes for a dental dysgenesis syndrome and the "piebald trait" of an inherited disease that results in spotty skin pigmentation due to lack of melanoblast migration and differentiation (JJHoo et al., 1986, Human Genet. 231), binding studies between AR and those located on chromosome 4q13-4q21

Disturbance allows the determination of the signifier of this co-localization. Current cytogenetic analyzes indicate that the 4q21 region comprises genes involved in lymphocytic differentiation. These relationships may be indicative of other biological activities or applications for the growth-regulating molecule of the present invention.

9.3 Genomic cloning

Southern blot analyzes (section 9.1.) Of HindIII-EcoRI or BamHI-digested MCF-7 DNA, when hybridized with an 830 bp probe ( ³² P-labeled Pvull / Bsml-digested pAR1), yield the 5 'region of the AR gene contains single bands of 12 kb, 8 kb and 20 kb, indicating that the AR gene is in a single copy. A 170 bp probe (AR 170, Baml-Pvull) from the З 'end of the mature AR containing the transmembrane domain and cytoplasmic domain coding regions hybridizes to the same HindIII, EcoRI and BamHI fragments and also to another 7.5kb HindIII fragment, indicating that most of the AR coding region is shared between two Hindk fragments of 12 and 6.5 kb. Identical banding can be seen in Southern blot analyzes with digested DNA from human placenta, brain, melanoma (SK-MEL 28), breast cancer (НТВ36), epidermoid carcinoma (A431) and lung cancer. This is considered an indication that the AR gene has not undergone extensive rearrangements or amplifications. The two HindIII fragments of the MCF-7 DNA are cloned because they may contain most of the AR gene or the entire AR gene and the adjacent sequences. The HindIII digested MCF-7 DNA is fractionated by size and the appropriate fractions are ligated with XL47.1. From a variety of positive clones, the two clones XARH12 and XARH6 are selected for more detailed investigations. The 12 and 6.4 kb inserts are subcloned into pEMBL18 and mapped to various restriction sites. Using exact oligonucleotide primers and direct sequencing of several smaller subclones, the sequences of all exons and their intron compounds are determined, with no discrepancies to the cDNA sequence.

9.4 Characterization of the AR-5'-regulatory region

Genomic cloning of AR results in isolation of 6.5kb of the 5 'flanking sequence contained in the 12kb HindIII fragment. The 688 bp fragment of the first EcoRI site in the 5 'direction from exon 1 to the SmaI site in exon 1 (position 40 in the cDNA) is subcloned and sequenced on both strands. These results are summarized in FIG. 17.

Primer extension is performed on MCF-7 RNA with two separate oligonucleotides from exon 1. One major transcript start point (confirmed by both oligonucleotides) is located 1 bp 5 'to the longest cDNA clone. Two subordinate points are found in the +1 and +2 positions in the cDNA sequence, confirming that many of the cDNA clones are mostly full-length. For functional confirmation of the regulatory region of the AR gene, a chimeric gene (pXAREI CAT) is constructed which covers the 688 bp Eco RI to SmaI AR 5 'flanking region (sequences 648 to +40, with +1 as the main starting point of the Transcription) and controls the expression of a promotor-free chloramphenicol transferase (CAT) gene. This construction has the ability to stimulate transcription of the CAT gene following insertion into MCF-7 cells, with a 6- to 7-fold stimulation of activity by the addition of TPA. This confirms both structurally and functionally the cloning of the 5 'end of the AR gene. In addition, a series of 5'-CAT deletion mutants are constructed containing the AR sequences -539 to +40 (EIa), -387 to +40 (EIb), -277 to +40 (Eic), -148 to +40 (EId), -77 to +40 (EIe), -79 to +40 (EIg), or +19 to + 40bp (EIH) in the same vector. For deletion below the range -77, the basal activity is lost, i. H. EIh shows no measurable activity, and all of these constructions show 3.5- to 7-fold enhanced expression as a result of 40 hours treatment with 100 ng / ml TPA. These constructions can be further used in experiments to determine the effects of morphogens, mitogens, growth factors, drugs or fermentation crude extracts on the regulation of AR expression and thus to identify new therapeutic agents.

"Nuclear run-off" experiments show 3 to 5-fold increased transcription of AR as a result of TPA treatment, and indicate that increased promoter activity is at least partially responsible for TPA induction of AR. Analysis with MCF-7 cells treated with TPA in the presence or absence of actinomycin D indicates relatively stable AR RNA, with a half-life of greater than 4 h, induction of AR expression as a result of TPA Treatment is therefore multi-faceted and involves increased transcription of a fairly stable RNA.

9.5 Intron / exon organization of the AR gene, AR protein domains, evolutionary and functional relationships

The entire genomic sequence of human amphiregulin is shown in FIG. The relationship between exons and the protein domains is shown schematically in FIG.

Primer extension analyzes of AR mRNA and studies with chimeric AR / CAT promoter constructions (CAT-chloramphenicol acetyl transferase, a marker gene) localize a functional promoter in the 64bp region 5 'to the end of the longest cDNA clone. Furthermore, a consensus TATA box 29bp is located upstream from the 5 'end of the cDNA sequence. Prior to the З 'end of the gene, a consensus polyadenylation signal sequence is located 64 nucleotides away from the poly (A) -TaU in the cDNA. The primary transcript of the AR gene has a size of about 10.2kb.

Six exons encode the human AR precursor and comprise 10.2kb of genomic DNA. The AR exons are 112 to 270 bp long and are separated by introns between 1.25 kb and 2.1 kb in size. The five introns of AR disrupt the coding sequence such that the protein domains are products of the individual exons. Exon 1 encodes the 5 'untranslated domain and the signal peptide domain, exon 2 encodes the N-terminal precursor, exon 5 contains the cytoplasmic region, and exon 6 represents the 3' untranslated region. Exon 3 and exon 4 together encode the mature AR protein and include both the hydrophilic and EGF-like sequences as well as the likely transmembrane domain. The connection between exons 3 and 4 can be found between the second and third loop of the EGF-like region.

If one superimposes the exon junctions of the amino acid sequences of AR, EGF and TGF-a, it is clear that all these proteins are encoded by two exons and that the intervening intron is located in the same position. In addition, each of the З 'exons encodes the transmembrane domain of the corresponding precursor protein. In contrast, the EGF-like region is located on a single exon in all the other mammalian EGF homologs for which the exon structure was determined: including the new EGF-like repeats in the EGF precursor (Gray, 1983, Nature, 303, 722-725): the three repeats in the LDL receptor (Yamamoto, 1984, CeI., 39, 27-38): and one repeat of each in rat fibronection (Patel, 1987, Embo J., 6, 2565-2572). and in human coagulation factor XII (Cool, 1987, J. Biol. Chem. 262, 13662-13673) invertebrate homologs such as the Drosophilia Notch gene and lin-12 of C. Elegans also contain multiple EGF-like repeats ( Kidd, 1986, Molec Cell, Biol., 6, 3094-3108; Greenwald, 1985, Cell, 43, 583-590). In contrast to the mammalian genes, most EGF-like repeats in Notch are contained in a single coding region, with only four of the 36 structures being separated by intervening sequences. Remarkably, two of these four structures show greater agreement with AR than with the notch repeat consensus, and a structure is even disrupted by an intron in the same CXC sequence as in EGF, TGF-a, and AR. The nematode lin-12 gene contains at least 11 EGF-like units, nine of which are present in the first exon and the remaining two units are found in other exons, flanking the intact EGF-like unit by introns.

Differences in the structure of the exons from different organisms coding for the EC-like repeats indicate their different origin. One group contains an EGF-like structure bounded by introns, and the other group to which AR belongs has an interrupted structure. The similarities in sequence between the two groups could be as a result of a converging evolution or intron insertion after their diverging from a common gene ancestor.

The intron arrangement within the same CXC sequence of EGF, TGF-a, and AR and one of the notch repeats indicates a common origin, but a closer examination reveals a different "intron phasing." "Intronphasing" indicates whether the intron interrupts the reading frame after the first (I), second (II) or third (0) nucleotide of a codon. It is believed that "phasing" is evolutionary in that it allows the shifting of exons containing functional domains within different proteins, EGF and TGF have a "Phase II" intron in the EGF-like unit while AR and Notch possess "phase g introns. A similar shift of one nucleotide is found in the penultimate intron within the protease domain of complement factor B and elastase (Cambell, 1983, PNAS, 80, 4464, Swift, 1984, J. Biol. Chem., 259, 14271). The non-random positions of these introns may indicate that a specific intron insertion sequence is present in these genes. On the other hand, a selection pressure may have existed for a division of the coding region at this point. A comparison of the sequences at the exon-intron binding site within the EGF-like unit for human EGF, TGF-a and AR and the first Notch repeat does not yield any direct repeats which are often observed in transposable elements. However, all four structures have the sequence "gtaagt" which limits the binding site, which sequence may have some function in splicing this intron.

Viral EGF homologs contain no introns; however, a comparison between VGF, EGF, TGF-α and AR results in homology in the transmembrane domain, while growth factors from myxoma and "shoot viruses" terminate before this hydrophobic region. Recent studies indicate that the TGF-α precursor is synthesized as a transmembrane protein, differential proteolytic cleavage leading to secretion of larger forms which may be cell type specific (Teixido, 1987, Nature, 326, 883-885).

Other EGF homologs that contain transmembrane domains include the coagulation factors, LDL, and the homeotic genes Notch and lin-12, although they are not adjacent to an EGF-like repeat. The presence of the GF structure in multiple membrane-bound precursors indicates that in some cases they are processed as secreted growth factors or remain in a membrane-associated state and have a function in intercellular and / or intracellular communication.

Some of the AR homologs include homeotic invertible genes. Homeotic gene products are involved in the regulation of cell development. For example, the Notch gene product mediates the correct progression of an ectodermal cell into a neuroblast or a dermoblast. In Notch mutants, all these cells become neuronal and the offspring, all brain and no skin, are destroyed. A homeotic gene may have an autocrine function to produce or stabilize a particular state in the cells that express the gene product, and may also be involved in the regulation of such conditions in neighboring cells via cell-cell interactions. It remains to be investigated whether AR has a regulatory influence on the developmental status of certain cell types.

9.6 Processing of mature amphiregulin

Characterization of the AR cDNA reveals that the 78- and 84-amino acid forms of AR are synthesized as the midportion of a 252 amino acid transmembrane precursor form (Section 8.2). The sites for the proteolytic cleavage of the AR precursor that would result in the release of the mature AR do not match any cleavage site of any known protease. At the N-terminal end of the interfaces are found Asp-Asp / Ser-Val and Glu-Gln / Val-Val, while the C-terminal sites have the following amino acids Glu-Lys / Ser-Met. The two forms of AR appear to be the result of further proteolytic processing of a common precursor, since all cDNA clones and genomic clones have the same sequence in this region. Interestingly, an intron lies between the two N-terminal junctions and it is possible that "intron-sliding" is responsible for these differences: MCF-7 cells produce 80% of the AR in the larger 84-amino acid form, while the choriocarcinoma cell line is НТВ 36 To determine whether these differences are associated with changes at the DNA level, the polymerase chain reaction technique (Scharf, 1986, Science, 233, 1076-1078 ) are used to isolate the AR exon 3 from MCF-7 cells and NTP-36 cells that produce AR mRNA in large quantities, an AR-intron-specific sense oligonucleotide and an antisense The oligonucleotide of the coding region of exon 3 was designed to specifically amplify the intermediate 220 bp fragment of the AR gene from both

DNA sources used. Direct sequence analysis did not reveal any discrepancies in the intron-exon binding sites or in the exon-3 coding sequence between these two human DNA sources. This is another indication that the two forms of AR are formed by differential proteolytic cleavage of the precursor.

9.7 Structural comparison of AR and EGF-like growth factors

AR shows a clear sequence homology with other EGF-like proteins, whereby 6 cysteines forming 3 disulfide bridges are conserved and determine the secondary structure of the mature growth factor. Previous computer-assisted comparisons of amino acid sequences result in categorization of EGF-like structures into two distinct groups: growth factors and coagulation factors (see Figure 13). Two sequence models were selected for comparison with AR and other known DNA or protein sequences. The selection is based on the fact that these sequences can be distinguished between the two protein groups and that very few gaps are required for an optimal match. The exact sequences are given in Table 1. The first region corresponds to the first 11 amino acids of the second loop in EGF and the second region corresponds to the third cysteine loop in EGF. Four members of each group of growth factors and coagulation factors are selected to produce consensus sequences based on their proven functional and structural homology. If possible, human sequences will be selected, as a comparison with human AR is sought. The growth factors include: human EGF, TGF-a, VGF and Shope growth factor; coagulation factors include: human factors IX, X, XII and protein C. AR was also compared to the database for both of these homology blocks. The consensus sequence was weighted based on the frequency of occurrence of a residue at a particular location.

Structural functional analyzes of various TGF-α VGF and EGF derivatives have recently led to the identification of several residues necessary for the biological activity of these growth factors. Recombinant proteins, synthetic peptides, site-specific chemical derivatives and proteolytic degradation reactions have been used to create altered molecules. Some generalizations include (the positions are given in relation to the arrangement in Figure 15): the 6 cysteine residues (positions 1, 19, 15, 26, 28, and 37) and their disulfide loops (1-15, 9-26, 28- 37) are required for biological activity; N-terminal extensions have little effect on activity; an aromatic radical (F, Y) is required at position 8; non-conservative replacement of Y ³² , D ^41, or L ⁴² results in loss of activity and / or marked decrease in receptor binding or autophosphorylation.

AR meets all the criteria that define those residues required for EGF receptor binding and / or for mitogenic activity except for the last two criteria. Matured AR terminates immediately before D ⁴¹ and has neither this nor the critical residue leucine 42 and yet interacts with EGF receptor binding and substitutes EGF in some mitogen assays. However, these differences may be responsible for the non-saturable receptor binding kinetics as well as their functional differences from EGF. These differences can be observed in the study of TGF-β synergism, the differential effect on selected A431 subclones, cross-linking, phosphorylation, and the ability to bind to DNA. The extremely hydrophilic region and the N-terminus of mature AR may contribute some of these differences in receptor binding or biological activity.

9.8 AR subcategory of the EGF superfamily

Based on the comparison in Figure 13 and on computer analyzes based on Table I (page 25), the evolutionary distance is calculated. This calculation is used to derive a dendrogram for the EGF superfamily. This search requires the presence of the cysteines, with one point added for each remainder to be found in the weighted consensus sequence. This evolutionary family tree predicts that AR can be classified into a new subset of EGF-like growth factors based on structural and functional homology. Such a model reveals that there are additional members of this growth family and that they can be identified on the basis of homology with the above sequence patterns based on three criteria: a combined score for the two growth factor regions greater than 20, a combined coagulation factor region score "less than 20, and a combined AR region score" greater than 20. All new molecules that meet these criteria are likely to have some functional homology with EGF, TGF-a, VGF, or AR molecules with a "combined AR Region score greater than 40 are classified as members of the AR family within the EGF superfamily and would thus be the subject of the present invention.

Example 10: Bacterial expression of amphiregulin

10.1 Materials and Methods

10.1.1 Plasmid constructions

Plasmid pARD1

Plasmid pARD1 contains the 1.4kb EcoRI cDNA fragment (pAR1) in pEMBLE 18 with a T / C exchange at nucleotide position 532 in the cDNA, which matured the valine valine sequence at the amino terminal end of the AR corresponds.

This base exchange is generated by site-directed mutagensis and confirmed by sequence analysis, which allows access to the junction between the AR amino-terminal prcessory domain and the hydrophilic region by creating a Ddel site (CTAAG).

Plasmid pARSTOP

The plasmid pARSTOP is an intermediate construction for the production of an AR secretion vector. pARD 1 is digested with Sspl and Xb = I and the 515 bp fragment is isolated which codes for the carboxy-terminal 9 amino acids of the mature AR, the transmembrane and cytoplasmic domain and for the З'-unsaturated region. The 4.7 kb (Sspl-XbaI) fragment: containing the remaining amino-terminal region of the AR cDNA was gel-purified and ligated with the kinased annealed complementary oligonucleotides ARSTOP1 and ARSTOP2 (Figure below). These oligonucleotides have a 5'-smooth end which is compatible with an Sspl site, followed by that sequence common to the carboxy-terminal 9 amino acids of the mature AR sequence, a TAA stop codon, and an EcoRV and XbaI site coded.

YFGERCGEK * EcoRV / XbaI AESTOPl 5 'ATTTCGGTGAACGGTGTGGGGAAAAGTAAGATATCT ARST0P2 3' TAAAGCCACTTGCCACACCCCTTTTCATTCTATAGAGTC

Plasmid pbAR

Plasmid pbAR contains a promoter-free TGF-β leader linked to the mature 78 amino acid form of AR. The plasmid is prepared by ligating the oligonucleotides bLARN 3 and bLARN 4 with the 240 bp Ddel-XbaI fragment from pARSTOP in EcoRI / XbaI-digested pEMPL 18. bLARN 3 and bLARN4 are complementary and produce a 5'EcoRI and a 3'Ddel overhang and a single Nael site compatible with the one interface near the carboxyterminal end of the TGF-β leader sequence. Ligation destroys the Ddel site and generates the correct amino acid sequence valine-valine at the amino terminus of mature AR.

SstII

AGVVKPPQNKTESE GGGAGTAGTTAAGCCGCCCCAAAACAAGACGGAAAGTGA

CGCCCn ¹ CATCAATTCGGCGGGGTT ¹ TTGTTCTGCCTTTCACT

NTSDKPKRKKKGG AAATACTTCAGATAAACCCAAAAGAAAGAAAAGGGAGG TTTATGAAGTCTATTTGGGTTTTCrTTCTTTTTCCCTCC

EcoRI

KNGKNRRNRKKKN CAAAAATGGAAAAATCGAAGAAACAGAAAGAAGAAG

rITR

Phill 5 ' PHIL2 3 ' Phill 5 ' PHIL2 3 ' Phill 5 'С PHIL2 3 'G

Plasmid pDCHBARI

Plasmid pDCHBARI is a mammalian expression vector (Figure 19) designed to secrete the processed, mature 78 amino acid form of AR. It contains the TGF-β leader / mature AR sequence from pbAR under the control of the CMV / HIV promoter flanked by an SV40 polyadenylation signal. The vector also contains Sv2dhfr in the same transcriptional orientation. PSVDR / bOM is digested with Nael and XbaI and the 6kb vector is separated from the excised OncoM coding sequence. pbAR is also digested with Nael and XbaI and the 260bp fragment responsible for the carboxy-terminal 5 amino acids of the TGF-β signal sequence and the mature amino acid sequence of 78 followed by a termination codon and an EcoRV and XbaI site coded, were isolated; the compounds are confirmed by sequence analysis.

Plasmid pDCHBPHILE

Plasmid pDCHBPHILE is a mammalian expression vector and is used to secrete a chimeric AR / EGF protein. The plasmid is also generated to facilitate future fusion constructions between the hydrophilic AR domain and other growth factors. This construction is generated by ligation of the Sstll / XbaI digested 6.5kb fragment of the pDCHBARI vector, the 175kp EcoRI / XbaI fragment containing the synthetic human EGF gene, and the oligonucleotides PHILI and PHIL2. These oligonucleotides are complementary to the 5'-SstII and 3'EcoRI extensions and encode the last residues of the TGF-β signal sequence and the entire hydrophilic AR domain. pDCHBARI contains the CMV / HIV promoter, all amino acids of the TGF-β signal sequence (except for the last amino acid) and the SV40 polyadenylation sequence. The EGF fragment was prepared by Dr. med. Timothy M. Rose (Oncogen) provided and contains suitable interfaces for further manipulation.

10.1.2 Preparation of the pTAC Vector

The plasmid TacPak / EGF is manufactured by Dr. med. Timothy M.Rose (Oncogen) and consists of the following units: trp-lac hybrid promoter and Cro-gene Shine-Delgarno sequence isolated from a BglII / BamHI fragment from the expression vector ρ 135-1, which in turn is derived from pDR540 (Pharmacia); the alkaline phosphatase signal sequence derived from synthetic oligonucleotides TacPaki and TacPak2 (Dr.Rose, Oncogen); the synthetic EGF sequence derived from plasmid pBM22 / PAK / EGF; derTranskriptionsterminations region; the pBR322 scaffold with the neomycin resistance gene derived from plasmid ρ 135-1. TacPak / EGF is digested with BamHI, filled with dATP and dGTP in "2-basic" to obtain an interface that is compatible with the "2-basic" filled Sall site and then digested with Pvul. This digestion removes the synthetic EGF gene and most of the alkaline phosphatase signal sequence leaving the Tac promoter, the Shine-Delgarno sequences and the initiation ATG intact. The 2.8kb fragment is gel purified.

10.1.3 Preparation of a modified amphiregulin cDNA fragment

Plasmid pARSTOP is digested with Sail, "2-basic" filled in with TTP and dCTP to create an interface compatible with the 2-basic-filled (dATP, dGTP) BamHI extension and then digested with Ddel and BgII subsequent digestion was required to separate concurrent DNA from the desired 242 bp fragment encoding the short form of the mature AR beginning with VKPP and ending at CGEK followed by a synthetically inserted stop codon The 243 bp fragment is gel cleaned.

10.1.4 Preparation of Synthetic Oligonucleotides ALKPAR1 and ALKPAR 2

The complementary synthetic oligonucleotides ALKPAR1 and ALKPAR 2 are prepared with a 5'Pvul overhang compatible with the partially filled TacPak / EGF Pvul / BamHI fragment and a 3'Ddel extension compatible with the partially filled pARSTOP Ddel / SalI Fragment. They are synthesized on an Applied Biosystems Oligonucleotide Synthesizer and purified on an acrylamide gel. Phosphates are added to the oligonucleotides with the T4 kinase and equimolar amounts are annealed with slow cooling after heat denaturation.

Pvul Ddel

I ALALLPLLFTPVTKAVV

ALKPARl 5 'ССОХТОЗСАСПСІТХЗДЛОСТ ^ 3 ¹

ALKPAR2 3 ¹ TAGa ^ GGAGCGTCAAGAGGGTCAO ^ ACA ^ 5 '

10.1.5 Ligation and isolation of pTacAPARI

The 243 bp partially filled Ddel-Sall AR fragment, the partially filled 2.8 kb Pvul / BamHI TacPak / EGF vector fragment, and the kinased and annealed ALKPAR1 + 2 oligonucleotides are ligated using DNA ligase. transformed into competent E. coli JM109 cells and selected on LB / neomycin plates. The correct construction is confirmed by restriction digestion and DNA sequencing. The sequence of pTacAPAR is shown in FIG.

10.1.6 Preparation of chimeric AR-hydrophilic domain / EGF gene fragment

The plasmid pDCHBPHILE is digested with Sstll and XbaI to generate the 286bp fragment that is responsible for the last two residues of the TGF signal sequence (AG), for the 37 residues of the hydrophilic domain of AR (VVKP ... RKKK), and for the 53rd Residues large synthetic sequence from the mature human sequence (NSDS ... WELR) encoded. The 286bp fragment is gel purified.

10.1.7 Preparation of Synthetic Oligonucleotides APAREGF1 and APAREGF 2

The complementary synthetic oligonucleotides APAREGF1 and APAREGF2 are generated with a 5 'Pvul overhang, compatible with the pTacAPAR 1 Pvul / Xbal fragment and a 3' Sst II extension, compatible with the pDCHBPHILE SstII / XbaI fragment. The oligonucleotides are synthesized on an Applied Biosystems Oligonucleotide Synthesizer and purified using an acrylamide gel. The phosphates are added to the oligonucleotides using T4 kinase and equimolar amounts are annealed with slow cooling after heat denaturation.

Pvul Sstll

IALALLPLLFTPVTK

APAREGFl 5 'CGCCCTCGCACTTCTCCCACTGCTGTTCACTCCAGTGACACC 3' APAREGF2 3 'TAGCGGGAGCGTGAAGAGGGTGACGACAAGTGAGGTCACTGTGGCG 5'

10.1.8 Ligation and isolation of pTACAPHILE

The 286bp Sstll / XbaI fragment encoding the hydrophilic domain of AR linked to the mature EGF sequence, the 2.8kbPvul / XbaI pTacAPAR 1 vector fragment, and the kinased annealed APAREGF1 + 2 oligonucleotides are synthesized using the DNA Ligase ligated, transformed into competent E.CoIi JM109 and selected on LB / neomycin plates. The correct construction is confirmed by restriction analyzes and DNA sequencing. The nucleotide sequence of pTacAPHILE is shown in FIG. The expected translation product should be cleaved between the dipeptide Ala-Gty following the alkaline phosphatase signal sequence, with an additional residue (glycine) added to the hydrophilic domain of AR. The nucleotide sequences that differ from the normal human sequence are highlighted (Figure 20).

10.1.9 Purification of Recombinant Amphiregulin

The plasmids pTacAPAR 1 and pTacAPHILE are transformed into E. coli complex JM 109 cells. These are grown to confluence in 10 ml of LB at 37 ° C until an ado of 0.7. The cultures are then induced with 100 μΜ EPTG and cultured for 24 to 72 h. The cultures are centrifuged twice at 5000 rpm for 15 minutes each. The pellet is set aside and purification is continued with the supernatant. Samples are concentrated in an Amicon ultrafiltration apparatus with YM5 membranes (excl. Molecular weight 5000). The concentrate is diluted with 5 volumes of MiIIiQ water and reconcentrated to ¹ Ao of the original culture volume. Glacial acetic acid is added to a concentration of 1 M and the samples are kept for 2 to 24 hours at 4 ° C. The samples are centrifuged at 19000 rpm at 4 ° C in Oakridge tubes in an SS34 rotor for 20 min, the pellet is extracted with 20 ml of 1M acetic acid, and the supernatants are pooled. The clear supernatants are then dialysed against 0.1 M acetic acid for 2 days, lyophilized and stored at -20 ⁰ C. The cell pellets and the crude dried supernatant are tested for AR protein by immunoblotting and growth inhibition assay (GIAs). The cell pellet of 50μΙ confluent cells or 100 to 200μΙ dried supernatant is analyzed on a 16% tricine polyacrylamide gel. Stain the gel with Fast Green, transfer to nitrocellulose, and perform Western blots as described in Section 7.1.6.

10.2 Results and discussion

The cell pellets of transformants containing pTacAPAR 1 contain large amounts of an immunoreactive protein which migrates at 10 kd, as well as some degradation products and possible dimers. The supernatants contain approximately 100 to 200 ng / ml of the secreted immunoreactive AR. By GIA with the supernatants an activity of approximately 150 ng / ml AR on the A431-A3 indicator cell line is determined in comparison with purified native AR. Large quantities of immunoreactive protein are produced in this system with the majority aggregated within the cell. Tests with periplasmic preparations and supernatants reveal secretion of active protein after 2 to 3 days.

pTacAPHILE provides a convenient tool for attaching the hydrophilic domain of AR to the N-terminus of any engineered gene. This region can alter the binding characteristics to the normal receptor of the ligand, can function as a nuclear localization sequence, can bind to DNA, or be transported through a lipid membrane or other diaphragms that are normally impermeable to the attached factor.

11. Deposit of microorganisms

The following microorganisms have been deposited with the Agricultural Research Culture Collection, Northern Regional Research Center (NRRL) under the following accession numbers:

Micro organism plasmid Deposit no. Escherichia coli HB101 PAR1 B-18438 EscherichiacoliHBIOI pARH12 B-18439 Escherichia coli HB101 pARH6 B-18440 Escherichia coli JM109 pTacAPARI B-18441 Escherichia coli JM109 pTacAPHILE B-18442

Claims (2)

1 Introduction The present invention relates to a method for the production of antibodies against an epitope of amphiregulin. Antibodies to the bifunctional growth regulator amphiregulin (AR) can be used for the qualitative and quantitative detection of mature AR and its precursor and its subcomponents, in the affinity purification of AR proteins and in the detection of AR biosynthesis, its metabolism and function. Antibodies to AR can also be used in diagnostic and therapeutic agents. Characteristic of the known state of the art A process for the preparation of antibodies which recognize an epitope of amphiregulin, which comprises immunizing a host animal with amphiregulin or a fragment or derivative thereof or inducing a cell clone capable of producing amphiregulin antibodies to produce the antibodies and recovering the antibodies , 2. The method according to claim 1, characterized in that the epitope comprises the following amino acid sequence: DTYSGKREPFSGDHSADGFE. 3. The method according to claim 1, characterized in that the epitope comprises the following amino acid sequence: SSSEPSSGADYDYSEEYDNE. 4. The method according to claim 1, characterized in that the epitope comprises the following amino acid sequence: VKPPQNKTESENTSDKPKRKKKG. 5. The method according to claim 1, characterized in that the epitope comprises the following amino acid sequence: EAVTCKCQQEYFGERCGEK. 6. The method according to claim 1, characterized in that the epitope comprises the following amino acid sequence: VQLRRQYVRKYEGEAEERKK. Field of application of the invention 2. State of the art Cellular growth and differentiation seems to be accelerated, sustained, and regulated by a variety of stimulatory, inhibitory, and synergistic factors and hormones. The change and / or collapse of the cellular homeostatic mechanism seems to be the fundamental cause of growth-related diseases, including neoplasia. Growth modulating factors are involved in a variety of pathological and physiological processes such as signal transduction, cell communication, growth and development, embryogenesis, immune response, hematopoesin, cell survival and differentiation, inflammation, tissue repair and remodeling, atherosclerosis and cancer. Because of their potential use in the diagnosis, prognosis and treatment of cancer, there is a great deal of interest in their isolation and characterization as well as in the elucidation of the functional mechanisms of growth-regulating factors. In addition, understanding these factors may lead to a better understanding of basic mechanisms of normal growth control and their loss in cancer cells. Factors such as epidermal growth factor (EGF), transforming growth factor-α (TGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), nerve growth factor (NGF), transforming growth factor-ß (TGFβ), Insulin growth factor I and II (IGF I, IGF II), hematopoietic growth factor, such as erythropoietin, colony stimulating factors (CSF 1 and 2), interleukins (IL-1 to 6), interferons (IFNa, ß, y), Tumor necrosis factor α and β (TNFα, β), leucoregulin, oncostatin M and other less defined factors are proteins that modulate growth and differentiation and are produced by different cell types either under normal physiological conditions or in response to exogenous stimulation. Most of these factors appear to be autocrine or paracrine. (Goustin et al., 1986, Cancer Res. 46: 1015-1029; Rozengurt,