FI89061C - Method for producing therapeutically active antibodies of biologically active polypeptides and polypeptides and oligopeptides used in the process - Google Patents

Description

1 89061

Method for Making Therapeutically Active Antibodies to Biologically Active Polypeptides and Polypeptides and Oligopeptides Used in the Method 5

By division separated from application 883765

The invention relates to a method of making antibodies to biologically active polypeptides or oligopeptides derived therefrom, to biologically active polypeptides used in the manufacture of these antibodies, which are produced from naturally occurring or synthetic sources, and to oligopeptides derived from these polypeptides. These polypeptides have a general polypeptide structure that defines proteins with cell growth promoting activity and are novel transformation factor (TGF) polypeptides that have the property of reversibly delivering a modified phenotype to normal cells in vitro and thus appear to be immediate of the malignant phenotype. effectors, and in particular antigenic oligopeptides derived from TGF polypeptides.

The antibodies of the present invention are useful in the treatment of malignancies, especially cancer and other proliferative diseases, as well as in the treatment of bone loss-related diseases such as osteoporosis and hypercalcemia.

A number of polypeptide hormone and hormone chalks ... growth factors have been found in tissue fluids and their association with the regulation of 30 normal cell growth or mitosis has been confirmed. These mitogenic polypeptide growth factors include insulin, insulin-like growth factors, platelet-derived growth factor, nerve growth factor, fibroblast growth factor, and epidermal growth factor (EGF). At least joil-35 of these known growth factors has an effect on the growth of modified cells, but from in vitro tests it appears that the modified cells need less of these known growth factors for optimal growth and proliferation than normal cells. In particular, it has been shown to experiment in cell culture that the addition of exogenous growth factors such as insulin and EGF can cause normal cells to mimic certain changes in cellular properties analogous to conversion. However, they are not able to produce all the changes associated with the modified phenotype, cf. e.g., Sporn et al., The New Eng. J. of Med. 15 (1981) 878-880.

New types of polypeptide growth factors, termed transformation growth factors, or TGFs, have recently been found in certain human and animal cancer and sarcoma cells with more properties that appear to be essential for phenotypic alterations [Roberts et al., Proc. Natl. Acad. Sci. USA 77 (1980) 3494-3498 and Todaro et al., Proc. Natl. Acad. Sci. USA 77 (1980) 5258-5262]. TGF polypeptides, as a class, are characterized by the changes they cause when applied to unmodified normal indicator cells that grow in culture. These changes include a) a lack of inhibition of cell growth-dependent growth in monolayer culture, b) cell overgrowth in monolayer culture, c) a change in cell shape resulting in the uptake of tumor phenotype by the indicator cells, and d) attachment sr dripping. resulting in the ability to grow in soft agar. The property of growth independent of cell attachment in culture is particularly strongly correlated with neoplastic growth in vivo. At least certain TGF polypeptides are somewhat similar to EGF in that they are both heat- and acid-resistant, reducing, and protease-sensitive peptides that appear to specifically interact with and produce biological effects through cell membrane EGF receptors. wherein TGF competes with EGF for binding to the cellular EGF receptor. However, TGF is distinguishable from EGF in several important relationships. In particular, EGF does not induce attachment-independent cell growth in culture, and antibodies raised against EGF do not detect TGF in radioimmunoassay or immunoprecipitation experiments. In addition, EGF has only a minor effect on the phenotype of cultured cells, whereas TGF produces a more pronounced change in phenotype in cultured cells and gives them the ability to behave like transformed cells. Most interestingly, TGF-induced conversion is not permanent but reversible in the absence of TGF, and there is no evidence that TGF itself acts as a complete carcinogen [Todaro et al., J. of Supramolecular Structure and Cell Biochem. 15 (1981) 287-301].

Thus, TGF polypeptides constitute a unique class of proteins that can be distinguished from other growth factors, such as EGF, in terms of both biological properties and chemical structure. These TGFs, in turn, have a number of different, valuable properties, such as potent but reversible cell growth or mitogenic properties. In addition, production of TGF polypeptide or: increased levels of production is characterized, if not essential, by morphological modification of certain cell lines in both human and mouse tissue and / or fluids; thus TGF polypeptides or fragments antigenic thereto ... are valuable in distinguishing normal cells from tumor cells and antibodies raised against them have utility in the diagnosis of malignancies. In addition, the realization that certain TGF polypeptides specifically interact with and produce their biological effects through cell membrane EGF receptors allows, once the basic structure of a TGF polypeptide has been determined, to align the structure with the structure of EGF to develop oligopeptides. having chemical properties that allow binding to EGF receptors without concomitant cell phenotypic modification.

For the preparation of antibodies in accordance with the present invention, a method has first been developed for producing TGF polypeptides in sufficient amounts and purity to allow determination of complete structure, and as a result of this assay and other findings, including finding substantial homology between human and mouse TGFs, found a basic peptide structure with extensive use in the area of cell mitosis and cell growth. In addition, homogeneous TGF polypeptides are obtained against which the antibodies raised have utility both in the area of cell growth and in the detection and treatment of cancer and other proliferative diseases. Antibodies raised against TGF polypeptides and oligopeptides can be used to detect and treat bone loss-related diseases such as osteoporosis, hypercalcemia, and bone resorption. In addition, antibodies are generated to antigenic oligopeptides and oligopeptides that have the ability to bind to cell growth factor receptors derived from the basic peptide construct and defined TGF polypeptide constructs.

Marquardt and Todaro, J. of Bio. Chem. 257 (9) (1982) 5220-5252 (published May 10, 1982) describe the isolation of low molecular weight human TGF from serum-free medium treated with a human metastatic melanoma tumor line using a series of method steps comprising extraction into 1 M acetic acid and sequential purification by reverse phase high pressure liquid chromatography eluting first with acetonitrile solvent and then with 1-propanol to give purified TGF having the characteristic biological activity of TGF, e.g., induction of attachment-independent cell growth. Twardzik et al., Science 216 (1982) 894-897 (published May 21, 1982) disclose the use of the same purification methodology to purify TGF from virus-modified rat cells. The biological activity of the purified substances in cell culture is also demonstrated. Pike et al., J. of Bio. Chem. 257 (24) (1982) 14,628-14,631 (published December 25, 1982) disclose that both partially purified rat and human TGF have the ability to activate protein kinase in human tumor cell membranes and thus stimulate phosphorylation of a synthetic tyrosine-containing peptide. . The only other molecules described to date with this activity are EGF, insulin, and platelet-derived growth factor, all of which are believed to have important physiological functions in humans and animals. Other interesting literature references are presented in the above-mentioned articles.

To prepare antibodies in accordance with the present invention, a basic protein construct has been developed that defines a new class of biologically active molecules. The discovery of this backbone polypeptide, which confers biological activity, particularly in the area of cell growth promotion, is based on the finding that there is a clear correlation between the three-dimensional structure of certain multi-disulfide bonded polypeptides, including TGFs, and the biological activity of the polypeptide. Thus, the present invention relates to a method for producing antibodies which are formed by ---. biologically active polypeptides of formula 6 89061 5 10

Val-Val-Ser-His-Phe-Asn-R-Cys-Pro-Asp-Ser-His-Thr- 15 20 25

Gln-R'-Cys-Phe-His-Gly-Thr-Cys-Arg-R "-Leu-Val-Gln- 5 30 35

Glu-R1-Lys-Pro-Ala-Cys-Val-Cys-His-Ser-Gly-R '"- 40 45 50

Val-Gly-R2-Arg-Cys-Glu-His-Ala-Asp-Leu-Leu-Ala 10 wherein R is Asp or Lys, R 'is Phe or Tyr, R "is Ser or Phe, R, M is Phe or Tyr, R 1 is Asp or Glu and R 2 is Ala or Val, or oligopeptide fragments comprising the amino acid sequence of these polypeptides 1-13, 34-43, 34-50 or 39-50, wherein method a) for the preparation of polyclonal antibodies is hyperimmunized a vertebrate, typically a domestic animal, with said biologically active polypeptide or oligopeptide fragment thereof or an immunogenic conjugate thereof, and the blood is collected shortly after repeated immunizations and the gamma globulin is isolated, or b) hyperimmunizing a small animal, typically a mouse, to produce monoclonal antibodies; biologically active polypeptide or oligopeptide fragment thereof or an immunogenic conjugate thereof, the spleen is removed and the lymphocytes are combined with myeloma cells by a suitable fusion promoter. in the presence of and the resulting hybrid cells are screened to isolate individual clones secreting a single antibody species to the antigen.

According to a preferred aspect of the invention, there is provided a method of making antibodies to polypeptides of the above formula wherein R is Asp, R 'is Phe, R' 'is Tyr, R1 is Asp and R2 is Ala.

Methods for making polyclonal antibodies are well known per se and are set forth in the Handbook of Experimental Immunology, 3.

7,89061 edition, Weir, editor, Blackwell Scientific Publications, Oxford and London, 1978. Similarly, methods for preparing monoclonal antibodies are well known to those skilled in the art and a general method for obtaining mono-5 clonal antibodies has been described by Kohler and Milstein, Nature. 256 (1975) 495-497. Each monoclonal antibody species obtained is a B cell product of a single immune animal that has developed in response to a specific antigenic site recognized in 10 immunogenic agents.

The polypeptides and antigenic oligopeptides used to produce antibodies in accordance with the invention may be used directly in the immunization process or may be advantageously bound to a suitable carrier protein using methods known in the art; see, for example, U.S. Patent 4,341,761 to Ganfield et al. The use of a carrier protein is particularly preferred when immunization is performed using the antigenic oligopeptides described above.

Antibodies raised against the above polypeptide and oligopeptide structures of the invention can be used to detect and treat malignancies. Antibodies are optionally labeled with a label, for example, a cytotoxic agent for use in the treatment of cancer or proliferative disease.

As described above, the antibodies prepared by the method of the invention are both monoclonal and polyclonal antibodies raised to TGF polypeptides of the above formula, antigenic oligopeptides derived from TGF polypeptides of the above formula, as well as homogeneous TGF polypeptides obtained using the isolation method described below from various modified cell lines and body fluids or modified cells of mammals with β 89061 Ia malignancies.

The present invention also relates to biologically active polypeptides of formula 5 for use in the method of making antibodies of the invention.

Val-Val-Ser-His-Phe-Asn-R-Cys-Pro-Asp-Ser-His-Thr- 15 20 25

Gln-R'-Cys-Phe-His-Gly-Thr-Cys-Arg-R "-Leu-Val-Gln- 10 30 35

Glu-R1-Lys-Pro-Ala-Cys-Val-Cys-His-Ser-Gly-R '"- 40 45 50

Val-Gly-R2-Arg-Cys-Glu-His-Ala-Asp-Leu-Leu-Ala 15 wherein R is Asp or Lys, R * is Phe or Tyr, R "is Ser or Phe, R '" is Phe or Tyr, R 1 is Asp or Glu and R 2 is Ala or Val, or oligopeptide fragments comprising the amino acid sequence 1-13, 34-43, 34-50 or 39-50 of these polypeptides Preferably R is Asp, R 'is Phe, R , M is Tyr, R1 is Asp and R2 is Ala.

According to the invention, antibodies are raised against homogeneous transformation factor polypeptides of the above formula isolated from pure aqueous media or body fluids containing these polypeptides obtained from tumor-producing tumor-bearing mammals. Biologically active polypeptides of the above formula can be isolated from an aqueous medium containing this growth factor polypeptide in impure form by a method comprising the steps of: (1) dialyzing an aqueous medium containing the growth factor peptide in impure form and an aqueous solvent containing an anti-solvent which phase is concentrated and optionally clarified, (2) the concentrated solvent phase of step 1) is reconstituted with aqueous acetic acid and the reconstituted solution is subjected to gel filtration chromatography by applying the reconstituted solution to an aqueous acidic elution column, eluting with an equilibrated aqueous column to obtain selected fractions containing a purer growth factor polypeptide, and the selected fractions the partially combined purified growth factor polypeptide product, 10 (3) subjecting the partially purified transforming factor polypeptide product of step (2) to sequential reverse phase high column acidic acid chromatography, transfer the product after reconstitution to aqueous silica gel, recrystallization, and aqueous hydrogenation. equilibrated with aqueous trifluoroacetic acid under high pressure liquid chromatography conditions, the initial elution of the column being carried out using a linear gradient of acetonitrile in aqueous trifluoroacetic acid and then the pyl-20 fat elution using gradient in aqueous acetic acid to give 1-propanol gr The α-25 orientation is raised in small enough increases in the concentration of 1-propanol to give the conversion growth factor polypeptide as a single clear peak as a homogeneous polypeptide.

The peptide sequence that characterizes the basic protein structure of the formula shown contains six cysteine residues located at critical positions in the polypeptide backbone. It is believed that the placement of the cysteine residues allows the polypeptide to fold in a special way due to the disulfide bridges formed between the paired cysteines and thus produces a three-dimensional structure that contributes to the biological activity of the resulting protein. There is some evidence to suggest a specific disulfide bridge sequence in which (by numbering Cys residues 1 to 6 from left to right in the above formula) Cys-1 is bound to Cys-3, 5 Cys-2 is bound to Cys-3 4 and Cys-5 is bound to Cys-6 in biologically active forms of the basic protein structure of disulfide bonds.

As described in more detail below, TGF polypeptides of the above formula are suitably obtained from a variety of modified human and mouse cell lines, certain fetal cell lines, and tumor-bearing mammalian body fluids using the isolation method described below or a conventional synthetic or recombinant method of polypeptide synthesis. Typically, TGF polypeptides of said formula and oligomers thereof have a molecular weight in the range of about 5,000 to 35,000. Preferred in this regard are TGF polypeptides having a molecular weight of about 5,000 to 8,000.

According to the invention, antibodies (both polyclonal and monoclonal) can be raised against the entire TGF polypeptide molecule, but it is possible and advantageous in terms of cost and technical effort to prepare antibodies also for haptens containing probable determinants of different TGF polypeptide sequences. regions and containing at least about 8 amino acids, typically at least about 10 and no more than about 20 amino acid oligopeptides.

In the method of the invention, the oligopeptide is bound to a suitable immunogen and introduced into a vertebrate to generate the desired antibodies. Thus, according to the present invention, there is also provided a series of antibodies to oligopeptides corresponding to antigenic regions in TGF polypeptides. Examples of antigenic oligopeptides useful in preparing the antibodies of this invention are listed below.

A) Val-Val-Ser-His-Phe-Asn-Lys-Cys-Pro-Asp-Ser-His-Thr 5 B) Cys-His-Ser-Gly-Tyr-Val-Gly-Val-Arg-Cys C ) Cys-His-Ser-Gly-Try-Val-Gly-Val-Arg-Cys-Glu-His-Ala-Asp-Leu-Leu-Ala- or D) Val-Gly-Val-Arg-Cys-Glu- His-Ala-Asp-Leu-Leu-Ala.

Polypeptides and oligopeptides of the above formulas can be prepared using synthetic methods, methods in which the peptide is isolated from a naturally occurring source, e.g., cell lines and body fluids, and methods using hybrid DNA-15 technology. Thus, for polypeptides and oligopeptides, conventional solid phase peptide synthesis is suitably used. This general synthetic method for preparing peptides, described, for example, in U.S. Patent 4,341,761 to Ganfield et al., Uses known side chain protecting groups and conventional polystyrene resin matrices, e.g., chloromethylated resins, hydroxymethyl resins, benzene resins, or benzhydryl resins. . For polypeptides, the method mentioned above and described in more detail below may also be suitably used to isolate homogeneous TGFs from natural sources to obtain pure forms of the desired peptide. Particularly suitable sources of TGF polypeptides in this regard are serum-free medium treated with retrovirus-modified Fischer rat fetal fibroblasts, especially fibroblasts modified with Snyder-Theile feline sarcoma virus, Moloney murine sarcoma virus-modified human mouse and 3T3 metastatic melanoma cell lines A2058 and A375. Sources and methods for suitable mouse cell lines have been described by 12,889,061

DeLarco et al., J. Biol. Chem. 255 (1980) 3685-3690 and Ozanne et al., J. Cell. Physiol. 105 (1980) 163-180. Sources and methods for human cell lines have been similarly described by Todaro et al., (1980) Proc.

5 Natl. Acad. Sci. USA 77, 5258-5262 and Giard et al., J. Natl. Cancer Inst., 51 (1973) 1417-1423. The isolation method described in more detail below can also be used to obtain TGF polypeptides from various body fluids, such as urine, serum, plasma, whole blood, or cerebrospinal fluid from human or mouse malignancies, or modified cells that produce TGF polypeptides. In this regard, a suitable source of TGF polypeptides is urine or other body fluid from mice transplanted with tumor cells (human melanoma or modified rat cells) known to produce TGF polypeptides. In all cases, the identification and purity of the TGF polypeptide can be monitored by a radioreceptor assay method based on cross-reactivity with EGF upon binding to the receptor (see experimental examples below). In methods using recombinant or hybrid DNA technology, oligopeptides or segments of polypeptides containing up to, for example, 20 amino acids can be used to derive a codon sequence for single-stranded 25 nucleotide (DNA) probes. These nucleotide probes can then be synthesized using known synthetic methods and used as a probe to obtain messenger RNA (mRNA) encoding growth factor-type polypeptides in both normal and modified cells or body fluids containing these peptides. Once the messenger RNA is obtained, conventional methods can be used to reverse transcribe the mRNA into the cDNA and then clone the cDNA into a suitable vector to effect expression of the desired polypeptide.

13 8 9 061

The method can be advantageously used to isolate homogeneous TGFs from serum-free media treated with modified TGF-producing cell lines. In this case, the treated medium is preferably clarified, for example by centrifugation and concentrated before dialysis, and the solvent phase from dialysis containing TGF is suitably clarified, for example by centrifugation, and also concentrated before gel filtration chromatography. In all cases, the dialysis is conveniently performed using an aqueous acetic acid solvent having an acetic acid content of 0.01 to 1 M, with 0.1 M acetic acid being preferred. Gel filtration chromatography can be performed using a variety of gels conventionally used to separate proteins or polypeptides based on molecular size. Suitable gels include dextran gels, agarose gels and polyacrylamide gels. In this regard, polyacrylamide gel filtration resins (Bio-GelR resins) such as Bio-Gel P-10, Bio-Gel P-30 and Bio-Gel P-60 are preferred, of which Bio-Gel P-10 is particularly preferred. The acetic acid content of the aqueous acetic acid 20 used to equilibrate the column and elute the TGF-containing fractions is suitably 0.2 to 2.0 M, with 1.0 M acetic acid being preferred. The TGF-containing fractions eluting from the column can be identified by determining their EGF-competitive activity and growth-promoting activity in soft agar (see experimental examples below). Fractions from gel filtration chromatography. Containing TGF polypeptides in improved purity are combined and concentrated, for example with a cold fiber dowel, as a preparatory step for further purification by reverse phase high pressure liquid chromatography (HPLC).

The final step of the purification process comprises successive HPLCs eluting with acetonitrile and 1-propanol in the presence of aqueous trifluoroacetic acid. These sequential HPLC runs can be performed using one or more 1, 89061 HPLC columns, but it is preferred to perform successive HPLC steps using a single HPLC column. The column packing used is suitably a porous silica matrix to which a long chain hydrocarbon is bound, for example a hydrocarbon containing from 5 to 16 carbon atoms. Preferred packings are pBondapak hydrocarbon columns, especially a pBondapak C10 column (particle size 10 μm, 0.39 x 30 cm, Waters Associates). The process is typically carried out under pressure, preferably at a pressure between about 3.45 x 10 105 and 3.45 x 107 Pa (50-5,000 psi). Prior to loading onto the column, the concentrated TGF-containing fractions are reconstituted in 1-10% trifluoroacetic acid and adjusted to pH 2-5, preferably 3-5, by the addition of trifluoroacetic acid. The column is suitably equilibrated with 0.01 to 0.1% aqueous trifluoroacetic acid, preferably 0.05% aqueous trifluoroacetic acid, before spraying the sample. The first e-elution is performed with acetonitrile in 0.01 to 0.1%, preferably 0.05% trifluoroacetic acid using a linear gradient of acetonitrile (the concentration of acetonitrile increases linearly with a gradient of about 0.1% / min to about 1 % / min). The elution is carried out for a period of 0.2 to 3 hours at a flow rate of about 0.2 to 2 ml / min and at a temperature of about 10 to 50 ° C, preferably at about 40 ° C. The combined fractions containing TGF activity as determined by competition with EGF and soft agar analysis are concentrated, for example, by lyophilization before the second HPLC step using a 1-propanol solvent. For the second step of successive HPLC, the combined and concentrated fractions from the first HPLC elution are reconstituted in 0.01 to 0.1% trifluoroacetic acid and rechromatographed on the same column or on a second column equilibrated with trifluoroacetic acid in the same manner as in the first column. for. This second elution is performed with 1-propanol, 0.01 to 8,961 in 0.1%, preferably 0.035%, trifluoroacetic acid using a linear gradient of 1-propanol. For optimal results, it is important to use a low linear 1-propanol gradient at this stage. In particular, the 5 1-propanol content should be increased linearly with a gradient not exceeding 0.1% / min, and preferably the linear 1-propanol gradient should be kept between 0.01% / min and 0.05% / min during elution. This second elution is suitably carried out over a period of one to five hours at a flow rate of about 0.5 to 5 ml / min and at a temperature in the range from 10 to 60 ° C, preferably at about 40 ° C. By adjusting the linear 1-propanol concentration gradient at the low levels given above, it is possible to elute TGF polypeptides as well-defined peaks of TGF activity as homogeneous polypeptides.

With this isolation method, it is possible to recover up to about 70% of the initial TGF activity in the impure starting material, achieving more than 200,000-fold purification rates. Homogeneous TGF polypeptides obtained by the isolation method typically have molecular weights in the range of about 5,000 to 35,000 and are sufficiently pure to allow peptide sequencing. Preferred homogeneous TGF polypeptides obtained by this method are TGFs having apparent molecular weights of 7,400, ... 20,000 and 30,000-35,000. These homogeneous TGF polypeptides are characterized by the biological properties of the TGF polypeptides. - ·· properties when used in culture on non-transformed, normal indicator cells, including the achievement of attachment independence, resulting in the ability to grow in soft agar. When these homogeneous TGF polypeptides are used to raise antibodies, it is not essential that each form of TGF be purified to homogeneity to produce antibodies to different TGF peptides.

35 16 89 061

Depending on the nature of the treatment and whether the foreign antibodies elicit an immune response, either xenogeneic or allogeneic antibodies may be used for therapeutic purposes. Many methods for making human antibodies have been described in the literature when it has been found that murine or other mammalian antibodies are not satisfactory. Antibodies can be used in many different ways. By using an appropriate IgG (other than IgGx), cell lysis can be achieved through the natural complement process. Alternatively, the toxin degrading moiety can be coupled to antibodies, particularly a Fab fragment. Antibodies can be bound to liposomes to direct liposomes to malignant cells, which are eaten by the cells by fusing the membranes. Other labels, such as radionuclides, can also be bound to antibodies. When antibodies are introduced into the body, they direct the label to a malignant cell where the malignancy can be treated.

The formulation of the antibodies varies widely depending on the nature of the marker and the site to which the antibodies are to be directed, etc. Usually, the antibodies are formulated in a physiologically acceptable carrier, e.g., physiological saline or phosphate buffered saline, and injected into the host at the desired site. is possible, and when this is not possible, to the circulatory system, such as blood.

Antibodies raised against TGF oligopeptides and polypeptides by the method of the invention are effective in detecting diseases associated with bone loss. For example, TGF activity is present in a material responsible for the bone resorption activity of tumors associated with hypercalcemia [Ibbatson et al., Science 221 (1983) 1292]. Increased bone resorption may be an endocrine effect of TGF molecules secreted by tumor cells.

17 89 061

Elevated TGF levels further promote bone resorption by dissolving calcium, which is associated with diseases such as osteoporosis. Osteoporosis is a disease that usually occurs in older people and causes bone-5 toa. TGF antibodies can be used to treat this important disease. The antibodies may be administered as compositions in association with a pharmaceutically acceptable carrier.

The following examples are provided to illustrate and not to limit the invention in any way:

Example I

Production, Purification and Characterization of Human Plenomolecular Weight Transformation Factor (hTGF) A. Experimental Methods Source of hTGFs hTGFs were purified from serum-free medium treated with human metastatic melanoma line alin2058 [Todaro et al., Proc. Acad. Sci. USA 77 (1980) 5258-5262], which was derived from brain metastasis in 20 43 year old men. Cells were grown to 90% confluence in round-bottomed flasks containing Dulbecco's modified Eagle's medium (Grand Island Biological Co., 430-2100) supplemented with 10% calf serum (Colorado Serum Co.) at 37 ° C. in. The cells were washed for 25 hours with 50 ml of serum-free Waymouth's medium (Grand Island Biological Co., MD 705/1). This and another supernatant collected 24 hours later were discarded. Subsequent supernatants were collected every other day or every three days for two weeks.

The medium was collected by decantation, stored for up to 24 h at 4 ° C in the presence of protease inhibitor phenylmethanesulfonyl fluoride (1 pg / ml) and clarified by continuous flow centrifugation at 32,000 rpm at 4 ° C. Flow rates of 5 1 / h were used in a CF-32 continuous flow rotor (Beckman) in an L5-50 ie 89061 ultracentrifuge (Beckman). The supernatant obtained after the high-speed centrifugation is called A2058-treated medium.

The A2058-treated medium was immediately concentrated in 5 hollow fiber dialysis / concentrators (Model DC10,

H1995-20 type cartridge Amicon Corp.) at 10 ° C. The concentrate was drained after a 150-fold decrease in volume. The cartridge was washed with 1000 ml of Waymouth's medium. The ultrafiltrate was rejected.

Purification of 10 hTGFs

Dialysis and centrifugation

The combined retentate fluid and cartridge wash after ultrafiltration of the A2058-treated medium were dialyzed against 0.1 M acetic acid in a Spec-15 trapor 3 dialysis tubing (Spectrum Medical Industries) for 60 h.

The retentate was centrifuged at 1,000,000 x g for 1 h at 4 ° C. The pellet was discarded. The supernatant was concentrated by freeze-drying and dissolved in 0.15 ml of 1 M acetic acid per liter of the original A2058-treated medium.

Chromatography on a Bio-Gel P-10 column After concentration, dialysis and centrifugation, the supernatant containing hTGF activity was further purified by gel filtration chromatography on a Bio-Gel P-10 column 25, 2.5 x 8.5 cm, column volume 420 ml, 200 - 400 mesh, Bio-Rad Laboratories). The column was equilibrated with 1 M acetic acid at 22 ° C. Protein samples (65-115 mg) were applied to the column in 1 M acetic acid (5 mL). To ensure a constant flow rate, the outflow of liquid from the 30 columns was set at 12 ml / h with a peristaltic pump. 4.8 ml fractions were collected. A small number of fractions were lyophilized for subsequent determinations of EGF-competitive activity and growth-promoting activity in soft agar. The fractions representing the major portions of a given peak were pooled and concentrated by freeze-drying.

Reverse Phase High Pressure Liquid Chromatography The final purification of hTGF was performed by reverse phase 5 HPLC using the general method described by Marquardt et al., J. of Biol. Chem., 256 (1981) 6859-6865. All separations were performed on a pBondapak C18 column (particle size 10 μm, 0.39 x 30 cm, Waters Associates) at a flow rate of 1 ml / min at 40 ° C. The lyophilized samples were reconstituted with 0.05% (v / v) trifluoroacetic acid in water, adjusted to pH 2 with 10% (v / v) trifluoroacetic acid, and applied to a column equilibrated with 0.05%: trifluoroacetic acid. Column 15 was then eluted with a linear gradient of acetonitrile in 0.045% trifluoroacetic acid. The effluent from the column was collected in 1.5 ml fractions. Small amounts of fractions were lyophilized with EGF for competition and growth stimulation assays. The combined fractions, which contained most of the hTGF activity, were concentrated by lyophilization.

The combined batches containing hTGF were dissolved in 0.05% trifluoroacetic acid and rechromatographed on the same column previously equilibrated with 0.05% trifluoroacetic acid in water. The column was then eluted with a linear 1-propanol gradient in 0.035% trifluoroacetic acid. The liquid flowing out of the column was collected in 1.5 ml fractions. Small aliquots of fractions were lyophilized with EGF for competition and 30 growth stimulation assays.

SDS-polyacrylamide gel electrophoresis SDS-polyacrylamide gel electrophoresis was performed as described by Laemmli. Nature (Lond) 227 (1980) 680-685. A 15-30% acrylic amine gradient plate (140 x 120 x 0.75 mm) was prepared with a 4% 20 85 061 concentration gel. . Gels were run at a running voltage of 30 V using an electrode buffer containing Tris (0.05 M), glycine (0.38 M) and SDS (0.1% w / v) until the tracer (bromophenol blue) had passed away from the gel. to be released.

After electrophoresis, the gels were fixed in 50% methanol containing 10% acetic acid for 2 hours, washed in 5% methanol containing 7% acetic acid overnight, and stained with silver [Oakley et al., Anals. Biochem. (1980) 361-363].

10 Protein Regulation

Total protein was determined using bovine serum albumin as a standard. Prior to protein assay, the starting material was dialyzed against phosphate-buffered saline to remove components of the culture medium that interfered with color reactions, or lyophilized if the samples were dissolved in volatile acids. Protein was also determined by amino acid analysis. The lyophilized samples were hydrolyzed at 110 ° C for 24 h in emptied Pyrex tubes using 0.1 mL of 6 M HCl containing 0.1% liquid phenol and analyzed on a Durrum D-500 analyzer equipped with PDP 8 / A - with a computer integrator, using o-phthalaldehyde for fluorogenic detection of primary amines [Bensen et al., Proc. Natl. Acad. Sci. USA 72 (1975) 619-622].

25 Radioreceptor regulation

Purified EGF was labeled with Na125I by modification of the chloramine-T method as described by DeLarco et al., Proc. Natl. Acad. Sci. USA 75 (1978) 4001-4005.125I-EGF binding assay was performed using nearly congruent monolayers of formalin-fixed A431 human cancer cells, as previously described [DeLarco et al., J. of Biol. Chem. 255 (1980) 3685-3690]. The fixed cells were washed twice with 0.5 ml of binding buffer (Dulbecco's modified Eagle's medium containing 35 1 mg / ml bovine serum albumin and 50 mM 2- [bis (2-hydroxy-89061 roxyethyl) amino] ethanesulfonic acid). , pH 6.8). The reaction was initiated by the addition of 0.2 ml of binding buffer containing 0.4 ng of 125 I-EGF with or without a potential inhibitor. After 1 hour of incubation at 22 ° C, the specifically bound 125 I-EGF was determined. The TGF content was expressed by the degree of inhibition of its 125 I-EGF to the EGF receptor. One unit of activity competing with EGF is defined as the amount of protein that inhibits 125 I-EGF binding to its receptor by 50%.

10 Growth assay with soft agar

Colony growth assay in soft agar using normal rat renal fibroblasts, clone 49F, was performed as described by Todaro et al. have reported in Proc. Natl. Acad. Sci. USA 77 (1980) 5258-5262. The lyophilized samples to be tested were reconstituted in 0.5 ml of Dulbecco's modified Eagle's medium supplemented with 10% calf serum. 1.5 ml of 0.5% (w / v) agar (Difco) in supplemented medium and 0.5 ml of supplemented medium containing 2.3 x 10 4 cells were added. 2.3 ml of the resulting mixture was pipetted onto a 2 ml base layer (0.5% agar in supplemented medium) in 60 mm petri dishes (Falcon). The cells were incubated at 37 ° C in a humidified atmosphere of 5% CO 2 and 95% air. The assay was read fixed and stained -... 25 days on day 5 and days 10-14.

B. Results Source, Concentration, and Initial Fractionation of hTGFs hTGFs were isolated from serum-free treated medium of a highly modified meta-static melanoma cell line, A2058. Quantitation of hTGFs was based on two of its properties: the ability to induce the attachment-independent growth of normal rat renal fibroblasts in soft agar and the ability to compete with 125 I-EGF for EGF receptor sites in human cancer cells 35 A431. A summary of the steps leading to the isolation and recovery of hTGFs is presented in Table I.

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ti 4Ϊ 23 8 9061

To remove serum proteins, A2058 cells were washed extensively with Waymouth's medium before culturing in serum-free medium. Superna tantalum fluids were collected every other day for two 5 weeks. The culture conditions were such that at the end of the culture period, more than 90% of the cells were still viable and adhered in unicellular layers. A first clarified volume of 136 L of A-2058-treated medium containing 1.02 g of total protein and 4525 units of 10 EGF-competitive activity was concentrated to about 900 ml using a hollow fiber concentrator with a 5,000 molecular weight cartridge. Competing activity with EGF was retained and the yield was over 95%.

15 Dialysis of the concentrated A2058-treated medium against acetic acid and subsequent centrifugation yielded 95% of the initial total activity competing with EGF. 18% of the protein was acid insoluble and was discarded. Acid-soluble, partially purified hTGF was subjected to gel filtration chromatography using a Bio-Gel P-10 gel. The column was eluted with 1 M acetic acid. The main mass of contaminating protein was eluted with a single column volume and differed well from EGF-competing activity and growth-stimulating activity. The two activity peaks were found to be well separated from each other. Fractions with both EGF-competitive and growth-stimulating activity (P-10-A and P-10-B) had apparent molecular weights of 10,500 and 680, respectively. Fractions with only 30 of these two activities were not observed.

Fractions containing hTGF were pooled as described, lyophilized and further purified. Higher molecular weight TGF eluted from the column as a broad peak (P-10-A) and appeared to be associated with polypeptides of different sizes.

.. 35 P-10-A contained 46% of the initial activity competing with EGF 24,89061. Low molecular weight TGF eluted from the column as a sharp peak (P-10-B) and represented 45% of the original total activity competing with EGF. The overall yield of column-competing EGF activity from step 1 through the gel filtration chromatography step was 91% (Table I). hTGF eluted as two distinct major peaks that varied quantitatively from preparation to preparation. In some preparations of A2058-treated medium, substantially all of the growth-promoting activity was in the region of hTGFs. Purification of hTGFs hTGFs were further purified by reverse phase HPLC. After gel-15 filtration chromatography of the acid-soluble EGF-competing activity of the concentrated A2058-treated medium using a Bio-Gel P-10 gel, the combined batch of P-10-B was reconstituted with 0.05% trifluoroacetic acid in water. and then chromatographed on a pBondapak Cie column. The EGF-competing and growth-stimulating activity of the individual fractions in the soft agar was determined. hTGFs were well separated from the bulk of the contaminating protein, which eluted at higher concentrations of organic solvent. Fractions containing hTGFs were pooled, lyophilized and rechromatographed. 57-25-fold purification of hTGFs was obtained after gel filtration chromatography. 80% of the initial EGF-competing activity in the P-10-B batch was recovered (Table I).

Rechromatography of fractions containing hTGFs on yBondapak C18 support was chosen as the final purification step because only relatively small losses of EGF-competing activity were observed on these columns. To achieve a clear separation of hTGFs from impurities, it was necessary to use a gentle linear gradient of 1-propanol in 0.035% trifluoro-35 acetic acid. The main mass of contaminating peptide material 25 89 061 differed from the well-defined peak of activity. Competing with EGF and growth stimulating activity were purified in parallel and had distinct absorption peaks in 13% 1-propanol. Fractions containing hTGFs were pooled and further analyzed. The purification of hTGFs was approximately 7,000-fold after gel filtration chromatography, with a yield of 33% of the initial total activity competing with EGF. The total yield of hTGFs from step 3 through the final reverse phase HPLC-10 step was 73% and the yield per step was 80-100% (Table I).

Characterization of hTGFs

The purity of the final hTGFr-containing preparation was determined by analytical SDS-polyacrylamide gel electrophoresis. The gel was stained with silver. One major polypeptide band with an apparent MP of 7,400 was observed. The same pattern was obtained when samples were run by electrophoresis under non-reducing conditions, indicating that TGF is a single chain molecule.

Receptor reactivity of 20 hTGFs was compared to EGF by a radioreceptor assay. Quantitative determination of hTGFs was based on amino acid analysis of a duplicate sample. Both hTGFs and EGF competed with 125 I-EGF for EGF receptor sites in human A431 cancer cells. The competitive activity of hTGFs with 25 specific EGFs was found to be 1 to 1.5 x 10 6 units / mg; 1.1 ng of hTGF or EGF was required to inhibit EGF binding by 50%.

hTGFs allowed the growth of normal attachment-dependent rat kidney cells, clone 49F, in its soft agar. The half-maximal response of hTRFs in soft agar was achieved with 1 unit competing with EGF or 1.1 ng of hTGF, whereas EGF did not stimulate the growth of these cells in soft agar even when tested at up to 10 pg.

26 8 9061

Example II

Large-scale production, purification and amino acid sequencing of human (hTGF), rat (rTGF) and mouse (mTGF) transformation factors. A. Experimental methods TGF source rTGF, mTGF and hTGF were purified from serum-free medium treated with Fisher rat. fibroblasts, FRE CLIO, a subclone of FRE 3A [Sacks et al., Virology 97 (1979) 231-240] modified to be non-productive by Snyder-Theilen feline sarcoma virus [Snyder et al., Nature (Lond ) 221 (1969) 1074-1075], in the Moloney murine sarcoma virus-modified 3T3 cell line 3B11-IC [Bassin et al., Int. J. Cancer 6 (1970) 95-107], and two human metastatic melanoma cell lines, A2058 (see Example I) and A375 [Girad et al., J. Natl. Cancer Inst. 51 (1973) 1417-1423], respectively. Cells were grown in 2 L plastic vials containing Dulbecco's modified Eagle's medium supplemented with 10% calf serum and then maintained in serum-free Weymouth's medium as described by DeLarco et al., Proc. Natl. Acad. Sci. USA 75 (1978) 4001-4005. Serum-free, treated medium was collected every 24 hours for 3 to 25 days, clarified by continuous flow centrifugation, and the supernatant was concentrated [Marquardt et al., J. of Biol. Chem. 255 (1980) 9177-9181]. The treated medium concentrate was the starting material for the purification of TGFs.

Purification of TGF TGFs were prepared essentially as described above for the purification of melanoma-derived hTGF in Example I. The retentate after ultrafiltration of the treated medium was dialyzed against 0.1 M acetic acid and the supernatant obtained after centrifugation was concentrated by freeze-drying and redissolved in 1 M acetic acid for subsequent gel filtration chromatography on a Bio-Gel P10 column (2.5 x 85 cm, 200 400 mesh, Bio-Rad

Laboratories). The column was equilibrated with 1 M acetic acid. Fractions containing the major portion of EGF-competitive activity and an apparent molecular weight of 7,000 were pooled and lyophilized.

The final purification of rTGF, mTGF and hTGF was performed by reverse phase HPLC using the chromatography system described in Example I. Separations were performed on a 10 pBondapak C18 column (particle size 10 μm, 0.39 x 30 cm, Waters Associates). The mobile phase was 0.05% trifluoroacetic acid and the mobile phase modifier was acetonitrile containing 0.045% trifluoroacetic acid. The concentration of acetonitrile was increased linearly over 15 hours (0.083% / min) at a flow rate of 1 ml / min at 40 ° C to elute the peptides. The combined TGF-batches were lyophilized and redissolved in 0.05% trifluoroacetic acid and rechromatographed on the same column using 1-propanol containing 0.035% trifluoroacetic acid as the mobile phase modifier. The concentration of 1-propanol was increased linearly (0.05% / min) over 2 hours at a flow rate of 1 ml / min at 40 ° C. The pooled batches of fractions, which contained most of the EGF-competitive activity, were lyophilized.

TGF Assay Method TGF was quantified by a radioreceptor assay based on the cross-reactivity of the receptor with murine submandibular epidermis (mEGF). Purified mEGF was labeled with Na125I by a modification of the chloramine-T method as described in Example I. 125 I-EGF binding assay was performed on formalin-fixed human A431 cancer cells, 8 x 103, on Micro Test II plates (Falcon). The concentration of TGF was measured as ng equivalents / ml of mEGF and was based on the amount of TGF 28,89061 required to provide the same inhibition of 125 I-EGF binding to A431 cells as a known amount of unlabeled mEGF.

Amino Acid Sequence Determination of TGF 5 For amino acid sequence analysis, rTGF (3 μg) was reduced with dithiothreitol (20 mM) in 100 μl of Tris-HCl buffer (0.4 M) containing guanidine HCl (6 M) and Na 2-EDTA (0 , 1%), pH 8.5, 2 h at 50 ° C, and then S-carboxyamidomethylated with iodoacetamide (45 mM) for 30 min at 22 ° C. S-carboxyamidomethylated rTGF was desalted on a pBondapak C18 column. The peptide was eluted with a gradient of aqueous acetonitrile containing 0.045% trifluoroacetic acid. The concentration of acetonitrile was increased linearly (1% / min) for 1 h at a flow rate of 1 ml / min at 40 ° C.

Automated sequence analyzes of S-carboxyamidomethylated rTGF and unmodified mTGF and hTGF [Edman et al., Eur. J. Biochem. 1 (1967) 80-91] was performed on a gas-liquid solid phase microsequencer 20 [Hewick et al., J. of Biol. Chem. 256 (1981) 7990-7997]. Sequencer fractions were analyzed by reverse phase HPLC [Hunkapiller et al., Science 219 (1983) 650-659].

B. Results Purification of TGF Purified preparations of low molecular weight rTGF, mTGF, and hTGF were obtained from treated medium of retrovirus-modified rat and mouse fibroblasts and two human melanoma cell lines, respectively. Purification was performed with acid-soluble EGF by gel filtration chromatography on Bio-Gel P-10 gel in 1 M acetic acid, followed by reverse phase HPLC on pBondapak C18 using a linear gradient of aqueous acetonitrile sequentially followed by 1-propanol. contained 0.035% trifluoroacetic acid, linear gradient. Elution patterns of rTGF, mTGF, and 29,89061 hTGF in the final purification step show that EGF-competing activity was cleared in parallel with a distinct absorbance peak and effectively separated from contaminating, UV-absorbing α-5. The major protein peak in rTGF, mTGF, and hTGF eluted from the μΒοη-dapak C18 column under standard conditions in 48-55 minutes.

Gel filtration chromatography on a Bio-Gel P-10 gel caused the separation of low molecular weight TGFs from 10 higher molecular weight TGFs and reduced the amount of protein added to the pBondapak C18 column in the next purification step. Low molecular weight TGFs represented 45-80% of the initial total activity competing with EGF. Reverse phase HPLC of TGFs on pBondapak 15 C18 in the next two purification steps was very effective, each giving a typical preparation yield of 80-100% per step. The final yield of low molecular weight TGFs was approximately 70% based on the maximum total activity observed with EGF 20 during purification. The mean yield of purified rTGF was 90 ng / L, mTGF 50 ng / L, and hTGF 10 ng / L treated medium. This calculation is based on the specific activity determined for isolated TGFs, assuming that the activity competing with EGF, as measured by the radioreceptor assay, reflects only the total concentrations of low and high molecular weight TGFs. No immunoreactive mEGF was detected in the treated medium.

Purity of TGFr The purity of 30 rTGF, mTGF and hTGF obtained by chromatographic elution profiles was determined by EGF radioreceptor assay and amino acid sequence analysis. rTGF, mTGF, and hTGF competed with 125 I-EGF for EGF receptor sites on human A431 cancer cells and were 30,89061 qualitatively and quantitatively nearly indistinguishable from mEGF. Thus, the final TGF preparations were believed to be highly pure and substantially homogeneous. A single amino-terminal sequence was determined for 5 rTGF, mTGF, and hTGF by automated Edman digestion. Any unprotected minor peptide sequence present in excess of 5% could have been detected by the methods used. The homogeneity of hTGF was further confirmed by analytical sodium dodecyl sulfate polyacrylic-10 amide gel electrophoresis. The purified preparation gave one large polypeptide band.

Amino Acid Sequencing of TGF Complete sequencing of rTGF, mTGF, and hTGF was performed, and the amino acid sequences of these three polypeptides are given below. From the sequences shown, it is found that rTGF and mTGF are chemically identical and, in addition, that substantial homology exists between murine TGFs and hTGFs and that different amino acid residues occur at only a few positions in the 20 (7, 15, 23, and 38) sequences.

3i 89061 (1) rTGF 5 10

Val-Val-i> er-His-Phe-Asn-Lys-Cys-Pro-Asp-Ser-His-15 20

Thr-Gln-Tyr-Cys-Phe-His-Gly-Thr-Cys-Arg-Phe-Leu- 25 30 35 5 Val-Gln-Glu-Glu-Lys-Pro — Ala-Cys-Val-Cys-His- Ser- 40 45

Gly-Tyr-Val-Gly-Val-Arg-Cys-Glu-His-Ala-Asp-Leu- 50

Leu-Ala (2) tnTGF 5 10 i Λ Val-Val-Ser-His-Phe-Asn-Lys-Cys-Pro-Asp-Ser-His- ° 15 20

Thr-Gln-Tyr-Cys-Phe-His-Gly-Thr-Cys-Arg-Phe-Leu-25 30 35

Val-Gln-Glu-Glu-Lys-Pro-Ala-Cys-Val-Cys-His-Ser-40 45

Gly-Tyr-Val-Gly-Val-Arg-Cys-Glu-His-Ala-Asp-Leu- 50 15 Leu-Ala (3) hTGF 5 10

Val-Val-Ser-His-Phe-Asn-Asp-Cys-Pro-Asp-Ser-His-15 20

Thr-Gln-Phe-Cys-Phe-His-Gly-Thr-Cys-Arg-Phe-Leu-25 30 35 is Val-Gln-Glu-Asp-Lys-Pro-Ala-Cys-Val-Cys-His- Ser- 40 45

Gly-Tvr-Val-Gly-Ala-Arg-Cys-Glu-His-Ala-Asp-Leu-: '· Leu-Ala

Example 111 Production of TGF in vivo, 1a isolation thereof

Tumor cell lines (1 x 10 6) known to produce (human melanoma and modified rat) TGF were transferred to thymus-deprived "hairless" mice and ... tumors were allowed to develop. Urine from tumor-bearing mice was collected and analyzed for the presence of TGF the isolation method and analytical methods given above in Example I. TGF having the same size and survival characteristics in HPLC as the cell culture TGF described above in Examples I and 32 89 061 II was observed in the urine of tumor-bearing mice. The methods described I showed that TGFs present in the urine of mice were characterized by the characteristic biological properties of TGF in that it stimulated cell-independent growth and binding to the EGF receptor, after which tumors were removed from tumor-bearing mice and the urine of mice was tested for tumor removal. after the presence of TGF using the above methods. In this case, no TGF having intrinsic elution properties by HPLC or TGF-like biological activity was detected. These results indicate that tumor cells produce TGF in whole animals as well as in cell cultures, and that TGF can be detected and isolated from body fluids using the methods described above. Similar experiments were also performed in rats with chemically carcinogen-induced tumors and found to have TGF in their urine based on the biological and biochemical properties listed above, whereas untreated rats did not.

Example IV

Inhibition of Retroviral-Modified Cell Growth by In Vitro Antibodies to Antigenic TGF-Oligopeptide 25 An oligopeptide having the following amino acid sequence (corresponding to amino acid sequences 34-50 of rat TGF):

Cys-His-Ser-Tyr-Gly-Val-Gly-Val-Arg-Cys-His-Ala

Asp-Leu-Leu-Ala 30 was synthesized using the method of Ohgak et al., Journal of Immunol. Meth. 57 (1983) 171-184, solid phase method. This oligopeptide was then coupled to volcanic hemocyanin according to the method of Baron et al., Cell 28 (1982) 395-404 and used to immunize rabbits 35 [Baron et al., Cell 28 (1982) 395-404] and sheep 33 89061 [Lerner, Nature 299 (1982) 592-596] for immunization. Antisera were analyzed against the peptide by peroxidase-coupled immunoassay (Kirkegaard and Perry Laboratories Gaithersberg, MD) and against homogeneous rat TGF (purified according to Example 11 above) by immunoprecipitation [Bister et al., J. Virol. 36 (1980) 617-621] and by Western blotting methods [Burnetti,

Analyt. Biochem. 112 (1981) 195-203]. Binding of 125 I-labeled rat TGF and mouse EGF (Bethesda Research Labs, Bethesda 10 MD) to A431 cells grown in 96-well microtiter plates was as described by Pruss et al., Proc. Natl. Acad. Sci. USA 74 (1977) 3918-3921.

Antisera prepared in one rabbit and two sheep were reacted with the corresponding peptide in peroxidase-coupled immunoassays at titers of at least 104. Reactions of rabbit antiserum with rat TGF were confirmed by immunoprecipitation and Western blotting. Antipeptide anti-20 sera did not immunoprecipitate iodinated mouse EGF in this study. Human A431 epidermal cancer cells have 106 additional EGF receptors per cell [Fabricant et al., Proc. Natl. Acad. Sci. USA 74 (1977) 565-569]. TGF competes with EGF for binding to these receptors. Binding of 125 I-labeled rat TGF to these cells was blocked with excess unlabeled EGF and antiserum against the peptide. The inhibitory effect of antiserum on TGF binding was also observed, although the antibody-TGF complex was not removed from the medium surrounding A431 cells by facilitated immunoprecipitation with '30 S. aureus protein A: 11. Inhibition by the antibody was due to an interaction with the TGF molecule and not with the receptor, as the antisera did not react with the purified iodinated EGF receptor in immunoprecipitation assays. As expected from the immunoprecipitation results, antiserum generated against the peptides did not interfere with the binding of murine EGF to A431 cells.

With the availability of antisera that inhibited TGF but not EGF cellular binding, it was possible to test whether TGF acts by an autocrine mechanism to stimulate the growth of malignant cells and thus the antibody can inhibit such growth in vitro. Thus, unmodified, normal rat kidney cells (NRK) and a number of different retroviral cell lines were plated at low densities (500-2,000 cells / plate) in serum-containing medium and changed to serum-free medium after a short adhesion time. Sheep or rabbit antibodies to TGF-peptide or various irrelevant cross-reactive peptides were added to the medium and the effect on cell growth, microscopic and macroscopic colony formation, and colony morphology was monitored. All antibodies were equally purified on peptide columns, thoroughly dialyzed, concentrated, and reconstituted to an anti-peptide titer of 103 to 104. The results are given in Table II below.

35 8 9 O 61 *

Table II

Effect of anti-peptide antibodies on cell growth in vitro

Colony formation

Antibody Source Volume Other inhibition of proliferation 5% (pg / l) Substance NRK-CL10- socio- Ki- src- luil- luil- NRK: 3T3: _______la la 11a 11a

Anti-TGF

oligopeptide Rabbit 5 - 0 85 NT 53 10 5 TGF peptide NT 36 NT 6

Anti-TGF

oligopeptide Sheep 2 0 50 49 NT

5 0 68 85 77 15 10 0 79 73 100 5 TGF peptide NT 9 20 43

5 relevant NT NT NT NT

maton peptides

Anti-hetero- Sheep & 2Q Peptides (4) rabbit 0 <15 <5 0 "As can be seen from Table II above, neither rabbit nor sheep anti-TGF oligopeptide inhibited colony formation on NRK cells, but prevented Kirsten-modified Growth of NRK cells, feline sarcoma virus-modified rat fetal fibroblasts (CLIO) cells, and Rous sarcoma virus-modified 3T3 cells, CLIO cells were known to be productive TGF producers [Marquardt et al., Proc. Natl. Acad. Sci. USA 80 30 (1983) 4684-4688] A few surviving colonies in anti-TGF antibody-treated cells appeared to be smaller and lacked the solid appearance of normal colonies.Intractor, undigested cells floated free in medium.This inhibition was partially reversed by TGF-peptide. , but not cross-reactive matter- 36 89061

peptide was added at the same time as the antibody raised against TGF. Rabbit and mouse antibodies to four different non-cross-reactive peptides had no effect on the growth of NRK colonies 5 or retrovirus-modified cell lines. Replacing the antibody-containing medium with fresh, antibody-free medium after 72 hours failed to undo the inhibition of colony formation, but the number of surviving colonies increased dramatically. 10 Example V

Synthesis and Characterization of Rat TGF Chemical synthesis of rat TGF (rTGF) having the chemical formula given in Example II was performed manually by a stepwise solid phase method according to Merrifield, J. Amer. Chem. Soc. 85 (1963) 2149-2156, in accordance with the general principles described. The differential acid-labile protecting group strategy was applied to this synthesis in conjunction with the conventional combination of N-amino-terminal tert-butyloxycarbonyl and side chain benzyl alcohol derivatives. A more acid-resistant benzyl ester bond that attached the protected amino acids to the polymer support was used to minimize peptide losses during repeated acid treatments [Mitchell et al., J. Amer. Chem. Soc. 92 (1976) 7357-7362]. Complete deprotection and removal of the peptide from the resin occurred according to Tam et al. Tetrahedron Lett. 23 (1982) 4435-4438, according to the Low-High HF method, which differed from the conventional HF deprotection method and removed the benzyl protecting groups by the SH2 mechanism in a dilute 30 HF solution to minimize severe side reactions from carbon cations that develop. in a conventional SN1 deprotection method. In addition, it is also designed to reduce the synthesis of many cysteine yl side reactions that often inhibit proteins containing many disulfide solids.

37 8 9 0 61 After HF treatment and before any purification, the crude and reduced synthetic rTGF were oxidized and regenerated by the mixed disulfide method in the presence of a combination of reduced and oxidized glutathione [Ahmed et al., J. Biol. Chem. 250 (1975) 8477-8482]. This avoided the formation of polymeric materials during purification. Regenerated, crude rTGF-Ι contained 40-50% EGF radioreceptor and tyrosine-specific protein kinase activities compared to native rTGF-I. The crude synthetic rTGF was purified to homogeneity in three steps: (1) by gel filtration on a Bio-Gel P-10 column; (2) ion exchange chromatography on a CM-Sephadex column; and (3) preparative high pressure liquid chromatography (HPLC) on a C-18 reverse phase column. The total yield, calculated from the starting amount of Ala to the resin, was 31%.

Synthetic rTGF purified under reducing or non-reducing conditions was found to give a single band with an apparent molecular weight of 7,000 by SDS-PAGE electrophoresis. Amino acid analysis with 6 mol / L HCl and enzymatic hydrolysis gave the expected theoretical molar ratio of the proposed sequence. No free thio was observed by the Ellman sulfhydryl assay for synthetic rTGF, but thiolytic reduction gave the expected theo-25 value of six cysteines. These findings support the conclusion that synthetic rTGF is a single chain polypeptide containing six cysteines in disulfide bonds, consistent with the expected chemical properties of natural rTGF. In addition, synthetic rTGF co-eluted with native rTGF as a single symmetric peak in C-18 reverse phase HPLC.

Example VI

In vivo studies with anti-TGF antibody

Anti-TGF serum was prepared by immunizing rat 35 with the Glu-taraldehyde conjugate of the 17 amino acid sequence 38 8 9 061 of rat rTGF described above in Example IV and KLH (volcanic conifer hemocyanin), and the Ig fraction was then prepared by doping the serum twice with 45% , with saturated ammonium sulfate solution, redissolving it back to the original volume of al-5 and dialyzing against PBS.

Anti-TGF serum and reference serum were diluted from an initial volume of 0.5 ml to 3 ml. The resulting solutions were injected intraperitoneally into mice at doses of 0.1 ml / mouse. Ten approximately 3-month-old hii-10 reens were each implanted subcutaneously with two small pieces of rat tumor derived from Snyder-Theile feline sarcoma virus. (Each implant was counted as one subject, resulting in a total of 20 subjects, of which 10 were in the control group and 10 in the treated group.) 15 Mice were injected the day after implantation and then on days 4, 7, and 11.

From day 7, tumor diameters were measured with a push gauge in two directions. Tumors were measured at regular intervals on days 9, 11, 14, 16, 18, 20, and 24.

At the first measurement, signs of tumor growth were observed in 7 of 10 sites in the control group, whereas growth was seen in only 4 of 10 sites in the treated group. In addition, in the control group, 6 out of 10 out of 10 subjects had a tumor diameter of at least 3 mm, whereas in only 3 subjects in the treated group, the tumor size 011 was so large. The control group had 4 subjects at least 5 mm in diameter on day 9, while only 2 subjects in the treated group were of this size. By day 11, tumors in the 30 control and treated groups were similar.

39 8 9 0 61

Example VII

Detection of TGF by monospeptic antiserum raised against synthetic peptide

Peptide Synthesis Peptides corresponding to amino acids from portions of the rTGF amino acid sequence described above in Example II were synthesized commercially (Peninsula Labs) by a conventional solid phase method [Ohgak et al., Journal of Immunol. Meth., 57 (1983) 171-184]. Peptides were purified, if necessary, by reverse phase high performance liquid chromatography (RP-HPLC) before use.

The sequences used were:

Peptide I - Val-Val-Ser-His-Phe-Asn-Lys-Cys-Pro-Asp-Ser-His-Thr Peptide II - Cys-His-Ser-Gly-Tyr-Val-Gly-Val-

Arg-Cys

Peptide III - Cys-His-Ser-Gly-Tyr-Val-Gly-Val-

Arg-Glu-Cys-His-Ala-Asp-Leu-Leu-Ala

Peptide IV - Val-Gly-Val-Arg-Cys-Glu-His-Ala-20 Asp-Leu-Leu-Ala

Conjugation and immunization of peptides

A sample of the peptide (10 mg) was mixed with volcanic conch hemocyanin (10 mg, Calbiochem) in 3.5 ml of 0.1 M sodium phosphate (pH 7.4). 4 ml of 25 mM glutaraldehyde was added and the mixture was incubated with shaking for 1 hour at 23 ° C. Glycine was then added to a final concentration of 0.1 mol / L and the mixture was shaken overnight at 23 ° C. The resulting conjugate was mixed with an equal volume of Freund's complete excipient before injection.

Rabbits were immunized by subcutaneous injection of 1 mg of conjugate at four different sites. After the first injection, two boosters were given two to two weeks apart. The serum used in this experiment was collected 80 days 40 8 9 061 after the first injection. An immunoglobulin fraction was prepared from this serum by ammonium sulfate precipitation. An antibody product specific for the immunizing peptide was monitored by solid phase ELISA.

5 Labeling of peptides with radioactive iodine

Peptides were labeled with Na125I using a chloramine-T procedure essentially as described by Dasin et al. [Proc. Natl. Acad. Sci. 74 (1977) 2790-2794]. Solutions containing 10 mouse submandibular EGF (mEGF, 170 pmol), synthetic rTGF (4 pmol) or peptide III (400 pmol) were mixed with 1-2 mCi Na125I (Amersham 2 mCi / nmol) 2 M in potassium phosphate (pH 7.5). 50-100 pg of chloramine-T was added and incubation was continued at 4 ° C for 15 min (mEGF and rTGF) or 7 min (peptide III). Reactions were stopped by the addition of 10-20 pg Na 2 S 2 O 5 and the labeled proteins were separated from unreacted Na125I chromatographically on Sephadex G-10 (Pharmacia). The specific activities of the peptides labeled in this way were: 1 x 20 1010 cpm / nmol (EGF), 6 x 108 cpm / nmol (rTGF) and 1 to 109 cpm / nmol (peptide III).

Radioceptor Assay The binding of 125 I-EGF to its receptor by monolayers of A431 cells attached to formalin was measured as described in Example I above. 125 I-EGF was added to a final concentration of 0.33 nmol / L in the presence or absence of competing agents. Addition of unlabeled EGF to a concentration of 0.3 to 0.5 nmol / L resulted in half of the maximal inhibition of 125 I-EGF binding. TGF levels were expressed as the amount required to produce 125 I-EGF binding inhibition corresponding to a known amount of TGF.

RIA (radioimmunoassay)

The reagents were mixed in a final volume of 50 microliters of a solution containing 20 mmol / l sodium phosphate 35 (pH 7.4), 200 mmol / l NaCl, 40 mmol / l dithiothreitol, 4i 8 9 061 0.1% (w / v) BSA, 0.1% (w / v) NaN3, 125 I-peptide III (104 cpm), an amount of antiserum corresponding to a final dilution of 1 / 15,000, and other agents. The reaction was initiated by the addition of antiserum and allowed to proceed at 23 ° C for 90 min. An equal volume of 10% formalin-fixed Staphylococcus A (Pansorbin, Calbiochem) was then added and incubation was continued for another 30 min at 23 ° C. The immunoadsorbent was removed by sedimentation through a 10% 10 (w / v) sucrose solution and the amount of bound 125 I-peptide III was determined using a gamma counter. Under these conditions, the antibody bound approximately 25% of the 125 I-: peptide in the absence of a competitor. The amount of bound peptide III was corrected for non-specific binding, which was measured in the absence of antibody (less than 5% of total binding) and expressed as a percentage of maximum binding. Competitor concentrations were expressed as the amount of peptide III required to achieve a corresponding inhibition of precipitation.

20 Chromatography

Gel filtration chromatography was performed according to the manufacturer's instructions on Bio Gel P-10 columns (BioRad) equilibrated with 1 N acetic acid. HPLC was performed on a Novapak C1B column (0.39 x 10 cm, Waters Associates) using a flow rate of 1 ml / min at 23 eC.

Manufacture of treated substrate

Serum-free modified medium of Snyder Theile-treated Fischer rat embryonic cells (ST-FrSV-RFE clone 10) was collected by Twardzik et al. as described [Virology, 30 124 (1983) 201-207]. The medium was clarified by low speed centrifugation and lyophilized. The residue was then suspended in 1 N acetic acid and dialyzed extensively against 0.1 N acetic acid. Insoluble protein was removed by centrifugation and the supernatant was lyophilized. Finally, the residue was resuspended to one hundredth volume of the original 1 N acetic acid and stored at 4 ° C.

42 8 9 061

Immunoblotting analysis

The samples to be analyzed were first subjected to sodium 5-dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using an acrylamide gradient of 15 -> 20% and the electrophoresis was performed under reducing conditions as described by Laemmli [Nature 227 (1970) 680-682]. After separation, the proteins were electrophoretically transferred to nitrocellulose (Schleicher and Schuell, BA85, 0.45 μ) as described by Burnette [Anal. Biochem. 112 (1981) 195-203]. The transfer was performed at 5 V / cm 3 -4 hours at 23 ° C. The resulting protein blot was incubated overnight in milk-based BLOTTO 15 (Johnson et al., Gene Anal. Techn. 1 pp. 3-8). The antiserum was diluted in BLOTTO and added to grind in the presence or absence of excess peptide III. Incubation with the antibody was continued frequently with stirring for 2-3 hours at 23 ° C. The blot was then washed, incubated with 125 I protein (4 x 10 6 cpm / ml, 4 x 10 7 cpm / pg) for 1 hour at 23 ° C and washed with BLOTTO. Antibody binding sites were visualized by autoradiography on Kodak XAR-5 film at -70 ° C using image intensifier plates.

In the radioimmunoassay described above, the added 125 I-: peptide III precipitated almost quantitatively with a low degree of antiserum dilution; a titer> 10,000 was obtained for the antiserum. Antibody affinity was measured by incubating the antiserum at a ratio of 1/5,000 diluted in the presence of varying concentrations of 125 I-peptide III. Analysis of these results by the Scatchard method yielded one group of binding component (s) with an affinity constant (Ka) for 125 I-peptide III of 8.3 x 10® M'1.

43 8 9 061 Specificity of the RIA To determine the specificity of the RIA, various peptides were tested for their ability to inhibit the precipitation of 12S1 peptide III. Unlabeled peptide III prevented the precipitation of 125 I-peptide III approximately approximately linearly in the range of 0.13 to 11 nmol / L; the concentration of unlabeled peptide III, which caused half of the maximum inhibition of precipitation, was 0.7 nmol / l. Peptide IV was only slightly less effective as an inhibitor of precipitation than peptide III, while peptide II was completely ineffective up to concentrations of 1.1 pmol / l. Peptide 1, which corresponds to 11 amino-terminal groups, was also ineffective as a competitor. These results indicate that the epitope detected under standard experimental conditions was localized to the 11 carboxy-terminal amino acids of peptide III. Table III summarizes the relative inhibitory concentrations of the various peptides.

Table III

20 Addition Relative inhibitory activity peptide III 1 peptide IV 3.5 peptide I> 1500 peptide II> 1500 25 rTGF 0 dithiothreitol 1 rTGF - dithiothreitol 20 mEGF> 1500

The reported added substances were tested for their ability to inhibit the standard RIA as described above. For each substance added, the concentration required to achieve half of the maximum inhibition of precipitation was determined and is reported relative to the concentration of peptide III required to achieve the corresponding inhibition. The notation ">" 35 indicates the highest concentration tested. (Peptides I-IV

44 8S061 inhibitory activity was not affected by the presence of dithiothreitol in the assay. )

The ability of rTGF to inhibit the precipitation of 125 I-labeled peptide III was also examined. Synthetic rTGF, which corresponded to 5 molecules with completely native biological activity, was as effective (as judged by molar concentration) as an inhibitor as peptide III. The relative inhibitory capacity of rTGF was significantly reduced when the reducing agent was omitted from the reaction mixture (Table). mEGF 10 was completely ineffective as a competitor up to concentrations above 1 pmol / l. This indicates that the R1A described herein differs from other available TGF assays in that it is specific for TGF but not EGF.

To directly demonstrate that antiserum si-15 produces rTGF, 125 I-peptide III was replaced with 125 I-labeled rTGF in a conventional RIA. Dose-dependent precipitation of 125 I-TGF was observed, which was substantially prevented by the addition of excess peptide III. Scatchard analysis of the binding results obtained in this way showed that the ratio of anti-20 serum K. 125 I-TGF was 2.3 x 108 M'1. This value is reasonably consistent with the Ka determined for 125 I-peptide III (8.3 x 10 8 M'1) and indicates that the antiserum binds to rTGF, the detection of rTGF from the treated medium of the transformed cells.

One source of TGF is the medium of cells transformed with RNA tumor viruses. The following procedure was used to detect rTGF in cultured modified cell culture medium. Modification of feline sarcoma virus strain Snyder-30 Theilen (ST-FeSV FRE clone 10)

The Fischer rat embryonic cell line has previously been shown to produce elevated levels of rTGF [Gray et al., Nature 303 (1983) 722-725]. The serum-free treated medium was collected, processed and concentrated as described above. A portion of the medium was then subjected to gel filtration chromatography 45,89061

Bio-Gel with P-10 under acidic conditions. The fraction was analyzed by rTGF EGF receptor competition assay or RIA.

Under these conditions, competitive activity with EGF was observed in two molecular size groups, one at about MP = 10,000 and the other at MP <= 20,000; both species of molecules eluted clearly after the bulk of the protein in the sample. Both size classes with EGF-competitive activity were also immunologically active. In addition, immunological activity was observed in the column column volume in which no activity competing with EGF was observed. These findings indicate that the rTGF size groups described above are active in R1A. The ratio of activity competing with EGF to immunological activity was strongly reduced in the high molecular weight TGF fractions, indicating that the biological activity of these TGF molecules is lower than that of the small molecule fractions.

To confirm that the immunological activity 20 was present in the same molecular species as the activity competing with EGF, RP-HPLC was performed on pooled fractions obtained by repeating this experiment on a preparative scale and containing the rTGF size groups MP = 10,000 and MP = * 20,000 ; the conditions used were similar to those used to purify rTGF by Marquardt et al. according to [Proc. Natl. Acad. Sci. 80 (1983) 4684-4688]. For the combined TGF fractions with an MP of 10,000, both EGF-competitive and immunological activity were purified in parallel in approximately quantitative yield. The parallel purification of both activities in both gel filtration chromatography and HPLC strongly suggested that both activities are present in the same molecular species. When HPLC was performed on a size group competing with EGF of 35 MP 20,000, a similar co-purification of EGFr competing with immunological activity and 46 89051 was observed. These experiments show that much of the immunological activity observed in the treated medium of St-FeSV-FRE clone 10 was caused by rTGF 5.

Immunoblotting Analysis of Different rTGF Size Groups The larger TGF size groups present in the treated medium of retrovirus-modified rat cells could represent either aggregated states or 10 distinct molecular forms of the TGF molecule. To distinguish between the two alternatives, synthetic rTGF and native TGF size classes MP 10,000 and MP = 20,000 were subjected to immunoblotting analysis after separation of the polypeptide components by SDS-PAGE under reducing conditions. Immune-15 reactive, synthetic rTGF, and small molecule non-rTGF each co-migrated as a single polypeptide chain with an MP of 6,000, and incubation of antiserum with excess peptide III blocked the immunological reactivity of these molecules. In contrast, high molecular size size 20 contained three immunoreactive peptides, MP = 24,000, MP - 40,000, and MP = 42,000; the addition of an excess of peptide III also blocked the immunological reactivity of these peptides. A radioactive zone with a rate of movement corresponding to a molecular weight of 43,500 was observed in all bands. The addition of excess peptide III incompletely removed this material and was observed equally in all experiments regardless of the sample to be analyzed; therefore, it appears to represent the artificial product formed in the experiment. It is noteworthy that no TGF with an MP of 6,000 was detected in the high molecular size class. These results indicate that the larger molecular rTGF size classes represent distinct forms of TGF.

47 89 061

Example VIII

A synthetic rTGF fragment that binds to receptors and is antigenic

peptides

5 EGF was isolated from mouse submandibular salivary glands

extraction with 1 M HCl containing 1% trifluoroacetic acid (TFA), 5% formic acid and 1% NaCl, concentration on Sep-Pak columns (Waters) and RP-HPLC on Bondapak C-18 columns (Waters) ) As described by Elson et al. 10 [Biochemistry Int., 8 (1984) 427-435]. The total synthesis of natural rat TGF is described above in Example V.

Synthetic peptide fragments were prepared with chloromethyl-polystyrene-nih resin (Bio-Rad) containing 1% divinylbenzene using Na-t-butoxycarbonyl protection. The peptides were deprotected and cleaved from the resin using HF or, in the case of C-terminal protected analogs, the peptide was first cleaved by ammonolysis (NH 3 / MeOH). The deprotected crude peptides were diluted and cyclized to the disulfide form by oxidizing with 0.01 M K 3 Fe (CN) 6. The peptide solution was applied to a Bio-Rex 70 cation exchange column, washed with H 2 O (300 mL) and eluted with an AcOH gradient (-> 50%). The peptide was purified by preparative HPLC on a column (2.5 x 25 100 cm, Altex) packed with Vydac 218TP C-18 using a 20% CH 3 CN solution containing 0.03 mol / L NH 4 OAc as eluent. (pH 4.5) (10). Peptides 1 and 2 represent amino acid sequences 34-43 of rTGF with unprotected or correspondingly protected ends (N-Ac, C-30 amide). Peptide 3 is the methyl ester of the corresponding hTGF loop (Ac-Cys-His-Ser-Gly-Tyr-Val-Gly-Ala-Arg-CysOMe) prepared by transesterification from a resin (base / MeOH).

Immunogen Preparation and Immunological Assay TGF-peptide 1 was coupled to volcanic conifer hemosya-.35in (KLH) or bovine serum albumin by a thiol / ma- 8 4861 906 leimide bond [King et al., J. Inununol. Methods 28 (1979) 201-206]. The carrier-peptide complexes were purified by gel filtration. An average of 60 mol peptide / 1 mol KLH and 20 ml peptide / 1 mol BSA were obtained. Rabbits were immunized with a conjugate of KLH and peptide 1 by multiple injections of 2 mg of protein in complete Freund's vehicle subcutaneously (s.c.) or intramuscularly (i.m.). Rabbits were boosted at two-week intervals by injecting s.c. immunogen in incomplete 10 Freund's adjuvant. The IgG fraction of the antiserum collected after five boosts was used for immunoassays and labeled with a radionuclide using Na125I (NEN) and Enzymobeads beads (Bio-Rad) to a count of 4 x 10 5 cpm / μg protein. 94-well polyvinyl chloride plates (Costar) were coated with a solution containing 200 pg / ml conjugate of BSA and peptide 1 and counter-coated with phosphate buffered saline containing 10% normal rabbit serum. The plates were incubated for 4 hours at 20 ° C with 37 ° C [125 I] anti (KLH - peptide 1) IgG (50,000 cpm / well) in the presence or absence of inhibitors, washed and the radioactivity of the individual wells counted.

Radio Receptor Assay

Human epidermal cancer cells A-431 or human pre-25 dermal fibroblasts (HFF) collected from primary cultures were grown to a uniform surface in 24-well group plates (Costar) in Dulbecco's minimal essential medium, DMEM, Gibco % fetal bovine serum (FCS, Hyclone). EGF 30 was labeled with radioiodine as described above at a pulse rate of 4 x 104 to 8 x 104 cpm / mg protein. Cells were incubated at 4 ° C for 60 min in DMEM (pH 7.4, 20 mmol / l Hepes) containing 1 mmol [125 I] -EGF and 0.1% BSA in the presence of inhibitor peptides. The cells were washed three times with cold buffer, lysed with 49 89061 0.1 M NaOH, and the radioactivity bound to the cells was measured (ganun counter) .Non-specific binding, determined in the presence of 100-fold excess cold EGF, was less than 5% and less than 10% of the specific binding to A431 cells and HFF cells, respectively.

Cell Proliferation Assay HFF cells were grown to a homogeneous layer in 48-well group plates in DMEM containing 10% FCS, and proliferation was stopped by keeping cells at 2 for 10 days in DMEM containing 0.5% FCS. Mitogens and peptides were incubated with cells at 37 ° C for 18 hours before 4 hours of treatment with 1 pCi [3H] -methylthymidine (NEN) and the radioactivity precipitated with trichloroacetic acid was determined.

Rabbit antibodies raised against the KLH-peptide 1 conjugate reacted with the BSA-TGF peptide according to solid phase RIA. This reaction was inhibited in a concentration-dependent manner by peptide 1 and slightly less by natural TGF. In contrast, EGF did not cause significant inhibition.

Rat TGF competitively displaces EGF binding to its receptors. The ability of TGF peptides to compete with [125 I] -EGF for binding to either A-431 cells or HFF cells was assessed. Peptide 1 partially inhibited the binding of [125I] -25 EGF to A-431 cells at a concentration of> 10-6 mol / L. The ability of peptides 2 and 3 to inhibit binding was better; The IC50 values were 4 x 10 * 6 mol / l and 4 x 10'7 mol / l, respectively (i.e. about 0.02 and 0.2% of the binding efficiency of EGF or TGF-α). Similar observations were made for the inhi-30 bit of EGF in HFF cells (Table IX). The receptor specificity of these interactions is illustrated by the inability of these peptides to inhibit endothelial cell growth factor binding or mitogenic effect on HFF cells (not shown).

None of the TGF peptides had intrinsic Mito-35 genetic properties up to a concentration of 10 55 mol / L when treated with incubated 89,061 HFF cells (data not shown). However, TGF-peptides inhibited the induction of DNA synthesis in non-proliferating HFF cells in a concentration-dependent manner by EGF. These 5 antagonists were equally effective when using TGF as a mitogen (Table IV). As well as the binding efficiency, the antagonistic potency of the TGF-peptides was improved by closing the amino and carboxy termini.

Table IV

10 Biological activity of synthetic TGF fragments ---— —— 'b

Inhibition of mitogenesis

Binding3 IC ^ g (M) * ^ 50 ^ _A-431_HFF_EGF_TGF <> C_ 15 Peptide 18 + 2 x 10-5 6 + 2 x 10 5> 10 5> 10 5

Peptides 2 4 + _ 2 x 10-6 2 ± 2 x 10 -6 5 + 2 x 10 6 3 t 1 χ 10 6

Peptide 3 4 ♦ 1 x 10 3 _ + _ 1 x 10 6 + 2 x 10 5 + _ 3 x 10 20 * IC50 values calculated from inhibition curves depicting [125 I] -EGF (1 nM) binding to cells. bIC50 values are concentrations that halve the ability of a control to promote uptake of [3H] -methylnimethide into non-proliferating HFF cells induced by EGF or TGF (1 nM).

Claims (14)

A process for the preparation of therapeutically active antibodies, which antibodies are formed against biologically active polypeptides of formula 5 Val-Val-Ser-His-Phe-Asn-R-Cys-Pro-Asp-Ser-His-Thr 15 20 25 Gln-R'-Cys-Phe-His-Gly-Thr-Cys-Arg-R "-Leu-Val-Gln-35 Glu-R ^ Lys-Pro-Ala-Cys-Val-Cys-His-Ser -Gly-R '"- 40 45 50 Val-Gly-R2-Arg-Cys-Glu-His-Ala-Asp-Leu-Leu-Ala wherein R is Asp or Lys, R' is Phe or Tyr, R" is Ser or Phe, R 'is Phe or Tyr, R R1 is Asp or Glu and R₂ is Ala or Vai, or against oligopeptide fragments comprising amino acid sequences 1-13, 34-43, 34-50 or 39-50 of these polypeptides, characterized in that a) for the production of polyclonal antibodies, a vertebrate, typically a pet, is hyper-immunized with said biologically active polypeptide or oligopeptide fragment thereof or with an immunogen conjugate thereof, and blood is collected shortly after repeated immunizations and gamma globulin is isolated In the preparation of monoclonal antibodies, a small animal, typically a mouse or a mouse, is hyperimmunized with said biologically active polypeptide or oligopeptide fragment thereof or with an immunogen conjugate of In these, the spleens are removed and the lymphocytes are joined to polydoma cells in the presence of a suitable fusion promoter and the emerging hybrid cells are harvested for isolation of single clones secreting a single antibody species against the antigen. 55 89061 2. A process according to claim 1, characterized in that R is Asp, R 'is Phe, R' is Tyr, R 1 is Asp and R 2 is Ala. Method according to claim 1, characterized in that the antibodies are formed against an oligopeptide fragment comprising the amino acid sequence A) Val-Val-Ser-His-Phe-Asn-Lys-Cys-Pro-Asp-Ser-His-Thr B) Cys-His-Ser-Gly-Tyr-Val-Gly-Val-Arg-Cys C) Cys-His-Ser-Gly-Tyr-Val-Gly-Val-Arg-Cys-Glu-His Ala-Asp-Leu-Leu-Ala or D) Val-Gly-Val-Arg-Cys-Glu-His-Ala-Asp-Leu-Leu-Ala. 4. A method according to claim 1, characterized in that the antibodies are formed against an oligopeptide fragment comprising the amino acid sequence. Cys-His-Ser-Gly-Tyr-Val-Gly-Ala-Arg-Cys. 5. A method according to any one of claims 1-4, characterized in that the antibody is polyclonal. 6. Biologically active, substantially purified polypeptide 20 of formula 5 Val-Val-Ser-His-Phe-Asn-R-Cys-Pro-Asp-Ser-His-Thr-20 ···. Gln-R '-Cys-Phe-His-Gly-Thr-Cys-Arg-R "-Leu-Val-Gln-35 ... * Glu-R1-Lys-Pro-Ala-Cys-Val-Cys -His-Ser-Gly-R '"- 40 45 50 Val-Gly-R2-Arg-Cys-Glu-His-Ala-Asp-Leu-Leu-Ala wherein R is Asp or Lys, R' is Phe or Tyr, R "is Ser or Phe, R" is Phe or Tyr, R 1 is Asp or Glu and R 2 is Ala or Vai, or a fragment of this polypeptide comprising the amino acid sequence 1-13, 34-43, 34 - 50 or 39 - 50. 56 89061 A polypeptide according to claim 6, characterized in that R is Asp, R 'is Phe, R' is Tyr, R 1 is Asp and R 2 is Ala. A polypeptide according to claim 6, characterized in that it is an oligopeptide fragment comprising the amino acid sequence A) Val-Val-Ser-His-Phe-Asn-Lys-Cys-Pro-Asp-Ser-His-Thr B) Cys -His-Ser-Gly-Tyr-Val-Gly-Val-Arg-Cys C) Cys-His-Ser-Gly-Tyr-Val-Gly-Val-Arg-Cys-Glu-His- Ala-Asp-Leu-Leu-Ala or D) Val-Gly-Val-Arg-Cys-Glu-His-Ala-Asp-Leu-Leu-Ala. A polypeptide according to claim 6, characterized in that it is an oligopeptide fragment comprising the amino acid sequence. Cys-His-Ser-Gly-Tyr-Val-Gly-Ala-Arg-Cys.