FI81497C - ANTIKROPPAR MOT BIOLOGISKT AKTIVA POLYPEPTIDER OCH DERAS ANVAENDNING VID DIAGNOSTISKA FOERFARANDEN. - Google Patents

Description

1 81497

Antibodies to biologically active polypeptides and their use in diagnostic methods

Divided by application 841 649 5

The invention is based on biologically active polypeptides and their production from naturally occurring or synthetic sources and oligopeptides derived from these polypeptides. In particular, this invention 10 is based on a general polypeptide structure defining proteins with cell growth promoting activity and novel transforming growth factor (TGF) polypeptides that have the property of reversibly delivering a modified phenotype to normal cells in vitro and thus appear to have immediate effects on the malignant phenotype. in particular to antigenic oligopeptides derived from TGF polypeptides.

The present invention relates to antibodies formed to the aforementioned antigenic oligopeptides and related TGF polypeptides useful in the detection of malignancies. Other specific objects of this invention are methods for detecting cancer and other proliferative diseases, as well as diseases associated with bone loss, such as osteoporosis and hypercalcemia.

A number of polypeptide hormone- and hormone-like growth factors have been found in tissue fluids and their association with the regulation of 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 some of these known growth factors have an effect on the growth of the transformed cells, but based on in vitro tests, it appears that the transformed cells need 81,497 2 less of these known growth factors for optimal growth and proliferation than normal cells. In particular, it has been shown 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) 10 878-880.

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, which have 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 growth rate-dependent growth in monolayer culture, b) cell overgrowth in monolayer culture, c) a change in cell form resulting in induction of tumor phenotype by the indicator cells, and d) adherence to adhesion independence. resulting in the ability to grow in soft agar. The property of cell-independent growth 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, reductant- and protease-sensitive peptides that appear to specifically interact with and produce biological effects through cell membrane EGF receptors, resulting in 81,497 3 TGF competes with EGF for binding to the cellular EGF receptor. However, TGF is distinguishable from EGF in several important respects. 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 form 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 properties that are valuable or potentially valuable in the health sciences, such as strong 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 antigenic fragments thereof 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 exert their biological effects through cell membrane EGF receptors allows, once the basic structure of a TGF polypeptide has been determined, to bring the structure into line with the structure of EGF. to develop oligopeptides with chemical properties that allow binding to EGF receptors without concomitant cell phenotyping.

To prepare the antibodies of this invention, a method has first been developed to produce TGF polypeptides in sufficient amounts and purity to allow determination of complete structure, and as a result of this assay and other observations, including the discovery of essential homologues between human and mouse TGFs, a basic peptide structure has been found. , which has extensive use in the area associated with cellulosis and cell growth. In addition, homogeneous TGF polypeptides are obtained that have utility both in the area of cell growth and in the detection of cancer and other proliferative diseases. Antibodies raised against TGF polypeptides and oligopeptides can also be used to detect diseases associated with bone loss, such as osteoporosis, hypercalcemia, and intelligence sorption. 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. Accordingly, the present invention provides antibodies raised to TGF-25 polypeptides and antigenic oligopeptides derived therefrom for use in the detection of TGF- and EGF-dependent diseases.

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 micro-35 chromatography eluting first with acetonitrile solvent and

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5,891,497 thereafter 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 for purifying 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 10, 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 so far that have 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 the antibodies of this invention, a basic protein structure 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-linked polypeptides, including TGFs, and the biological activity of the polypeptide. Thus, the present invention provides antibodies formed to biologically active polypeptides of formula

Val-Val-Ser-His-Phe-Asn-R-Cys-Pro-Asp-Ser-His-Thr- 15 20 25 35 Gln-R'-Cys-Phe-His-Gly-Thr-Cys-Arg-R "-Leu-Val-Gln- 30 35

Glu-R ^ Lys-Pro-Ala-Cys-Val-Cys-His-Ser-Gly-R '”- 6 81497 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 Val, or oligopeptide fragments comprising the amino acid sequence of these polypeptides 1-13, 34-43, 34-50 or 39-50.

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

The invention also relates to methods for detecting malignancies using antibodies of the invention raised against the polypeptide and oligopeptide structures set forth above. The antibodies are optionally labeled with a label capable of providing a detectable signal for use in diagnostic methods. Other methods utilizing antibodies generated to the cell growth promoting polypeptides of the above formula also form part of this invention.

The invention relates to antibodies raised to homogeneous growth factor poly-peptides of formula I isolated from pure aqueous media or body fluids containing these polypeptides obtained from tumor-producing tumor-bearing mammals. The polypeptides of formula I above can be isolated from an aqueous medium containing this transformation factor polypeptide in impure form by a method comprising the steps of: (1) dialyzing an aqueous medium containing the transformation factor in impure form against an aqueous solvent of acetic acid; which contains a growth factor polypeptide and which phase is concentrated and. . optionally clarified, 81 497 7 (2) reconstitute the concentrated solvent phase of step 1) with aqueous acetic acid and subject the reconstituted solution to gel filtration chromatography by applying the reconstituted solution to a column of gel filtration chromatography eluting with ethyl acetate, equilibrating with aqueous acetic acid. containing the growth factor polypeptide purer, and the selected fractions are combined and concentrated to give a partially purified transformation factor polypeptide-containing product, through one or more hydrocarbon-bonded silicon-acid matrix columns equilibrated with aqueous with 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 column elution carried out on the propellant a gradient in aqueous acetic acid, wherein the gradient of 1-propanol is increased 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 location of the cysteine residues allows the polypeptide to fold in a special way due to 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 dlsulfldi bridge sequence in which (by numbering Cys residues 1 to 6 to 5 from left to right in the above formula) Cys-1 is bound to Cys-3, Cys-2 is bound to Cys-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, 10 TGF polypeptides of the above formula are suitably obtained from various modified human and mouse cell lines, certain fetal cell lines, and body fluids of tumor-bearing mammals using the isolation method described below or a conventional synthetic or recombinant polypeptide method.

Typically, TGF polypeptides of said formula and their oligomers have a molecular weight in the range of n.

5,000 to 35,000. Preferred in this regard are TGF polypeptides having a molecular weight of about 5,000 to 8,000.

As mentioned above, the TGF polypeptide antibodies of this invention have value in detecting malignancies in mammals because the production and / or elevated production levels of TGF polypeptides are characteristic of the morphological modification of certain human and mouse cell lines. In this regard, antibodies to TGF polypeptides have utility in the diagnosis of malignancy because they can be used to detect extremely low levels of TGF polypeptide present in tumor cells or body fluids. While the entire TGF polypeptide molecule 30 can be used to generate antibodies (both polyclonal and monoclonal), it is also possible and advantageous in terms of cost and technical effort to determine the various regions of the TGF polypeptide sequence that are likely to be determinants and use these at least n Oligopeptides of 8 amino acids, typically at least about 10 and not more than about 20 amino acids, are defined as a hapten that can be used to induce antibody formation. As further discussed below, the oligopeptide is bound to a suitable immunogen and introduced into the vertebrate to detect the desired antibodies. Thus, the present invention also provides a series of antibodies to oligopeptides corresponding to antigenic regions in TGF polypeptides. Examples of antigenic oligopeptides useful in generating the antibodies of this invention are listed below.

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-Try-Val-Gly-Val-Arg-Cys-Glu-His- 15 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 formula, as well as the above peptide sequences, 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 technology. Thus, for polypeptides and oligopeptides, conventional solid phase peptide synthesis is suitably used. In this general synthetic method for the preparation of peptides, which method is described, for example, by Ganfleld et al. In U.S. Patent 4,341,761, known side chain protecting groups and conventional polystyrene resin matrices, e.g., chloromethylated resins, hydroxymethyl resins, or benzhydrylamine resins, are used to effect amino acid coupling. For polypeptides, the method described below for isolating homogeneous TGFs from natural sources can also be suitably used to obtain pure forms of the desired peptide. Particularly suitable sources of 35 TGF polypeptides in this regard are serum-free medium treated with retrovirus-modified Fischer rat 81 497 ίο fetal fibroblasts, in particular fibroblasts modified with Snyder-Theile feline sarcoma virus, Moloney mouse sarcoma virus 3 and human metastatic melanoma cells-5 with A2058 and A375. Sources and methods for suitable mouse cell lines are described by 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 in Todaro et al., (1980) Proc. Natl. Acad. Sci. USA 77, pages 5258-5262 and Giard et al., J. Natl. Cancer Inst., 51 (1973) 1417-1423. The isolation method described below can also be used to obtain TGF polypeptides from a variety of body fluids, such as urine, serum, plasma, whole blood, or cerebrospinal fluid from human or murine malignancies, or from modified cells that produce TGF polypeptides. In this regard, a suitable source of TGF polypeptides is urine or other body fluids 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 25 for receptor binding (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 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 has been obtained,

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81 497 11 conventional methods for reverse transcribing mRNA into cDNA and then cloning the cDNA into a suitable vector to achieve expression of the desired polypeptide.

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, e.g. by centrifugation and concentrated before dialysis, and the solvent phase 10 from dialysis containing TGF is suitably clarified, for example by centrifugation, and also concentrated before gel filtration chromatography. In all cases, the dialysis is suitably carried out 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. Preferred in this regard are polyacrylamide gel filtration resins (Bio-Gel *) resins such as Bio-Gel P-10, Bio-Gel P-30 and Bio-Gel P-60, of which Bio-Gel P-10 is particularly preferred. The aqueous acetic acid used to equilibrate the column and elute the TGF-containing fractions suitably has an acetic acid content of 0.2 to 2.0 M, with 1.0 M acetic acid being preferred. 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 obtained from Gee-30 additional filtration chromatography, which contain TGF polypeptides in improved purity, are combined and concentrated, for example by freeze-drying, 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 successive HPLC runs can be performed using one or more HPLC columns, but it is preferred to perform successive HPLC steps using a single HPLC column. The 5-column packing used is suitably a porous silica matrix to which a long chain hydrocarbon is bound, for example a hydrocarbon containing 16 to 22 carbon atoms. Preferred packings are pBondapak hydrocarbon columns, especially a pBondapak C1B column (particle size 10 μm, 0.39 x 30 cm, Waters Asso-10 dates). The process is typically carried out under pressure, preferably at a pressure between about 3.45 x 10 5 and 3.45 x 10 7 Pa (50-5000 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 elution is performed with acetonitrile in 0.01 to 0.1%, preferably 0.05% trifluoroacetic acid using a linear acetone triil gradient (acetonitrile concentration increases linearly with a gradient of about 0.1% / min to about 1% / min). The elution is carried out over 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 and soft agar analysis with EGF are concentrated, for example by freeze-drying before the second HPLC step using 30 L of propanol solvent. For the second step of sequential HPLC, the combined and concentrated fractions from the first HPLC elution are reconstituted to 0.01-0.1% trifluoroacetic acid and rechromatographed on the same column or on a second column equilibrated with trifluoroacetic acid in the same manner as used. for the first column. This second elution 81 497 13 is carried out with 1-propanol, in 0.01 to 0.1%, preferably 0.035%, trifluoroacetic acid using a linear 1-propanol gradient. For optimal results, it is important to use a low linear gradient of 1-5 propanol at this stage. In particular, the 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 10 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-β1 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 to 35,000. These homogeneous TGF polypeptides have the characteristic biological properties of TGF polypeptides when used. used in culture to grow a transformer tower, normal indicator cells, including achieving attachment independence, resulting in the ability to grow in soft agar. These homogeneous TGF polypeptides can be used to raise antibodies (see below) of value in the detection of cancer and other proliferative diseases. In this regard, it is not essential that each of the TGF forms be purified to homogeneity to produce antibodies to different TGF peptides. All antibodies raised to the various TGF polypeptides are considered to be within the scope of the invention.

The antibodies of the invention include both monoclonal and polyclonal antibodies raised to TGF polypeptides of the above formula, antigenic oligopeptides derived from TGF polypeptides, and homogeneous TGF polypeptides obtained using the isolation method described above in mammals having: there are malignancies, from a variety of 10 modified cell lines and body fluids or modified cells. The antibodies of the invention can be prepared in a variety of ways known in the art, depending on whether monoclonal or polyclonal antibodies are desired. For polyclonal antibodies, a vertebrate, typically a domestic animal, is hyperimmunized with antigen and blood is collected shortly after repeated immunizations and gamma globulin is isolated. Suitable methods for making polyclonal antibodies are set forth in the Handbook of Experimental Immunology, 3rd Edition, Weir, edited by Blackwell Scientific Publications, Oxford and London, 1978. For monoclonal antibodies, a small animal, typically a mouse or rat, is hyperimmunized with antigen, the spleen is removed and the lymphocytes are combined with myeloma cells in the presence of a suitable fusion promoter. The resulting hybrid cells, or hybridomas, are screened to isolate individual clones, each of which secretes a single antibody species to the antigen. Each individual antibody species thus obtained is the product of a single immune animal B-30 cell that has developed in response to a specific antigenic site recognized in an immunogenic agent. A general method for obtaining monoclonal antibodies, including those of the invention, has been described by Kohler and Milstein Nature 256 (1975) 495-497. The polypeptides and antigenic oligopeptides used to produce the antibodies can be used directly in the immune recognition process or can be bind 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.

The antibodies of the invention can be used in many ways. In a preferred embodiment, they can be used in the diagnosis of malignancy and other proliferative diseases. In cases where the antigen is present in physiological fluid or at a different concentration only when malignancy or other proliferative disease exists, a physiological fluid such as serum, plasma, whole blood, or cerebrospinal fluid is analyzed. The antibodies used in the assays may be labeled or unlabeled. Unlabeled antibodies can be used in agglutination; Labeled antibodies can be used in a wide variety of methods, using a wide variety of markers, such as radionuclides, enzymes, fluorescent agents, enzyme substrates or cofactors, or the like. These methods have been extensively described in the literature and exemplary illustrative methods can be found in U.S. Patents 3,817,834; 3,935,074; 4,233,402 and 4,318,980.

In some methods, it is useful to label an antigen or fragment thereof rather than an antibody and to create competition between the antigen labeled by the antibody and the antigen in the sample. In this situation, it is common to prepare test kits containing an amount of a labeled antigen or a combination of a labeled fragment and an antibody in an amount that provides optimum sensitivity and accuracy. In other cases, it is desirable to have a solid support to which either the antigen or antibody is bound. The multi-epitope antigen can act as a bridge between the antibody bound to the carrier and the labeled antibody in the assay medium. Alternatively, there may be 16 81 497 competition between the labeled antigen and an antigen in the sample for a limited amount.

When the antigen cannot be found in physiological fluid or, if found, is not diagnostic of the malignancy or proliferative target disease, the cells must be isolated and analyzed for the presence of the antigen. For antigen detection, the tissue sample can be digested by conventional methods, e.g., with base, detergents, etc., the cell debris separated by filtration or centrifugation, and the filtrate or supernatant isolated and analyzed.

Markers can also bind to markers, such as radionuclides, fluorescent agents, enzymes, etc. When the antibodies are introduced into the body, they direct the marker to a malignant cell, allowing the presence of the malignancy to be diagnosed.

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

The antibodies of this invention can also be used to isolate cells that express TGF polypeptides and to remove cells in vitro from a heterogeneous cell population containing cells that express TGF polypeptide. Separation can be performed with a fluorescence activated cell sorter (FACS). This same method can be used to identify and isolate cells expressing TGF polypeptide. To remove cells expressing a TGF polypeptide from a cell-35 mixture, target antibodies can be combined with complement, or a toxin-degrading fragment (A fragment 81 497 17) (see EPO Patent Publication No. 17,507 and U.S. Patent Application Publication No. 2,034,324) or the cell can be agglutinated and separated by physical methods.

The antibodies raised against TGF oligopeptides and polypeptides of the invention are effective in detecting diseases associated with bone loss. For example, TGF activity is present in a material responsible for bone resorption activity in tumors associated with hypercalcemia [Ibbatson et al., Science 221 (1983) 1292]. 10 Elevated bone resorption may be an endocrine effect of TGF molecules secreted by tumor cells.

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 loss. TGF antibodies can be used to detect osteoporosis and thus are useful in determining this important disease. The invention relates to a method for detecting bone loss in a human, comprising contacting cells or body fluids with a TGF antibody and determining the level of binding of said antibody to cells or cell products in the body for diagnosing a host suffering from bone loss.

Example I

Production, Purification and Characterization of Human Low Molecular Weight Transformation Factor (hTgFs) A, Experimental Methods Source of hTGFs hTGFs were purified from serum-free medium treated with the human metastatic melanoma line Ά2058 [Todaro et al. Proc. Natl. Acad. Sci. USA 77 (1980) 5258-5262], which was derived from brain metastasis in a 43-year-old man. Cells were grown to 90% confluence in round bottom flasks containing Dulbecco's 35 modified Eagle's medium (Grand Island Biological Co., 430-2100) supplemented with 10% calf se- χβ 81 49? ugly (Colorado Serum Co.) at 37 ° C. The cells were washed for 1 hour with 50 ml of serum-free Weymouth'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 ultracentrifuge (Beckman). The supernatant obtained after high-speed centrifugation is called A2058-treated medium.

The A2058-treated medium was immediately concentrated in a hollow fiber dialysis / concentrator (Model DC10, H1095-20 type cartridge Amicon Corp.) at 10 ° C. The concentrate was drained when the volume had decreased 150-20 times. The cartridge was washed with 1000 ml of Waymouth's medium. The ultrafiltrate was rejected. Purification of hTGFs Dialysis and centrifugation

The combined retentate fluid and cartridge wash fluid after ultrafiltration of the A2058-treated medium were dialyzed for 60 h against 0.1 M acetic acid in a Spectrapor 3 dialysis tubing (Spectrum Medical Industries). 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 Blo-Gel P-10 gel After concentration, dialysis and centrifugation, the supernatant containing hTGF activity was further purified by gel filtration chromatography on a Bio-Gel li 19 81497 P-10 column 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 ml) in 1M acetic acid (5 ml) were applied to the column.

5 To ensure a constant flow rate, the liquid outflow from the column was set at 12 ml / h with a peristaltic pump. 4.8 ml fractions were collected. Small amounts of fractions were lyophilized for subsequent determinations of EGF-competitive and growth-promoting activity in soft agar. Fractions representing the major parts 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 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 cold-20 dried 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 a sample injector. With .0.5% trifluoroacetic acid. The column was then eluted with a linear gradient of acetonitrile in 0.045% trifluoroacetic acid. The liquid flowing out of 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 gradient of 1-propanol 20 81 497 in 0.035% 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.

5 SDS-polyacrylamide gel electrophoresis SDS-polyacrylamide gel electrophoresis was performed as described by LaemmII, Nature (Lond) 227 (1980) 680-685. A 15-30% acrylamide digradient plate (140 x 120 x 0.75 mm) was prepared 4%: with con-10 centrifugation gel. Gels were run at a running voltage of 30 V using an electrode buffer containing Trls (0.05 M), glycine (0.38 M) and SDS (0.1% w / v) until the tracer (bromophenol blue) had passed. away from the gel end. After electrophoresis, gels were fixed in 50-15% 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].

Protein Assay 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 culture medium components that interfered with color reactions, or lyophilized if 25 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 evacuated 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 a PDP 8 / 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). Radioreceptor Assay 35 Purified EGF was labeled with Na125I with a modification of the chloramine-T method as described by DeLarco et al.

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81 497 21 et al., Proc. Natl. Acad. Sci. USA 75 (1978) 4001-4005. 125 I-EGF binding assay was performed using nearly congruent monolayers of formalin-fixed A431 human cancer cells as previously described (DeLarco 5 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 1 mg / ml bovine serum albumin and 50 mM 2- [bis (2-hydroxyethyl) amino] ethanesulfonic acid, pH 6). , 8). Races 10 were started by adding 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, specifically bound 125 I-EGF was determined. The TGF content was expressed by the degree of inhibition of its 125 I-EGF binding 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%.

Growth assay with soft agar

Colony growth assay in soft agar using normal-20 Iin renal fibroblasts, clone 49F, was performed as described by Todaro et al. have been reported by Proc. Natl. Acad. Sci. USA 77 (1980) 5258-5262. Test lyophilized samples were reconstituted in 0.5 ml of Dulbecco's modified Eagle's medium supplemented with 10% 25 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 30 (Falcon). The cells were incubated at 37 ° C in a humidified atmosphere of 5% CO 2 and 95% air. The assay was read unattached and unstained on day 5 and days 10-14.

B. Results Source, Concentration, and Initial Fractionation of 35 hTGFr hTGFr was isolated from serum-free 8145 of the highly modified human meta-static melanoma cell line, A2058? 22 treated media. 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-5 EGF for EGF receptor sites in human cancer cells A431. A summary of the steps leading to the isolation and recovery of hTGFs is presented in Table I.

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To remove serum proloteins, A2058 cells were washed thoroughly 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. The first clarified volume of 136 L of A-2058-treated medium containing 1.02 g of total sprotel and 4525 10 units of EGF-competitive activity was concentrated to about 900 ml using a hollow fiber concentrator with a molecular weight cut-off of cartridges. 5000. The activity competing with the whole 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 pools 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 6,800, 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 original EGF competing activity. Low molecular weight TGF eluted, 81497 25

from the column as sharp ice (P-10-B) and represented 45% of the original total activity competing with the EGF. The overall yield of column-competitive EGF activity from step 1 through the gel filtration-5 chromatography step was 91% (Table I). -hTGF

eluted as two distinct major peaks that varied quantitatively from preparation to another. 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 filtration chromatography of the acid-soluble EGF-competing activity of the concentrated A2058-treated medium using Bio-Gel P-10 gel, a combined 15-portion of P-10-B was reconstituted with 0.05% trifluoroacetic acid in water and chromatographed. was then on a pBondapak C18 column. The EGF-competing and growth-stimulating activity of the individual fractions on 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. A 57-fold purification of hTGFs was obtained after gel filtration chromatography. 80% of the 25 initial EGF-competing activities in the P-10-B batch were recovered (Table I).

Rechromatography of hTGF-containing fractions on pBondapak C18 support was chosen as the final purification step because only relatively small losses of EGF-competitive activity were observed on these columns. In order to achieve a clear separation of hTGFs from impurities, it was necessary to use a gentle linear gradient of 1-propanol in 0.035% trifluoroacetic acid. The main mass of contaminating peptide material 35 differed from the well-defined peak of activity. The EGF-competing and growth-stimulating activity cleared the floods 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 step was 73% and the yield per step was 80-100% (Table I).

10 Characterization of hTGFs

The purity of the final hTGF preparation was determined by analytical SDS-polyacrylamide gel electrophoresis. The gel was stained with silver. One major polypeptide band with an apparent Mr of 7400 was observed. The same pattern 15 was obtained when the samples were electrophoresed under non-reducing conditions, indicating that TGF is a single-chain molecule.

Receptor reactivity of hTGFs was compared to EGF by 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 specific EGF 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, on soft agar. The half-maximal response of hTRFs in soft-mytagar was achieved with 1 EGF-competing unit 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.

li 35 81 497 27

Example II

Large-scale production, purification and amino acid sequencing of human (hTGF), rat (rTGF) and mouse (mTGF) transformation factors. robots, FRE CLIO, a subclone of FRE 3A [Sacks et al., Virology 97 (1979) 231-240], which was transformed into non-productive Snyder-Theilen feline sarcoma virus [Snyder et al. Nature (Lond) 221 (1969) 1074-1075], in the Molony 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 round bottom flasks containing Dulbecco's modified Eagle's medium supplemented with 10% calf serum and then maintained in serum-free Waymouth'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 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 TGFs 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 on 35 freeze-dried columns and redissolved in 1 M acetic acid for subsequent gel filtration chromatography on a Bio-Gel 28 81 497 P-10 column (2.5 x 85 cm). 200-400 mesh, Rio-Rad Laboratories). The column was equilibrated with 1 M acetic acid. Fractions containing most of the activity competing with EGF and having 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 1. Separations were performed on a pBondapak C18 column (particle size 10 μm, 0.39 x 30 10 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 acetone tril was increased linearly (0.083% / min) over 2 hours 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 activity competing with EGF, were lyophilized.

Method for Determining TGF TGF was quantified by a radioreceptor assay based on the cross-reactivity of the receptor with murine submandibular epidermal growth factor (mEGF). Purified mEGF was labeled with Na125I by modification of the chloro-30 amine-T method as described in Example I. 125 I-EGF binding assay was performed on formalin-fixed human A431 cancer cells, 8 x 103, in Micro Test II plates (Falcon). The concentration of TGF was expressed in mEGF ng equivalents / ml and was based on the amount of TGF required to produce an equal amount of 125 I-EGF.

II

81 497 29 inhibition of binding to A431 cells by a known amount of unlabeled mE6F.

Amino Acid Sequence Determination of TGF For amino acid sequence analysis, rTGF (3 pg) was reduced with dithiothreitol (20 mM) in 100 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-carboxamidomethylated 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 [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 of TGF purification

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 by gel filtration chromatography of acid-soluble EGF on Bio-30 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. % trifluoroacetic acid, straight gradient. The lifetime models of the final purification step of rTGF, mTGF, and 35 hTGF show that the activity competing with EGF was purified in parallel with a clearly distinct absorbance peak and effectively separated from the contaminating, UV-absorbing material. The major protein peak in rTGF, mTGF, and hTGF steps eluted from the pBondapak 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 higher molecular weight TGFs and reduced the amount of protein added to the pBondapak 10 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 C18 in the next two purification steps was very efficient, each giving a typical preparation yield of 80-100% per step. The final yield of low molecular weight TGFs was about 70% of the maximum total activity competing with EGF observed during purification. The mean yield of purified 20 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 TGF The purity of 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 qualitatively and quantitatively nearly indistinguishable from 35 mEGF. Thus, the final TGF preparations were believed to be highly pure and substantially homogeneous. A single amino-terminal sequence was determined for 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 polyacrylamide gel electrophoresis. The purified preparation gave one large polypeptide band.

10 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 (7, 15, 23 and 38) in the sequences.

(1) rTGF 5 10 20 Val-or-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-25 50

Leu-Ala: X; (2) mTGF 5 10

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- 30 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

Leu-Ala (3) hTGF 5 10 '···' 35 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 '' Val-Gln-Glu-Asp-Lys-Pro-Ala-Cys-Val-Cys-His-Ser- 40 45

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

Leu-Ala 32 81497

Example III

Production of TGF in vivo and its isolation

Tumor cell lines (1 x 10 6) known to produce (human melanoma and modified rat) TGF were transplanted into thymus-deprived "hairless" mice and tumors were allowed to develop. Urine from tumor-bearing mice was collected and analyzed for the presence of TGF using the isolation method and analytical methods given in Example 1 above. TGF of tumor-bearing mice was found to have TGF having the same size and elution characteristics by HPLC as TGF from cell culture described in Examples I and II above. Furthermore, using the methods described in Example I above, TGF present in the urine of mice was found to have the characteristic biological properties of TGF in that it stimulates cell-independent growth and binds to the EGF receptor. The tumors were then removed from the tumor-bearing mice, and the urine of the mice was tested for the presence of TGF after tumor removal using the above-mentioned methods. In this case, no TGF with characteristic 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 in body fluids using the methods described above. Similar experiments were also performed in rats with chemically carcinogen-induced tumors and found to have urinary TGF based on the biological and biochemical properties listed above, whereas untreated rats did not.

35

II

81 497 33

Example IV

Inhibition of Retroviral-Modified Cell Growth by In Vitro Antibodies to Antigenic TGF-Oligopeptide 5 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 10 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 conifer hemocyanin according to the method of Baron et al., Cell 28 (1982) 395-404 and used to immunize rabbits [Baron et al., Cell 28 (1982) 395-404] and sheep [Lerner , Nature 299 (1982) 592-596]. Antisera were analyzed against the peptide by peroxidase-coupled immunoassay (Kirkegaard and Perry Laboratories Gaithersberg, MD) and by immunoprecipitation against homogeneous rat TGF (purified according to Example II above) [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 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 confirmed by Western blotting. An-35 tipeptide antisera 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 12SI-labeled rat TGF to these cells was blocked by an excess of 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 S. aureus protein A. 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, the antiserum raised 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 (NRKs) and a variety of retroviral cell lines were plated at low densities (500-2000 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 redissolved to an antipeptide titer of 103 to 104. The results are given in Table II below.

„81497 35

Table II

Effect of antipeptide antibodies on cell growth in vitro

Colony formation ® Antibody Source Volume Other% inhibition of proliferation (μg / l) substance NRK-CL10-sol- Ki-src-luilluil-NRK: 3T3 lala 11a 11a

Anti-TGF

10 oligopeptide Rabbit 5 - 085NT53 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 & 20 peptides (4) rabbit 0 <15 <5 0

As shown in Table II above, neither rabbit nor sheep anti-TGF oligopeptide inhibited colony formation on NRK cells, but prevented Kirsten-modified NRK cells, feline sarcoma virus-modified rat fetal fibroblasts (CLIO) cells, and Rous sarcoma virus-modified 3T3 cell growth.

CLIO cells were known to be productive TGF producers [Marquardt et al., Proc. Natl. Acad. Sci. USA 80 (1983) 4684-4688]. A few surviving colonies in anti-TGF antibody-treated cells tended to be smaller and lacked the stout appearance of normal colonies. Non-adherent, non-disrupted cells floated free in the medium. This inhibition was partially reversed when 36 81 497 TGF peptide, but not unreacted irrelevant peptide, was added simultaneously with the antibody raised against TGF. Rabbit and mouse antibodies to four different unreacted peptides had no effect on the growth of NRK colonies or retrovirus-modified cell lines. Replacing the antibody-containing medium with fresh, antibody-free free medium after 72 hours failed to undo the inhibition of colony formation, but the number of surviving colonies increased dramatically.

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 Merri-field, 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 benzyl alcohol derivatives of side chains. A more acid-resistant benzyl ester bond, which attached the protected amino acids to the polymeric 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 was performed by Tam et al., Tetrahedron Lett. 23 (1982) 4435-4438, according to the Low-High HF method, 30 which differed from the conventional HF deprotection method and removed the benzyl protecting groups by the S2 mechanism in dilute HF solution to minimize the severe side reactions caused by carbon cations evolving in the conventional SNL Shield demolition method. In addition, it 35 is also designed to reduce many cysteinyl side

II

81 497 37 reactions that often inhibit the synthesis of proteins containing many disulfide bonds.

After HF treatment and before any purification, crude and reduced synthetic rTGF was oxidized and re-generated 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, unpurified rTGF-Ι contained 40-50% of EGF radioreceptors and tyrosine-specific protein kinase activities compared to native rTGF-Ι. 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 15 (3) by preparative high pressure liquid chromatography (HPLC) on a C-18 reverse phase column. The total yield calculated from the starting amount of Ala in the resin was 31%.

Under reduced or non-reducing conditions, purified synthetic rTGF was found to give a single band of 20,000 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 thiol was detected by the Ellman sulfhydryl assay for synthetic rTGF, but thiolytic reduction gave the expected theoretical 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 native rTGF. In addition, synthetic rTGF co-eluted with native rTGF as a single symmetric peak in C-18 reverse phase HPLC.

35 38 81 497

Example VI

Detection of TGF activity in the urine of cancer patients

Various urine samples were kept at 90 ° C for 15 min after the addition of 1/9 volume of a solution containing 20% SDS, 0.4 mol / L DTT, 0.4 mol / L Hepes, 0, 08 mol / l sodium chloride and 1 mmol / l EDTA (pH 7.4). A sample (20 μΐ) was added to the incubation mixture (total volume 50 μΐ) supplemented with 1% NP-40. A sample, 10 antibodies to the 17 amino acid sequence of rat TFG of Example IV, and a 125 I-labeled synthetic 17 amino acid sequence were incubated in 96-well plates for 60 min and then for 30 min with Pansorbin. The antibody-bound peptide was pelleted on a dibutyl-15 phthalate medium, and the pellets were counted in a gamma counter using 3 mm thick lead shields to protect the non-cleaved isotope.

Synthetic sequence and human melanoma (cell line A375) -TGF from concentrated serum-free modified growth medium 20 were used as TGF standards. In each experiment, one or more normal samples were analyzed and the mean normal value was considered to correspond to one relative TGF equivalent.

A. Sample Preparation 25 Fresh, unclear urine samples were stored in 25 ml aliquots at -70 ° C for up to 6 months. In most cases, samples were thawed rapidly and treated immediately with protease inhibitors (0.5 m / mol phenylmethylsulfonyl fluoride, 0.05 30 m / mol pepstatin A, Sigma Chemical Co.) for 30 min at 4 ° C. Ten milliliter samples were then dialyzed at 4 ° C against either 0.1 M acetic acid or 0.1 M ammonium bicarbonate. Three dialysis tubing of different pore sizes were used: 3,500, 6,000-8,000; or 35 12,000 to 14,000. (In some cases, urine is a cold

II

39 81 497 were boiled and extracted first, but the neutralization and ethanol / ether precipitation steps were omitted.) After 2-4 days of dialysis, the urine was clarified (acceleration 10,000 g, 10 min), lyophilized, dissolved to 50-fold concentration. 5 m / mol formic acid containing 50 mmol / l sodium chloride relative to the original and then clarified rapidly (12,000 g, 1 min) before pretreatment.

B. Creatine Analysis 10 Creatinine was tested in duplicate in each sample using a manual colorimetric assay.

C. Results Samples were normalized to creatinine levels. 15 In each of the six experiments, statistical analysis (T-test) showed a significant difference between cancer patients and normal samples (P <0.001, 0.001, 0.001, 0.001, 0.01, 0.05). In these six experiments, the mean urinary TGF concentration of normal individuals was 0.18 ng TGF / mg creatine-20, while the mean urinary concentration of lime cancer patients was 0.64 ng TGF. Elevated TGF levels were observed in several cancer patients. The results of each experiment were also normalized to the mean normal value obtained in that experiment. The results are shown numerically in Table III. Using twice the TGF-β concentration as an optional cleavage site relative to the mean normal value, an abnormal TGF-β concentration was observed in 81% of the various cancer cases studied.

30 81497 40

Table lii: Detection of alpha-TGF antigen in urine

Patient Group Positive Sample Processing pH

total_and RIA concentration factor pH 2_pH 8_ ___9x_18x 9x 18x Seemingly healthy controls 1/18 (6%) 0/6 1/12 1/15 0/15

Patients with benign condition 1/3 0/3 1/3 0/1 0/1 colon (hairy adenoma) 0/1 0/1 0/1 breast (connective tissue vesicle tumor) 0/1 0/1 0/1 0/1 0/1 pregnancy (normal, 38 weeks) 1/1 0/1 1/1

Cancer patients 26/32 17/28 22/31 9/16 12/16 (81%) (61%) (71%) (56%) (75%)

Lung 11/13 11/13 12/13 3/4 4/4 (85%) (85%) (92%) (75%) (100%) 2o Gastrointestinal tract 4/7 4/7 4/7 3/7 3/7 (57%) (57%) (57%) (43%) (43%)

Urinary and genital organs 3/3 1/3 1/3 2/3 3/3 (100%) (33%) (33%) (67%) (100%)

Chest 5/5 1/1 5/5 25 (100%) (100%) (100%)

Imukudos 3/4 0/4 1/4 2/2 2/2 (75%) (0%) (25%) (100%) (100%)

2Q * Breakpoint> 2 x mean normal value Example VII

Detection of TGF-β activity in the urine of cancer patients The immunization of 35 50 subjects (25 normal and 25 advanced cancers) was immunologically

II

81497 using the antibody generated for TGF and the procedure described in Example VI above. In this study, 100 ml of urine was collected from subjects, dialyzed against 1.0 m / mol acetic acid, and concentrated approximately 100-fold. This concentrated urine was then immunoassayed using a rabbit polyclonal antibody raised against a 17 amino acid oligopeptide as described in Example IV above, and the results are given in the table below.

10 Table IV

Detection of TGF-activity in the urine of cancer patients Diagnosis Positive LKM / Tested LKM

15 Lung cancer 4/5

Breast cancer 4/5

Colon cancer 3/5

Melanoma 5/5

Leukemia 0/5 20 Normal status 1/25 In this experiment, the majority of cancer patients had urinary TGF levels at least 5-fold higher than the normal individuals studied.

25 Example VIII

Detection of TGF by monospeptic antiserum raised against synthetic peptide Peptide synthesis

Peptides corresponding to amino acids from portions of the amino acid sequence of rTGF-30 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 reversed-phase high-pressure liquid chromatography (RP-HPLC) before use.

42 81497 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-5 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-Asp-Leu-Leu-Ala 10

Conjugation of peptides, 1a immunization

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 glutaralde-15 hyd 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 at two-week intervals. The serum used in this experiment was collected 80 days 25 after the first injection. From this serum, an immunoglobulin fraction was prepared by ammonium sulfate precipitation. An antibody product specific for the immunizing peptide was monitored by solid phase ELISA.

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

Radioreceptor Assay The binding of 125 I-EGF to its receptor by monolayers of A431 cells formalized in 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 concentrations were expressed as the amount required to produce 125 I-EGF binding inhibition corresponding to the known amount of TGF-20.

RIA (radlolmunune analysis)

The reagents were mixed in a final volume of 50 microliters of a solution containing 20 mmol / l sodium phosphate (pH 7.4), 200 mmol / l NaCl, 40 mmol / l dithiothreitol, 0.1% (w / v) BSA , 0.1% (w / v) NaN 3, 125 I-peptide III (104 cpm); an amount of antiserum corresponding to a final dilution of 1/15000, and other substances. The reaction was initiated by the addition of antiserum and allowed to proceed at 23 ° C for 90 min. An equal volume of Staphylococcus A (Pansorbin, Calbiochem) fixed with 10% formalin was then added and incubation was continued for a further 30 min at 23 ° C. The immunoadsorbent was removed by sedimentation through a 10% (w / v) sucrose solution, and the amount of bound 125 I-peptide 35 III was determined using a gamma counter. Under these .. 81497 44 conditions, the antibody bound approximately 25% of the 125I peptide in the absence of a competitor. The amount of bound peptide III was corrected for non-specific binding, 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.

Chromatograph 10 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 C18 column (0.39 x 10 cm, Waters Associates) using a flow rate of 1 ml / min at 23 ° C.

15 Manufacturing the treated substrate

Serum-free modified medium of Snyder Theilen-transformed Fischer rat aliquots (ST-FrSV-RFE clone 10) was collected by Twardzik et al. as described [Virology, 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. The non-soluble protein was removed by centrifugation, and the supernatant was lyophilized. Finally, the residue was resuspended in 25 parts by volume of the original 1 N acetic acid and stored at 4 ° C.

Immunoblotting analyysl

The samples to be analyzed were first subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis 30 (SDS-PAGE) using an acrylamide gradient of 15 -> 20% and the electrophoresis was performed under reducing conditions as described by Laemm [Nature 227 (1970) 680-682]. After separation, the proteins were electrophoretically transferred to nitrocellulose (Schleicher and Schuell, BA85, 0.45 35 μ) as described by Burnette [Anal. Biochem. 112 (1981)

II

81 497 45 195 - 203]. The transfer was performed at 5 V / cm for 3-4 hours at 23 ° C. The resulting protein blot was incubated overnight in a milk-based mixture BLOTTO [Johnson et al., Gene Anal.Techn., Is. 3-8]. The antiserum was diluted in BLOTTO and added by grinding 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® cpm / ml, 4 x 107 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 amplification plates.

In the radioimmunoassay described above, 125 I-peptide III added almost precipitated almost quantitatively with a low degree of antiserum dilution; a titer of> 10,000 was obtained for the antiserum. Antibody affinity was measured by incubating the antiserum diluted 1/5000 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 108 M'1.

RIA Specificity To determine the specificity of the RIA, various peptides were tested for their ability to inhibit the precipitation of 125 I-peptide III. Unlabeled peptide III prevented the precipitation of 125 I-peptide III approximately linearly in the range of 0.13-11 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 I, which corresponds to 11 amino-terminal groups, was also ineffective as 35 competitors. These results indicate that the epitope detected under standard experimental conditions was localized to the 11 carboxy-terminal amino acids of peptide III. Table V summarizes the relative inhibitory concentrations of the various peptides.

5

Table V

Addition Relative inhibitory activity peptide III 1 10 peptide IV 3.5 peptide I> 1500 peptide II> 1500 rTGF 0 dithiothreitol 1 rTGF - dithiothreitol 20 15 mEGF> 1500

The reported added substances were tested for their ability to inhibit standard R1A 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 ">" indicates the highest concentration tested. (The inhibitory activity of peptides I-IV 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 a molecule entirely native in biological activity, was as potent (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 was completely ineffective as a competitor up to concentrations above 1 ymol / l. This indicates that the RIA described herein differs from the other 35 available TGF assays in that it is specific for TGF but not for EGF.

ti 81 497 47

To directly demonstrate that antiserum binds 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 anti-serum Ka with respect to 125 I-TGF was 2.3 x 10 8 M -1. This value is reasonably consistent with the Ka determined for 125 I-peptide III (8.3 x 108 M'1), and shows that antisee-10 rum binds to rTGF.

Detection of rTGF from modified medium of 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 the modified medium of cultured transformed cells. A Fischer rat embryonic cell line transformed with feline sarcoma virus strain Snyder-Theilen (ST-FeSV FRE clone 10) has previously been shown to produce elevated levels of rTGF [Gray et al., Nature 303 (1983) 722-725]. Serum-free modified medium was collected, treated, and concentrated as described above. A portion of the medium was then subjected to gel filtration chromatography on Bio-Gel P-10 under acidic conditions. The fraction was analyzed for rTGF EGF receptor competition assay or RIA.

Under these conditions, EGF-competitive activity was observed in two molecular size groups, one approximately Mr - 10,000 and the other Mr> = 20,000; both species of molecules clearly elute 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 RIA. The ratio of activity competing with EGF to immunological 48 8 1 4 9 7 activity was strongly reduced in the high molecular weight TGF fractions, indicating that the biological activity of these TGF molecular species is lower than that of the small molecule fractions.

To confirm that immunological activity was present in the same molecular species as EGF-competitive activity, RP-HPLC was performed on pooled fractions obtained by repeating this experiment on a preparative scale and containing rTGF size groups of 10 Mr to 10,000 and Mr 20,000; the conditions used were similar to those used to purify rTGF according to Marquardt et al. [Proc. Natl. Acad. Sci., 80 (1983) 4684-4688]. For the combined TGF fractions with a Mr 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 the size group Mr -20,000 competing with EGF, a similar co-purification of EGF-competing and immunological activity was observed. These experiments show that much of the immunological activity observed in the treated medium of St-FeSV-FRE-25 clone 10 was caused by rTGF.

Immunoblotting Analysis of Different rTGF Size Groups The larger TGF size groups present in the treated medium of rat cells transformed with retroviruses could represent either aggregated states or distinct molecular forms of the TGF-Molecule. To distinguish between the two alternatives, synthetic rTGF and native TGF size classes were subjected to Mr = 10,000 and Mr * 20,000 immunoblotting analysis after polypeptide components 35 were separated by SDS-PAGE under reducing conditions. Im-trans-reactive synthetic rTGF and small molecule

II

49 8 1 4 9 7 natural rTGF each co-migrated as a single polypeptide chain with a Mr of 6000, and incubation of the antiserum with excess peptide III blocked the immunological reactivity of these molecules. The large molecular size class, in contrast, contained three immunoreactive peptides, Mr = 24,000, Mr = 40,000, and Mr = 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 equal to Mr 43,500 was observed in all lanes. 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 was detected in the 15 large molecular size classes by

Mr - 6000. These results indicate that the larger molecular rTGF size classes represent distinct forms of TGF.

Example IX

A synthetic rTGF fragment that binds to receptors and is antigenic

peptides

EGF was isolated from mouse submandibular salivary glands by 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 a Bondapak C-18 - columns (Waters) as described by Elson et al [Biochemistry Int., 8 (1984) 427-435]. The total synthesis of native rat TGF is described above in Example V.

Synthetic peptide fragments were prepared with chloromethyl polystyrene 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 Blo-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 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. a (pH 4.5) (10). Peptides 1 and 2 represent amino acid sequences 34-43 of rTGF, which have unprotected or correspondingly protected ends (N Ac, C-amide), respectively. 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 hemocyanin (KLH) or bovine serum albumin by a thiol / maleim disfusion [King et al., J. Immunol. Methods 28 (1979) 201-206]. The carrier-peptide complexes were purified by gel filtration. An average of 60 mol peptide / 1 mol 20 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 every two weeks by injecting s.c. immunogen in incomplete 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. 92-well polyvinyl chloride plates (Costar) were coated with a solution containing 200 pg / ml of BSA and peptide 1 conjugate and counter-coated with phosphate buffered saline containing 10% normal 35 rabbit serum. The plates were incubated for 4 hours at 37 ° C with [125 I] anti (KLH - peptide 1) IgG (50,000 μl 81,497 51 cpm / well) in the presence or absence of inhibitors, washed and counted in individual wells.

Radloreseptorimäärltys

Human epidermoid carcinoma cells A-431 or human foreskin 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) containing 10 % fetal bovine serum (FCS, Hyclone). EGF 10 was radioiodinated as described above to 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 mM Hepes) containing 1 mM [125 I] -EGF and 0.1% BSA in the presence of inhibitor peptides. . The cells were washed three times with cold buffer, lysed with 0.1 N NaOH, and the radioactivity bound to the cells was measured (gamma counter). Non-specific binding, determined in the presence of a 100-fold excess of cold EGF, was less than 5% and less than 10% of the specific binding to A431 cells and HFF cells, respectively.

Cell Proliferative Assay HFF cells were grown to a uniform layer in 48-well group plates in DMEM containing 10% FCS, and proliferation was stopped by keeping cells for 2 25 days in a diet deficient 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 a conjugate of 30 KLH and peptide 1 reacted with 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.

52 81 497

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] -5 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; 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 EGF inhibition of 10 by HFF cells (Table VI) 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-15 genetic properties up to a concentration of 10-5 mol / L when incubated with non-proliferating 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 20 antagonists were equally effective when using TGF as a mitogen (Table VI). As with binding efficiency, the antagonistic potency of TGF-peptides was improved by closing the amino and carboxy termini.

Table VI

25 Biological activity of synthetic TGF fragments ------— b ~ '-

Inhibition of mitogenesis

Binding3 IC5Q (M) IC50 ^ 30 _A-431_HFF_EGF_TGF PC_

Peptides 18 + 2 x ΙΟ-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 + 1 x io_6

Peptides 3 4 + 1 x 10 ^ 3 + 1 x 10 ^ 6 + 2 x 10 ~ ^ 5 + 3 x 10 ~ ^

II

35 81 497 53 * IC50 values calculated from the inhibition curve describing the binding of [125 I] -EGF (1 nM) to cells. bIC50 values are concentrations that halve the ability of a control to promote uptake of [3H] -methylthymide into proliferating HFF cells induced by EGF or TGF (1 nM).

Claims (9)

1. Antibodies to Biologically Active Polypeptides of Formula 5 Val-Val-Ser-His-Phe-Asn-R-Cys-Pro-Asp-Ser-His-Thr-15 Gln-R'-Cys-Phe-5 His-Gly-Thr-Cys-Arg-R "-Leu-Val-Gln-35 10 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 Val, or against oligopeptide fragments comprising the amino acid sequence 1-13, 34-43, 34-50 or 39-50 of these polypeptides. An antibody according to claim 1, characterized in that R is Asp, R 'is Phe, R' is Tyr, R1 is Asp and R2 is Ala. An antibody according to claim 1 against an oligopeptide fragment containing 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. An antibody according to claim 1 against an oligopeptide fragment containing the amino acid sequence Cys-His-Ser-Gly-Tyr-Val-Gly-Ala-Arg-Cys. Antibodies according to any one of claims 1-4, characterized in that they have been labeled with a label which is capable of producing a detectable signal. The in vitro use of an antibody according to any of claims 1-4 for the detection of malignancy in a human host. Il 81 497 A method for detecting malignancy in a human host, characterized in that cells or body fluids from that human host are contacted in vitro with an antibody according to any of claims 1 to 4 and the level of binding of the antibody to these cells or cell products in said body fluids is analyzed. for the diagnosis of host malignant tumors. A method for detecting osteoporosis in a human host, characterized in that cells or body fluids from said human host are contacted in vitro with an antibody according to any of claims 1-4 and the level of binding of the antibody to said cells or cell products in said body fluids is analyzed. for the diagnosis of host osteoporosis. 15 Method according to claim 7 or 8, characterized in that the antibody has been labeled with a label which is capable of producing a detectable signal.