USE OF PROLACTTN AS A TGF-BETA ANTAGONIST
Background of the Invention
Transforming growth factor-β (TGF-β) belongs to a family of polypeptide factors that share certain structural and functional characteristics (Massague et al, Cell, 49:437 (1987)). Other members include the activins, the inhibins, Mύllerian inhibiting substance (MIS), bone morphogenic proteins (BMPs) and the decapentaplegic gene product (DPP-C) found in Drosophila.
Transforming growth factor-α is an unrelated peptide that shares a high degree of homology with epidermal growth factor (EGF), and binds the same receptor (Todaro et al, Proc. Natl. Acad. Sci. USA, 77:5258 (1980)).
TGF-β was discovered as a product of murine sarcoma virus- transformed cells (Delarco et al, Proc. Natl. Acad. Sci. USA, 75:4001 (1978)). TGF-β is produced by a large number of cell types, including fibroblasts, myocytes, chondrocytes (Jakowlew et al, J. Cell. Physiol, 150:377 (1992)), astrocytes (da Cunha et al, J. Neuroimmunol., 36:157 (1992)), and epithelial cells (Steigerwalt et al., Mol. Carcin., 5:32 (1992)). Named in accordance with its ability to stimulate anchorage- independent growth of normal fibroblasts, TGF-β has also been isolated from other sources, including a human glioblastoma, where it was identified as tumor-inducing factor-1 (TIF-1) (Iwata et al., Cancer Res., 45:2689 (1985)) and bovine bone, from which two cartilage- inducing factors (CIF-A and CIF-B) have been purified (Seyedin et al, Proc. Natl. Acad. Sci. USA, 82:2267 (1985)). Sequence information has Proc. Natl. Acad. Sci. USA, 82:2267 (1985)). Sequence information has established that CIF-A is identical to TGF-βl and that CIF-B represents a second isoform, TGF-β2. TGF-β is likely to be identical to Dicidual Suppressor Factor (DSF) (Lea et al, J. Immunol., 148:778 (1992)).
There are several well-defined isoforms of TGF-β; these disulfide- linked homodimers are designated numerically as TGF-βl through TGF-β5. A heterodimer, TGF-β 1.2, has been identified in porcine platelets. TGF-bl and TGF-β2 are produced in many cell types, while TGF-β3 is mainly expressed by cells of mesenchymal origin (Roberts and Sporn in Human Cytokines, Blackwell Scientific, p. 399 (1992); Lyons and Moses, Eur. J. Biochem., 187:467 (1990); Derynck et al, EMBO J., 7:3737 (1988); and Massague, Ann. Rev. Cell Biol., 6:597 (1990)). The amino acid homology between isoforms ranges from 70% (βl versus β2) to 79% (β2 versus β3). Each of the TGF-βl, TGF-β2 and TGF-β3 isoforms displays greater than 98% amino acid sequence homology between species (Massague (1990); and Kondiah et al, J. Biol. Chem.,
265:1089 (1990)). Detection of the TGF-β4 and TGF-β5 isoforms have been limited to chick embryo chondrocytes and Xenopus embryos, respectively (Roberts and Sporn in Human Cytokines, (1992)).
The isoforms of TGF-β are synthesized as larger protein precusors. Proteolytic cleavage at a site of basic amino acid residues yields the mature monomer consisting of the C-terminal 112-114 amino acids. A biologically inactive latent complex is formed from the non-co valent association of the mature TGF-β dimer and two prosegments that interact non-covalently (the latency associated protein, LAP) (Roberts and Sporn in Human Cytokines, (1992); Lyons and Moses (1990)). Subsequent to secretion from the cell source, activation must occur to release the biologically active dimer form. Whereas acidification can release the TGF-β dimer in vitro, in vivo activation is still a subject of research.
The biologically active TGF-β isoforms are all 25 kD dimers, which, under reducing conditions, yield monomers of 11.5 to 12.5 kD. Whereas the TGF-b precursor contains 3 to 4 N-glycosylation sites, none are present on the monomers constituting the mature TGF-β dimer. There are nine cysteines per monomer of mature TGF-β (Roberts and Sporn in Human Cytokines (1992)). Chou-Fasman analysis of TGF-β suggests an extensive b-sheet structure with very little a-helical character (Gamier et al., J. Mol. Biol, 120:97 (1978)).
A number of TGF-β receptors mediate the biological effects of the TGF- β dimer. Five TGF-β receptors have been identified A type I (53-65 kD), type II (83-110 kD), type III (250-310 kD), type IV (60 kD), and type V (400 kD) receptors (Segarini, Clinical Applications of TGF-b. Wiley, p. 29 (1991); O'Grady et al, J. Biol. Chem., 266:8563 (1991); Massague, Cell, 69:1067 (1992).; and Cheifetz et al, J. Biol. Chem., 263:17225 (1988)). The type I, II, III and V receptors are co-expressed on most cells examined, with the exception of a few tumor lines. The type IV receptor has been identified only on pituitary cells (Cheifetz (1988)). Since loss of cellular response to TGF-β correlates with loss of type I and /or type II receptors (Laiho et al., J. Biol. Chem., 266:9108 (1992)), these have been studied in the greatest detail.
Differential affinities of the receptors for TGF-βl, TGF-β2 and TGF-β3 exist between receptor types. For example, the majority of type I and II receptors bind TGF-βl and TGF-β3 with greater affinity than TGF-β2
(Massague (1992)). However, there is no direct relationship between binding affinity and biological potency since TGF-β2 is equipotent to
TGF-βl in many biological assays. Isoform potency is linked to many factors, including the combination of receptor types present, the number of each receptor type present, and the presence of type I and type II receptor subsets that bind all three isoforms with equal affinity (Massague (1992); Cheifetz (1988); Laiho (1992); and Cheifetz et al., J. Biol. Chem., 265:20533 (1990)).
The cloning of the type II receptor demonstrated that the cytoplasmic domain contains a functional serine/threonine kinase (Lin et al., Cell, 68:775 (1992)). Cloning of the type III receptor demonstrated that the betaglycan structure contains a short cytoplasmic domain without a signaling motif (Wang et al, Cell, 67:797 (1991)). The type III receptor may function as a reservoir for surplus TGF-b or as a regulator of ligand-binding ability or surface expression of the type I or II receptors
(Wang (1991)). Cloning of the other TGF-β receptors is necessary to determine whether they share a similar pattern of phosphorylation with the type II receptor. Other observations providing insight to the TGF-b signaling mechanism include:
1. induction of changes in Jun B, c-fos, and c-myc expression (Ohtsuki et al., Mol. Cell. Biol., 12:261 (1992));
2. prevention of cell-cycle dependent phosphorylation of the retinoblastoma protein (Ohtsuki (1992));
3. involvement of the guanine nucleotide-binding proteins (Diaz- Meco et al., Mol. Cell Biol, 12:302 (1992));
4. phosphatidyl inositol turnover (Segarini (1991)); and 5. translocation of protein Kinase C from the cytosol to the cell membrane (Segarini (1991)).
TGF-β functions both as an inhibitory and stimulatory factor. TGF-βl and TGF-β2 exert similar effects with only a few exceptions (Roberts and Sporn in Human Cytokines (1992); Rizzino, Dev. Biol., 130:411 (1988); Weinberg et al., J. Immunol., 148:2109 (1992)).
Stimulatory activities include:
1. stimulation of fibroblast and osteoblast proliferation;
2. enhancement of matrix protein synthesis by fibroblasts, osteoblasts, and endothelial cells;
3. induction of cytokine production by monocytes;
4. promotion of fibroblast, monocyte and neutrophil chemotaxis;
5. enhancement of in vivo effector function and memory phenotype of antigen-specific T helper cells; and
6. stimulation of IgA secretion from B cells.
Inhibitory activities include:
1. growth inhibition of lymphocytes, endothelial cells, hepatocytes, keratinocytes and certain tumor cell lines;
2. inhibition of IgG and IgM secretion from B cells;
3. inhibition of respiratory burst capacity in monocytes;
4. suppression of hematopoietic progenitors dependent on IL-3;
5. inhibition of megakaryotopoiesis;
6. inhibition of steriodogenesis in adrenocortical and Leydig cells; and,
7. inhibition of adipocyte and myocyte differentiation.
Expression of TGF-β isoforms in a time-dependent fashion during development and the ability to induce mesodermal marker in the developing Xenopus (Rosa et al, Science, 239:783 (1988)) may demonstrate a role for TGF-β in embryogenesis. Released at the site of a wound by platelet degranulation, TGF-β is reported to cause infiltration of other effector cells, protein matrix synthesis and secretion of other factors that combine with TGF-β to mediate in angiogenesis, and fibrosis associated with wound healing. Similarly TGF-β is thought to stimulate chongrogenesis and mediate bone fracture healing.
TGF-β has been associated with different forms of cancer (Macias et al..,
Anticancer Res., 7:1271 (1987).; Shirai et al, Japan J Cancer Res., 83:676 (1992); Reed et al , Am J Pathology, 145:97 (1994)) and the hepatic and pulmonary disease that often developes as a complication of chemotherapy and bone marrow transplantation procedures used to treat cancer patients (Kong et al, Ann Surg 222:155 (1995); and Murase et al, Bone Marrow Transplantation, 15:173 (1994); Anscher et al. N
Eng J Med, 328:1592 (1993). Increased TGF-β secretion by peripheral blood mononuclear cells (PBMC) from HIV-infected donors has also been reported (Kekow et al, J Clin Invest 87:1010 (1991). Therefore, any antagonist of TGF-β may be useful in treating these forms of cancer, as well as, may be useful in countering any of the numerous effects caused by TGF-β.
Summary of the Invention
This invention is based upon the discovery that prolactin has been found to counter the effect that TGF-β has on cells. An object of the present invention is to claim a method for treating a patient that may be suffering from a disease or disorder which is associated with the presence of TGF-β, by administering an effective amount of a pharmaceutically acceptable composition containing prolactin.
Another object of the present invention is to claim a method of preventing cell growth inhibition by TGF-β by administering an effective dose of a composition containing prolactin.
Another object of the present invention is a method of enhancing cell growth by administering an effective amount of a composition containing prolactin.
Brief Description of the Drawings
Figure la illustrates the effect of TGF-β on hybridoma Clone 5C6.
Figure lb illustrates the effect of prolactin on hybridoma Clone 5C6.
Figure lc illustrates prolactin's ability to overcome the effects of TGF-β on hybridoma Clone 5C6.
Figure 2 is a table containing the results of example 2.
Figure 3 illustrates prolactin's ability to overcome the effects of TGF-β on stimulated human PBL.
Detailed Description of the Invention
This invention is based upon the discovery that prolactin has been found to counter the effect of TGF-β on cells. It was discovered that prolactin countered TGF-β inhibitory effect on both mouse hybridoma cells and human peripheral blood mononulcear cells.
Definitions
As used herein, "prolactin" refers to a polypeptide obtained from tissue cultures or by recombinant techniques and other techniques known to those of skill in the art, exhibiting the spectrum of activities characterizing this protein. The word includes not only human prolactin (hPRL), but also other mammalian prolactin such as, e.g., mouse, rat, rabbit, primate, pig and bovine prolactin. The recombinant PRL (r-PRL) is preferred herein.
The term "recombinant prolactin", designated as r-PRL, preferably human prolactin, refers to prolactin having comparable biological activity to native prolactin prepared by recombinant DNA techniques known by those of skill in the art. In general, the gene coding for prolactin is excised from its native plasmid and inserted into a cloning vector to be cloned and then inserted into an expression vector, which is used to transform a host organism. The host organism expresses the foreign gene to produce prolactin under expression conditions.
As used herein, the term "patient" has its conventional meaning, i.e., one who is suffering from a disease or disorder and is under treatment for it (Stedmans Medical Dictionary, (1987)).
As used herein, the term "disease" has its conventional meaning, i.e., morbus: illness; sickness; an interruption, cessation, or disorder of the body functions, systems or organs (Stedmans Medical Dictionary, (1987)).
As used herein, the term "disorder" has its conventional meaning, i.e., a disturbance of function, stucture or both (Stedmans Medical
Dictionary, (1987)).
General Method
Formulations or compositions containing prolactin for counteracting the effects of TGF-β are most conveniently administered by injection, although other methods of administration are possible.
Standard formulations are either liquid injectables or solids which can be taken up in suitable liquids as suspensions or solutions for injection. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, and so forth. Nontoxic auxiliary substances, such as wetting agents, buffers, or emulsifiers may also be added.
Sustained and continuous release formulations are of considerable variety and could be used in the method of the present invention, as is understood by those skilled in the art.
In addition, because we have demonstrated that prolactin can counteract the effects of TGF-β, it is believed that the exogenous administration of the prolactin gene would result in the expression of prolactin in vivo which would be available to function to counteract
TGF-β whether administered through conventional means or via gene inoculation. The gene sequence for prolactin is disclosed in (U.S. Patent No. 4,725,549). This could be produced by inserting prolactin cDNA into a DNA delivery vehicle (e.g., plasmid vectors, liposomes, viral vectors). This could be accomplished as described by Pellegrini I., et al, Molec. Endocrinolgy, 6, 1023 (1992), Maniatis T., et al, Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Press (1989) and Felger P., et al, Proc. Natl. Acad. Sci., 84, 7413, (1991). EXEMPLIFICATION
Example 1: Dose Response of TGF-β and PRL
Mouse hybridoma cell lines (including 5C6) were maintained in Hybridoma Growth Media (HGM) consisting of 25% DMEM (Gibco), 40% NSO conditioned media, 10% NCTC 109 (Gibco), 15% FCS (Gibco), 0.1% ITS (Sigma 1-1884), 0.05 mM 2-mercaptoethanol, 2 mM L- glutamine (Gibco), 10% ORIGIN (Igen, Inc.). The cells were fed the day before use to ensure log phase growth.
The day of assay, cells were pipetted into 15 mL conical tubes and centrifuged at 1000 rpm for 8 minutes. The media was aspirated from the cells and they were washed twice with 5 mL of assay media consisting of 88% RPMI-1640 (Gibco), 10% FCS (Gibco), 2 mM L- Glutamine, 0.05 mM 2-mercaptoethanol, 100 U/mLPen/Strep (Gibco). The cells were resuspended in 2 mL of RPMI-1640 assay media, counted, and diluted with assay media to 2 x 10-^ cells/mL. A volume of lOOμL of cells was pipetted into wells of a 96-well sterile tissue culture plate. The plate was placed into a 37°C incubator until use.
A dilution series of recombinant human prolactin (Genzyme Corporation, lot 02 A, 800 μg/mL) and TGF-b (Genzyme Corporation, lot 94F003, 96μg/mL) were prepared in assay media. Twenty-five microliters of each reagent was added to appropriate wells. In all cases, the TGF-β was added first.
The cells were incubated at 37°C/5% CO2 for 72 hours and the amount of proliferation measured by tritiated thymidine incorporation. Tritiated thymidine (0.5μCi) was added for the last 18 hours. Cell- associated radioactivity was measured by scintillation counting (1205 Betaplate LSC, Wallac) after harvesting the cells onto glass fiber filters using a TOMEC Harvestor 96 .
The results are shown in Figures la-c. Figure la provides a graph demonstrating the dose-response for TGF-β on the proliferation of hybridoma cell. TGF-β can significantly inhibit the growth of these cells in a dose dependent manner.
Figure lb demonstrates the dose response of r-hPRL on this cell line. The molecule clearly stimulates the proliferation of these cells in a dose dependent manner.
Figure lc demonstrates the effect of incubating various concentrations of r-hPRL with 0.4 μg/mL TGF-β, a concentration of TGF-β which was demonstrated to inhibit proliferation by > 50% on these cells. r-hPRL antagonized the suppressive effect of TGF-β on these cells in a dose related manner.
Example 2: Effect of TGF-β and PRL on Murine Hybridoma Clones
Mouse hybridoma cell lines were maintained in Hybridoma Growth Media (HGM) as described above. The day of assay, cells were pipetted into 15 mL conical tubes and centrifuged at 1000 rpm for 8 minutes. The media was aspirated from the cells and they were washed twice with 5 mL of assay media consisting of 88% RPMI-1640 (Gibco), 10% FCS (Gibco), 2 mM L-Glutamine, 0.05 mM 2-mercaptoethanol, 100 U/mLPen/Strep (Gibco). The cells were resuspended in 2 mL of RPMI- 1640 assay media, counted, and diluted with assay media to 2 x 10^ cells /mL. A volume of lOOμL of cells was pipetted into wells of a 96- well sterile tissue culture plate and were placed into a 37°C incubator until use.
A ten-fold dilution series of TGF-β (Genzyme Corporation, lot 94F003, 96μg/mL) was prepared in assay media and twenty five microliters added to appropriate wells. Subsequently, lμg of PRL was added to the wells and the cells were incubated at 37°C/5% CO2 for 72 hours.
Tritiated thymidine (0.5μCi) was added for the last 18 hours. The amount of proliferation measured by tritiated thymidine incorporation was measured by scintillation counting (1205 Betaplate LSC, Wallac) after harvesting the cells onto glass fiber filters using a TOMEC Harvestor 96 .
The results are shown in the Table (Figure 2). The hybridomas varied in their sensitivity to TGF-β, but in all cases, a decrease in cell proliferation was observed in the presence of TGF-β. Prolactin's ability to overcome TGF-β suppressive effects was in part dependent upon the concentration of TGF-β. Prolactin was observed to overcome TGF-β's suppressive with all the hybridomas (8/8) incubated with 0.1 μg TGF-β, with 7/8 hybridomas incubated with 1 μg TGF-β, and with 5/8 hybridomas incubated with 10 μg TGF-β.
Example 3: Effect of TGF-β and PRL on Human Peripheral Blood Mononuclear Cells
Human peripheral blood mononuclear cells (PBMC) were isolated by Ficoll Paque (Pharmacia) gradient centrifugation. The cells were washed twice in PBS and resuspended in RPMI-1640 (Gibco) media containing 1% FCS (Gibco), 2mM glutamine (Gibco), 15mM HEPES
(Gibco), and 100 units/mL penicillin (Gibco) and lOOμg/mL streptomycin (Gibco). The cells were adjusted to a final cell density of 2xl06 cell/mL and 0.1 mL was plated into wells of microtiter tissue culture plates. The cells were incubated overnight in a 37°C, 5% CO2 humidifying incubator.
The next day, 20μg/mL PHA was added to the wells to stimulate the resting human peripheral blood lymphocytes (PBL). Immediately,
TGF-β was added to the cultures at concentrations of 0, 0.5 and lOng/mL TGF-β, followed by 0, 10, and 20μg/mL PRL.
The cultures were incubated for 96 hours and were pulsed for the last 8 hours with 0.5μCi Tritiated thymidine/well. The cells were harvested on a glass fiber filter using a TomTec Mach II cell harvester and incorporated radiolabel counted using a Wallac 1205 Betaplate Counter.
The results are shown in Figure 3. Prolactin antagonized the TGF-β suppression of PHA stimulated human PBL.