HK1213781B - POLYPEPTIDES DERIVED FROM TGFβ AND USES THEREOF - Google Patents

Description

Polypeptides derived from TGF β and uses thereof

Technical Field

In particular, the invention relates to the use of mutant variants of the TGF β molecule (antagonists of signalling to its natural homologues), and their therapeutic applications.

Background

The cytokine TGF β was discovered for its ability to stimulate Cell colony formation (Roberts AB et al, ProcNatl Acad Sci USA,78:5339-43,1981), and since this process is a typical marker of Cell transformation, this molecule is called transforming growth factor β. nowadays, ligand TGF β (TGF β 1, TGF β 2, TGF β 3) is considered the prototype of a multifunctional growth factor almost any Cell type in an organism produces them and expresses its receptor complex. these molecules are potent inhibitors of epithelial, endothelial and hematopoietic Cell proliferation (Ravitz MJ et al, Adv Cancer Res,71:165-207,1997), are potent inhibitors of extracellular matrix production and deposition (Massague J. the Annu Rev Biol,6:597-641,1990) and tissue repair cascades (Roberts et al, Plenum 275, 1996, Amnu Rev-Rev Biol, 1998; various embryonic developmental mechanisms of which also regulate the growth of cells, such as T-Cell differentiation, 35, J.35, 1998; Cell proliferation, J.75. J..

One of its most prominent functions in the immune system is the maintenance of lymphocyte homeostasis and immune tolerance by inhibiting the proliferation of naive T cells induced by self-antigens in a lymphocytopenic environment (Bevan M. et al, Nat Immunol, 27,13(7):667 73, 2012). this molecule also abolishes or modifies natural killer cells (Laouar, Y. et al, Nature Immunol,6: 600. Ampullo 607,2005), dendritic cells (Luo, X. et al, Proc. Natl Acad. Sci. USA,104: 2821. 2826, 2007; Bekeredjian-Dionce, I. et al, Mumnology, 128: 439. 2009, 450,2009), macrophages (Sina, A. et al, Semin. 18,349, I. et al, J. orgen. J. 11, 2009, 22. mura. J. 22. Ehrysol. 22. J. Ehrn. Ehryshula. 10, J. Ehrn. J. Ehrn. J. Ehrn. J. Ehrn, J. Ehrn. J. Ehrn, J. Ehrn, J. Ehrn. E. Ehrn. J. E. J. Ehrn, J. E. Ehrn. E. H. J. T.K, J. H. E. H. E. H.

Mature ligand TGF β is a homodimer with 112 amino acid residues derived from a precursor molecule formed by a latency associated propeptide towards the N-terminus (LAP: latent associated pro-peptide) and an active domain towards the C-terminus, these two domains are separated proteolytically within the cell and the ligand is secreted as an inactive precursor (formed by a prodomain reversibly linked to the active domain), thereby regulating its access to the cellular receptor (Geofrey D.Young and Joane E.Murphy-Ullrich, JBC, Vol.279, No.36: 38032-.

The three isoforms (TGF β, TGF β, TGF β) interact with the receptors T β RI, T β RII and T β RIII on the plasma membrane this last (also known as β glycan) is not expressed in all Cell types and although it may be absent for signalling mediated by the ligands TGF 6351 and TGF 6363, it constitutes a reservoir for these ligands when T β RII is saturated (Wang, X.F. et al, Cell,67:797-805, 1991; Lebrin F. et al, Cardiovasc. Cre., Res.,65:599-608, 2005. T β RIII forms a complex with the receptors T β 9 and T β RII to present them as such ligands bind first to T β RII and T β/RII complexes cooperate with the receptors T β RI 9 and T β -19-III to form a complex with the TGF-. 21-19-III binding to the TGF-19-III-23-three isoforms (TGF-three isoforms (TGF-three) may be present as separate isoforms).

It has not been reported so far that ligands TGF β, TGF β and TGF β 03 can bind to other type II receptors that are members of the same protein family to which T β RII belongs (Huang F and YG, Chen, Cell Biosc, Mar 15,2:9,2012), but they can bind to several type I receptors, ALK5 was described as a receptor of this ligand subfamily T β 2 RI. which, after its recruitment into complex T β RII/TGF- β, induced phosphorylation of the protein sm3672/3 (Huang F and YG Chen, Cell Biosc, Mar 15,2: β), ALK β was activated in response to the formation of complex TGF 72/T β RII in endothelial cells, and signaling through SMAD β and SMAD β was highlighted by the TGF receptor 3, TGF β 72, TGF β 3, TGF β 72, and b β 72, and TGF β 16, and b 16, and TGF β 72, which are related to the endothelial Cell receptor, further related (TGF β 3, TGF β 72, 3, and k3, TGF β 3, 7, 3, and k3, 7, 3, 7, respectively, and 3, and 3, which are related to the processes, respectively, related to which are also related to the biochemical, respectively, and 5, and 3, respectively, and the biochemical, a further related to the biochemical, a cardiovascular receptor, a cardiovascular protein, a further related to which are involved in vitro, a biochemical, a.

The abrogation of tumor progression operates primarily during the early stages of tumorigenesis, however, its role as a tumor-developing agent occurs in the late stages of carcinogenesis by inducing invasive and metastatic phenotypes and abrogating anti-tumor immune responses (Miyazono K., nat. Rev. Cancer,10: 415. 24, 2010; Miyazono K., Cancer Sci,101(2): 306. 12, 2010; Hawinkels LJ. et al, Growth Factors,29(4): 140. 52, 2011.) other examples where TGF β also has a deleterious effect are those caused by pathogens such as HIV, HBV, HCV, mycobacteria and trypanosoma (Triplosis trypanosoma), which contribute to the protective effects of these pathological conditions in the liver cirrhosis, especially the chronic sclerosis, TGF-induced by the pathogens affecting the liver cirrhosis, TGF-S-.

Most inhibitors have been evaluated in preclinical models, although some of them have begun to be tested in clinical studies in several types of cancer and fibrosis (Flavell RA., Nat Rev immunol.,10(8): 554-:

1-neutralization of the ligand, using soluble forms of the extracellular domain of its receptor (US 2002/0004037 and US 2007/0244042), anti-TGF β antibodies (US 2002/076858, US 2005/0276802) or oligonucleotides that block translation of the gene of the cytokine (US 2004/0006036 and US 2007/0155685);

2-block of signaling using small molecules that bind to the kinase domain of T β RI/ALK5 (US 2006/0057145, US 2005/0136043, US 2006/0234911);

3-blocking of receptor II, using antibodies that recognize its extracellular domain (US 2010/0119516, US 2009/7579186).

Inhibition strategies using muteins of the ligand TGF β as antagonists of signaling have not been reported to date.

Brief description of the invention

The present invention is based on the scientific discovery that mutant variants of the TGF β (TGF β, TGF β and TGF β) family can inhibit the function of their natural homologues the inventors have found for the first time in vitro experiments that mutant variants of TGF β can inhibit signalling induced by natural variants to a considerable extent and thereby neutralise their biological effects.

The present invention relates to polypeptides that share a primary sequence with human TGF β except for some amino acids that have been mutated to eliminate or substantially reduce their ability to signal through the type I receptor ALK5 these mutant variants of TGF β retain their ability to bind with high affinity to the receptors T β RII and T β RIII, but are unable to signal because they do not interact with the type I receptor ALK5, thereby negatively modulating the signaling of the natural variant by competing with the natural variant via binding to the high affinity receptors T β RII and T β RIII.

The novelty of the present invention is to provide a new therapeutic strategy to modulate TGF β signaling and thus the invasion and metastatic capacity of some tumors and the activity of some immune system and connective tissue cells in diseases where TGF β function reduces protective immune response (either native or induced by vaccination) or induces excessive tissue repair and fibrosis. none of the above mentioned inhibition strategies use a mutein of the ligand TGF β 0 as antagonist of signaling. mutants of TGF β 1 of the present invention are virtually self-proteins and therefore have low immunogenicity which reduces the risk of antibody response thereto to a minimum.

Detailed Description

The polypeptides of the invention have at least one mutation in the primary amino acid sequence thereof at one of the residues selected from the group consisting of Y6, W30, W32, I51, L51, Q67 and L101.

For position 6: a;

for position 30: n, R, K, D, Q, L, S, P, V, I, G, C, T, A, E, respectively;

for position 32: a;

for position 51: q, W, Y, A, respectively;

for position 67: H. f, Y, W, respectively; and

for position 101: A. and E.

Another object of the invention is a polypeptide wherein mutations are added at the interaction interface with the receptors TGF β RII and/or TGF β RIII to increase its affinity for said receptors.

The invention also relates to a pharmaceutical composition for treating cancer, diseases associated with fibrosis and chronic infectious diseases, characterized in that it comprises a polypeptide or a mixture of polypeptides as described in the invention and a pharmaceutically suitable carrier for the use thereof. The polypeptide may be covalently linked to a carrier protein, and the protein may be albumin or the Fc region of a human immunoglobulin. In another embodiment, the polypeptide of the invention may be pegylated.

Finally, the invention relates to the use of the described polypeptides for the preparation of a pharmaceutical composition useful in the treatment of cancer, diseases with fibrosis and chronic infectious diseases, and for enhancing the cellular and/or humoral response to a vaccine in place of native TGF β, especially when the vaccine to be enhanced is a therapeutic vaccine for cancer.

Obtaining similar Polypeptides from TGF β

The present invention relates to polypeptides of size 112 amino acids which retain high sequence identity, greater than 90% identity, to a variety of different native TGF β molecules comprising 1 to 6 mutations in one region of their sequence, relative to native TGF β the choice of residues to replace the original residues is selected so as to have very different physicochemical properties from the original amino acids, the substitution of non-polar residues to polar residues, charged residues to uncharged residues, large residues to small residues, acidic residues to basic residues.

These polypeptides (also referred to as TGF β muteins) were designed starting from the three-dimensional structure of native TGF β 1, TGF β and TGF β 3 in complex with its receptors T β RII and ALK5 (deposited in database PDB.) mutations were introduced only at the positions of TGF β that correspond to amino acids that are clearly exposed to solvents and that constitute part of the binding region with the receptor ALK5 (but not T β RII.) by using the well-known field bioinformatics procedures, these residues are predicted to be important in the interaction with ALK 5.

TABLE 1 amino acids of TGF β sequence that are important for interaction with T β RI/ALK5 and mutations that would make binding unstable according to theoretical predictions.

The polypeptides of the invention can be obtained by a variety of different routes, in particular by chemical synthesis of proteins. They can also be obtained by genetic engineering techniques, for example as inclusion bodies in bacteria such as e.coli (e.coli) or by expressing them in mammalian cells such as CHO and HEK293, using any transfection protocol accumulated in the prior art. Point mutations at specific positions can also be obtained by directed mutagenesis techniques by means of the polymerase chain reaction.

Analogous polypeptides of native TGF β selected by their biological activity

The polypeptides of the invention are selected starting from in vitro and in vivo experiments to have both the following properties:

these mutant variants of TGF β (also referred to as muteins in the present invention) retain their ability to bind to the receptor T β RII the binding ability can be assessed either directly by a commercially available ELISA assay for the chain of the receptor T β RII or indirectly on a population of cells positive for the receptor the level of recognition for the receptor T β RII should be comparable to native TGF β.

These muteins should block the binding of ligand TGF β (TGF β 1, TGF β 2, TGF β 3) to the receptor T β RII this can be measured directly by a competitive ELISA, in which plates are coated with each of the ligand TGF β (commercially available) and the ability of the muteins to inhibit the binding of T β RII to the ligand is evaluated.

These mutant variants of TGF β lose or substantially reduce their ability to signal through the receptor T β RI this property can be assessed directly in an immunodetection assay by Western, where the level of phosphorylated Smad2 and Smad3 is quantified in lysates of murine lineage tumor cells 4T1 and human lineage tumor cells MDA-MB231 treated with the mutant proteins or with native TGF β these mutant proteins should induce phosphorylation of Smad2 and Smad3 at least 100 fold less than native TGF β the mentioned properties can also be assessed in vitro in an inhibition assay for IL-2 induced proliferation of the cell line CTLL-2 these mutants should have at least 100 fold less inhibitory activity than native TGF β.

This property can be assessed directly in an immunoassay by Western, where the levels of phosphorylated Smad2 and Smad3 in lysates of murine lineage tumor cells 4T1 and human lineage tumor cells MDA-MB231 treated with the mutein or with native TGF β are quantified, the mutein should exhibit at least a 100-fold ability to inhibit signaling induced by a commercial ligand over a range of mutein concentrations of 50ng/mL to 10 μ g/mL.

These mutant variants of TGF β have an in vitro anti-tumor effect, they can inhibit the migration of several tumor cell lines, treated or not with natural ligands examples of these tumor cell lines are the murine cell line 4T1 and the human cell line MDA-mb231.

These mutant variants of TGF β lose or substantially reduce their ability to induce differentiation of CD4+ naive T cells towards the Treg foxp3+ or TH17 phenotype in the presence of IL-2 or IL-6 and IL-23, respectively.

These mutant variants of TGF β have the ability to inhibit differentiation of CD4+ naive T cells induced by their natural homologues into the Tregfoxp3+ or TH17 phenotype this property can be assessed directly in an in vitro induction assay of Treg and TH17 cells the muteins should exhibit at least a 100-fold ability to inhibit signaling induced by commercial ligands over a range of mutein concentrations from 50ng/mL to 10 μ g/mL.

These mutant variants of TGF β have an in vivo anti-tumor effect, they can inhibit tumor growth and metastasis ability of several tumor strains in vivo in a transplantable tumor model this property can be evaluated by using an orthotopic primary tumor model of the breast cell line 4T1 in BALB/c mice and an orthotopic primary tumor model of the human tumor cell line MDA-MB231 in immunodeficient mice.

These mutant variants of TGF β have the ability to increase the natural or vaccination-induced anti-tumor immune response in vivo in transplantable tumor models this property can be assessed by using orthotopic primary tumor model of the breast cell line 4T1 and subcutaneous primary tumor model of the colon tumor cell line CT26 in BALB/c mice.

The present invention includes additional modifications of the muteins of TGF β, which may mean the generation of mature domains lacking LAP without affecting their secretion, this implies that the muteins may be obtained in their active form for interaction with T β RII.

Among these modifications, fusion, PEGylation of any of the immunomodulatory polypeptides described above with a carrier protein (which may be albumin or the Fc region of a human immunoglobulin) is specifically included.

In a more particular embodiment, the invention relates to mutant variants of TGF β fused to the Fc fragment of human IgG1 (the specific mutations mentioned in Table 2) selected to exhibit the properties mentioned above the Fc region selected for coupling has a set of mutations in domain C γ 2 (ala234, ala235) that prevent interaction with Fc γ receptors (except neonatal Fc receptors) thereby silencing its ability to induce immune effector mechanisms (Labrijn AF et al, Currin Immunol., Aug,20(4):479 @ 85,2008, Epub 2008, 7/9).

However, they retain their ability to bind to T β RII and to bind to T β RIII intact, thus achieving the inhibitory ability of the activity of native TGF β.

TABLE 2 constructed mutants in which mutations are referred to according to the numbering of human TGF β.

Mutations	Reference name
		·W30E、L101E、I51Q	·G_M1
·W30E、L101A、L51Q	·G_M2
		·W30E、L101E、I51Q、Q67H	·G_M3
·W30E、L101A、K97D、E12H、I51Q、Q67H	·G_M4

In another aspect, the invention also includes additional modifications of the TGF β muteins, either to increase the affinity of the muteins for T β RII and T β RIII, but not to affect or even improve their inhibitory characteristics, or to improve the in vivo pharmacodynamics of the muteins by increasing longevity.

Therapeutic use of TGF β analogous polypeptides

The invention also includes pharmaceutical compositions comprising as active ingredients the muteins of TGF β and analogues or fusion proteins thereof disclosed by the invention, as well as their possible therapeutic uses, aimed at modulating the invasion and metastatic capacity of some tumors and the activity of some immune systems and connective tissue cells in diseases where the function of TGF β reduces the protective immune response (either natural or induced by vaccination) or induces excessive tissue repair and fibrosis.

For its therapeutic use, the polypeptide of the invention should be administered to a subject carrying the disease, either alone or in combination with other polypeptides or other substances that facilitate or enhance its therapeutic effect. The route of administration may be any of the routes of administration described in the prior art for parenteral administration of drugs. Administration may preferably be by intravenous, intramuscular, subcutaneous or intratumoral route.

The polypeptides or fusion proteins described by the present invention may also be administered by being part of a pharmaceutical composition useful in the treatment of cancer, diseases with fibrosis and chronic infectious diseases. Preferably, the present invention includes compositions, including pharmaceutical compositions, comprising a pharmaceutically acceptable carrier.

The composition comprises a pharmaceutically acceptable carrier. Among the pharmaceutically acceptable carriers include, but are not limited to: saline, sterile water, phosphate buffered saline, and the like. Other buffers, dispersing agents and non-toxic inert materials suitable for administration to a patient may be included in the compositions of the present invention. The composition may be a solution suitable for administration, and is generally sterile and free of undesirable particles.

In one aspect, the invention also relates to a method of treatment comprising administering to a subject having cancer, a disease with fibrosis, or a chronic infectious disease a therapeutically effective amount of a polypeptide, fusion protein, or composition described in the invention. In a particular embodiment, the subject is a human.

The polypeptides or fusion proteins described by the present invention can also be administered in combination with traditional oncology therapies (chemotherapy and radiotherapy) as enhancers of their effects.

In order to obtain the desired therapeutic effect, the polypeptide of the invention should be administered at a dose high enough to ensure its concentration at the lymph nodes or peripheral sites important for the disease under investigation, within such a range of concentrations for which the mutein shows an inhibitory effect against native TGF β.

The number of administrations to be applied should also be adjusted to suit the biodistribution of the mutein under consideration. In general, it should be achieved that the above-mentioned effective concentrations last from 2 to 30 consecutive days. If the mutein is coupled to a carrier protein, the frequency of its administration should be adjusted accordingly.

When the compounds or compositions of the invention are used in combination with other anti-cancer producing agents, the invention provides, for example, simultaneous, staged or alternating treatment. Thus, the compounds of the invention may be administered simultaneously in separate pharmaceutical compositions, and wherein the compounds of the invention may be administered before or after the other anti-cancer producing agent, e.g., by seconds, minutes, hours, days or weeks.

Therapeutic effect is to be understood as a complete or partial relief of the symptoms of the disease. In cancer, a decrease in tumor volume or an increase in time to recurrence is especially a criterion for remission of the disease.

The advantages of this new therapeutic strategy over other proposals for modulating TGF β signaling are multiple.

This factor minimizes the risk of an antibody response thereto, since the mutant of TGF β is actually a self protein (except for few mutations) and thus has low immunogenicity.

A high degree of specificity for the receptor system reduces unwanted toxicity (this is common in strategies based on small-size inhibitors).

These mutant variants of TGF β retain a binding affinity to the receptor T β RII of at least the order of the affinity (6-10pM) of native TGF β 1 and TGF β 3, which affinity is difficult to exceed with receptor or ligand inhibition strategies, monoclonal antibodies or other drugs.

These mutant variants of TGF β have a smaller size than monoclonal antibodies and Fc-coupled receptors this ensures greater permeability to tumors in the therapeutic context of oncological diseases than the drugs described above which inhibit TGF β signalling.

These mutant variants of TGF β inhibit signaling of any ligand that signals through the receptor system T β RII/T β RI (ALK5), a theoretical advantage over pan-ligand antibodies (pan-ligand antibodies) in that these mutant variants are directed only against the known TGF β isoforms, which may not be inhibited by these antibodies if other isoforms are present.

These mutant variants of TGF β retain the ability to interact with the receptor T β RIII this constitutes an advantage over antibodies against the receptor T β RII because the muteins block all possible binding sites of the natural ligand to the cell surface.

Linking to the Fc fragment increases the half-life of the mutein, which gives it an advantage over small-sized drugs, since the dose and its frequency will be less. This reduces undesirable toxicity.

Linking to the Fc fragment of human IgG1, which does not interact with Fc γ receptors (except for neonatal Fc receptors), avoids triggering immune effector mechanisms for non-tumor host cells, thereby reducing the potential for unwanted toxicity compared to antibodies against receptor II, which retain their ability to interact with Fc γ receptors intact.

The invention will be further elucidated with the following examples and figures. However, these examples should not be construed as limiting the scope of the invention.

Brief Description of Drawings

FIG. 1 immunodetection assay by Western of mutein G _ M1 and native TGF β SDS-PAGE of these proteins was performed under non-reducing and reducing conditions and stained with an antibody against human TGF β 1A first lane corresponds to low molecular weight standards, a second lane corresponds to mutein G _ M1, a third lane corresponds to native TGF β 1, and a fourth lane corresponds to unrelated antibody hR 3.

Figure 2. mutant protein G _ M1 retained its ability to bind to receptor T β RII, similar to that of native TGF β 1, as a negative control, using the unrelated antibody hr3.

FIG. 3. inhibition assay of proliferation of IL-2 dependent cell line (CTLL-2) induced by mutated or native TGF β it was demonstrated that the ability of the mutein to inhibit proliferation of cell line CTLL-2 is much lower than native human TGF β 1 data are expressed as mean percent inhibition of 3 independent experiments.

FIG. 4. assay for evaluating the ability of mutein G _ M1 to neutralize inhibition of the IL-2 dependent cell line (CTLL-2) induced by native TGF β 1 the graph shows the average percent cell proliferation of 3 independent experiments.

FIG. 5 mutein G _ M1 inhibited the migration of the tumor cell line 4T1 in vitro. As indicated in the figure, photographs of healing were taken at different time intervals, which shows that healing was less at each time point when cells were treated with the mutant protein.

FIG. 6. mutein G _ M1 inhibited the differentiation/transformation of CD4+ CD25-Foxp 3-naive T cells induced by native TGF β into CD4+ CD25+ Foxp3+ regulatory cell phenotype when cells were incubated with 500ng/ml of the mutein, the percentage of regulatory T cells recovered from the T cell culture dropped almost to zero (almost complete reduction) — this concentration was only 30-fold that of recombinant human TGF β 1 used to induce transformation.

Examples

Example 1 design of a mutein of TGF β

The mutein was designed in silico starting from bioinformatics technology using the reported structure of the ternary complex of human TGF β 1 and TGF β 3 with the TGF β receptor as a basis the binding energy of the natural isoform and all possible mutant variants was determined by using bioinformatics programs in the public domain the mutein G _ M1 was expressed in CHO-K1 cells starting from a gene construct in the lentiviral vector PLW and including at the C-terminus the hinge region and domains C γ 2 and C γ 3 of human IgG1 and the histidine tail G _ M1 was purified by protein A affinity chromatography (FIG. 1) and obtained in high purity (> 95%).

Example 2 ability of mutein G _ M1 to bind to T β RII

An ELISA assay was performed in which T β RII (1. mu.g/ml) was coated and revealed with an anti-human Fc antibody coupled to alkaline phosphatase FIG. 2 shows that the mutein G _ M1 retains its ability to bind to the receptor T β RII, similar to that of native TGF β 1, using the irrelevant antibody hR3 as a negative control.

Example 3 mutant protein G _ M1 has reduced ability to signal through the receptor T β RI (ALK5) and thus mimics the biological activity of native TGF β

Inhibition of proliferation of the IL-2 dependent cell line CTLL-2 induced by mutated or native TGF β was compared cells/well of 5000 cell lines CTLL-2 were stimulated with 50U/ml of commercial IL-2 and a defined concentration of commercial rTGF β 1 or mutein G _ M1 for 48 hours then Almar Blue (Alamar Blue) reagent was added to the cultures and reduction was measured at 540 and 630 nm.

The mutein G _ M1 was unable to inhibit the proliferation of the cell line CTLL-2, at least at a concentration 200 times that of commercial TGF β 1 used as a positive control for the experiment (FIG. 3), so the mutein was much less able to inhibit the proliferation of cells of the cell line CTLL-2 than commercial TGF β 1.

Example 4 mutein G _ M1 is an antagonist of TGF β signaling in vitro

Cells/well of 5000 cell lines CTLL-2 were stimulated with 50U/ml of commercial IL-2 and cultured in the presence of 2pM of commercial TGF β 1 and a defined concentration of mutein G _ M1, isotype control antibody hR3 or positive control, i.e. anti-TGF β 1 antibody after 48 hours alamar blue reagent was added to the culture and reduction was measured at 540 and 630 nm.

Mutein G _ M1 (but not hR3) neutralizes the inhibition of the proliferation of the cell line CTLL-2 induced by commercial TGF β 1 (fig. 4).

Example 5 mutein G _ M1 has an in vitro antitumor Effect

The ability of the mutein G _ M1 to inhibit migration of the murine tumor cell line 4T1 was evaluated in an in vitro assay using the method of wounding in a monolayer of adherent cells (Liang CC et al, Nat Protoc.,2(2):329-33, 2007.) confluent cells were injured by scraping cell monolayers with a sterile pipette and either the mutein G _ M1 or commercial TGF β 1 (negative control) was added to the wells. it was observed in FIG. 5 that the rate of closure was significantly reduced when the cells were treated with the mutein (p <0.05, Kruskal-Wallis test and Dunn's post hoc test at each time point).

Example 6 mutein G _ M1 inhibits differentiation of conventional naive T cells

The CD4+ CD 25-GITR-naive cell population of the spleens of 4C 57/BL6 mice was purified by flow cytometry 5X 10 stimulated with 5ng/mL IL-2 and 5ng/mL TGF β in the presence of 3. mu.g/mL anti-CD 3 attached to the plate and 3. mu.g/mL soluble anti-CD 28⁴And (c) a plurality of such cells. The muteins were evaluated at concentrations of 250 and 500 ng/mL. Under these conditions, the percentage of conversion to induced regulatory T cells was 55.6%. The percentage of recovered Foxp3+ regulatory T cells was measured in the presence or absence of the mutein. Figure 6 shows how the percentage of regulatory T cells recovered in T cell culture in the presence of the mutein G _ M1 dropped almost to zero.

Claims (8)

1. A mutant TGF β 1 polypeptide that antagonizes the activity of ligand TGF β 1 mediated by receptor ALK5, wherein the mutations in the polypeptide are W30E, L101E and I51Q.

2. The polypeptide according to claim 1, characterized in that it is fused to the Fc fragment of human IgG1 in which the amino acids at positions 234 and 235 in domain C γ 2 are alanine.

3. A pharmaceutical composition for the treatment of cancer comprising at least one polypeptide according to claim 1 in the range of 50ng/mL to 10 μ g/mL and a pharmaceutically suitable carrier.

4. The pharmaceutical composition of claim 3, wherein the polypeptide is covalently linked to a carrier protein.

5. The pharmaceutical composition of claim 4, wherein the carrier protein is the Fc region of a human immunoglobulin.

6. The pharmaceutical composition of claim 3, wherein said polypeptide is PEGylated.

7. Use of a polypeptide according to claim 1 for the preparation of a medicament useful in the treatment of cancer and diseases associated with fibrosis.

8. Use of a polypeptide according to claim 1 in the manufacture of a medicament useful for enhancing cellular and/or humoral responses to a vaccine that is a therapeutic vaccine for cancer.