HK1182403B - Polypeptides derived from il-2 having agonist activity, for the therapy of cancer and chronic infections - Google Patents

Description

IL-2-derived polypeptides with agonist activity for the treatment of cancer and chronic infections

Technical Field

The present invention relates to immunology. In particular, the invention relates to the therapeutic use of modulating the immune system by analogues of natural molecules which have agonist effects of the original molecule, yet surprisingly show superior therapeutic efficacy.

Background

Interleukin 2 (IL-2) was the first growth factor described for T cells. Since its discovery, its ability to promote proliferation and survival of T cells in vitro was observed (Smith, K.A. (1988) science.240, 1169-76), and its ability to enhance T immune responses in the context of viral infections (Blattman, J.N. et al, (2003) Nat Med.9, 540-7) or vaccines (Fishman, M. et al, (2008) JImmunether.31, 72-80; Kudo-Saito, C. et al, (2007) Cancer Immunol Immunol.56, 1897-910; Lin, C.T. et al, (2007) Immunol Lett.114, 86-93). However, this classical role of IL-2 as a promoter of the T immune response has recently been suspected by a number of experimental data (Almeida, A.R. et al, (2002) J Immunol.169, 4850-60; de la Rosa, M. et al, (2004) Eur J Immunol.34, 2480-8; Malek, T.R. et al, (2004) NatRev Immunol.4, 665-74), which show that this cytokine is an internationally stable growth factor for naturally regulated T cells CD4+ CD25+ FoxP3+ T (Tregs).

Interleukin 2 is also proposed to be a significant role in the mechanism by which T cells are regulated to inhibit the activity and expansion of other effector cells, such as helper CD4T cells, cytotoxic CD8T cells and Natural Killer (NK) cells. In particular, it has recently been proposed that regulatory T cells inhibit other T cells by inducing a local decrease in IL-2 levels (Pandiyan, p. et al, (2007) Nat immunol.8, 1353-62). The inhibitory effect is based on: a) it directly inhibits the ability of effector T cells it inhibits to produce new IL-2 (Almeida, a.r. et al, (2002) J immunol.169, 4850-60; takahashi, T. et al, (1998) Int Immunol.10, 1969-80; thornton, A.M. et al, (1998) JExp Med.188, 287-96; wolf, M. et al, (2001) Eur J Immunol.31, 1637-45); b) the ability to rapidly sequester, internalize and degrade IL-2 present in its microenvironment (Pandiyan, p. et al, (2007) Nat immunol.8, 1353-62); and c) its ability to overexpress the alpha-chain of the IL-2 receptor (Kuniyasu, Y., et al, (2000) IntImmunol.12, 1145-55), which enables more efficient use of IL-2 when having low concentrations of IL-2.

In summary, IL-2 is a cytokine with highly pleiotropic properties that is of great importance in the biological activities of different cell populations. This property makes IL-2 an important node in the regulation of immune responses, which turns it into an attractive and complex target for immunomodulatory therapies.

IL-2 has been used in the treatment of cancer for several years. In particular, its use at high doses is a therapy approved in several countries for the treatment of metastatic melanoma and renal cancer. However, the direct use of IL-2 in patients is severely limited by its toxic effects and low potency. So that only 20% of eligible patients receive the treatment and only 17% of treated patients show significant objective responses. A possible explanation for this dramatic failure in the clinic is that treatment with native IL-2 also stimulates the regulatory T cell population (Ahmaddzadeh, M. et al, (2006) blood.107, 2409-14), which acts inversely on the immune stimulation it is intended to use. Now, a number of preclinical evidence support this idea. In particular, experiments in murine models have shown that the primary activity of in vivo injected IL-2 is an expansion of the homeostasis of natural regulatory T cells.

Several strategies have been developed with the aim of reducing the toxic effects of treatment with IL-2. Some of these strategies are based on the use of mutant variants of IL-2 designed to increase the ability of the molecule to signal primarily through high affinity receptors (alpha, beta and gamma chains) rather than through intermediate affinity receptors (beta and gamma chains). The basic idea is to promote signaling in T cells rather than in NK cells, which are cells thought to be responsible for the observed toxic effects. In this series of work, the following inventions were included: us patent 7,186,804, us patent 7,105,653, us patent 6,955,807, us patent No. 5,229,109, us patent application 20050142106. In any event, it is important to note that none of these inventions relates to muteins of IL-2 that have greater therapeutic efficacy in vivo than native IL-2, based on their reduced ability to stimulate native regulatory T cells.

Other mutant variants of IL-2 have been made with the aim of increasing their pharmacological activity. For example, improve their folding or increase their survival in blood. The following invention is particularly in this series of work: U.S. Pat. No. 4,959,314, U.S. Pat. No. 5,116,943, U.S. Pat. No. 4,853,332. Again, none of these muteins have a reduced ability to activate regulatory T cells, nor show greater therapeutic efficacy.

Finally, it should be mentioned that there are a number of proposals for therapeutic agents in the literature (adjustment of activity of cells in Kreitman, R.J. (2009) Curr Pharm Des.15, 2652-64; Litzinger, M.T., Fernando, R., Curiel, T.J., Grosenbach, D.W., Schlom, J. and Palena, C. (2007) blood.110, 3192-201; Morse, M.A., Hobeika, A.C., Osada, T.Serra, D., Niedzwiecki, D.D., Lyerly, H.K. and Clay, T.M. (2008) blood.112, 610-8; Tawarka, S.a, I., Shikazu, J.J., Saguchi, S.M., Fujita, T.M.112, K. 1999), and Klein.19, T.J.19, Onzawa, S.84, C., and C. (1937) in vivo, regulation of cells in Kreitan, Katsuwa, I., Shikazu, Shirkia, J.S.59, and Reiz. These therapeutic agents have been tested in animal models and even in patients, either for direct treatment of cancer or for enhancing vaccine effect. There are also reports that suggest modulating the activity of IL-2, in particular more effective immunization with monoclonal antibodies (Boyman, o., Kovar, M., Rubinstein, m.p., Surh, c.d., and Sprent, J. (2006) science.311, 1924-1927; Boyman, o. et al, (2006) Expert Opin Biol ther.6, 1323-31; Kamimura, d. et al, (2006) jimmuol.177, 306-14; Murakami, M., Sakamoto, a., Bender, J., Kappler, J. and Marrack, P. (2002) Proc Natl Acad usa.99, 8832-7; Tomala, J., chlevela, h., mkvan, t., rivava, b. and jw. (2009), kommu.04), or more effective to promote responses. However, to our knowledge, there is no report in the literature of any mutant variant of IL-2 based on which it is shown that the possibility of obtaining greater therapeutic efficacy is based on its reduced ability to stimulate natural regulatory T cells.

Brief description of the invention

The present invention relates to the acquisition of mutant variants of IL-2 that exhibit greater therapeutic efficacy than native IL-2 in murine transplantable tumor models. These muteins are characterized in that: it is an incomplete agonist of the activity of IL-2 and is selected for its particularly low ability to stimulate natural regulatory T cells (CD 4+ CD25+ FoxP3+ T) in vitro and/or in vivo. The in vivo therapeutic efficacy of these muteins represents a practical solution for improving the treatment with IL-2 in malignancies. In particular, these muteins make it possible to overcome the limitations observed in therapy with native IL-2, which arise from their demonstrated ability to expand native regulatory T cells in vivo.

The present invention relates to polypeptides having the same primary sequence as human IL-2, except that several amino acids are mutated. The introduced mutations substantially reduce the ability of these polypeptides to stimulate regulatory T cells (CD 4+ CD25+ FoxP3+ T) in vitro and in vivo and confer greater efficacy in the treatment of murine transplantable tumors. The invention also includes the therapeutic use of these mutant variants (alone or in combination with a vaccine) for the treatment of diseases such as cancer or infections in which the activity of regulatory T cells (Tregs) is relevant.

The present invention allows substantial improvement of the current IL-2 based immunomodulating strategies, both in the direct treatment of cancer and in its combination with different vaccines. In particular, the replacement of native IL-2 with the mutant variants described in the present invention enables the avoidance of expansion of regulatory T cells which significantly diminish the desired therapeutic effect.

Detailed Description

Obtaining analogous polypeptides to IL-2

The present invention relates to polypeptides of 100 to 500 amino acids, preferably 140 amino acids in size, having an apparent molecular weight of at least 15 kD. These polypeptides retain high sequence identity, greater than 90% identity, to native IL-2, comprising 3 to 6 mutations in one region of their sequence (compared to native IL-2). At said positions, these polypeptides are mutated by introducing an amino acid residue that is different from the amino acid residue present at the same position in the native IL-2. Residues that replace the original residues are chosen for having very different physicochemical properties than the original amino acid, in particular polar residues to non-polar residues, charged residues to uncharged residues, large residues to small residues, acidic residues to basic residues.

The polypeptides of the invention may be referred to indiscriminately, in particular as immunomodulating polypeptides, analogues of IL-2 or muteins of IL-2. These polypeptides were designed starting from the 3D structure of the IL-2-receptor complex (available in public database PDB) by introducing mutations preferably at positions of IL-2 corresponding to amino acids that are clearly exposed to the solvent and highly conserved among IL-2 of different species (sequences obtained from the database Swissprot). Solvent-exposed amino acids of the type mentioned above are identified by using bioinformatic procedures for visualization of protein structure, such as RASMOL, swisspdbeviewer, etc. Conserved positions in the sequence of IL-2 are identified by using bioinformatic programs such as Fasta, ClusterW, etc., for multiple alignments of sequences.

The polypeptides of the invention can be obtained by different routes, in particular by protein synthesis. They can also be obtained by genetic engineering techniques, for example by expressing them in bacteria, such as in particular escherichia coli (e.coli), in mammalian cells, such as in particular NSO cells. Point mutations at specific positions can also be obtained by targeted mutagenesis techniques with the aid of the polymerase chain reaction.

Surprisingly, the inventors have found substantial advantages of these muteins over the use of traditional native IL-2. This advantage lies in improving its efficacy in antitumor therapy, which results from its ability to avoid the expansion of regulatory T cells.

Selection of analogous polypeptides for IL-2 with regard to their biological activity

The polypeptides of the invention are selected by the following properties:

1) agonist action of native IL-2. This property can be assessed directly in vitro proliferation assays using IL-2 dependent cell lines (e.g., CTLL2 or Kitt 225), or in assays using a mixture of mouse and/or human T lymphocytes. In these experiments, these mutant proteins should have 5 to 50 times lower specific stimulation activity than native IL-2.

2) (ii) a loss of ability to stimulate a population of regulatory T cells in vitro and/or in vivo, as compared to native IL-2. This property can be assessed, for example, by studying the ability of the muteins of the invention to directly induce the expansion of CD4+ CD25+ T cells (which were purified from naive mice and stimulated in vitro with anti-CD 3 antibody) compared to native IL-2. The effect of intraperitoneal or subcutaneous injection of these muteins or native IL-2 in mice over a five day period in terms of expansion or increase in the proliferation rate of the regulatory T cell population (CD 4+ CD25+ FoxP3+ T) can also be evaluated. In these assays, the activity of mutant IL-2 on regulatory T cells should be at least 1000-fold less than native IL-2.

3) Increased therapeutic effect compared to native IL-2 in an animal model. This property can be assessed, for example, by comparing the anti-tumor or anti-metastatic effects of the mutant protein and native IL-2 alone in an implantable tumor model (e.g., B16 melanoma). It can also be assessed by enhancing the effect on the cellular and/or humoral response of the vaccine of interest. These muteins should exhibit greater therapeutic efficacy than native IL-2 at doses containing the same total protein mass of IL-2 and the muteins.

The invention relates in particular to the muteins described in detail in table 1. These muteins comprise a plurality of amino acid substitutions which in their entirety confer the above-mentioned properties. Table 1: constructed muteins having the three basic properties described in this patent.

The mutations are indicated by the numbering of human IL-2.

Mutations
	R38K、F42I、Y45N、E62L、E68V
R38K、F42Q、Y45E、E68V
	R38A、F42I、Y45N、E62L、E68V
R38K、F42K、Y45R、E62L、E68V
	R38K、F42I、Y45E、E68V
R38A、F42A、Y45A、E62A

The present invention also includes additional modifications in addition to the classes of IL-2 muteins mentioned above, in particular, described in table 1. Or to increase its affinity for specific components of the IL-2 receptor without affecting or even improving its agonist characteristics of not stimulating regulatory T cells; or to improve its pharmacodynamics in vivo: increase survival or decrease internalization thereof by T cells. These additional mutations can be obtained via rational design using bioinformatic tools, or by using combinatorial molecular libraries of different nature (phage libraries, gene expression libraries in yeast, or gene expression libraries in bacteria).

In another aspect, the invention relates to a fusion protein comprising any one of the immunomodulatory polypeptides described above coupled to a carrier protein. The carrier protein may be albumin or the Fc region of a human immunoglobulin.

Therapeutic use of IL-2-like polypeptides

The invention also includes pharmaceutical compositions comprising as active ingredients the muteins of IL-2 and analogs thereof disclosed herein; as well as their possible therapeutic use, with the aim of enhancing the immune response native or induced by vaccines in diseases such as cancer or chronic infections in which regulatory T cells are particularly relevant.

For its therapeutic use, the polypeptides of the invention should be administered to a subject carrying the disease, independently or in combination with other polypeptides or with other substances which contribute to 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 a drug. Preferably, administration may be by intravenous, intramuscular, subcutaneous or intratumoral routes.

The polypeptides described in the present invention may also be administered by replacing native IL-2 as part of a pharmaceutical composition useful in the treatment of cancer and chronic infectious diseases or for enhancing the cellular and/or humoral response to a vaccine. The polypeptides of the invention may be used in combination with a therapeutic vaccine for cancer or a prophylactic vaccine in infectious diseases in which regulatory T cells are associated.

To obtain the desired therapeutic effect, the polypeptide of the invention should be administered in a sufficiently high dose to ensure its concentration in the lymph nodes or peripheral sites of great significance for the disease under investigation, within the concentration range at which the mutein shows an immunostimulatory effect. Thus, the dosages considered should be adjusted according to the type of disease studied and the route of administration. For example, in the case of tumor treatment, the dose should be adjusted to achieve a concentration of the mutein within the tumor and/or in regional lymph nodes that ensures stimulation of the anti-tumor immune response. The dosage range to be explored may vary from hundreds of micrograms/dose to hundreds of milligrams/dose. For those applications in which the mutein replaces traditional therapies using native IL-2, the mutein dose to be used should be smaller than or equivalent in activity (determined by using an assay using the CTLL2 cell line) to the dose traditionally used for native IL-2.

The number of administrations to be performed should also be adjusted according to the biodistribution of the mutein under consideration. In general, it should be achieved that the above-mentioned effective concentration is maintained for a period of 2 to 30 consecutive days. For example, it should be noted that if a mutein is coupled to a carrier protein, its frequency of administration should be adjusted accordingly. For applications in which native IL-2 is replaced, the mutein may be administered in a regimen similar to that administered in conventional therapy.

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

The polypeptides of the invention are particularly useful in the treatment of tumors, such as, inter alia, melanoma and renal tumors.

Brief Description of Drawings

FIG. 1 acquisition of mutant proteins. a) Expression of the mutant protein in E.coli BL21DE3 strain as assessed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), lane 1: total protein of BL21DE3 strain, negative control for expression, lanes 2 and 3: two examples of expression levels obtained in this strain, the arrow indicating the band corresponding to the mutein; b) a reverse phase chromatogram showing the main final purification step of the protein, arrows indicating the peaks corresponding to the protein of interest; c) purification of the mutant protein assessed by SDS-PAGE, 1: results of the semi-purification process by washing the cell pellet, 2: the mutant protein obtained after purification by reverse phase chromatography.

FIG. 2 evaluation of agonist characteristics of muteins of IL-2. a) The ability of the mutant proteins to bind to the surface of the CTLL2 cell line was measured by flow cytometry. Both IL-2 and the mutant protein were detected by using mAbs specific for the 6His sequence present in the recombinant protein. b) The figure shows the ability of the mutein to induce the proliferation of the IL-2 dependent CTLL2 cell line compared to native IL-2. The proliferation was measured by incorporation of MTT.

FIG. 3 mutant proteins do not induce proliferation of regulatory T cells in vitro. a) A flow cytometry plot showing the purity of the CD3+ CD4+ CD25+ population purified from the lymph nodes of C57BL6 mice; b) treg cells were stimulated in vitro with anti-CD 3 mAb and were administered either native IL-2 at a concentration of 0.5ng/mL or mutein at a concentration of 32ng/mL over a 72 hour period, which shows the number of viable cells recovered after each treatment compared to controls where no cytokine was added. The concentrations chosen correspond to the concentrations at which each molecule induced proliferation of the same CTLL2 cell line.

FIG. 4 evaluation of the effect of treatment with the mutant proteins on the proliferation of lymphocyte populations. a) Quantification of relative weights of spleens of mice treated with the mutein for five days. The weight of the spleen of the treated mice was statistically greater than the control group. The Kruskal-Wallis nonparametric test and Dunn multiple comparison test were used. b) Measurement of the CD8+ T lymphocyte population, the figure shows the percentage of this population.

FIG. 5 mutant proteins were more effective in reducing metastasis than native IL-2 in an experimental metastatic model of the MB16F0 melanoma cell line. a) Representative photographs of the lungs corresponding to each treatment. b) Quantification of lung metastasis in each group.

Figure 6 administration of the muteins in combination with the OVA/VSSP vaccine enhanced the anti-tumor effect of the vaccine. EG7 tumor bearing mice were treated with OVA/VSSP vaccine (alone or in combination with mutant proteins). The figure shows tumor growth, and the group treated with the combination of vaccine and mutein shows a reduction in tumor size that is statistically different from the control group.

Examples

Example 1 design of muteins of IL-2

Mutant proteins were designed with the aid of a computer in accordance with bioinformatics techniques by using as a basis the structure of human IL-2 reported in database PDB (protein Data Bank) and the amino acid sequences of IL-2 of different species available in database Swissprot. Several mutant proteins were designed which contained 3 to 6 mutations (introducing non-conservative amino acid substitutions) at residues that were solvent-exposed and highly conserved in evolution. In E.coli, these muteins were expressed from among the gene constructs in the vector pET28a, which contained a recognition sequence with 6 histidines at the amino terminus. The mutein was purified by reverse phase chromatography (fig. 1) obtained in high purity (> 95%). The mutant proteins obtained were selected according to their properties in vitro and in vivo experimental tests to show the 3 basic properties described in the main body of the invention. Among all the mutant proteins constructed, a whole set of specific mutations are described in Table 1, which have the desired property of being agonists of IL-2 activity without significantly stimulating regulatory T cells and showing greater therapeutic efficacy than native IL-2 in the treatment of murine transplantable tumors. Table 2 indicates other mutant proteins constructed, but they do not show the desired properties.

Table 2: a constructed mutein which does not have the essential characteristics described in this patent.

The mutations are indicated by the numbering of human IL-2.

Mutations
	Q22V、Q126A、I129D、S130G
L18N、Q126Y、S130R
	Q13Y、Q126Y、I129D、S130R
L18N、Q22V、T123A、I129D、S130R
	R38A、F42A、Q126Y、I129D
Q126Y、I129D、E62L、E68V

Example 2 demonstration of the agonist characteristics of the muteins of the designed IL-2

FIG. 2 illustrates how the muteins mentioned in Table 1 bind to components of the IL-2 receptor on the surface of the CTLL2 cell line (FIG. 2 a). The constructed mutant proteins bound to CTLL2 cells, which are known to have high and medium affinity receptors for IL-2 on their surface. As a result, the binding detected in our assay was similar to that obtained with native IL-2. FIG. 2b illustrates the ability of the muteins given in Table 1 to stimulate the growth of the CTLL2 cell line (FIG. 2 b). As a result, in this test, these muteins were not full agonists of the activity of IL-2. Their specific activity is less than native IL-2 to a 5 to 50 fold extent.

Example 3 Effect of muteins of IL-2 on regulatory T cells

The muteins described in table 1 showed a very low capacity to stimulate regulatory T cells in vitro (fig. 3). As observed in this figure, native IL-2 was nevertheless able to significantly proliferate regulatory T cells (CD 4+ CD25+ FoxP3+ T) stimulated with anti-CD 3 antibody adhered to the bottom of the culture plate. The muteins described in table 1 at significantly higher mass concentrations than native IL-2 did not stimulate regulatory T cells. It should be noted that the results described above are valid even if the amount of mutein to be used is increased so as to employ an amount capable of exhibiting an activity equivalent to native IL-2 in a proliferation assay carried out with the CTLL2 cell line. In general, the mutant proteins described in Table 1 exhibit an ability to stimulate regulatory T cells that is at least 1000-fold less than native IL-2.

Example 4 characterization of in vivo immunostimulatory Activity of the designed muteins

The muteins described in table 1 show increased in vivo immunostimulatory capacity. FIGS. 4a, b show how the mutein induces splenomegaly greater than native IL-2 in naive mice after five days of treatment with two intraperitoneal daily doses of 20. mu.g mutein. This stimulation was associated with a significant increase in the effector cell population (e.g., CD8+ T lymphocytes). As a significant observation, this treatment with the mutant protein did not stimulate the expansion of regulatory T cells (CD 4+ CD25+ FoxP3+ T), which is different from that observed for native IL-2 (FIG. 4c, d).

Example 5 measurement of therapeutic efficacy of mutant proteins in murine models of transplantable tumors

The increased therapeutic efficacy of the designed muteins was demonstrated in a murine model of transplantable tumors. The muteins described in table 1 showed increased efficacy for the treatment of lung metastases induced in the MB16 murine melanoma model. FIG. 5 shows how treatment with two intraperitoneal daily doses of 20. mu.g of one of the muteins of Table 1, for 5 days, showed a strong anti-metastatic effect, which was not observed in the group treated in the same manner with the same dose of native IL-2.

Example 6 measurement of the ability of the muteins to enhance the Effect of an anti-tumor vaccine

The ability of the designed muteins to enhance the anti-tumor effect of the vaccine was demonstrated. A primary tumor model of the EG7 cell line was used, the EG7 cell line being a tumor cell line engineered to express the antigen OVA. Tumor bearing mice were immunized with antigen OVA adjuvanted with VSSP, either alone or in combination with the mutein. Figure 6 shows that the reduction of tumor growth was greater in the group of mice treated with the combination of vaccine and mutein than in the group treated with vaccine alone.

Claims (8)

1. An agonist polypeptide of IL-2, wherein the polypeptide is an IL-2 mutein, and wherein the polypeptide is selected from the group consisting of:

   (i) the sequence difference from native IL-2 is that of the polypeptides of mutations R38K, F42I, Y45N, E62L, E68V;

   (ii) the sequence difference from the native IL-2 is that of the polypeptides of mutations R38K, F42Q, Y45E, E68V;

   (iii) the sequence difference from native IL-2 is that of the polypeptides of mutations R38A, F42I, Y45N, E62L, E68V;

   (iv) the sequence difference from native IL-2 is that of the polypeptides of mutations R38K, F42K, Y45R, E62L, E68V;

   (v) the sequence difference from the native IL-2 is that of the polypeptides of mutations R38K, F42I, Y45E, E68V;

   (vi) the sequence difference from the native IL-2 is that of the polypeptides of mutations R38A, F42A, Y45A, E62A.
2. 2. A fusion protein comprising the polypeptide of claim 1 coupled to a carrier protein.
3. 3. The fusion protein of claim 2, wherein the carrier protein is albumin.
4. 4. The fusion protein of claim 2, wherein the carrier protein is the Fc region of a human immunoglobulin.
5. 5. A pharmaceutical composition useful in the treatment of cancer and chronic infectious diseases, characterized in that it comprises the polypeptide of claim 1 as an active ingredient.
6. 6. A pharmaceutical composition useful in the treatment of cancer and chronic infectious diseases, characterized in that it comprises as an active ingredient a fusion protein as described in any one of claims 2 to 4.
7. 7. Use of the polypeptide of claim 1 for the preparation of a medicament useful in the treatment of cancer.
8. 8. Use of the polypeptide of claim 1 for the preparation of a medicament useful in the treatment of chronic infectious diseases.