Patent Application
20120315245
The present invention is related to the field of biotechnology and particularly immunology. The invention is related to technical solutions with therapeutic applications for human health. It particularly relates to the therapeutic modulation of the immune system using analogs of natural molecules.
Interleukin 2 (IL-2) was the first growth factor described for T cells. Since its discovery it showed a strong capacity to promote proliferation and survival of T cells in vitro (Smith, K A (1988) Science. 240, 1169-76) and to enhance T cells immune response in vivo, in the context of viral infections (Blattman, J N, et al. (2003) Nat Med 9, 540-7) or vaccines (Fishman, M., et al. (2008) J Immunother. 31, 72-80, Kudo-Saito, C., et al. (2007) Cancer Immunol Immunother. 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 T immune response has been questioned recently by numerous 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) Nat Rev Immunol. 4, 665-74) showing that this cytokine is a homeostatic growth factor for natural regulatory T cells T CD4+CD25+FoxP3+(Tregs).
Interleukin-2 is a major player in the mechanism by which regulatory T cells suppress the activity and expansion of other effector cells such as CD4 helpers T cells, CD8 cytotoxic T cells and NK cells. Specifically, it has been recently proposed that regulatory T cells suppress other T cells, inducing the local decrease in the levels of IL-2 (Pandiyan, P., et al. (2007) Nat Immunol. 8, 1353-62). This suppressive effect is based in: a) their ability to inhibit directly the production of IL-2 by the effector T cells that they suppresses: (Almeida, A. R., et al. (2002) J Immunol. 169, 485060; Takahashi, T., et al. (1998) Int Immunol. 10, 1969-80; Thornton, A. M., et al. (1998) J Exp Med. 188, 287-96; Wolf, M., et al. (2001) Eur J Immunol. 31, 1637-45); b) The ability to consume fast and efficiently the IL-2 in their microenvironment (Pandiyan, P., et al. (2007) Nat Immunol. 8, 1353-62); and c) Its capacity to over-express IL-2 alpha chain receptor (Kuniyasu, Y., et al. (2000) Int Immunol. 12, 1145-55), which enables them to use the IL-2 more efficiently when its concentrations are low.
Summarizing, IL-2 is a highly pleiotropic cytokine which is very significant for the biological activity of different cell populations. This property makes IL-2 an important node in the regulation of the immune response, making it an attractive and complex target for immune modulation therapies. In particular, the pleiotropic nature of the action of this cytokine, makes it very significant for the design of therapeutic strategies that modulate in a selective/preferential way the activity of this cytokine in different cell populations.
The IL-2 has been used for several years in cancer therapy. In particular, its use in high doses is an approved therapy in several countries for the treatment of melanoma and renal cell carcinoma. However, the direct use of IL-2 in patients is severely limited by is toxic effects. So much so that only 20% of eligible patients received further therapy and only 17% of patients show relevant objective response. One possible explanation for this dramatic failure in the clinical stage is that therapy with native IL-2 also stimulates regulatory T cell populations (Ahmadzadeh, M., et al. (2006) Blood. 107, 2409-14) that hamper the immunestimulation pursued with it.
Several strategies have been developed to mitigate the toxic effects of IL-2 therapy. Some of these strategies are based on the use of mutated variants of IL-2, designed to increase the capacity of signaling of this molecule mainly by means of the high affinity receptor (alpha, beta and gamma chains) and not by means of the intermediate affinity receptor (beta and gamma chains). The basic idea is to promote preferential signaling on T cell versus signaling in NK cells which are the cells believed to be responsible for the observed toxic effects. The following inventions are in the same line of work: U.S. Pat. No. 7,186,804, U.S. Pat. No. 7,105,653, U.S. Pat. No. 6,955,807, U.S. Pat. No. 5,229,109, U.S. Patent application 20050142106. It is important to note anyway that none of these inventions is related to IL-2 mutants with the capacity to differentially modulate the activity of regulatory T cells. Moreover, the mutants in these inventions are agonists of the IL-2 and not antagonist/inhibitors such as those described in this application.
Other mutated variants of the IL-2 have been created with the aim of increasing their pharmacological activity. For example, improving its folding or increasing their lifetime in blood. Among others, the following inventions are related to this line 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 mutants has demonstrated ability to differentially modulate the activity of regulatory T cells.
Other existing inventions relate to inhibitors of the activity of IL-2, primarily for the treatment of autoimmune diseases or to prevent organ transplant rejection. Among these inventions are: U.S. Pat. No. 5,876,717, U.S. Pat. No. 5,635,597, U.S. Pat. No. 6,906,170, U.S. Pat. No. 6,168,785.
Finally, it should be referred that in the literature there are many proposals of therapeutic agents (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., Lyerly, H. K. and Clay, T. M. (2008) Blood. 112, 610-8; Onizuka, S., Tawara, I., Shimizu, J., Sakaguchi, S., Fujita, T. and Nakayama, E. (1999) Cancer Res. 59, 312833; Quezada, S. A., Peggs, K. S., Curran, M. A. and Allison, J. P. (2006) J Clin Invest. 116, 1935-45) that propose to modulate or reduce the activity of regulatory T cells in vivo. These therapeutic agents have been tested in animal models and even in patients for direct cancer therapy or to enhance the effect of vaccines. There are also some reports that propose to modulate the activity of IL-2, particularly 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) J Immunol. 177, 306-14; Murakami, M., Sakamoto, A., Bender, J., Kappler, J. and Marrack, P. (2002) Proc Natl Acad Sci USA. 99, 8832-7; Tomala, J., Chmelova, H., Mrkvan, T., Rihova, B. and Kovar, M. (2009) J Immunol. 183, 4904-4912), to promote better or more effective immune responses. However, to the best of our knowledge there is no report in the literature, on mutated variants of the IL-2, which support the possibility of their use to modulate, selectively or preferentially the activity of regulatory T cells. In particular IL2 muteins capables of selectively/preferentially antagonize the activity of IL2 on regulatory T cells, thus affecting its function and promoting in consequence a therapeutic potentiation of immune responses.
The present invention is based on the scientific finding that proves that mutant variants of IL-2 can exert a preferential inhibition on regulatory T cells. Inventors found out for the first time in in vitro experiments that mutated variants of IL-2 can substantially inhibit the activity of regulatory T cells (T CD4+CD25+FoxP3+), while hardly affecting the activation and/or proliferation of other lymphocytes with effector functions This finding provides the basis for a new strategy of immunomodulation of regulatory T cells in diseases such as cancer or chronic infections where these cells are relevant.
The present invention relates to polypeptides which share their primary sequence with human IL-2, except in the fact that several amino acids have been mutated by eliminating or substantially reducing their capacity to signal through the different forms of the IL-2 receptor
These mutant variants of IL-2 maintain their ability to bind to one or more components of IL-2 receptor, and have an inhibitory activity preferentially seen on regulatory T cell populations, where they negatively modulate their function. Several specific variants of IL-2 mutants with preferential inhibitory property on regulatory T cells are protected. The invention also includes the therapeutic uses of these mutated variants, used alone or in combination with vaccines for the therapy of diseases such as cancer or chronic infections where the activity of regulatory T cells (Tregs) is relevant.
The present invention proposes a new strategy to modulate the activity of regulatory T cells in diseases in which suppression by means of these cells reduces the protective immune response, induced naturally or by vaccination. The advantages of this new therapeutic strategy over other proposals to modulate the activity of Tregs are numerous. For instance:
The present invention is related to polypeptides from 100 to 500 amino acids, preferably those whose size is 140 amino acids and whose apparent molecular weight is at least 15 kD. These polypeptides maintain a high level of sequence identity with native IL2, more than 90% identity, in an area of their sequence, they include 2 to 6 mutations with respect to native IL-2.
In these positions, these polypeptides are mutated by inserting amino acid residues different to those aminoacids placed in the same position in the native IL-2. The residues that replace the original residues are selected because they have physicochemical properties very different from those in the original amino acid, residues changed from polar to apolar, from charged to uncharged, from large to small, from acid to base, among others.
The polypeptides of the present invention can also be called indistinctly immunomodulator polypeptides, analogs of IL-2 or muteins of IL-2, among others. These polypeptides are designed from the 3D structure of the IL-2 (deposited in the PDB database), introducing mutations only in the positions of the IL2 that correspond to amino acids significantly exposed to the solvent, which are identified using bioinformatics programs of the public domain such as RASMOL, SwissPDBviewer and others.
The polypeptides of this invention can be obtained in several ways, among others by protein synthesis. They could also be obtained by genetic engineering techniques, such as expressing them in inclusion bodies in bacteria such as E. coli. Punctual mutations at the specific positions may also be obtained by directed mutagenesis techniques by using the polymerase chain reaction.
The polypeptides of the present invention are selected by performing in vitro or in vivo experiments to simultaneously have the following properties
1) These mutant variants of IL-2 loose or substantially reduce their signaling capacity to the different forms of the IL-2 receptor. This property can be assessed directly in in-vitro proliferation assays with cell lines, which are IL-2 dependent like CTLL2 or Kitt225, or with T lymphocytes or NK cells of murine and/or human origen. These mutants should have a stimulatory activity in these assays at least 100 times lower than that of native IL-2.
2) These mutated variants of IL-2 (muteins) maintain their ability to bind to one or more molecular components of the IL-2 receptor. This binding capacity can be assessed directly by ELISA against chains of the receptor commercially available such as alpha and beta chains of the receptor or indirectly on cell populations positive to the receptor. The recognition rates of IL-2 muteins should be comparable to those of native IL-2 in these assays
3) The mutated variants of IL-2 have an inhibitory activity of the native IL-2 activity on lymphocytes, which is preferential on regulatory T cell populations (at least in T CD4+CD25+FoxP3+cells). The muteins of IL-2 included in this invention are capable in a certain range of concentrations to preferentially or selectively inhibit the activity or expansion of regulatory T cells, without affecting or only minimally affecting the activity and/or expansion of other lymphocytes with effector functions such as T helper cells, cytotoxic T cells or NK cells. The preferential or selective inhibitory activity of these muteins can be evidenced in several in vitro tests, which examine the response to stimuli of mixtures of effector and regulatory populations in the presence of increasing amounts of the muteins. In the appropriate range of concentration muteins should be able to inhibit at least three times more the growth or activity of regulatory T cells than they inhibit the activity or expansion of the effector populations used in the experiment, e.g. T helper cells, cytotoxic T cells or NK cells.
This invention also includes several specific variants of IL-2 muteins (specific mutations disclosed in Table 1), which have been selected to have the properties mentioned above. These muteins include multiple aminoacids substitutions that significantly reduce their ability to stimulate murine and human lymphocytes. However, their ability to bind to alpha and beta chains of the receptor remains intact, and they gain inhibitory (antagonist) capacities of IL-2 native activity. The most significant aspect of these muteins, is that they display a marked ability for, in a certain range of concentrations, to inhibit preferentially regulatory T cells (CD4+CD25+FoxP3+), in a culture of lymphocytes containing these cells and other effector T cells.
The present invention also includes additional modifications of the type of IL-2 mutants referred to above and in particular those disclosed in Table 1. Either to increase their affinity to specific components of IL-2, but without affecting or even enhancing its preferential inhibitor properties, or to improve their in-vivo pharmacodynamics: increased life span or reduce its internalization by T cells. These additional mutations may be obtained by rational design with bioinformatics tools, or by using combinatorial molecular libraries of different nature (phage display libraries, libraries of gene expression in yeast or bacteria).
This invention also includes pharmaceutical compositions comprising as active ingredient IL-2 muteins and its analogs, disclosed in the present invention, as well as its potential therapeutic applications with the aim of selectively modulate the activity of IL-2 on regulatory T cells. Particularly, this invention protect the use of these muteins to promote the immune response induced naturallly or by vaccines in diseases such as cancer or chronic infections where regulatory T cells are particularly relevant.
For therapeutic use, the polypeptide of the present invention should be administered to a subject carrier of the disease independently or in combination with other polypeptides or other substances that facilitate or enhance its therapeutic action. The route of administration may be any of the routes of administration described by the prior art for parenteral administration of drugs. It can be preferably administered by intravenous, intramuscular, subcutaneous or intratumoral routes.
The polypeptides or fusion proteins described by the present invention can also be administered as part of a pharmaceutical composition useful in the treatment of cancer and chronic infectious diseases.
To obtain the desired therapeutic effect, the polypeptide of the present invention should be administered at doses high enough to ensure an adequate concentration in the lymph node or in the relevant peripheral site for the disease under study, it should be in the adequate range of concentrations for the mutein to show a preferential inhibitory effect on regulatory T cells. The dose referred must therefore be adjusted according to disease type and route of administration in the study. For instance in the case of tumor therapy, the dose should be adjusted until the concentrations of the mutant within the tumor and/or loco-regional lymph node are adequate to ensure a preferential inhibitory effect on regulatory T cells. The dose ranges to be explored can range from dozens of micrograms to a few milligrams per dose.
The number of administrations to be applied shall also be adjusted according to the biodistribution of the mutein in question. In general, the aforementioned effective concentrations should be maintained for a period ranging from 2 days to 30 consecutive days. Note, for example, that if the mutein is coupled to a carrier protein, the frequency of administration should be adjusted accordingly. Therapeutic action is meant by total or partial remission of the disease symptoms. For cancer, a decrease in tumor volume or an increased time to relapse will be considered, among others, as remission criteria. Finally, it should be noted that the benefits of this new therapeutic strategy as compared to other proposals to modulate the activity of Tregs would be multiple. For example:
The mutants were designed computationally, from bioinformatics techniques, using as basis the reported structure of the quaternary complex of human IL-2 coupled to the receptor in line the report by Wang, X., Rickert, M. and Garcia, K. C. in Structure of the quaternary complex of interleukin-2 with its alpha, beta, and gamma receptors. Science, 2005. 310(5751): p. 1159-63 and energy calculation algorithms for the protein-ligand interaction in the public domain. Different variants of muteins were initially predicted to not affect the binding capacity of the alpha and beta chains of the receptor. These muteins were expressed in E. coli from a genetical construction in the pET28a vector including an identifying sequence of 6 histidines at the amino terminal. The muteins were purified using reverse phase (
The selected muteins retain the ability to bind to different components of the IL2 receptor, especially to the alpha and beta chains of the receptor.
The selected muteins significantly reduce their ability to signal by the IL-2 receptor.
The muteins selected preferentially inhibit the in vitro expansion of regulatory T cells (CD4+CD25+FoxP3+).
The selected muteins are sequestered preferentially by the regulatory T cells in a culture, reducing their ability to affect the activity of effector T cells. These muteins inhibit the signaling (stimulation) mediated by the IL-2 produced endogenously by CD4+CD25−FoxP3− helper T cell populations purified and stimulated with anti-CD3 antibodies. However, the addition of increasing amounts of CD4+CD25+FoxP3+ regulatory T cells to these cultures, paradoxically reduces the inhibition mediated by the mutant on T effector populations (
The selected muteins show antitumoral activity on a murine model of transplantable tumor.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2009-0203 | Nov 2009 | CU | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CU2010/000005 | 11/26/2010 | WO | 00 | 8/16/2012 |
