Patent Grant
12312388
This application is a National Stage Application that claims a benefit of priority from Application No. PCT/CU2017/050007 filed Nov. 10, 2017, which claims priority to Application No. CU 2016-0171, filed Nov. 15, 2016, the disclosures of which are herein incorporated in their entirety.
The instant application contains a Sequence Listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on May 24, 2018, is named IAC-00501_SL.txt and is 23,070 bytes in size.
The present invention relates to the field of Biotechnology. Particularly to a method for the introduction of mutations in the gene of interleukin-2 (IL-2) that leads to an increase in the secretion levels of said molecule and the family of immunomodulatory muteins derived thereof without affecting their biological functions.
IL-2, originally described as a growth factor for T cells (Smith, K. A. Immunol. Rev. 51: 337-357, 1980), has subsequently emerged as a regulator with dual functions within the immune response (Malek, T. R. Annu. Rev. Immunol. 26: 453-479, 2008; Hoyer K. K. et al, Immunol. Rev. 226: 19-28, 2008), which exhibits the ability to promote or negatively modulate the effector functions of the immune system. Its main role is currently considered related to the maintenance of immunological tolerance (Malek, T. R. & Bayer, A. L. Nat. Rev. Immunol. 4: 665-674, 2004) through the stimulation of regulatory T cells, which constitutively express high levels of the alpha chain of the IL-2 receptor. Although the beta and gamma subunits form the intermediate affinity dimeric receptor constitutively present in the effector cells of the immune system, the constitutive presence of high levels of the alpha chain gives the regulatory T cells a high affinity trimeric receptor that allows the preferential use of the cytokine by this cell population. (Malek, T. R. & Castro, I. Immunity. 33: 153-165, 2010).
The functional dichotomy of IL-2 has been exploited to produce opposite therapeutic effects on the immune system and modulate the immune response in the desired sense in different scenarios. Its immunopotentiating capacity has been used to stimulate anti-tumor responses (Klapper, J. A. et al, Cancer. 113: 293-301, 2008). On the other hand, the ability of IL-2 to stimulate preferentially T regulatory cells has been exploited through the application of low doses, insufficient to stimulate effector T cells or produce toxic effects, for the control of autoimmune disorders (Hartemann, A. et al, Lancet Diabetes Endocrinol. 1: 295-305, 2013) and inflammatory (Saadoun, D. et al, N. Engl. J. Med. 365: 2067-2077, 2011), and of graft versus host disease (Koreth, A. et al, N. Engl. J. Med. 365: 2055-2066, 2011).
The segregation of the interactions of IL-2 through the introduction of mutations in the different binding interfaces with the subunits of the receptor has been proposed as a way to obtain muteins with different immunomodulatory properties. The selective perturbation of the interface with the alpha chain by directed mutagenesis has allowed to obtain a molecule called no-alpha with reduced capacity to stimulate the regulatory T cells, but which retains its agonist action on the effector cells that carry the beta/gamma dimeric receptor (Carmenate, T. et al, J. Immunol. 190: 6230-6238, 2013; U.S. Pat. No. 9,206,243 B2). This molecule has a strong antitumor effect in mice. On the other hand, the disruption by mutagenesis of the IL-2 interface with the beta and/or gamma subunits can generate IL-2 receptor antagonists that selectively modulate the stimulation of different cell populations (Shanafelt, A. B. et al, Nat. Biotechnol. 18: 1197-1202, 2000; WO 2011/063770). Examples of this type of molecules are the muteins M1 and M2 described in U.S. Pat. No. 8,759,486 B2.
In addition to the muteins with loss of their interaction capacity, mutated variants of IL-2 with superagonist properties due to the increase of their binding capacity to one or another subunit of the receptor have also been described The increase in affinity for the beta subunit leads to the production of molecules that potently stimulate the effector cells and have a strong antitumor effect (Levin, A. M. et al, Nature. 484: 529-533, 2012). On the other hand, the increased affinity of IL-2 for the alpha subunit of the receptor has given rise to other superagonist variants with superior ability to stimulate the proliferative response of T cells in vitro (WO 2005/007121).
The IL-2-derived muteins described above have been obtained through rational design, in silico screening and the directed evolution of IL-2 displayed on the surface of yeast cells. Although the display of biologically active IL-2 on filamentous phages has been achieved (Buchli, P. J. et al, Arch. Biochem. Biophys. 339: 79-84, 1997; Vispo, N. S. et al, Immunotechnology 3: 185-193, 1997), this technological platform has not yet been exploited for the selection of new variants of the cytokine with modified properties.
Beyond the immunomodulatory properties of IL-2 and its derived muteins, an essential element for their therapeutic exploitation is the development of systems that allow it to be obtained in sufficient quantities. In particular, on a laboratory scale, on an industrial scale or by transfection or transduction of normal and/or tumor cells or tissues.
The predominant pathway for the recombinant production of IL-2 and other related molecules has been the expression in the cytoplasm of E. coli forming inclusion bodies, followed by in vitro re-naturalization procedures (Devos, R. et al, Nucl. Acids Res. 11: 4307-4323, 1983; Weir, M. P. & Sparks, J., Biochem. J. 245: 85-91, 1987). Despite the utility already demonstrated for this strategy, the exploration of other expression systems that lead from the beginning to obtaining correctly folded molecules very similar to natural IL-2, has continued. The secretion of IL-2 in some of these expression systems has been limited by the tendency of IL-2 to aggregate (Halfmann, G. et al, J. Gen. Microbiol. 139: 2465-2473, 1993; Cha, H. J. et al, Biochem. Eng. J. 24: 225-233, 2005).
Surprisingly, the inventors of the present invention found several mutations not previously described or predictable from the analysis of the crystal structure of human IL-2, whose introduction increases the ability of different cell types to secrete recombinant human IL-2 and multiple muteins derived from it that have specific immunomodulatory properties. This finding provides the basis for the use of these mutations at a productive scale.
In one embodiment, the present invention is related to method that leads to increased secretion levels of recombinant human IL-2 in different hosts without affecting their biological functions. Said method is based on the introduction of unique mutations in the genes encoding human IL-2 and other polypeptides derived from it, which include but are not limited to muteins derived from human IL-2 designed to act as antagonists, superagonistas or selective agonists. The increase in the secretion levels of said proteins when the method of the present invention is used is at least three times higher in relation to the unmutated counterparts. In the present invention derived muteins refers to those which have more than 90% identity with human IL-2.
The method of the present invention relates to mutations that lead to a non-conservative change of the amino acid occupying the position 35 of the primary protein sequence (Lys in the original sequence), preferably the K35E, K35D and K35Q substitutions.
Particularly, the present invention relates to the proteins obtained according to the method described here which are selected from the group comprising SEQ ID NO. 1 to 18.
Also the object of the present invention are genetic constructs that include the above-described mutated genes fused to other nucleotide sequences that encode the synthesis of fusion proteins formed by IL-2 or other immunomodulatory polypeptides derived therefrom and additional protein sequences. Additional protein sequences include but are not limited to the capsid proteins of filamentous phages, albumin, Fc region of the antibodies, whole antibodies or antibody fragments that include their variable domains.
In a particular embodiment, the hosts that are used to obtain the molecules described above include but are not limited to E. coli, yeast and mammalian cells such as HEK-293, CHO, NSO, among others. The method of the present invention is useful for improving the efficiency of the production of IL-2 and other polypeptides derived therefrom, both at the laboratory and industrial scale. The proteins obtained by the method of the present invention can be used for therapeutic purposes. In another embodiment, the objective of the present invention is to modify the physiology of normal and tumoral cells and/or tissues through the expression of human IL-2 and/or the family of immunomodulatory muteins derived therefrom (alone or fused to other proteins), both in vitro and in vivo. Example the transduction of T lymphocytes, B lymphocytes or NK cells for adoptive transfer therapies; or the direct transduction/transfection of a tumor tissue. The method of the present invention is useful for increasing the secretion of the molecules of interest in these contexts.
The present invention relates to a method that leads to increased levels of secretion of recombinant human IL-2 in different hosts without affecting their biological functions. Said method is based on the introduction of unique mutations in the genes encoding human IL-2 and other polypeptides derived therefrom, which include but are not limited to muteins derived from human IL-2 designed to act as antagonists, superagonists or selective agonists.
Identification of Mutations Increasing the Capacity of Proteins Derived from Human IL-2 to be Displayed on Filamentous Phages.
The selection of polypeptides derived from human IL-2, with unique mutations that lead to the increase of their display levels on filamentous phages can be made from libraries of more than 108 molecules presented on filamentous phages. Genes corresponding to said polypeptides can be inserted into phagemid type expression vectors (fused to one of the genes encoding filamentous phage capsid proteins) and used for the production of viral particles that display the protein variants on their surface. The starting libraries may include different degrees of diversification throughout the entire sequence or in a set of pre-defined positions. Each of the original residues in these positions can be replaced by a mixture of the 20 aminoacids or by a subset of selected residues. Diversification can be achieved through random or site-directed mutagenesis.
The selection of phages with increased levels of display of human IL-2 can be based on the incubation of phage mixtures from libraries in contact with a selector molecule immobilized on a solid surface, the elimination of unbound phages by washing, and the elution of bound phages under conditions that interfere with protein interactions. As a selector molecule, one of the recombinant IL-2 receptor subunits or a monoclonal antibody directed against IL-2 or against a peptide genetically fused thereto can be used. Several successive cycles of selection can be made under similar conditions. Analysis of the DNA sequences inserted in the selected phagemids can reveal regularities that lead to the identification of those more abundant substitutions and potentially related to the increase in display capacity on the phages.
The levels of display of the mutated variants of IL-2 can be evaluated through binding assays such as ELISA, on an immobilized capture molecule that recognizes indistinctively the different IL-2 mutated variants and the native reference. As a capture molecule for this type of assays, an antibody against a marker peptide sequence genetically fused to IL-2 variants, such as the c-myc peptide, which is fused to all foreign proteins in the expression system based on the phagemid vector pCSM, is preferred. The generality of the mutations' effects identified in the screening described above on the IL-2 derived muteins family can be demonstrated through the introduction of said changes in the sequence of the different mutated variants described for human IL-2, which include a variable number of substitutions of diverse nature throughout its sequence aimed at selectively affecting the interactions with the different subunits of the IL-2 receptor, with the subsequent modification of their immunomodulatory functions. All these modified muteins are displayed on filamentous phages, by inserting their coding genes in phagemid vectors. Evaluation of each mutein display levels on filamentous phages can be performed by ELISA as described for IL-2. As a reference for calculating the magnitude of phage display increase associated with the introduction of the changes identified as part of the present invention, the original muteins are used without any additional change and display on phages. Alternatively, the method of the present invention could be performed by exploiting other platforms of combinatorial biology, such as display on yeast or mammalian cells, in order to select variants of IL-2 and/or its derived muteins with increased levels of presentation on the cell membrane. From the selection process described above, recurrent non-conservative mutations can emerge at position 35 (particularly K35E, K35D and K35Q).
Use of Identified Mutations to Increase the Secretion Levels of Human IL-2 and Muteins Derived Thereof, as Soluble Proteins and their Re-Naturalization from Inclusions Bodies
Once a group of mutations that result in an increased display on filamentous phages of the human IL-2 and its derived muteins is identified, the effect of these same changes on the secretion of soluble proteins can be demonstrated, by introducing them into the corresponding coding genes cloned in soluble expression vectors for yeast or mammalian cells. Evaluation of concentrations of the proteins secreted to the supernatant by the host cells containing said expression vectors allows to demonstrate the increase in the secretion of IL-2 and its derived muteins associated with the introduction of the mutations that the method of the present invention uses, in comparison with its original counterparts that do not include said changes.
Alternatively, the increased production of human IL-2 and its derived muteins should be verified from transfection and/or transduction of normal and/or tumor cells and/or tissue in vivo or in vitro.
The studies described above that use the method of the present invention can be performed with IL-2 and its derived muteins alone or fused to additional polypeptide sequences, such as albumin, Fc region of human immunoglobulins, whole antibodies or antibody fragments based on its variable regions.
The mutations described in the present invention can also be used to improve the processes of in vitro re-naturalization of human IL-2 and its derived muteins, obtained as inclusion bodies in the cytoplasm of E. coli. The increase in the efficiency of re-naturalization can be evaluated by measuring the specific biological activity per protein mass by comparison to the unmutated variant.
Demonstration of Compatibility of the Used Mutations with Biological Functions of the IL-2 and the Selective Modulation of its Interactions with the Receptor Subunits.
The evaluation of biological activity of IL-2 variants modified by the present invention method can cover in vitro and in vivo techniques directed to evidence the preservation of their ability to induce proliferation, differentiation and activation of different cell types, such as T lymphocyte subpopulations, NK cells and cell lines of lymphoid origin dependent on IL-2 for their growth. The effect of native IL-2 on the proliferation of T lymphocytes expressing the trimeric receptor can be determined by the in vitro assay of CTLL-2 cell line proliferation using the colorimetric technique of Alamar blue reduction or by flow cytometry. The in vitro effect of native IL-2 on the differentiation of T CD4+ lymphocytes to T regulatory lymphocytes and the capacity of this molecule to expand and activate NK cells in vitro, are determined by flow cytometry.
The compatibility of mutations used in the method of the present invention with the selective modulation of IL-2 interaction with its receptor can be evidenced by introducing said changes on the framework of muteins previously designed and/or selected to increase or decrease their binding capacity to any of the subunits of the IL-2 receptor. The occurrence of the desired changes in the binding properties can be demonstrated through the direct determination of them in ELISA experiments on microtitre plates coated with each of the receptor subunits. The previously described assays used to characterize the immunomodulatory and/or antitumor activity of the different muteins in vitro and in vivo can be used as additional verification tools. In the case of no-alpha mutein (Carmenate, T. y otros, J. Immunol. 190: 6230-6238, 2013), it can be verified that it maintains the same capacity as native IL-2 to stimulate in vitro the proliferation of T CD8+ lymphocytes. In the case of a mutein with increased binding capacity to the beta subunit of the receptor and that has superagonist activity (super-beta mutein), it can be verified that it maintains higher capacity than native IL-2 to stimulate in vitro NK cell proliferation. In both cases proliferation can be determined by flow cytometry. The differential effect on the proliferation of populations in vivo can be determined by experiments of bromodeoxyuridine incorporation. It can be demonstrated that both muteins induce greater antitumor effect in vivo than native IL-2, in the experimental metastasis model that uses the MB16F0 melanoma line.
5
a. Photograph of the spleens of mice injected with IL-2 K35E variant and PBS.
5
b. Flow cytometry histograms of CD3+CD8+ memory phenotype (CD44hi) cell population in the spleens.
A soft randomization library targeting several positions of human IL-2 was constructed. Selected positions included those having residues with side chains contributing to the alpha subunit receptor interface (K35, R38, T41, F42, K43, F44, Y45, E61, E62, K64, P65, E68, V69, N71, L72, Q74 and Y107). Human IL-2 was diversified by Kunkel mutagenesis with spiked mutagenic oligonucleotides keeping 85% of the original nucleotide at each targeted position, plus 15% of the equimolar mixture of the remaining three nucleotides, in order to introduce a moderate degree of diversity in all the selected region. The resulting 109 clone's library thus contained as a whole the 20 amino acids at each position of the interface, while each molecule within the library only had a few replacements, restricting the search for new polypeptides to the functional sequence space closer to the starting molecule. Library phages were purified by precipitation with polyethylene glycol using established procedures (Marks, J. et al, J. Mol. Biol. 222: 581-597, 1991). Purified viral particles were incubated on immunotubes (Nunc, Denmark) coated with the recombinant alpha IL-2 receptor subunit (R&D), in order to isolate functional mutated IL-2 variants due to their ability to be displayed on phages. Two independent panning procedures were performed on human and mouse IL-2 receptor subunits. After washing non-bound phages, bound phages were eluted by adding a basic triethylamine solution. TG1 bacteria were infected with the selected phages, which were amplified using M13KO7 helper phage and used as starting material for a new selection round. Four phage selection rounds were performed. Sequencing of the inserts in the selected phagemids (from the third and fourth selection rounds) revealed similarities in the resulting mutated variants. Despite the predominance of the original non-mutated IL-2 gene (highly represented in the original library), there was a minor proportion of variants having the replacements K35E, K35D and K35Q, showing the influence of non-conservative changes at position 35 in the display of functional IL-2 on filamentous phages. K35E was the most frequent replacement. This finding was surprising, as the analysis of the crystal structure of the IL-2/receptor complex (PDB codes3B51 and 2ERJ) points to the involvement of the original K35 residue in ionic interactions in the polar peripheral region of the interface with the alpha subunit. The ability of the non-conservative replacements (charge inversion in two of the cases) to keep the interaction with the selector molecule was thus unexpected.
The ability of different IL-2 variants selected from the library to be secreted to E. coli periplasm and displayed on phages was compared. Native IL-2 was used as reference molecule. The K35R-containing variant (conservative change at position 35) was also constructed by Kunkel mutagenesis to be used as an additional control. All the proteins were obtained through the insertion of their coding genes in the phagemid vector pCSM (fused to M13 gene 3) and subsequent phage production from TG1 bacteria transformed with the resulting genetic constructs (Rojas, G. et al, Immunobiology. 218: 105-113, 2013). The levels of phage display of each variant were evaluated through an ELISA on microtitration plates coated with 9E10 monocional antibody. Bound phages were detected with an anti-M13 antibody coupled to horseradish peroxidase. It was shown that replacements K35E, K35D and K35Q result in an increase of the display of human IL-2 as compared with the original molecule (
K35E was introduced by Kunkel mutagenesis in the genes of several mutated variants of human IL-2 (in the phage-displayed format). The panel included four muteins already described to perform different immunomodulatory functions: one no-alpha mutein with selective agonist function on effector T cells (Carmenate, T. et al, J. Immunol. 190: 6230-6238, 2013; U.S. Pat. No. 9,206,243 B2), one antagonist mutein that loses its binding ability to the gamma IL-2 receptor subunit (no-gamma) (U.S. Pat. No. 8,759,486 B2), and two superagonist muteins with enhanced binding ability to either beta (super-beta) or alpha (super-alpha) IL-2 receptor subunits (Levin, A. M. et al, Nature. 484: 529-533, 2012; WO 2005/007121). Phages displaying each of these proteins were produced and purified (together with the original molecules without K35E), and the display levels of the foreign proteins were evaluated by ELISA on microtitration plates coated on with the 9E10 monoclonal antibody. A phage preparation displaying native IL-2 was used as reference (assuming the presence of 100 arbitrary units/ml in it) to construct a standard curve in order to calculate the relative display levels for each variant. Table 1 shows the increase in the display level of each mutein associated to the introduction of K35E.
Genetic constructs were designed to fuse the genes of human IL-2 and its derived muteins to the human IgG1 Fc region gene, in the context of the pCMX expression vector. An additional panel of equivalent constructs having the mutation K35E was also prepared. HEK 293 T cells (adapted to grow in suspension) were transfected with each of the above described genetic constructs properly mixed with polyethyleneimine. The transfection volume was 50 ml. Supernatants from transfected cells were collected after six days of culture. The presence of the recombinant IL-2-derived proteins was evaluated by ELISA on microtitration plates coated with IL-2.2 monoclonal antibody (directed against a linear epitope present on all the muteins). Captured fusion proteins were detected with an anti-human Fc antibody coupled to horseradish peroxidase. The levels of fusion proteins in the supernatants were higher for those molecules containing the replacement K35E as compared with their original counterparts (
The binding ability of recombinant mutated IL-2 (K35E) in the human Fc-fused homodimer format was evaluated by ELISA on microtitration plates coated with different molecules known to interact with native IL-2. The panel of coating molecules included four monoclonal antibodies that recognize different epitopes on IL-2, as well as the IL-2 receptor alpha subunit (of human or mouse origin). The captured fusion protein was detected with an anti-human Fc antibody coupled to horseradish peroxidase. A similar fusion homodimer including non-mutated IL-2, produced in the same expression system, was used as the control. Binding of the mutated homodimer to both the antibodies and the receptors was not affected by the presence of K35E, on the contrary it produced the opposite effect. Reactivity of the mutated variant towards all the coating molecules was higher than that of its non-mutated recombinant counterpart (
The ability of mutated IL-2 (K35E) in the Fc-fused homodimer format (purified from HEK 293 T cells transfected in suspension) to induce CTLL-2 proliferation was evaluated. Recombinant human IL-2 was used as the control. Cells were grown in the presence of different concentrations of both proteins, and proliferation was measured through the colorimetric Alamar blue reduction assay (
C57BL/6 mice received five daily doses of 4×104 IU of Fc-fused mutated IL-2 (K35E) during 5 consecutive days to study the ability of this protein to stimulate in vive proliferation of IL-2-dependent cell populations. The animals were sacrificed after the treatment and their spleens were observed. Additionally, the size of the population of CD3+CD8+ cells having memory phenotype (CD44hi) was determined by flow cytometry. The control of the experiment was a group of mice injected with phosphate buffered saline (PBS). The recombinant Fc-fused IL-2 (K35E) had the expected effect on memory CD8 T cell population, as judged by the enlargement of spleens (
The binding properties of both human IL-2 and of a no-alpha mutein previously described (Carmenate, T. et al, J. Immunol. 190: 6230-6238, 2013; U.S. Pat. No. 9,206,243), which contains the replacements R38A, F42A, Y45A and E62A resulting in a loss of ability to bind the IL-2 receptor alpha subunit aimed at reducing its stimulatory potential on T regulatory cells without affecting the action on effector cells having the heterodimeric beta/gamma receptor, were compared. Both recombinant proteins had the additional K35E mutation and were produced as fusion proteins containing the Fc domain of human immunogiobulins. Microtitration plates were coated with the recombinant IL-2 receptor alpha subunit of human (a) and mouse (b) origin. Captured fusion proteins were detected with an anti-human Fc antibody coupled to horseradish peroxidase. The introduction of K35E gave rise to a new no-alpha molecule with expression levels higher than those of its original counterpart (
The binding ability of IL-2 and a super-beta mutein containing the mutations L80F, R81 D, L85V, 186V and 192F (both with the additional mutation K35E and fused to the Fc domain of human IgG1) was evaluated by ELISA on plates coated with the IL-2 receptor beta subunit. Captured fusion proteins were detected with an anti-human Fc antibody coupled to horseradish peroxidase. The introduction of K35E gave rise to a new molecule with higher expression levels as compared to the original super-beta mutein (
| Number | Date | Country | Kind |
|---|---|---|---|
| CU2016-0171 | Nov 2016 | CU | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CU2017/050007 | 11/10/2017 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/091003 | 5/24/2018 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 4738927 | Taniguchi | Apr 1988 | A |
| 8759486 | Monzón | Jun 2014 | B2 |
| 9206243 | Monzón | Dec 2015 | B2 |
| 20070014765 | Elias | Jan 2007 | A1 |
| 20120244112 | Ast | Sep 2012 | A1 |
| Number | Date | Country |
|---|---|---|
| WO 2005007121 | Jan 2005 | WO |
| WO 2011063770 | Jun 2011 | WO |
| WO 2005086798 | Sep 2015 | WO |
| Entry |
|---|
| Tokuriki et al, 2009, Current Opinion in Structural Biology. 19: 596-604. |
| Bhattacharya et al, 2017. Plos One. 12(3): e0171355, pp. 1-22 as printed. |
| Buchli, Pamela J., et al., “The Functional Display of Interleukin-2 on Filamentous Phage”, Archives of Biochemistry and Biophysics, vol. 339, No. 1, pp. 79-84, (Mar. 1, 1997). |
| Carmenate, Tania, et al., “Human IL-2 Mutein with Higher Antitumor Efficacy Than Wild Type IL-2”, The Journal of Immunology, vol. 190, pp. 6230-6238 (2013). |
| Cha Hyung Joon, et al., “Comparative Production of Human Interleukin-2 fused with Green Fluorescent Protein in Several Recombinant Expression Systems”, Biochemical Engineering Journal 24, pp. 225-233 (2005). |
| Devos, R., et al., “Molecular Cloning of Human Interleukin 2 cDNA and Its Epression in E. coli”, Nucleic Acids Research vol. 11, No. 13 (1983). |
| Drew, Yvette, “The Development of PARP Inhibitors in Ovarian Cancer: From Bench to Bedside”, British Journal of Cancer 113, S3-S9 (2015). |
| Halfmann, Gabriele, et al., “Targeting of Interleukin-2 to the Periplasm of Escherichia coli”, Journal of General Microbiology vol. 139, pp. 2465-2473 (1993). |
| Hartemann, Agnes, et al., “Low-dose interleukin 2 in Patients with Type 1 Diabetes: A phase 1/2 Randomised, Double-Blind, Placebo-Controlled Trial”, Lancet diabetes Endocrinology vol. 1, pp. 295-305 (Dec. 2013). |
| Hoyer, Katrina K., et al., “Interleukin-2 in the Development and Control of Inflammatory Disease”, Immunological Reviews, 226: pp. 19-28 (2008). |
| Klapper, Jacob A., et al., “High-dose Interleukin-2 for the Treatment of Metastatic Renal Cell Carcinoma”, Cancer vol. 113, No. 2, pp. 293-301 (Jul. 15, 2008). |
| Koreth, John, et al., “Interleukin-2 and Regulatory T Cells in Graft-versus-Host Disease”, The New England Journal of Medicine, vol. 365, No. 22, pp. 2055-2066 (Dec. 1, 2011). |
| Levin, Aron M., et al., “Exploitig a natural Conformational Switch to Engineer and Interleukin-2 ‘Superkine’”, Nature vol. 484, pp. 529-533 (Apr. 26, 2012). |
| Malek, Thomas R., et al., “Tolerance, Not Immunity, Crucially Depends on IL-2”, Nature Review Immunology, vol. 4, pp. 665-674 (Sep. 2004). |
| Malek, Thomas R., “The Biology of Interleukin-2”, Annual Review of Immunology, vol. 26, pp. 453-479 (2008). |
| Malek, Thomas R., et al., “Interleukin-2 Receptor Signaling: At the Interface between Tolerance and Immunity”, Immunity Review, 33: 153-165, (Aug. 27, 2010). |
| Robbens, J., et al., “Improved Periplasmic Production of Biologically Active Murine Interleukin-2 in Escherichia coli Through a Single Amino Acid Change at the Cleavage Site”, Process Biochemistry 41:1343-1356 (2006). |
| Saadoun, David, et al., “Regulatory T-Cell Responses to Low-Dose Interleukin-2 in HCV-Induced Vasculitis”, The New England Journal of Medicine, 365: 2067-2077 (Dec. 1, 2011). |
| Sauve, K., et al., “Localization in Human Interleukin 2 of the Binding Site to the a Chain (p55) of the Interleukin 2 Receptor”, Proc. Nati. Acad. Sci USA, vol. 88, pp. 4636-4640 (Jun. 1991). |
| Shanafelt, Armen, B., et al., “A T-cell-selective Interleukin 2 Mutein Exhibits Potent antitumor Activity and is Well Tolerated in Vivo”, Nature Biotechnology vol. 18, pp. 1197-1202 (Nov. 2000). |
| Smith, Kendall, A., “ T-Cell Growth Factor”, Immunological Review, vol. 51, pp. 337-357 (1980). |
| Vispo, N.S., et al., “Displaying Human Interleukin-2 on the Surface of Bacteriophage”, Immunotechnology 3: 185-193 (1997). |
| Weigel, Ulrich, et al., “Mutant Proteins of Human Interleukin 2 Renaturation Yield, Proliferative Activity and Receptor Binding”, Eur. J. Biochem., 180: 295-300 (1989). |
| Weir, Malcolm P., et al., “Purification and Renaturation of Recombinant Human Inteleukin-2”, Biochemistry Journal vol. 245, pp. 85-91 (1987). |
| Extended European Search Report for EP Application No. 17817648.3 dated Apr. 28, 2020. |
| International Search Report and Written Opinion for International Application No. PCT/CU2017/050007 dated May 24, 2018. |
| Number | Date | Country | |
|---|---|---|---|
| 20190315826 A1 | Oct 2019 | US |
