Interleukin-2 variants with modified biological activity

Abstract

The present invention relates to interleukin-2 (IL-2) variants for use in the prevention or treatment of immune disorders, including but not limited to allergic diseases, autoimmune diseases, chronic or acute inflammation, and infectious diseases; graft versus host disease; transplant rejection and cancer. The present invention also relates to the use of said IL-2 variant for screening for anti-IL-2 antibodies having T effector promoting cell or T regulatory promoting cell activity.

Description

[0001] This application is a divisional application of the invention patent application with application date of March 27, 2020, application number 202080034172.4, and invention name “Interleukin-2 variant with modified biological activity”. Technical Field

[0002] The present invention relates to interleukin-2 (IL-2) variants, which are used to prevent or treat immune disorders, such as but not limited to acute or chronic inflammatory diseases, allergic diseases, autoimmune diseases or infectious diseases, graft-versus-host disease, transplant rejection and cancer. The present invention also relates to the use of the IL-2 variants for screening anti-IL-2 antibodies with pro-T effector cell or pro-T regulatory cell activity. Background Art

[0003] Interleukin-2 (IL-2) is a key cytokine of the immune response that promotes the activation, proliferation and survival of T and B lymphocytes (T and B cells). The IL-2 receptor (IL-2R) is a heterotrimeric protein complex composed of α (IL-2-RA or CD25), β (IL-2RB or CD122) and γ (IL-2RG or γc or CD132) chains. The α chain binds IL-2 with low affinity and does not participate in signal transduction. The combination of the β chain and the γ chain forms a medium affinity receptor expressed on effector T cells (conventional T cells (Tconv; CD4+Foxp3-)), CD8+ and NK cells responsible for cell-mediated immune responses; and all three receptor chains form a high affinity receptor constitutively expressed on regulatory T cells (T regulatory cells or Treg; CD4+Foxp3+), which has an immunosuppressive function and is transiently or transiently expressed by activated effector T cells and NK cells.

[0004] In fact, IL-2 is a cytokine with both immunostimulatory and inhibitory functions. Therefore, it has been used in the clinic to stimulate immune responses against cancer at high doses, and recently to block immune responses in various pathophysiological conditions at low doses, including autoimmune diseases (such as type 1 diabetes (T1D)), autoimmune vasculitis, inflammatory diseases (such as Parkinson's disease) and parasitic infections (such as Trypanosoma cruzi infection); and transplant rejection, including graft-versus-host disease (GVHD) (Rosenberg et al., Sci. Transl. Med., 2012, 4, 127-; Grinberg-Bleyer, Y. et al., J. Exp. Med., 2010, 207, 1871-1878; Tang et al., Immunity, 2008, 28, 687-697; Pilon et al., Am. J. Transplant., 2014, 14, 2874-2882; Saadoun ... al., N.Engl.J.Med., 2011, 365, 2067–2077; Koreth et al., N.Engl.J.Med., 2011, 365, 2055–2066; Kennedy-Nasser et al., Clin.Cancer Res.Off.J.Am.Assoc.Cancer Res., 2014, 20, 2215–2225; Baeyens et al., Diabetes, 2013, 62, 3120–3131; González et al., Brain. Behav. Immun., 2015, 45, 219–232; Pérol et al. al., Immunol. Lett., 2014, 162, 173–184.

[0005] However, high doses of IL-2 can be very toxic in cancer therapy, and its efficacy is suboptimal (5-20% of responders) due to deleterious effects of IL-2 on Tregs.

[0006] In humans, low doses of IL-2 have been successfully administered to promote Tregs and suppress inflammation in patients with autoimmune vasculitis and graft-versus-host disease (Saadoun et al.; Koreth, J. et al.; Kennedy-Nasser et al.). However, improvements are needed because IL-2 has a short half-life and requires repeated administration.

[0007] Interestingly, when IL-2 is complexed with anti-IL-2 antibodies (Abs), IL-2 efficacy is improved, its toxicity is reduced, and depending on the Ab, the complex can redirect IL-2 action to effector (pro-Teff anti-IL-2 Abs) or regulatory (pro-Treg anti-IL-2 Abs) immune cells, thus solving the major problems associated with IL-2-based therapies (Boyman et al., Science, 2006, 311, 1924–1927. Letourneau et al., Proc. Natl. Acad. Sci., 2010, 107, 2171–2176). Regarding the mechanism of action, it was shown that the formation of a complex with a pro-Teff anti-IL-2 antibody directly blocks the interaction of IL-2 with CD25 (Levin et al., Nature, 2012, 484, 529-533; Rojas, G. in Monoclonal Antibodies (eds. Ossipow, V. & Fischer, N.) 1131, 447-476 (Humana Press, 2014)).

[0008] Treg-directed IL-2 / anti-IL-2 complexes have given impressive results in different mouse inflammatory models: T1D, asthma, EAE, atherosclerosis, chronic kidney disease and transplantation, including solid organ transplantation (Webster et al., J. Exp. Med., 2009, 206, 751–760; Dinh, T. Net al., Circulation, 2012, 126, 1256–1266; Polhill, T. et al. J. Am. Soc. Nephrol. JASN, 2012, 23, 1303–1308; Satake et al., PLoS ONE, 2014, 9, e92888; Vokaer et al., Transplant. Proc., 2012, 44, 2840–2844; Goldstein et al., Front. Immunol., 2013, 4, 155).

[0009] Another strategy for fine-tuning the biological activity of IL-2 is to perform point mutations in the IL-2 molecule to generate IL-2 variants or mutant IL-2. A mutant IL-2 protein (IL-2 super factor) containing five point mutations has been disclosed, which confers a 300-fold higher affinity for CD122 than wild-type IL-2 (Levin et al., Nature, 2012, 484, 529–533). Immunosuppressive IL-2 variants with greater affinity for IL-2RA (CD25) and / or altered signaling through IL-2RBG have also been disclosed (WO2010 / 085495 and US2014 / 0286898).

[0010] In order to improve IL-2-based immunotherapy, IL-2 variants for the prevention or treatment of immune diseases are needed. Summary of the invention

[0011] The present inventors have engineered resurfaced variants of human IL-2 and identified IL-2 variants that are able to preferentially stimulate T-regulatory (Treg) cells or antagonize IL-2.

[0012] The examples of the present application show that a resurfaced variant of human IL-2 (Treg-agonist variant) preferentially stimulates Tregs in vitro ( Figure 6 ) and increased circulating Treg cells in mice in vivo ( Fig.10 ). Treg-agonist variants can be used to indirectly reduce immune activation by preferably activating Treg, thereby in turn inhibiting immune effector cells. Therefore, the Treg-agonist variants of the present invention can be used to treat diseases in which immunomodulation or immunosuppression is beneficial, such as but not limited to allergic and autoimmune diseases, and diseases comprising overactivation of the immune system, particularly including chronic or acute inflammatory diseases, graft-versus-host disease (GVHD) and transplant rejection.

[0013] The examples of this application also show that some human IL-2 resurfaced variants are IL-2 antagonists ( Fig. 9 ).

[0014] In the case of diseases involving overactivation of the immune system associated with excessive production of IL-2 (particularly including chronic or acute inflammatory diseases, graft-versus-host disease (GVHD) and transplant rejection), the IL-2 antagonist variants according to the present invention directly reduce immune activation by competing with endogenous IL-2, thereby blocking IL-2-mediated overactivation of the immune system.

[0015] In a murine model of acute GVHD, the IL-2 variants of the invention, whether Treg agonists or IL-2 antagonists, can control GVHD and delay the development of clinical GVHD. Compared with the tested Treg-agonist variants, the tested IL-2-antagonist variants were more effective in preventing weight loss and preferentially increasing circulating human Treg cells ( Figure 7 and Figure 8 ).

[0016] Furthermore, IL-2 variants that act as IL-2 antagonists inhibit Treg division in vitro by removing them from wild-type IL-2 signaling (similar to blocking anti-IL-2 antibodies; Fig.12 ). In vivo, it is expected that inhibition of Tregs with the IL-2 antagonist variants of the present invention will reduce tumor growth, as previously shown with other IL-2 antagonists (Carmenate et al., The Journal of Immunology, 2018, 200, 3475-3484). Inhibition of Tregs by the IL-2 antagonist variants according to the present invention will therefore promote immune responses (lymphocytes (B, NK, CD4+ or CD8+ T cells); dendritic cells (DC); macrophages, etc.) by releasing immune cells from Treg suppression. In addition, CD8+ T cell function is retained or moderately affected, depending on the dose of the IL-2 antagonist variant used ( Fig. 9 ; Fig.12 ). Therefore, the IL-2 antagonist variant further allows direct stimulation of CD8+ T cell immune responses, for example against tumors, pathogens or vaccines. For all these reasons, better immune responses to cancer, infectious agents and vaccines are expected with the IL-2-antagonist variants of the present invention.

[0017] In summary, the results presented in this application show that the effect of IL-2 antagonist variants in vivo can vary according to the immune environment, because when the immune system is overactivated and produces excessive IL-2, it reduces immune activation (by neutralizing excessive endogenous IL-2), and in different immune environments, it increases immune activation (by Treg inhibition and CD8+T cell activation). For these reasons, IL-2 antagonist variants can be used to treat diseases including overactivation of the immune system associated with excessive production of IL-2, such as but not limited to chronic or acute inflammatory diseases, graft-versus-host disease (GVHD) and transplant rejection, and are also used to treat cancer and infectious diseases and increase immune responses to vaccines.

[0018] The inventors have also solved the crystal structures of some variants, which provide some insights into their mechanism of action. The inventors observed that the Y31P substitution (IL2-V4) stabilized IL-2 in a conformation similar to that observed in the IL2-Rα subunit complex (hereinafter referred to as "α-induced conformation"). In fact, there is some recent evidence from mouse IL-2 that conformational changes in the loop AB where Tyr31 is located allosterically affect the interaction with IL-2Rβ and γ subunits (De Paula et al., PNAS, doi / 10.1073; March 17, 2020 and Spangler et al., Immunity, 2015, 42, 815-825). Thus, it is easy to speculate that the molecular mechanism behind the activity of IL2-V4 utilizes this allosteric loop. In fact, the inventors have also obtained the structure of IL2-V1 with the same activity as IL2-V4. Although none of the mutations present in this variant are located in the AB ring, it also shows the same "α-induced conformation".

[0019] Without being bound by theory, the inventors believe that the substitutions introduced in the IL-2 antagonist variants of the invention stabilize the bound form and induce conformational changes that allosterically affect the interaction with the IL-2Rβ and γ subunits.

[0020] These results show that these human IL-2 variants are useful for treating immune diseases and for increasing the potential of immune responses to vaccines. In particular, immune disorders include inflammatory diseases, allergic diseases, infectious diseases and autoimmune diseases, graft versus host disease (GVHD), transplant rejection and cancer. Vaccines can be directed against cancer or infectious diseases.

[0021] The inventors have also shown that resurfaced variants of IL-2 can also be used to screen for anti-IL-2 antibodies with pro-Teff or pro-Treg activity.

[0022] Thus, the present invention relates to interleukin-2 (IL-2) variants capable of preferentially stimulating T-regulatory cells or antagonizing IL-2, comprising at least one amino acid substitution at a surface position of IL-2 outside the region contacting the alpha receptor.

[0023] In some embodiments, the interleukin-2 variant according to the present invention comprises at least one amino acid substitution at a position selected from the group consisting of: 9, 12, 16, 19, 23, 26, 31, 87, 91 and 95, wherein:

[0024] - the amino acids at positions 9 and 12 are substituted by D or E, preferably E;

[0025] - the amino acids at positions 16, 19, 26, 91 and 95 are substituted by K or R; preferably, the amino acids at positions 16 and 19 are substituted by R, and the amino acids at positions 26, 91 and 95 are substituted by K;

[0026] - the amino acid at position 23 is substituted by E, Q, T, N, G, A, V, L or I, preferably L;

[0027] - the amino acid at position 31 is substituted by P or N, preferably P;

[0028] - the amino acid at position 87 is substituted by M, V, E, D, T, C, N or Q, preferably N; and

[0029] Wherein when the substitution is at position 91, the variant comprises at least one further substitution at position 9, 12, 16, 19, 23, 26, 31, 49, 52, 81, 84, 87, 95, 119, 123, 127, 131 or 132, and the indicated positions are determined by alignment with SEQ ID NO: 1.

[0030] In some preferred embodiments, the variants according to the invention comprise one or more amino acid substitutions at positions selected from the group consisting of: 9, 12, 16, 19, 23, 26, 87, 91 and 95, and preferably at least one additional substitution at a position selected from the group consisting of: 31, 49, 52, 81, 84, 119, 123, 127, 131 and 132, wherein:

[0031] - the amino acid at position 31 is substituted by P or N, preferably P, and,

[0032] - the amino acids at positions 49, 52, 84 and 132 are substituted by another amino acid selected from the group consisting of M, V, E, D, S, T, C, N and Q; preferably, the amino acid at position 49 is substituted by Q; the amino acids at positions 52 and 132 are substituted by S; and the amino acid at position 84 is substituted by N;

[0033] - the amino acid at position 81 is substituted by D or E, preferably E;

[0034] - the amino acids at positions 119, 127 and 131 are substituted by K or R; preferably, the amino acid at position 127 is substituted by K and the amino acid at position 131 is substituted by R; and

[0035] - the amino acid at position 123 is substituted by E, Q, N, M, G, A, V, L or I, more preferably A.

[0036] In some other preferred embodiments, the variants according to the invention comprise a substitution at position 31 and do not comprise any substitutions at positions 9, 12, 16, 19, 23, 26, 49, 52, 81, 84, 87, 91, 95, 119, 123, 127, 131 and 132.

[0037] In some embodiments, the variant according to the invention is a human IL-2 variant, which is preferably selected from the group consisting of:

[0038] - a variant comprising the substitutions K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, N119K, T123A, and S127K;

[0039] - variants comprising the substitutions K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, Y31P, K49Q, E52S, R81E, D84N, N119K, T123A, T131R and L132S;

[0040] - a variant comprising the substitution Y31P, wherein the variant does not comprise any substitutions at positions 9, 12, 16, 19, 23, 26, 49, 52, 81, 84, 87, 91, 95, 119, 123, 127, 131 and 132;

[0041] - variants comprising the substitutions K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, Y31P, K49Q, E52S, R81E, D84N, T131R and L132S;

[0042] - a variant comprising the substitutions K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, Y31P, N119K, T123A and S127K; and

[0043] - Variants comprising the substitutions K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, K49Q, E52S, R81E, D84N, N119K, T123A, T131R and L132S.

[0044] In some embodiments, the variant according to the present invention has at least 70% amino acid identity with any one of SEQ ID NOs: 1 and 3-8, and preferably it does not contain any substitutions in the following positions: 11, 13, 15, 18, 20, 22, 29, 30, 35, 37, 48, 68, 69, 71, 74, 75, 76, 80, 85, 86, 88, 92, 110, 125, 126, 129, 130 and 133.

[0045] In some embodiments, the variants according to the invention preferentially stimulate T regulatory cells.

[0046] In some embodiments, the variant according to the invention is an IL-2 antagonist, preferably wherein the IL-2 antagonist inhibits T regulatory cells and thereby stimulates an immune response, in particular a CD8+ T cell response.

[0047] In some embodiments, variants according to the present invention are combined with target agents in the form of molecular complexes, particles, conjugates or fusion proteins. In some preferred embodiments, IL-2 variants are compounded with anti-IL-2 antibodies, preferably with anti-IL-2 antibodies that promote T regulatory cells or promote T effector cell activity. In some preferred embodiments, IL-2 variants are fused with antibodies to Treg-specific surface molecules or functional fragments thereof that at least contain antigen binding sites. Antibodies to Treg-specific surface molecules are selected from the group consisting of anti-CTLA-4, anti-CD25, anti-CCR8, anti-ICOS, anti-IKZF2, anti-CD70, anti-GARP, anti-IL1R1, anti-CD39, anti-CCR4 and anti-CD177 antibodies.

[0048] The present invention relates to a polynucleotide encoding the variant in an expressible form, a vector comprising the polynucleotide, preferably an expression vector, and a host cell comprising the polynucleotide or the vector.

[0049] The present invention relates to a pharmaceutical composition comprising as an active substance an IL-2 variant as disclosed in the present invention ultimately combined with a target agent, a polynucleotide, a vector and / or a cell according to the present invention, and at least one pharmaceutically acceptable excipient and / or carrier.

[0050] The present invention relates to the use of the IL-2 variants, polynucleotides, vectors and / or cells according to the present invention for the treatment of immune disorders.

[0051] In some embodiments, the IL-2 variants that preferentially stimulate Tregs (Treg-agonist variants), polynucleotides, vectors and / or cells according to the present invention are used to expand Tregs ex vivo or in vivo to treat diseases in which immunomodulation or immunosuppression is beneficial, such as but not limited to allergic and autoimmune diseases, and diseases involving overactivation of the immune system, which particularly include chronic or acute inflammatory diseases, graft-versus-host disease (GVHD) and transplant rejection.

[0052] In some embodiments, the IL-2 antagonist variants, polynucleotides, vectors and / or cells according to the present invention are used to block IL-2-mediated overactivation of the immune system to treat diseases involving overactivation of the immune system associated with excessive production of IL-2, particularly chronic or acute inflammatory diseases, graft-versus-host disease (GVHD) and transplant rejection.

[0053] In some embodiments, the IL-2 antagonist variants, polynucleotides, vectors and / or cells according to the present invention are used to inhibit Tregs and thereby stimulate immune responses, including B, NK, CD4+ or CD8+ T cells, DCs, macrophages, etc., in particular CD8+ T cell immune responses, for treating cancer or infectious diseases or increasing the immune response to vaccines, in particular vaccines against cancer or infectious diseases.

[0054] In some embodiments, IL-2-antagonist variants, polynucleotides, vectors and / or cells according to the present invention are used in combination with chimeric antigen receptor T cell therapy, immunomodulatory monoclonal antibody therapy, or the use of anticancer agents or anti-infective agents including therapeutic agents and vaccines for cancer and infectious diseases. In some preferred embodiments, IL-2 antagonist variants, polynucleotides, vectors and / or cells according to the present invention are used in combination with at least one other cancer therapy selected from the group consisting of targeted therapy, immunotherapy (such as immune checkpoint therapy and immune checkpoint inhibitors), co-stimulatory antibodies, chemotherapy and / or radiotherapy.

[0055] The present invention also relates to the use of the IL-2 variant according to the present invention for screening anti-IL-2 antibodies having T effector cell-promoting or T regulatory cell-promoting activity. DETAILED DESCRIPTION

[0056] IL-2 variants

[0057] The present invention relates to interleukin-2 (IL-2) variants capable of preferentially stimulating T-regulatory cells or antagonizing IL-2, comprising at least one amino acid substitution at a surface position of IL-2 outside the region contacting the alpha receptor.

[0058] The term interleukin-2 or IL-2, also known as TCGF or lymphokine, refers to the protein encoded by the IL-2 gene in the mammalian genome. IL-2 is expressed as a precursor containing an N-terminal signal peptide (20 amino acids), which is cleaved to produce the mature protein (IL-2). A representative example of IL-2 is shown in Figure 1 , and includes but is not limited to human (gene ID: 35583558), rat (gene ID: 116562), cat (gene ID: 751114) and mouse (gene ID: 16183) forms. As used herein, IL-2 refers to wild-type IL-2. The human IL-2 precursor has a 153 amino acid sequence UniProtKB / Swiss-Prot: P60568.1. Mature IL-2 has a 133 amino acid sequence at positions 21-153 of the precursor and corresponds to SEQ ID NO: 1.

[0059] In the following description, the standard single-letter amino acid code is used to designate residues, and the designated positions are determined by alignment with SEQ ID NO: 1. For example, K9 is the lysine residue at position 9 of SEQ ID NO: 1. Substitutions are designated herein by the single-letter amino acid code followed by the substituted residue in the single-letter amino acid code; K9E is a substitution of the lysine (K) residue at position 9 of SEQ ID NO: 1 with a glutamic acid (E) residue.

[0060] "A", "an" and "the" include plural referents unless the context clearly indicates otherwise. Thus, the terms "a" (or "an"), "one or more" or "at least one" are used interchangeably herein; unless otherwise specified, "or" means "and / or".

[0061] The present invention provides interleukin-2 (IL-2) variants capable of preferentially stimulating T-regulatory cells (Tregs) or antagonizing IL-2, which can be used to treat immune disorders.

[0062] "Immune disorders" as used herein refer to diseases involving immune dysfunction or immune disorders. Immune dysfunction may include suppression, dysfunction or overactivation of the immune system. Immune disorders include diseases that can be prevented or treated by immunomodulation using immunomodulators, immunosuppressants and / or immunostimulants. Immunomodulation may include preferably stimulating Tregs or suppressing Tregs. Immunosuppression may include preferably stimulating Tregs, which in turn suppress immune effector cells or directly suppress immune effector cells. Similarly, immunostimulation may include suppressing Tregs, which in turn release immune cells from immunosuppression or directly stimulate effector immune cells. Immunomodulators and immunostimulators that suppress Tregs can be used to prevent or treat diseases in which suppression of Tregs and / or stimulation of immune responses are beneficial, such as, but not limited to, infectious diseases and cancers, including treatment of infectious diseases and cancers and increasing immune responses to vaccines. Vaccines are particularly vaccines for cancer or infectious diseases. Immunomodulators and immunosuppressants that stimulate Tregs can be used to treat diseases involving immune dysfunction, such as but not limited to allergic and autoimmune diseases and diseases associated with overactivation of the immune system, such as but not limited to chronic or acute inflammatory diseases, graft-versus-host disease (GVHD), including acute GVHD, and transplant rejection.

[0063] The IL-2 variants (Treg- agonist variants) of the present invention that preferably stimulate T- regulatory cells can be used for ex vivo or in vivo amplification Treg. The IL-2 variants (IL-2- antagonist variants) of the present invention that antagonize IL-2 can be used for blocking the overactivation of the immune system mediated by IL-2, or by depriving them of IL-2 signaling, and therefore stimulate immune response, including B, NK, CD4+ or CD8+ T cells, DC, macrophages, etc., particularly CD8+ T cell immune response in vivo, such as anti-tumor immune response or immune response for vaccines or pathogens for suppressing Tregs. As used herein, "preferably stimulating T- regulatory cells" refers to that IL-2 variants promote the proliferation or activation of T- regulatory cells relative to non-regulatory T cells. As used herein, "T- regulatory cells", "Treg" or "Tregs" refer to CD3+CD4+Foxp3+T cells, including CD3+CD4+Foxp3+CD25+ and CD3+CD4+Foxp3+CD25- cells. As used herein, "T-effector cells", "Teff" or "Teffs" refer to one or more Tconv cells (CD3+CD4+Foxp3-); CD8+T cells (CD3+CD8+) and NK cells (CD3-CD16+). As used herein, "inhibiting Treg" refers to that IL-2 variants can inhibit the proliferation, activation or inhibition of Treg cells by depriving Treg cells of IL-2 signaling. The inhibition of Treg includes the inhibition of Treg function or the loss of Treg function, particularly the loss of Treg immunosuppressive function and the elimination of Treg. The IL-2 variants of the present invention preferably stimulate the ability of T-regulatory cells to be measured by standard assays well known in the art and disclosed in the embodiments of the present application, including but not limited to STAT5 phosphorylation assays or flow cytometric analysis of T cell populations from subjects treated in vivo or peripheral blood samples treated in vitro. The ability of IL-2 variants to antagonize IL-2 can be determined by standard assays known in the art and disclosed in the Examples of this application, such as a STAT5 phosphorylation assay by competition with Proleukine on in vitro treated peripheral blood samples or quantification of proliferation using the CellTrace Violet (CTV) dilution assay.

[0064] Treg-agonist variants can be used to indirectly reduce immune activation by preferably activating Treg, which in turn inhibits immune effector cells. Therefore, the treg-agonist variants of the present invention can be used to treat diseases in which immunomodulation or immunosuppression is beneficial, such as but not limited to allergic and autoimmune diseases, and diseases comprising overactivation of the immune system, including but not limited to chronic or acute inflammatory diseases, graft-versus-host disease (GVHD) and transplant rejection.

[0065] The IL-2 variants according to the present invention as IL-2 antagonists can be used to inhibit Treg and thus stimulate immune responses (B, NK, CD4+ or CD8+T cells, DC, macrophages, etc.). The IL-2-antagonist variants according to the present invention can be used to stimulate anti-tumor immune responses or immune responses to pathogens or vaccines (the pathogens or vaccines include vaccines for cancer or infectious diseases), especially anti-tumor CD8+T cell responses and anti-CD8+T cell responses to pathogens or vaccines. In addition, since the application results show that the effect of IL-2 antagonists in vivo may vary depending on the immune environment, IL-2 antagonists can also be used in diseases including over-activation of the immune system associated with excessive production of IL-2 (such as, but not limited to, chronic or acute inflammatory diseases, graft-versus-host disease (GVHD) and transplant rejection) to reduce immune activation by blocking the excess IL-2 present in the patient.

[0066] The present invention provides interleukin-2 (IL-2) variants comprising at least one amino acid substitution at a surface position of IL-2 outside the region that contacts the α receptor. A residue that contacts the α receptor is defined as one in which at least one atom is 100% away from any atom of the α receptor. Surface residues are defined as residues with >50% of their side chain surface area exposed, as determined by standard methods well known in the art (Fraczkiewicz et al., J. Comp. Chem, 1998. 19, 319-333).

[0067] In some embodiments, the IL-2 variant comprises at least one amino acid substitution at a position selected from the group consisting of 9, 12, 16, 19, 23, 26, 31, 87, 91, and 95, as determined by alignment with SEQ ID NO:1.

[0068] "Comprising at least one substitution" means that the IL-2 variant has one or more amino acid substitutions as shown in the amino acid sequence SEQ ID NO: 1, but may have other modifications, including but not limited to substitutions, deletions or additions of amino acid residues. The IL-2 variant may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or all of the substitutions listed above. All these possible combinations are specifically contemplated.

[0069] In some embodiments, the IL-2 variant comprises one or more amino acid substitutions at positions selected from the group consisting of 9, 12, 16, 19, 23, 26, 87, 91, and 95. The IL-2 variant may comprise 1, 2, 3, 4, 5, 6, 7, 8, or all of the substitutions listed above. All of these possible combinations are specifically contemplated.

[0070] In some preferred embodiments, the IL-2 variant comprises one or more amino acid substitutions at positions selected from the group consisting of 31, 49, 52, 81, 84, 119, 123, 127, 131 and 132, as determined by alignment with SEQ ID NO: 1. The IL-2 variant may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or all of the substitutions listed above. All of these possible combinations are specifically contemplated.

[0071] In some more preferred embodiments, the variant comprises additional substitutions at the following positions: 119, 123 and 127; 31, 119, 123 and 127; 31, 49, 52, 81, 84, 119, 123, 131 and 132; 31, 49, 52, 81, 84, 131 and 132; or 49, 52, 81, 84, 119, 123, 131 and 132.

[0072] In some preferred embodiments, when the substitution is at position 91, the variant comprises at least one further substitution at position 9, 12, 16, 19, 23, 26, 31, 49, 52, 81, 84, 87, 95, 119, 123, 127, 131 or 132. The IL-2 variant may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or all of the additional substitutions listed above. All of these possible combinations are specifically contemplated. Preferably, the variant comprises substitutions at positions 9, 12, 16, 19, 23, 26, 87, 91 and 95, and finally comprises at least one further substitution at a position selected from the group consisting of 31, 49, 52, 81, 84, 119, 123, 127, 131 and 132. IL-2 variants may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9 or all of the additional substitutions listed above. All of these possible combinations are specifically contemplated. In some more preferred embodiments, the IL-2 variant comprises additional substitutions at the following positions: 119, 123 and 127; 31, 119, 123 and 127; 31, 49, 52, 81, 84, 119, 123, 131 and 132; 31, 49, 52, 81, 84, 131 and 132; or 49, 52, 81, 84, 119, 123, 131 and 132.

[0073] In some other embodiments, the IL-2 variant comprises a substitution at position 31; the IL-2 variant comprises only one of said substitutions, which means that the IL-2 variant does not comprise any substitutions at the other specified positions (9, 12, 16, 19, 23, 26, 87, 91, 95 and 49, 52, 81, 84, 119, 123, 127, 131 and 132).

[0074] In some preferred embodiments, the amino acids at positions 9, 12 and 81 are substituted with D or E, preferably E.

[0075] In some preferred embodiments, the amino acids at positions 16, 19, 26, 91, 95, 119, 127 and 131 are substituted with K or R; preferably, the amino acids at positions 16, 19 and 131 are substituted with R, and the amino acids at positions 26, 91, 95 and 127 are substituted with K.

[0076] In some preferred embodiments, the amino acids at positions 23 and 123 are substituted by another amino acid selected from E, Q, T, N, G, A, V, L, I and M; more preferably, the amino acid at position 23 is substituted by L, and the amino acid at position 123 is substituted by A.

[0077] In some preferred embodiments, the amino acid at position 31 is substituted with N or P, preferably P.

[0078] In some preferred embodiments, the amino acids at positions 49, 52, 84, 87 and 132 are substituted by another amino acid selected from the group consisting of: M, V, E, D, S, T, C, N and Q; preferably, the amino acids at positions 87 and 84 are substituted by N; the amino acid at position 49 is substituted by Q, and the amino acids at positions 52 and 132 are substituted by S.

[0079] IL-2 variants may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or all of the substitutions listed above. All of these possible combinations are specifically contemplated.

[0080] In some preferred embodiments, the IL-2 variant comprises at least one or more (i.e., 1, 2, 3, 4, 5, 6, 7, 8, or 9) selected from the following substitutions: substitutions K9E or K9D, L12E or L12D, H16R or H16K, L19R or L19K, M23L, N26K or N26R, S87N, V91K or V91R, E95K or E95R; preferably one or more (i.e., 1, 2, 3, 4, 5, 6, 7, 8, or 9) selected from the following substitutions: K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, and E95K. Preferably, the IL-2 variant comprises all of the substitutions listed above. In some preferred embodiments, the IL-2 variant comprises an additional substitution selected from the group consisting of: N119K or N119R, T123A, and S127K or S127R; Y31P, N119K or N119R, T123A, and S127K or S127R; Y31P, K49Q, E52S, R81E or R81D, D84N, N119K or N119R 9R, T123A, T131R or T131K and L132S; Y31P, K49Q, E52S, R81E or R81D, D84N, T131R or T131K and L132S; or K49Q, E52S, R81E or R81D, D84N, N119K or N119R, T123A, T131R or T131K and L132S. Preferably, it is selected from the following group: N119K, T123A, and S127K; Y31P, N119K, T123A, and S127K; Y31P, K49Q, E52S, R81E, D84N, N119K, T123A, T131R and L132S; Y31P, K49Q, E52S, R81E, D84N, T131R and L132S; or K49Q, E52S, R81E, D84N, N119K, T123A, T131R and L132S.

[0081] In some preferred embodiments, the IL-2 variant is a human IL-2 variant.

[0082] According to said preferred embodiment, said IL-2 variant, preferably a human IL-2 variant comprises:

[0083] a) substitutions K9E or K9D, L12E or L12D, H16R or H16K, L19R or L19K, M23L, N26K or N26R, S87N, V91K or V91R, E95K or E95R; preferably substitutions K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K and E95K; and additional substitutions selected from the group consisting of:

[0084] a1) N119K or N119R, T123A, and S127K or S127R; preferably N119K, T123A and S127K;

[0085] a2) Y31P, N119K or N119R, T123A, and S127K or S127R; preferably Y31P, N119K, T123A and S127K;

[0086] a3) Y31P, K49Q, E52S, R81E or R81D, D84N, N119K or N119R, T123A, T131R or T131K and L132S; preferably Y31P, K49Q, E52S, R81E, D84N, N119K, T123A, T131R and L132S;

[0087] a4) K49Q, E52S, R81E or R81D, D84N, N119K or N119R, T123A, T131R or T131K and L132S; preferably K49Q, E52S, R81E, D84N, N119K, T123A, T131R and L132S; and

[0088] a5) Y31P, K49Q, E52S, R81E or R81D, D84N, T131R or T131K and L132S; preferably Y31P, K49Q, E52S, R81E, D84N, T131R and L132S; or

[0089] b) substituted with Y31P or Y31N; preferably Y31P;

[0090] And the indicated position is determined by alignment with SEQ ID NO:1.

[0091] According to the invention, IL-2 variants having substitutions in a) and additional substitutions in a3) or a5) are capable of preferentially stimulating T-regulatory cells (treg-agonist variants), whereas IL-2 variants having substitutions in a) and additional substitutions in a1), a2) or a4) and IL-2 variants having substitutions in b) are capable of antagonizing IL-2 (IL-2 antagonist variants).

[0092] In some more preferred embodiments, the human IL-2 variant is selected from the group consisting of:

[0093] - a variant comprising substitutions K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, N119K, T123A and S127K; this variant is named IL2-v1 (IL2V1, IL2-V1, IL-2V1, IL-2-V1 or IL-2V1) in the examples;

[0094] - a variant comprising substitutions K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, Y31P, K49Q, E52S, R81E, D84N, N119K, T123A, T131R and L132S; the variant is named IL2-v2 (IL2V2, IL2-V2, IL-2V2, IL-2-V2 or IL-2V2) in the examples;

[0095] - a variant comprising substitutions K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, Y31P, K49Q, E52S, R81E, D84N, T131R and L132S; the variant is named IL2-v3 (IL2V3, IL2-V3, IL-2V3, IL-2-V3 or IL-2V3) in the examples;

[0096] - a variant comprising the substitution Y31P, wherein the variant does not comprise any substitutions at the other specified positions (9, 12, 16, 19, 23, 26, 87, 91, 95 and 49, 52, 81, 84, 119, 123, 127, 131 and 132); the variant is named IL2-v4 (IL2V4, IL2-V4, IL-2V4, IL-2-V4 or IL-2V4) in the examples;

[0097] - a variant comprising substitutions K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, Y31P, N119K, T123A and S127K; the variant is named IL2-v5 (IL2V5, IL2-V5, IL-2V5, IL-2-V5 or IL-2V5) in the examples; and

[0098] - A variant comprising substitutions K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, K49Q, E52S, R81E, D84N, N119K, T123A, T131R and L132S; the variant is named IL2-v6 (IL2V6, IL2-V6, IL-2V6, IL-2-V6 or IL-2V6) in the examples.

[0099] In various embodiments, the IL-2 variant (Treg-agonist variant or IL-2 antagonist) may or may not include at least one additional amino acid mutation (insertion, deletion, substitution). In some embodiments, the IL-2 variant does not include additional amino acid mutations. In some other embodiments, the IL-2 variant includes at least one additional amino acid mutation (insertion, deletion, substitution). The IL-2 variant preferably includes at least one amino acid deletion, more preferably at least one amino acid deletion at a position selected from S4S5 or S6, and the position shown is determined by comparing with SEQ ID NO: 1. The IL-2 variant may include 1, 2 or all of the above-listed deletions and the above-listed substitutions. All these possible combinations are particularly contemplated.

[0100] In various embodiments, the IL-2 variant may be derived from wild-type IL-2 or a wild-type IL-2 construct. In some embodiments, the IL-2 variant is derived from a wild-type human IL-2 construct of SEQ ID NO: 2. In some preferred embodiments, the IL-2 variant comprises or consists of any one of SEQ ID NO: 3-8.

[0101] The size of the IL-2 variant is at least 125 amino acids. Preferably, the size of the IL-2 variant is at least 130 or more amino acids.

[0102] In some preferred embodiments, the IL-2 variant has at least 70% amino acid identity with SEQ ID NO: 1. Preferably, the IL-2 variant has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the sequence. More preferably, the IL-2 variant sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with the sequence, and even more preferably has at least 95%, 96%, 97%, 98% or 99%, 98% or 99% identity with the sequence.

[0103] The amino acid sequence identity percentage is defined as the percentage of amino acid residues in the comparison sequence identical to the reference sequence after the alignment sequence and, if necessary, introducing a gap to achieve maximum sequence identity and not considering any conservative substitution as a part of the sequence identity. Sequence identity is calculated over the full length of the reference sequence. Comparison for determining the amino acid sequence identity percentage purpose can be achieved in a variety of ways known to those skilled in the art, such as using publicly available computer software, such as BLAST (Altschul et al., J. Mol. Biol., 1990, 215, 403-). When using this software, preferably default parameters are used, such as for gap penalty and extension penalty. The BLASTP program defaults to using a word length (W) of 3 and an expected value (E) of 10.

[0104] In some preferred embodiments, the IL-2 variant does not comprise any substitutions at positions 11, 13, 15, 18, 20, 22, 29, 30, 35, 37, 48, 68, 69, 71, 74, 75, 76, 80, 85, 86, 88, 92, 110, 125, 126, 129, 130, and 133 or at positions 4, 8, 10, 11, 13, 15, 18, 20, 22, 29, 30, 35, 37, 38, 42, 45, 48, 62, 67, 68, 69, 71, 74, 75, 76, 80, 85, 86, 88, 90, 92, 110, 125, 126, 128, 129, 130, and 133.

[0105] In some preferred embodiments, the IL-2 variant is selected from the group consisting of sequences SEQ ID NO: 3-8 and sequences having at least 70% amino acid identity with said sequences. Preferably, the IL-2 variant has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with said sequences. More preferably, the IL-2 variant has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence, even more preferably at least 95%, 96%, 97%, 98% or 99%, 98% or 99% identity to the sequence; more preferably, the IL-2 variant has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence; , 69, 71, 74, 75, 76, 80, 85, 86, 88, 92, 110, 125, 126, 129, 130 and 133 or does not contain any substitution at position 4, 8, 10, 11, 13, 15, 18, 20, 22, 29, 30, 35, 37, 38, 42, 45, 48, 62, 67, 68, 69, 71, 74, 75, 76, 80, 85, 86, 88, 90, 92, 110, 125, 126, 129, 130 and 133.

[0106] In some embodiments, the IL-2 variant is capable of preferentially stimulating T-regulatory cells (Treg). Non-limiting examples of such variants include variants IL-2V2 and IL-2V3 as defined above. In some preferred embodiments, the IL-2 variant is selected from the group consisting of sequences SEQ ID NO: 4 and 5, and sequences having at least 70% amino acid identity with the sequences as defined above; more preferably, the IL-2 variant is selected from the group consisting of sequences SEQ ID NO: 11, 13, 15, 18, 20, 22, 29, 30, 35, 37, 48, 68, 69, 71, 74, 75, 76, 80, 85, 86, 88, 92, 110, 125, 126, 129, 13 0 and 133 or positions 4, 8, 10, 11, 13, 15, 18, 20, 22, 29, 30, 35, 37, 38, 42, 45, 48, 62, 67, 68, 69, 71, 74, 75, 76, 80, 85, 86, 88, 90, 92, 110, 125, 126, 128, 129, 130 and 133 do not contain any substitution. The IL-2 variants of the invention that are capable of preferentially stimulating Tregs (Treg-agonists) can be used to treat diseases in which immunomodulation or immunosuppression is beneficial, such as but not limited to allergic and autoimmune diseases, and diseases involving overactivation of the immune system, including but not limited to chronic or acute inflammatory diseases, graft versus host disease (GVHD) and transplant rejection.

[0107] In some embodiments, the IL-2 variant is an IL-2 antagonist. Non-limiting examples of such variants include variants IL-2V1, IL-2V4, IL-2V5 and IL-2V6 as defined above, preferably IL-2V1 or IL-2V4; more preferably IL-2V4. In some preferred embodiments, the IL-2 variant is selected from the group consisting of sequences SEQ ID NO: 3, 6, 7 and 8 and sequences having at least 70% amino acid identity with said sequences as defined above; preferably SEQ ID NO:3 or a sequence having at least 70% amino acid identity thereto; More preferably, the IL-2 variant does not comprise any substitution at position 11, 13, 15, 18, 20, 22, 29, 30, 35, 37, 48, 68, 69, 71, 74, 75, 76, 80, 85, 86, 88, 92, 110, 125, 126, 129, 130 and 133 or at position 4, 8, 10, 11, 13, 15, 18, 20, 22, 29, 30, 35, 37, 38, 42, 45, 48, 62, 67, 68, 69, 71, 74, 75, 76, 80, 85, 86, 88, 90, 92, 110, 125, 126, 128, 129, 130 and 133. The IL-2 antagonists according to the present invention can be used to suppress harmful immune responses in vivo or stimulate beneficial immune responses in vivo. Therefore, the IL-2 antagonists according to the present invention can be used to treat diseases including the overactive immune system associated with excessive production of IL-2, such as but not limited to chronic or acute inflammatory diseases, graft versus host disease (GVHD) and transplant rejection. IL-2 antagonists can also be used to deprive Treg from endogenous wild-type IL-2 signaling and thus stimulate immune responses (B, NK, CD4+ or CD8+T cells, DC, macrophages, etc.), particularly CD8+T cell immune responses to suppress Treg. Therefore, IL-2 antagonist variants can also be used to prevent or treat immune disorders in which Treg suppresses and / or stimulates immune responses, particularly CD8+T cell immune responses are beneficial, such as but not limited to cancer, infectious diseases and vaccinations.

[0108] The present invention encompasses IL-2 variants with one or more modifications at one or more amino acid residues, peptide bonds, N- and / or C-termini, as long as the modified variants are functional (i.e., able to preferably stimulate Tregs or antagonize IL-2). Preferably, the modification is to increase the stability or bioavailability of the IL-2 variant according to the present invention, in particular to increase its in vivo half-life; reduce its immunogenicity; or facilitate its purification, detection or targeting to a specific cell type or tissue. These modifications introduced into the variant by conventional methods known to those skilled in the art include, in a non-limiting manner: mutations (insertions, deletions, substitutions) of one or more amino acids in the amino acid sequence; fusion with a target amino acid portion (target protein for therapeutic use or a label (antibody epitope or tag) for purification, detection, or coupling with a target molecule or agent; substitution of natural amino acids with non-proteinogenic amino acids (D amino acids or amino acid analogs); modification of peptide bonds, in particular with retro or retro-inverso type bonds or bonds different from peptide bonds; cyclization, and addition of chemical groups to the amino acid side chains or N- and / or C-termini of the variant. Chemical groups, particularly for coupling a target molecule or agent to a variant. Modifications include, but are not limited to, esterification, glycosylation, acylation (such as acetylation) or attachment to myristic acid, amidation, phosphorylation, biotinylation, PEGylation, farnesyl coupling and similar modifications well known in the art. Modifications can be introduced at the N-terminus (acetylation), C-terminus (amidation) of the IL-2 variant, or if deemed appropriate, any amino acid other than the terminal amino acid (e.g., farnesyl coupled to a cysteine ​​side chain) can also be introduced. Conversion of the acid functional group at the C-terminus to an aldehyde and alkylation of the thiol functional group of the cysteine ​​residue are used for chemoselective attachment or formation of a reduced peptide bond.

[0109] In particular, the present invention encompasses IL-2 variants comprising or consisting of natural amino acid chains (20 gene-encoded amino acids in L- and / or D-configuration (A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, X and Y)) linked via peptide bonds, and also peptide mimetics of the variants, wherein the amino acids, peptide bonds N- and / or C-termini have been replaced by functional analogs. The functional analogs of these amino acids include all known amino acids except the amino acids encoded by the 20 genes.

[0110] In some embodiments, the IL-2 variant is provided in the form of a combination with at least one target agent, for example in the form of a complex (such as a molecular complex) or a particle; a conjugate or a fusion protein. The target agent includes, but is not limited to, any therapeutic agent, including cells (such as chimeric antigen receptor (CAR) T- cells of a patient), any agent that increases the stability or bioavailability of the IL-2 variant according to the present invention, in particular, any agent that increases its half-life in vivo; any agent that reduces its immunogenicity; or any agent that promotes its purification, detection or targeting of a specific cell type or tissue. The target agent can be a small or large compound, macromolecule or particle. Particles include, but are not limited to, liposomes, micelles and nanoparticles, including liposomes, micelles and nanoparticles carrying active agents (such as nanocarriers). For example, the IL-2 variant and ultimately other target agents can be encapsulated in the particle or grafted onto the particle by methods well known in the art. In a preferred embodiment, the target agent is selected from the group consisting of peptides; proteins, including antibodies; biologically active substances, such as drugs for treating human or animal diseases; labels, markers and particles.

[0111] In some more preferred embodiments, the target agent is an anti-IL-2 antibody, preferably an anti-IL-2 antibody having a Treg-promoting or T-effector cell function or a functional fragment thereof comprising at least an antigen binding site; more preferably a human or humanized anti-IL-2 antibody. Anti-IL-2 antibodies or fragments thereof having a Treg-promoting function can be used to treat diseases associated with overactivation of the immune system, such as, but not limited to, chronic or acute inflammatory diseases, graft-versus-host disease (GVHD), and transplant rejection. Anti-IL-2 antibodies or fragments thereof having a T-effector cell function can be used to prevent or treat immune disorders in which Treg inhibition and / or stimulation of CD8+T cell responses are beneficial, such as, but not limited to, cancer, infectious diseases, and vaccination. In some embodiments, an anti-IL-2 antibody, preferably an anti-IL-2 antibody having a Treg-promoting or T-effector cell function, is a complete antibody molecule.

[0112] In some more preferred embodiments, the target agent comprises or consists of a ligand of a Treg-specific surface molecule. Treg-specific surface molecules are known in the art and are disclosed in, for example, Bhairavabhotla et al., Human Immunol., 2016, 77, 201-13; van der Veeken et al., Cold Spring Harb. Symp. Quant Biol., 2013, 78, 215–22; Pfoertner et al., Genome Biol., 2006, 7, R54; Sugimoto et al., Int. Immunol., 2006, 18, 1197–. Non-limiting examples of Treg-specific surface molecules that can be used in the present invention include CD25, CTLA-4, CCR8, ICOS, IKZF2, CD70, GARP, IL1R1, CD39, CCR4, and CD177.

[0113] The target agent is preferably an antibody against a Treg-specific surface molecule or a functional fragment thereof containing at least an antigen binding site; preferably an anti-CTLA-4, anti-CD25, anti-CCR8, anti-ICOS, anti-IKZF2, anti-CD70, anti-GARP, anti-IL1R1, anti-CD39, anti-CCR4 or anti-CD177 antibody; more preferably a human or humanized antibody. In some embodiments, the antibody against a Treg-specific surface molecule is a complete antibody molecule, preferably a complete human or humanized antibody molecule.

[0114] In some more preferred embodiments, the target agent is selected from the group consisting of: a complete antibody or an antibody Fc region, preferably a complete human antibody or a human Fc region; a multispecific antigen binding protein, such as a bispecific antibody; an ankyrin and a designed ankyrin repeat protein (DARPin); an MHC-peptide multimer, such as a class I or class II MHC-peptide tetramer, in particular an MHC-peptide multimer. The peptide in the MHC-peptide multimer can be derived from an antigen (such as a self-antigen) that can induce an autoimmune or alloimmune response, or from an exogenous antigen (such as an antigen from a pathogen or a tumor), in particular a vaccine antigen against a pathogen or a tumor.

[0115] The complex, conjugate or fusion protein within the scope of the present invention comprises more than one IL-2 variant of the present invention, i.e. a plurality of such variants, wherein the plurality of variants may comprise a plurality of identical or different variants. In addition, the complex, conjugate or fusion protein according to the present invention may also comprise more than one target agent, wherein the plurality of agents may comprise a plurality of identical or different agents.

[0116] The target agent is covalently or non-covalently linked to the IL-2 variant of the present invention. The target agent can be coupled directly or indirectly to the IL-2 variant. The indirect coupling of the target agent to the IL-2 variant can be through a joint connected to the IL-2 variant of the present invention. Joints (also referred to as spacers) that can be used to physically separate the IL-2 variant of the present invention from the target agent are known in the art and include peptide bonds, amino acids, peptides of appropriate length or different molecules that provide the desired characteristics. The joint can be connected to the N-terminus, C-terminus of the IL-2 variant, or if deemed appropriate, it can also be connected to any amino acid other than the terminal amino acid. The IL-2 variant of the present invention can be chemically linked to the target agent by covalent bonds using standard conjugation techniques. The target agent can be connected to the N-terminus, C-terminus of the IL-2 variant, or, if applicable, to any amino acid other than the terminal amino acid. Functional groups, modifications (also referred to as derivatization) or joints can also be introduced into the IL-2 variant to conjugate the IL-2 variant to the target agent. This covalent bond is preferably formed between the suitable reactive group of IL-2 variant and the target agent, more preferably between the end of the IL-2 variant according to the present invention and the target agent. Depending on the chemical properties of the target agent, the part, group or free radical forming this covalent bond therewith is different, and producing this bond is within the skill range of those skilled in the art. The chemical bond can be via a disulfide bond, a thioether bond, a thiol-maleimide bond or an amide bond. Other ways of connecting IL-2 variant to cargo include forming an oxime using a C-terminal aldehyde, using a click reaction or forming a morpholine bond with a basic amino acid on a peptide.

[0117] When the target agent is a peptide or protein including an antibody or a functional fragment thereof, the IL-2 variant is advantageously provided as a chimeric fusion protein comprising a heterologous target agent (different from IL-2 or IL-2 fragment) fused directly or via a peptide spacer as described above to the N-terminus or C-terminus of the IL-2 variant of the present invention or directly or via insertion into the amino acid sequence of the IL-2 variant of the present invention. The fusion protein is expressed by a chimeric construct in which the nucleotide sequence encoding the IL-2 variant of the present invention is fused in frame to the nucleotide sequence encoding the target peptide / protein using standard recombinant DNA techniques. The resulting fusion protein / peptide is heterologous, i.e., it is different from a naturally occurring peptide or protein, such as IL-2 or other cytokines of the same family.

[0118] IL-2 variants can also be linked to target agents (molecules or particles carrying molecules) via non-covalent bonds (such as ionic bonds, hydrogen bonds or hydrophobic interactions or combinations of these bonds). Non-limiting examples include antigen-antibody interactions between IL-2 variants and anti-IL-2 antibodies; streptavidin-biotin interactions between biotinylated IL-2 variants and target agents (e.g., nanoparticles, such as quantum dots) conjugated to streptavidin or between biotinylated agents and IL-2 variants conjugated to streptavidin.

[0119] In a preferred embodiment, the IL-2 variant is provided in the form of a chimeric fusion protein comprising a heterologous protein or peptide of interest fused directly or via a peptide spacer to the N-terminus or C-terminus of the IL-2 variant of the present invention or inserted into the amino acid sequence of the IL-2 variant of the present invention. The protein or peptide of interest is preferably selected from the group consisting of an intact antibody or antibody Fc region, preferably an intact human antibody or human Fc region; a multispecific antigen binding protein, such as a bispecific antibody; ankyrin and a designed ankyrin repeat protein (DARPin); an MHC-peptide multimer, such as a class I or class II MHC-peptide tetramer, in particular an MHC-peptide multimer, wherein the peptide is derived from an antigen capable of inducing an autoimmune or alloimmune response, such as a self-antigen. In some more preferred embodiments, the antibody is an antibody against a Treg-specific surface molecule or a functional fragment thereof comprising at least an antigen binding site, preferably an anti-CTLA-4, anti-CD25, anti-CCR8, anti-ICOS, anti-IKZF2, anti-CD70, anti-GARP, anti-IL1R1, anti-CD39, anti-CCR4 or anti-CD177 antibody; more preferably a human or humanized antibody or a complete antibody molecule, and even more preferably a complete human or humanized antibody molecule.

[0120] The protein or peptide of interest is advantageously fused directly or via a peptide spacer to the N-terminus or C-terminus of the IL-2 variant of the invention.

[0121] In a preferred embodiment, the IL-2 variant or derived fusion protein as described above is complexed with an anti-IL-2 antibody. In some more preferred embodiments, the anti-IL-2 antibody is an IL-2 antibody with pro-Treg function, i.e., blocking IL-2RB / IL-2 interaction and inducing structural modifications on IL-2. These antibodies are well known in the art, including, for example, clone JES6-1A12 (rat IgG2A anti-mouse IL-2) and clone 5344.111 (mouse IgG1 anti-human IL-2). These Abs have shown the ability to expand Tregs in vivo in preclinical models of diabetes (Tang Q et al, Immunity, 2008, 28, 687-97; Grinberg-Bleyer Y et al, J Exp Med, 2010, 207, 1871-8), allergy (Smaldini PL et al, Allergy, 2018, 73, 885-895), multiple sclerosis (Webster KE et al, J Exp Med, 2009, 206, 751-60), rheumatoid arthritis (Lee-S Y, et al, Immunology, 2012, 137, 305-16) and transplantation (Vokaer B, et al, Transplant Proc., 2012, 44, 2840-4). In some other more preferred embodiments, the anti-IL-2 antibody is an IL-2 antibody with pro-Teff function. These antibodies are well known in the art, including, for example, clone S4B6 (rat IgG2A anti-mouse IL-2) and clone Mab602 (mouse IgG2a anti-human IL-2). These Abs have shown better ability to control tumor growth in melanoma (as a monotherapy or combination therapy) in vivo than IL-2 (Boyman O et al, Science 2006, 311, 1921-27, Krieg et al, PNAS, 2010, 107, 11906-11, Caudana T et al, 2019, Cancer Immunol Res, 7, 443-457) and lymphoma (Newman RG et al, 2019, Cancer Immunol Res, 7, 443-457). al, Blood, 2014, 123, 3045-55). The anti-IL-2 antibody is advantageously a human or humanized anti-IL-2 antibody, including a humanized antibody derived from the above-mentioned mouse or rat monoclonal antibody.

[0122] In a preferred embodiment, the IL-2 variant or derived fusion protein as disclosed above is complexed with an antibody, preferably a whole antibody, more preferably a whole human antibody, directed against a Treg-specific surface molecule as defined above.

[0123] In a preferred embodiment, the IL-2 variant is linked to a CART-cell, in particular a CART-cell of a patient.

[0124] The term "IL-2 variant" as used herein encompasses different forms of the IL-2 variants disclosed herein, such as modified or unmodified IL-2 variants with or without binding to at least one target agent in the form of a complex, conjugate or fusion protein as disclosed above.

[0125] The IL-2 variants according to the invention can be prepared by routine techniques in the art, in particular by expressing recombinant DNA in a suitable cell system (eukaryotic or prokaryotic) and screening for activity (i.e. the ability to preferentially stimulate T-regulatory cells) using the assays described herein or other similar assays.

[0126] Polynucleotides, vectors and host cells

[0127] The present invention also relates to isolated polynucleotides encoding an IL-2 variant in an expressible form.

[0128] A polynucleotide encoding an expressible form of an IL-2 variant refers to a nucleic acid molecule that produces a functional protein when expressed in a cell or a cell-free system.

[0129] The synthetic or recombinant polynucleotide can be single-stranded and / or double-stranded DNA, RNA or a combination thereof. The polynucleotide is operably linked to at least one transcriptional regulatory sequence and optionally operably linked to at least one translational regulatory sequence. Preferably, the polynucleotide comprises a coding sequence optimized for the host expressing the IL-2 variant; more preferably, the polynucleotide sequence is selected from the group consisting of SEQ ID NO: 9-14, which encodes the IL-2 variants of SEQ ID NO: 3-8, respectively.

[0130] The polynucleotides according to the present invention are prepared by conventional methods known in the art. For example, nucleic acid sequences are amplified by PCR or RT-PCR, by screening genomic DNA libraries by hybridization with homologous probes, or by complete or partial chemical synthesis.

[0131] Another aspect of the present invention is a recombinant vector comprising the polynucleotide. The recombinant vector is advantageously an expression vector capable of expressing the polynucleotide when it is delivered to a host cell (such as a prokaryotic or eukaryotic cell), for example a mammal or a bacterial cell. The recombinant vector includes conventional vectors for genetic engineering and gene therapy, including, for example, plasmids and viral vectors.

[0132] Recombinant vectors are constructed by conventional recombinant DNA, genetic engineering and gene therapy techniques known in the art and introduced into host cells.

[0133] Therefore, another aspect of the present invention provides a host cell comprising the polynucleotide or recombinant vector.

[0134] In some embodiments, the host cell is a patient cell (such as a T cell, NK cell or CART-cell), which is modified with a polynucleotide or vector according to the present invention for adoptive T cell therapy.

[0135] In some embodiments, the host cell of the present invention is a host cell deposited at the National Collection of Microorganisms (CNCM) of the Pasteur Institute (25 rue du Docteur Roux, 75724 Paris, FR) on November 15, 2018 under the deposit number CNCM I-5377 (IL2-V1).

[0136] In some embodiments, the host cell of the present invention is a host cell deposited at the National Collection of Microorganisms (CNCM) of the Pasteur Institute (25 rue du Docteur Roux, 75724 Paris, FR) on November 15, 2018 under the deposit number CNCM I-5378 (IL2-V2).

[0137] In some embodiments, the host cell of the present invention is a host cell deposited at the National Collection of Microorganisms (CNCM) of the Pasteur Institute (25 rue du Docteur Roux, 75724 Paris, FR) on November 15, 2018 under the deposit number CNCM I-5379 (IL2-V3).

[0138] In some embodiments, the host cell of the present invention is a host cell deposited in the Center for the Collection of Microorganisms (CNCM) of the Pasteur Institute (25 rue du Docteur Roux, 75724 Paris, FR) on November 15, 2018 under the deposit number CNCM I-5380 (IL2-V4).

[0139] In some embodiments, the host cell of the present invention is a host cell deposited in the Center for the Collection of Microorganisms (CNCM) of the Pasteur Institute (25 rue du Docteur Roux, 75724 Paris, FR) on November 15, 2018 under the deposit number CNCM I-5381 (IL2-V5).

[0140] In some embodiments, the host cell of the present invention is a host cell deposited in the Center for the Collection of Microorganisms (CNCM) of the Pasteur Institute (25 rue du docteur roux, 75724 Paris, FR) on November 15, 2018 under the deposit number CNCM I-5382 (IL2-V6).

[0141] The polynucleotides, vectors, and cells of the present invention can be used to produce the variants of the present invention using well-known recombinant DNA techniques.

[0142] Pharmaceutical compositions and therapeutic uses

[0143] The IL-2 variants, polynucleotides, vectors and / or cells of the invention are useful for treating immune disorders.

[0144] As used herein, the term "treatment" or "treat" refers to prophylactic or preventive treatment as well as curative or disease-modifying treatment, including treatment of patients at risk of contracting a disease or suspected of having contracted a disease and patients who are ill or have been diagnosed with a disease or medical condition, including inhibition of clinical relapse. The treatment can be administered to a patient who has a medical condition or may eventually have the condition in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate the symptoms of one or more symptoms of the condition or recurrent condition, or in order to prolong the patient's survival beyond the expected survival in the absence of such treatment.

[0145] In some embodiments, the IL-2 variants, polynucleotides, vectors and / or cells of the present invention are used to amplify Treg in vitro or in vivo or block IL-2-mediated immune system overactivation, for treating diseases associated with immune disorders in which immunomodulation or immunosuppression is beneficial, including diseases associated with immune system dysfunction or overactivation. In particular, IL-2 variants (Treg agonists) that can preferably stimulate T-regulatory cells are used to treat immune diseases associated with immune system dysfunction, such as allergic and autoimmune diseases, and immune diseases associated with immune system overactivation, such as acute or chronic inflammatory diseases, GVHD and transplant rejection. IL-2 antagonist variants are used to treat diseases associated with immune system overactivation, including overproduction of IL-2 (such as GVHD, particularly acute GVHD) and others (such as acute or chronic inflammatory diseases) and transplant rejection.

[0146] For ex vivo treatment, peripheral blood samples are collected from patients; T cells are expanded in vitro using IL-2 variants, polynucleotides and / or vectors according to the present invention, and the expanded Tregs are then injected into the patient. For in vivo treatment, IL-2 variants, polynucleotides and / or vectors according to the present invention are administered to patients and Tregs are expanded in the patient. Alternatively or additionally, IL-2 already present in the patient is antagonized by the IL-2 variant.

[0147] In some embodiments, IL-2 antagonist variants, polynucleotides, vectors and / or cells according to the present invention are used to inhibit Treg by depriving them of IL-2, and thus allow stimulation of immune responses (including B, NK, CD4+ or CD8+ T cells, DC, macrophages, etc.) for treating cancer or infectious diseases or increasing immune responses to vaccines, particularly vaccines for cancer or infectious diseases. In some preferred embodiments, IL-2-antagonist variants, polynucleotides, vectors and / or cells according to the present invention are used to stimulate anti-tumor CD8+ T cell responses or CD8+ T cell responses to pathogens or vaccines (including vaccines for cancer or infectious diseases).

[0148] For in vivo treatment, the IL-2 antagonist variants, polynucleotides and / or vectors according to the present invention are administered to a patient, and Tregs are suppressed or eliminated in the patient to release immune cells (B, NK, CD4+ or CD8+ T cells; DCs; macrophages, etc.) from Treg suppression.

[0149] The present invention relates to a pharmaceutical composition comprising as active substance an IL-2 variant, a polynucleotide, a vector and / or a cell according to the present invention and at least one pharmaceutically acceptable excipient and / or carrier.

[0150] The pharmaceutical compositions are formulated for administration by a variety of routes including, but not limited to, oral, parenteral, and topical. Pharmaceutical excipients are those well known in the art to be suitable for the intended route of administration.

[0151] The pharmaceutical composition may further comprise a carrier. Non-limiting examples of carriers suitable for the compositions of the present invention include unilamellar or multilamellar liposomes, ISCOMS, virosomes, viral pseudoparticles, saponin micelles, sugar (poly (lactide-co-glycolide)) or gold microspheres and nanoparticles.

[0152] The pharmaceutical composition comprises a therapeutically effective amount of IL-2 variants, polynucleotides, vectors and / or cells, which are sufficient to show a positive medical response in the individual to which it is applied. A positive medical response refers to a reduction in subsequent (preventive treatment) or determined (therapeutic treatment) disease symptoms. A positive medical response includes partial or complete inhibition of disease symptoms. A positive medical response can be determined by measuring various objective parameters or criteria (such as objective clinical signs of disease and / or survival increase). The medical response to the composition according to the present invention can be easily verified in an appropriate animal model of the disease well known in the art and illustrated in the embodiments of the present application.

[0153] The pharmaceutically effective dose depends on the composition used, the route of administration, the type of mammal (human or animal) being treated, the physical characteristics of the particular mammal under consideration, concurrent medications and other factors that will be recognized by those skilled in the medical arts.

[0154] "Therapeutic regimen" refers to the treatment mode of a disease, such as the mode of administration used during treatment. The therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction phase" refers to a therapeutic regimen (or part of a therapeutic regimen) used for the initial treatment of a disease. The general purpose of an induction regimen is to provide a high level of drug to the patient in the initial stage of the therapeutic regimen. The induction regimen may adopt (partially or entirely) a "loading regimen", which may include administering a larger dose of the drug than the physician would adopt during the maintenance regimen, administering a drug more frequently than the physician would administer during the maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance phase" refers to a therapeutic regimen (or part of a therapeutic regimen) that is used to maintain a patient during the treatment of a disease, such as to keep the patient in remission for a long time (months or years). The maintenance regimen may adopt continuous treatment (e.g., administering the drug at regular intervals, e.g., weekly, monthly, annually, etc.) or intermittent treatment (e.g., intermittent treatment, intermittent treatment, recurrent treatment, or treatment when a specific predetermined criterion [e.g., pain, disease manifestation, etc.] is reached).

[0155] In some embodiments, the pharmaceutical composition comprises another active agent, wherein the active agent is a medicament or therapeutic agent capable of preventing, treating or improving a human or animal disease. The active agent can be a protein including an antibody, an oligonucleotide including an antisense oligonucleotide, a peptide nucleic acid (PNA), a small interfering RNA, a locked nucleic acid (LNA), a phosphorodiamidate morpholino oligonucleotide (PMO) and a decoy DNA molecule, a plasmid, an aptamer including DNA, RNA or a peptide aptamer, a small molecule or a macromolecular chemical drug or a mixture thereof. In particular, the active agent can be an anti-inflammatory agent or an immunomodulator, such as rapamycin or a corticosteroid. The active agent can also be an anticancer agent or an anti-infective agent or an antigen (such as a tumor antigen or a pathogen antigen).

[0156] The present invention also provides an IL-2 variant, a polynucleotide, a vector, a cell or a pharmaceutical composition according to the present invention for use as a medicament.

[0157] The present invention also provides an IL-2 variant, a polynucleotide, a vector, a cell or a pharmaceutical composition according to the present invention for use in preventing or treating an immune disorder.

[0158] In some embodiments, the disease associated with immune dysfunction or dysregulation is a disease in which immunosuppression is beneficial, such as, but not limited to, allergic diseases, autoimmune diseases, and diseases associated with overactivation of the immune system, such as, but not limited to, chronic or acute inflammatory diseases, graft versus host disease (GVHD), or transplant rejection. Non-limiting examples of autoimmune diseases include: type 1 diabetes, rheumatoid arthritis, psoriasis and psoriatic arthritis, multiple sclerosis, systemic lupus erythematosus (lupus), inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), Addison's disease, Graves' disease, Sjögren's disease, alopecia areata, autoimmune thyroid disease (such as Hashimoto's thyroiditis), myasthenia gravis, vasculitis (including HCV-related vasculitis and systemic vasculitis), uveitis, myositis, pernicious anemia, celiac disease, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, scleroderma, hemolytic anemia, glomerulonephritis, autoimmune encephalitis, fibromyalgia, aplastic anemia, etc. Non-limiting examples of inflammatory and allergic diseases include: neurodegenerative disorders (such as Parkinson's disease), chronic infections (such as parasitic infections) or diseases (such as Trypanosoma cruzi infection), allergies (such as asthma), atherosclerosis, chronic kidney disease, etc. The disease may be allograft rejection, including transplant rejection, graft-versus-host disease (GVHD), and spontaneous abortion.

[0159] In some embodiments, diseases associated with immune dysregulation or dysfunction are diseases in which suppression of Tregs and / or stimulation of an immune response, particularly a CD8+ T cell immune response, is beneficial, such as, but not limited to, cancer and infectious diseases. The present invention includes treatment of infectious diseases and cancer and increasing immune responses to vaccines, particularly vaccines against cancer or infectious diseases.

[0160] As used herein, the term "cancer" refers to any member of a class of diseases or conditions characterized by the uncontrolled division of cells and the ability of these cells to invade other tissues by directly growing into adjacent tissues or implanting into distant sites by metastasis. Metastasis is defined as a stage in which cancer cells are transported through the bloodstream or lymphatic system. The term cancer according to the present invention also includes cancer metastasis and cancer recurrence. Cancer is classified according to the cell type similar to the tumor, and therefore, the tissue is considered to be the origin of the tumor. For example, carcinoma is a malignant tumor derived from epithelial cells. This group represents the most common cancers, including common forms of breast cancer, prostate cancer, lung cancer and colon cancer. Lymphoma and leukemia include malignant tumors derived from blood and bone marrow cells. Sarcoma is a malignant tumor derived from connective tissue or mesenchymal cells. Mesothelioma is a tumor derived from mesothelial cells lining the peritoneum and pleura. Glioma is a tumor of glial cells derived from the most common type of brain cells. Germ cell tumors are tumors derived from germ cells, usually found in the testicles and ovaries. Choriocarcinoma is a malignant tumor derived from the placenta.

[0161] As used herein, the term "cancer" refers to any cancer that may affect any of the following tissues or organs: breast; liver; kidney; heart, mediastinum, pleura; floor of mouth; lip; salivary glands; tongue; gingiva; oral cavity; palate; tonsillar tissue; larynx; trachea; bronchi, lungs; pharynx, hypopharynx, oropharynx, nasopharynx; esophagus; digestive organs such as stomach, intrahepatic bile ducts, bile ducts, pancreas, small intestine, colon; rectum; urinary organs such as bladder, gallbladder, ureters; rectosigmoid junction; anus, anal canal; skin Skin; bones; joints, articular cartilage of limbs; eyes and appendages; brain; peripheral nerves, autonomic nervous system; spinal cord, cranial nerves, meninges; and various parts of the central nervous system; connective tissue, subcutaneous tissue and other soft tissues; retroperitoneum, peritoneum; adrenal glands; thyroid gland; endocrine glands and related structures; female reproductive organs, such as ovaries, uterus, cervix; uterine body, vagina, vulva; male reproductive organs, such as penis, testicles and prostate; hematopoietic and reticuloendothelial systems; blood; lymph nodes; thymus.

[0162] The term "cancer" according to the present invention includes leukemia, seminoma, melanoma, teratoma, lymphoma, non-Hodgkin's lymphoma, neuroblastoma, glioma, adenocarcinoma, mesothelioma (including pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma and end-stage mesothelioma), rectal cancer, endometrial cancer, thyroid cancer (including papillary thyroid cancer, follicular thyroid cancer, medullary thyroid cancer, anaplastic thyroid cancer, multiple endocrine neoplasia type 2A, multiple endocrine neoplasia type 2B, familial medullary thyroid cancer, pheochromocytoma and paraganglioma), skin cancer (including malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, keratoacanthoma, nevus, dysplastic nevus, lipoma, hemangioma and dermatofibroma), nervous system cancer, brain cancer (including astrocytoma, medulloblastoma, glioma, low-grade glioma, Cancer of the brain (including squamous cell carcinoma of the head and neck and oral cancer (e.g., oral cavity cancer, lip cancer, tongue cancer, oral cancer, or pharynx cancer)), lymph node cancer, gastrointestinal cancer, liver cancer (including hepatoma, hepatocellular carcinoma, bile duct cancer, hepatoblastoma, angiosarcoma, hepatocellular adenoma, and hemangioma), colon cancer, stomach cancer or gastric cancer, esophageal cancer (including squamous cell carcinoma, larynx, adenocarcinoma, leiomyosarcoma, or lymphoma), colorectal cancer, intestinal cancer, small intestine cancer, bowel or small intestines cancer (e.g., adenocarcinoma, lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma, or fibroma), large bowel or large intestines cancer (e.g.,adenocarcinoma, tubular adenoma, villous adenoma, hamartoma or leiomyoma), pancreatic cancer (including ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid nasopharyngeal (ENT) cancer, breast cancer (including HER2-enriched breast cancer, luminal A breast cancer, luminal B breast cancer and triple-negative breast cancer), uterine cancer (including endometrial cancer (such as endometrial carcinoma), endometrial stromal sarcoma and malignant mixed Müllerian tumor, uterine sarcoma, leiomyosarcoma and gestational trophoblastic disease), ovarian cancer (including dysgerminoma, granulosa-theca cell tumor and Sertoli-Leydig cell tumor), cervical cancer, vaginal cancer (including vulvar cancer (including squamous cell vulvar cancer, verrucous vulvar cancer, vulvar melanoma, basal cell vulvar cancer, Bartholin's gland carcinoma, vulvar adenocarcinoma, and erythematosus proliferative), genitourinary tract cancer, renal cancer (including clear cell renal cell carcinoma, chromophobe renal cell carcinoma, papillary renal cell carcinoma, adenocarcinoma, Wilms' tumor, Wilms' tumor, lymphoma, or leukemia), adrenal cancer, bladder cancer, urethral cancer (e.g., squamous cell carcinoma, transitional cell carcinoma, or adenocarcinoma), prostate cancer (e.g., adenocarcinoma or sarcoma), tumours) and testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, teratoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenomatous carcinoma or lipoma), lung cancer (including small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), including squamous cell lung cancer, lung adenocarcinoma (LUAD) and large cell lung cancer, bronchial carcinoma, alveolar carcinoma, bronchiolar carcinoma, bronchial adenoma, pulmonary sarcoma, enchondromatous hamartoma and pleural mesothelioma), sarcoma (including Arkin's tumor, sarcoid, chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma and Soft tissue sarcomas), soft tissue sarcomas (including alveolar soft part sarcoma, angiosarcoma, cystic sarcoma phyllodes, dermatofibrosarcoma protruding, desmoid tumors, desmoplastic small round cell tumors, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, gastrointestinal stromal tumors (GIST), hemangiopericytoma, angiosarcoma, Kaposi sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant peripheral nerve sheath tumor (MPNST), neurofibrosarcoma, plexiform fibrohistiocytoma, rhabdomyosarcoma, synovial sarcoma and undifferentiated pleomorphic sarcoma), cardiac cancer (including sarcomas,Such as angiosarcoma, fibrosarcoma, rhabdomyosarcoma or liposarcoma, myxoma, rhabdomyomas, fibromas, lipomas and teratomas), bone cancer (including osteogenic sarcoma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma and reticulum cell sarcoma, multiple myeloma, malignant giant cell tumor chordoma, osteochondroma, osteochondral exostosis, benign enchondroma, chondroblastoma, chondromyxoid fibroma, osteoid osteoma and giant cell tumor), blood and lymphatic cancer, blood cancer (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myeloproliferative disorders, multiple myeloma and myelodysplastic syndrome), Hodgkin's disease, non-Hodgkin's lymphoma and hairy cell and lymphatic disorders, and their metastases.

[0163] Infectious diseases include viral, bacterial, fungal and parasitic diseases, for example HIV / AIDS, viral hepatitis, such as hepatitis A, B, C, measles, malaria and Chagas disease.

[0164] The present invention also provides a method for treating a disease associated with immune dysfunction or abnormal regulation, comprising: administering a therapeutically effective amount of the pharmaceutical composition according to the present invention to a patient.

[0165] The pharmaceutical compositions of the present invention are generally administered according to known methods at dosages and time periods effective to induce beneficial effects in individuals. Administration may be by injection or by oral, sublingual, intranasal, rectal or vaginal, inhalation or transdermal administration. Injection may be subcutaneous, intramuscular, intravenous, intraperitoneal, intradermal, etc.

[0166] The pharmaceutical composition of the present invention is advantageously used in combination with another therapy, in particular immunotherapy, such as CAR-T cell therapy; therapy with an immunomodulator, in particular an immunomodulatory monoclonal antibody or a functional derivative thereof; therapy with an anticancer agent or an anti-infective agent, including therapeutic agents and vaccines for anticancer and infectious diseases. The combined therapy may be separate, simultaneous and / or sequential.

[0167] In some embodiments, the pharmaceutical composition is for use in the prevention or treatment of humans.

[0168] In some embodiments, the pharmaceutical composition is used to treat an animal.

[0169] In some embodiments, the IL-2 variants, polynucleotides, vectors, cells and / or pharmaceutical compositions according to the present invention are co-administered with other cancer therapies. In particular, the IL-2 variants, polynucleotides, vectors, cells and / or pharmaceutical compositions of the present invention can be co-administered with targeted therapy, immunotherapy (such as immune checkpoint therapy and immune checkpoint inhibitors), co-stimulatory antibodies, chemotherapy and / or radiotherapy.

[0170] Immune checkpoint therapies (such as checkpoint inhibitors) include, but are not limited to, programmed death-1 (PD-1) inhibitors, programmed death ligand-1 (PD-L1) inhibitors, programmed death ligand-2 (PD-L2) inhibitors, lymphocyte activation gene 3 (LAG-3) inhibitors, T cell immunoglobulin and mucin domain containing protein 3 (TIM-3) inhibitors, T cell immunoreceptor with Ig and ITIM domains (TIGIT) inhibitors, B- and T-lymphocyte attenuator (BTLA) inhibitors, V-domain Ig inhibitor of T-cell activation (VISTA) inhibitors, cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitors, indoleamine 2,3-dioxygenase (IDO) inhibitors, killer immunoglobulin-like receptor (KIR) inhibitors, KIR2L3 inhibitors, KIR3DL2 inhibitors, and carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM-1) inhibitors. In particular, checkpoint inhibitors include antibodies anti-PD1, anti-PD-L1, anti-CTLA-4, anti-TIM-3, anti-LAG3. Co-stimulatory antibodies deliver positive signals through immunomodulatory receptors, including but not limited to ICOS, CD137, CD27, OX-40 and GITR.

[0171] Examples of anti-PD1 antibodies include, but are not limited to, nivolumab, cemiplizumab (REGN2810 or REGN-2810), tislelizumab (BGB-A317), spartalizumab (PDR001 or PDR-001), ABBV-181, JNJ-63723283, BI 754091, MAG012, TSR-042, AGEN2034, pidilizumab, nivolumab (ONO-4538, BMS-936558, MDX1106, GTPL735 or Opdivo), pembrolizumab (MK-3475, MK 03475, pembrolizumab, SCH-900475 or Keytruda) and the antibodies described in international patent applications WO2004004771, WO2004056875, WO2006121168, WO2008156712, WO2009014708, WO2009114335, WO2013043569 and WO2014047350.

[0172] Examples of anti-PD-L1 antibodies include, but are not limited to, LY 3300054, atezolizumab, durvalumab, and avelumab.

[0173] Examples of anti-CTLA-4 antibodies include, but are not limited to, ipilimumab (see, e.g., U.S. Pat. Nos. 6,984,720 and 8,017,114), tremelimumab (see, e.g., U.S. Pat. Nos. 7,109,003 and 8,143,379), single-chain anti-CTLA4 antibodies (see, e.g., International Patent Applications WO1997020574 and WO200712373), and antibodies described in U.S. Pat. No. 8,491,895.

[0174] Examples of anti-VISTA antibodies are described in U.S. patent application US20130177557.

[0175] Examples of inhibitors of the LAG3 receptor are described in US Pat. No. 5,773,578.

[0176] An example of a KIR inhibitor is IPH4102, which targets KIR3DL2.

[0177] Targeted therapy is a drug designed to interfere with specific molecules necessary for tumor growth and progression. For example, therapeutic monoclonal antibodies target specific antigens found on the cell surface, such as transmembrane receptors or extracellular growth factors. In some cases, monoclonal antibodies are conjugated with radioisotopes or toxins to allow these cytotoxic agents to be specifically delivered to the intended cancer cell target. Small molecules can penetrate the cell membrane to interact with the target within the cell. Small molecules are usually designed to interfere with the enzymatic activity of the target protein, such as proteasome inhibitors, tyrosine kinase or cyclin-dependent kinase inhibitors, histone deacetylase inhibitors. Targeted therapy can also use cytokines. Examples of such targeted therapies include, but are not limited to: Ado-trastuzumab emtansine (HER2), afatinib (EGFR (HER1 / ERBB1), HER2), Aldesleukin (Proleukin), alectinib (ALK), Alemtuzumab (CD52), Axitinib (Kit, PDGFRbeta, VEGFR1 / 2 / 3), Belimumab (BAFF), Belinostat (HDAC), Bevacizumab (VEGF ligand), Blinatumomab (CD19 / CD3), Bortezomib (Proteasome), Brentuximab Vedotin (CD30), Bosutinib (ABL), brigatinib (ALK), Cabozantinib (FLT3, KIT, MET, RET, VEGFR2), Canakinumab; (IL-1beta), Lenalidomide (Proteasome), Ceritinib (ALK), Cetuximab (EGFR), Cofimetinib (MEK), Crizotinib (ALK, MET, ROS1), Dabrafenib (BRAF), Daratumumab (CD38)), Dasatinib (ABL), Denosumab (RANKL), Denutusimab (B4GALNT1 (GD2)), Elotuzumab (SLAMF7), Enasidenib (IDH2), Erlotinib (EGFR), E Verolimus (mTOR), Gefitinib (EGFR), Ibrutinib (CD20), Sonidegib (Smoothened), Sipuleucel-T, Siltuximab (IL-6), Sorafenib (VEGFR, PDGFR, KIT, RAF), (Tocilizumab (IL-6R), Temozolomide (mTOR), Tofacitinib (JAK3), Trametinib (MEK), Tositumomab (CD20), Trastuzumab (HER2), Vandetanib (EGFR), Vemurafenib (BRAF), Venetoclax (BCL2), Vismodegib (PTCH, Smoothened), Vorinostat (HDAC), Aflibercept (PIGF, VEGFA / B).

[0178] In some embodiments, the IL-2 variants, polynucleotides, vectors, cells and / or pharmaceutical compositions of the invention are administered to a patient in combination with chemotherapy. As used herein, the term "chemotherapy" has its ordinary meaning in the art and refers to a treatment that includes administering a chemotherapeutic agent to a patient. Chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines, for example, benzodepa, carboquinone, metodepa and uredepa; ethyleneimines and methylmelamines, including hexamethylmelamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trihydroxymethylmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; carlistatin (carlistatin); CC-1065 (including its synthetic analogues adolesin, carzelesin and biszelesin); nostoc (particularly nostoc 1 and nostoc 8); pyralidin; duocarmycin (including the synthetic analogues KW-2189 and CB1-TM1 ); acanthopanacin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, naphthiazine, cholophosphamide, estramustine, ifosfamide, dichloromethyl diethylamine, methoxychlor hydrochloride, melphalan, nembixin, phenambucil, prednimustine, trolofosamide; uramustine; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics, such as enediynes (e.g., calicheamicins, particularly calicheamicin gamma and calicheamicin omega); anthracyclines, including anthracycline A; bisphosphonates, such as clodronate; ramycin;and new tumor suppressor protein chromophores and related chromophores of enediyne antibiotics, aclacinomycins, actinomycins, authrarnycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carcinophilin, chromomycinis, actinomycin D, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrroline-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, ephedrine, Ciromycin, mitomycins such as mitomycin C, mycophenolic acid, nogamycin, olive mycin, peplomycin, potfiromycin, puromycin, triferric doxorubicin, rhodorubicin, streptomycin, streptozotocin, tuberculocidin, ubenimex, netastatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as methotrexate, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiopurine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as caprotestosterone, propylthiocyanate, chloramphenicol ... (including drostanolone, cyclothiocarb, mestanol, testolactone; antiadrenal agents such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as folinic acid; acetoglucuronide; aldehyde phosphoramide glycoside; aminolevulinic acid; eniluracil; amsacrine; bespur; bisantrene; edatrexate; defosfamide; demeclocycline; diacronitrile; eflornithine; elitrilonium; epothilone; ethoxydimidine; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansine alkaloids such as maytansine and stalk-type streptocin; mitoguanidine; mitoxantrone; mopidarol; nitrilosine; pentostatin; methamine mustard; pirarubicin; losoxantrone; 2-ethylhydrazide; methylhydrazine derivatives, including N-methylhydrazine (MIH) and procarbazine; PSK polysaccharide complex) ; razoxane; rhizoxin; sizoran; germanium spiroamine; tricholomanic acid; triethyleneimine benzoquinone; 2,2',2"-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, verrucosporin A, baculosporin A, and serpentin); urethane; vindesine; dacarbazine; mannitol mustard; dibromomannitol; dibromodulanol; piperobroman; gatosine; cytarabine ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as paclitaxel and docetaxel; chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum coordination complexes, such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;Vinorelbine; mitoxantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; anthracyclines, nitrosoureas, antimetabolites, epipodophylotoxins, enzymes, such as L-asparaginase; anthracenediones; hormones and antagonists, including adrenocortical steroids antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestins such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogens such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogens such as tamoxifen; androgens including testosterone propionate and fluoxymesterone / equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and non-steroidal antiandrogens such as flutamide; and pharmaceutically acceptable salts, acids or derivatives of any of the above compounds. ;

[0179] In some embodiments, the IL-2 variants, polynucleotides, vectors, cells and / or pharmaceutical compositions of the present invention are administered to a patient in combination with radiotherapy. Suitable examples of radiotherapy include, but are not limited to, external beam radiotherapy (such as surface X-ray therapy, orthovoltage X-ray therapy, megavoltage X-ray therapy, radiosurgery, stereotactic radiotherapy, fractionated stereotactic radiotherapy, cobalt therapy, electron therapy, fast neutron therapy, neutron capture therapy, proton therapy, intensity modulated radiotherapy (IMRT), three-dimensional conformal radiotherapy (3D-CRT), etc.); short-distance radiotherapy; non-sealed source radiotherapy; tomotherapy; etc. γ rays are another form of photons used in radiotherapy. Gamma rays are naturally produced when certain elements (such as radium, uranium and cobalt 60) release radiation when they decompose or decay. In some embodiments, radiotherapy can be proton radiotherapy or proton microbeam radiotherapy. Proton radiotherapy is an ultra-precise form of radiation therapy that uses proton beams (Prezado Y, Jouvion G, Guardiola C, Gonzalez W, Juchaux M, Bergs J, Nauraye C, Labiod D, De Marzi L, Pouzoulet F, Patriarca A, Dendale R. Tumor Control in RG2Glioma-Bearing Rats: A Comparison Between ProtonMinibeam Therapy and Standard Proton Therapy.Int J Radiat Oncol BiolPhys.2019Jun 1;104(2):266-271.doi:10.1016 / j.ijrobp.2019.01.080; Prezado Y, Jouvion G, Patriarca A, Nauraye C, Guardiola C, Juchaux M, Lamirault C, Labiod D, Jourdain L, Sebrie C,Dendale R, Gonzalez W, Pouzoulet F. Proton minibeam radiation therapy widens the therapeutic index for high-grade gliomas. Sci Rep. 2018 Nov 7; 8(1): 16479. doi: 10.1038 / s41598-018-34796-8). Radiation therapy can also be FLASH radiotherapy (FLASH-RT) or FLASH proton irradiation.FLASH radiotherapy involves ultra-fast radiotherapy (ultra-high dose rate) at a dose rate several orders of magnitude higher than current routine clinical practice (Favaudon V, Fouillade C, Vozenin MC. The radiotherapy FLASH to save healthy tissues. Med Sci (Paris) 2015; 31: 121-123. DOI: 10.1051 / medsci / 20153102002); Patriarca A., Fouillade CM, Martin F., Pouzoulet F., Nauraye C., et al. Experimental set-up for FLASH proton irradiation of small animals using a clinical system. Int J Radiat Oncol Biol Phys, 102 (2018), pp. 619-626. doi: 10.1016 / j.ijrobp.2018.06.403. Epub 2018Jul 11).

[0180] Use for antibody screening

[0181] The IL-2 variants according to the present invention can be used to screen for anti-IL-2 antibodies having pro-Teff or pro-Treg activity using standard immunoassays well known in the art.

[0182] In this regard, the present invention relates to a method for screening an anti-IL-2 antibody having pro-Teff or pro-Treg activity, comprising at least the following steps: a) contacting an anti-IL-2 antibody with a variant according to the present invention, and b) measuring the level of bound antibody from step a), wherein if the level of bound antibody is greater than 70% (preferably 80%, 90% or more) of a reference value, the anti-IL-2 antibody may have pro-Teff activity; and wherein if the level of bound antibody is less than 50% (preferably 40%, 30%, 20%, 10% or less) of the reference value, the anti-IL-2 antibody may have pro-Treg activity.

[0183] The antibody having Teff promoting activity is particularly an antibody having CD25 mimotope binding.

[0184] Reference values ​​are usually determined by contacting the antibody with wild-type IL-2 under the same conditions. Step a is advantageously performed using an IL-2 variant fixed on a solid surface (plate, beads). Step b is performed using a labeled primary or secondary antibody. The assay may be an ELISA, fluorescent immunoassay (FIA) or chemiluminescent immunoassay (CLIA), chemiluminescent immunoassay (CLIA). The assay may be a high-throughput assay, for example using a microfluidic device.

[0185] The present invention also relates to a kit for carrying out the screening method of the present invention, comprising an IL-2 variant according to the present invention.

[0186] In various embodiments, the kit may further comprise instructions for use and / or a labeled antibody.

[0187] Unless otherwise indicated, the practice of the present invention will employ conventional techniques within the skill of the art, which are fully explained in the literature.

[0188] The invention will now be illustrated by the following non-limiting examples with reference to the accompanying drawings, in which: BRIEF DESCRIPTION OF THE DRAWINGS

[0189] Figure 1 : Multiple sequence alignment of IL-2 from different species used in the evolutive filter.

[0190] Homo sapiens (SEQ ID NO: 1); white-handed gibbon (SEQ ID NO: 15); macaque (SEQ ID NO: 16); pig-tailed monkey (SEQ ID NO: 17); crab-eating macaque (SEQ ID NO: 18); white-collared mangabey (SEQ ID NO: 19); Brazilian night monkey (SEQ ID NO: 20); baboon (SEQ ID NO: 21); house mouse (SEQ ID NO: 22); brown rat (SEQ ID NO: 23); long-clawed gerbil (SEQ ID NO: 24); guinea pig (SEQ ID NO: 25); rabbit (SEQ ID NO: 26); cattle (SEQ ID NO: 27); sheep (SEQ ID NO: 28); Eastern European red deer (SEQ ID NO: 29); goat (SEQ ID NO: 30); beluga whale (SEQ ID NO: 31); killer whale (SEQ ID NO: 32); wild boar (SEQ ID NO: 33); domestic horse (SEQ ID NO: 34); NO:34); domestic cat (SEQ ID NO:35); domestic dog (SEQ ID NO:36); gray seal ((SEQ ID NO:37); northern elephant seal (SEQ ID NO:38) and domestic chicken (SEQ ID NO:39).

[0191] Figure 2 : Shown is the structure of the quaternary complex of IL-2 with its α (CD25), β (CD122), and γC (CD132) receptors.

[0192] The locations of surface remodeling in IL-2-V2 are shown.

[0193] Figure 3 : Sequence alignment of the IL-2-WT construct and the resurfaced variants IL-2-V1 and IL-2-V2.

[0194] IL-2-WT construct (SEQ ID NO: 2). IL-2-V1 (SEQ ID NO: 3). IL-2-V2 (SEQ ID NO: 4).

[0195] Figure 4 : Size exclusion chromatography and SDS-PAGE analysis of recombinant wild-type and resurfaced variants of IL-2.

[0196] A. IL-2 wild type. B. IL-2-V1. C. IL-2-V2. Peaks used for experimental testing are indicated by arrows. D. SDS-PAGE analysis of IL-2 wild type (WT); IL-2-V1 (V1) and IL-2-V2 (V2). Gel: Laemli, 15% acrylamide. Samples: WT: 3.4 μg. V1: 3.6 μg. V2: 2-2.9 μg. Samples were denatured under reducing conditions (DTT).

[0197] Figure 5 : ELISA evaluated the specific binding of different anti-IL-2 antibodies to different IL-2 variants.

[0198] ELISA test is used to evaluate the selectivity of IL-2V for antibodies (Ab) with T effector or Treg function. To this end, a typical ELISA test was performed by using a plate coated with Proleukine, IL-2V1, IL-2V2, IL-2V3, IL-2V4, IL-2V5 or IL-2V6 (all 6 μg / mL). The Abs evaluated were: MAB605 (mouse anti-human IL-2Ab with T effector activity in vivo), 5344 (mouse anti-human IL-2Ab with Treg activity in vivo) and NARA (scFV antibody with T effector activity in vivo, derived from Arenas-Ramirez et all, Sci. Transl. Med., 2016, 8, 367ra166). In order to detect anti-IL-2Ab, anti-mouse IgG-hrpAb or anti-NARA M13-HRPAb was used to read the absorbance at 450nm. OD values ​​are shown, from which the absorbance of control wells (uncoated wells, incubated with the corresponding anti-IL-2 Ab plus anti-mouse IgG-hrpAb) was subtracted.

[0199] Figure 6 : Human IL-2V2 and IL-2V3, but not IL-2V1, IL-2V4, IL-2V5, and IL-2V6, selectively induce STAT-5 phosphorylation in Treg cells.

[0200] CD3+ cells were purified from human PBMC and incubated with different concentrations of Proleukine, IL-2V1, IL-2V2, IL-2V3, IL-2V4, IL-2V5 or IL-2V6 (0.0001nM-1000nM). Phosphorylation of STAT5 (STAT5-P) in human Treg cells (IL-2R), CD4+Teff cells (IL-2R) and CD8+T cells (IL-2R) was measured by flow cytometry. (AB) The frequency of STAT5-P+ cells in different T cell populations of two different healthy donors using Proleukine, IL-2V1, IL-2V2 and IL-2V3. (C) The frequency of STAT5-P+ cells in different T cell populations from a healthy donor using Proleukine, IL-2V4, IL-2V5 and IL-2V6. The results obtained in two independent experiments are shown.

[0201] Figure 7 :hIL-2V delays the development of clinical GVHD

[0202] NSG-SGM mice were given iv with 6 × 10 6Human PBMCs with 100 CD3+T cells were transplanted and treated with 6 μg / dose of Proleukine, IL-2V1 and IL-2V2 by ip route for 5 consecutive days. (A) Mean values ​​of the evolution of body weight changes, expressed as percentage (%) of the initial body weight of the different treatment groups, and (B) Kaplan-Meier survival curves of transplanted mice. Untreated n=4; Proleukine, n=4; IL-2V1, n=5; IL-2V2, n=5.

[0203] Figure 8 hIL-2V preferentially increases circulating human Treg cells in a murine model of acute GVHD

[0204] NSG-SGM mice were given iv with 6 × 10 6 Human PBMCs with 100 CD3+ T cells were treated with 6 μg / dose of Proleukine, IL-2V1, and IL-2V2 by ip for 5 consecutive days. Blood samples were collected on days 5, 10, and 21 after PBMC injection. (A) The frequency of human CD8+ T cells; (B) The frequency of human CD4+ Tconv cells (CD4+Foxp3- T effector cells) and human Tregs (CD4+CD45RA-Foxp3 高 )(C) and CD25+Tregs(CD4+Foxp3+CD25 高 ) (D). Squares: Donor 1. Circles: Donor 2. Untreated, n=4; Proleukine, n=4; IL-2V1 n=5; IL-2V2, n=5.

[0205] Fig. 9 : Human IL-2V1 competes with Proleukine in stimulating T cells.

[0206] CD3+ cells were purified from human PBMCs and incubated with Proleukine (0.0001nM-1000nM; black curve), Proleukine (0.0001nM-1000nM; blue curve) or Proleukine (0.0001nM-1000nM) in the presence of a fixed concentration of IL-2V1 (1000nM, competition curve, red curve). Phosphorylation of STAT-5 (STAT5-P) was measured by flow cytometry. (A) Frequency of STAT5-P cells in Treg cells (IL-2Rαβγ). (B) Frequency of STAT5-P cells in CD4+Foxp3-T cells (IL-2Rβγ). (C) Frequency of STAT5-P cells in CD8+T cells (IL-2Rβγ). (D) Frequency of STAT5-P+ cells in IL-2V1-induced Treg, CD4+Teff, and CD8+T cells. Schematic representation of high-affinity (top) and intermediate-affinity (middle) IL-2Rs interacting with IL-2V1 are shown.

[0207] Fig.10 : IL-2V2 and IL-2V3 preferentially increase the frequency of Treg cells over effector cells in vivo.

[0208] C57BL / 6 healthy mice were treated by ip injection. Proleukine (4 μM), IL-2V2 (40 μM) or IL-2V3 (40 μM) were injected. One group of mice was not treated. n = 3 mice in each group of treated or untreated mice. Mice received 4 doses of Proleukine or IL-2V and blood samples were taken for FACS analysis.

[0209] (A) Frequency: Tregs (CD4+Foxp3 + ); effector memory CD8+ T cells (CD8+CD44 高 ) and NK cells, among CD45+ cells; proliferating (Ki67+) Tregs in CD45+ cells, effector memory CD8+ T cells (CD44 高 Ki67+) and NK cells; (B) effector memory CD8+ T cells (CD8+CD44 高 ) to Treg; the ratio of NK cells to Treg.

[0210] Fig.11 : IL-2 variants bind to IL-2Rαβγ.

[0211] Binding assay for the IL-2-dependent human cell line Kit225 constitutively expressing IL-2Rαβγ. IL-2-depleted Kit225 cells were incubated for 2 days alone or with 10 μM of IL-2wt, IL-2V1, V2, V3, V4, V5, and V6 produced with streptavidin labeling. After incubation, cells were stained with anti-strep-tag antibody (A-488) and analyzed by flow cytometry.

[0212] Fig.12 : IL-2V antagonists compete with IL-2 for CD4+ T cell activation, leaving active signaling in CD8+ T cells.

[0213] CTV-labeled human PBMC and CD25+T cells (mixed at a 50:50 ratio) were incubated with anti-CD3 / anti-CD28 beads (1:5 beads: cell ratio) + IL-2V1, 4, 6, anti-hIL-2 mAb (Mab602) or PBS (endogenous IL-2 control) for 96 h. CTV dilutions of CD4+Tconvs, Tregs, and CD8+T cells were measured by flow cytometry. CTV is shown 低 Percentage (%) of cells (as a measure of dividing cells) relative to PBS conditions (maximum).

[0214] Example

[0215] Example 1. Computational design of IL-2 resurfacing variants

[0216] IL-2 binds to its α (also known as CD25), β (CD122), and γ c The atomic structure of the quaternary complex with the (CD132) receptor (PDB code 2B5I) was used to guide the computational design of new IL-2 variants that retain residues that contact the receptor but modify the surface outside this region ( Figure 2 The general pipeline of surface reconstruction design is as follows: First, residues contacting the α receptor are defined as having at least one atom away from any atom of the α receptor. Once the amino acids contacting CD25 were discarded, the set of candidate positions to be mutated was identified using accessibility criteria and only those residues with >50% exposed side chain surface area were selected (Fraczkiewicz et al., J. Comp. Chem, 1998. 19, 319-333).

[0217] Using this approach, 28 candidate positions were identified on the IL-2 surface. Next, to determine which set of mutations to allow at each position, orthologous sequence analysis was combined with solubility criteria. To include evolutionary information, a multiple sequence alignment of IL-2 sequences from 26 different species was performed ( Figure 1 ), and only mutations of residues present in any ortholog at a given position are allowed. The original set of mutations was further filtered by using solubility factors, so that when possible, the presence of hydrophobic residues was discarded and all polar and native residues of IL-2 were allowed. Finally, the Rosetta "fixed skeleton design application" was used to select low-energy sequences, maintaining natural rotational isomers in all unexposed residues (Kuhlman et al., Science, 2003.302, 1364-8). The lowest energy design of the surface remodeling position of a specific combination was selected for experimental testing. Using this method, different variants were designed, referred to as IL-2-V1 to IL-2-V6 (Table I). For ease of purification, a C-terminal Strep-tag was added to the construct. The wild-type human IL-2 construct and the derived IL-2 variants (IL-2-V1 to IL-2-V6) with a C-terminal Strep-tag have a 171 amino acid sequence (SEQ ID NO: 2-8; Figure 3 ).

[0218] Table I: IL-2-variants

[0219]

[0220]

[0221] Example 2. Protein expression and purification

[0222] The genes encoding human IL-2 and IL-2 variants were codon optimized for expression in mammalian cells and synthesized with a C-terminal Strep-tag by GENSCRIPT (Piscataway, NJ, USA). The corresponding DNA sequences (SEQ ID NOs: 9-14) were then cloned between the AgeI and XhoI restriction sites of the plasmid pHL-sec, which is a mammalian expression vector suitable for producing proteins in high yields (Aricescu et al., Acta Crystallogr D Biol Crystallogr., 2006, 62, 1243-50). E. coli Top10 transformed with different pHL-sec recombinant plasmids encoding each IL-2 variant was deposited at the National Collection of Microorganisms (CNCM) of the Pasteur Institute, 25 rue du Docteur Roux, 75724 Paris, FR on November 15, 2018, with accession numbers CNCM I-5377 to CNCM I-5382 (IL-2-V1 to IL-2-V6). The transformed bacteria (IL-2-V1 to IL-2-V6) deposited at the CNCM with accession numbers CNCM I-5377 to CNCM I-5382 were grown under standard conditions, such as in Luria-Bertani (LB) medium supplemented with 100 μg / mL ampicillin, at 37°C, with agitation of> 160 rpm. The viability of transformed bacteria can be verified by standard assays, such as by plating bacterial cultures on LB-agar plates supplemented with 100 μg / mL ampicillin and detecting the presence of bacterial colonies. The transformed bacteria can be stored under standard conditions, such as by freezing at -80°C a mixture of 0.5mL bacterial cultures and 0.5mL 50% (V / V) glycerol in 2mL Eppendorf tubes. The HEK293 FreeStyle cells (THERMO SCIENTIFIC) maintained in serum-free medium (FreeStyle 293 expression medium) by transient transfection produce protein. Culture supernatants were collected 3 days after transfection, and adding Tris pH 8.0 to a final concentration of 100mM. First, the protein was purified by Streptactin affinity chromatography using a StrepTrap column (GE HEALTHCARE, Piscataway, NJ), and then the eluted fraction was further purified by gel filtration using a Superdex75 column (GE HEALTHCARE, Piscataway, NJ), resulting in a profile with two peaks, one corresponding to monomeric protein and the other corresponding to oligomers, presumably dimers ( Figure 4 ). The fractions containing the monomer peak were concentrated and used for experimental testing.

[0223] Example 3. IL-2V is a new tool for specifically selecting anti-IL-2 Abs with pro-Teff or pro-Treg activity.

[0224] ELISA test is used to evaluate the selectivity of IL-2 variants to antibodies (Ab) with pro-T-effector or pro-Treg functions. For this reason, a typical ELISA test is performed by using a plate coated with Proleukine, IL-2V1, IL-2V2, IL-2V3, IL-2V4, IL-2V5 or IL-2V6 (all 6 μg / mL). The Ab evaluated is: MAB605 (mouse anti-human IL-2Ab with pro-T-effector activity in vivo), 5344 (mouse anti-human IL-2Ab with pro-Treg activity in vivo) and NARA (scFV antibody with pro-T-effector activity in vivo, derived from Arenas-Ramirez et all, Sci.Transl.Med., 2016, 8, 367ra166). In order to detect anti-IL-2Ab, anti-mouse IgG-HRP Ab or anti-M13-HRP Ab for NARA is used, and absorbance is read at 450nm. OD values ​​are shown, from which the absorbance of control wells (uncoated wells incubated with the corresponding anti-IL-2 Ab plus anti-mouse IgG-HRP Ab) was subtracted.

[0225] Figure 5 Abs (MAB605) that show pro-Teff effects bind to all IL-2 forms; and NARA antibodies, which also have pro-Teff effects, also bind to IL-2V1 and IL-2V2 (in these preliminary experiments, NARA antibodies were tested only against IL-2V1 and IL-2V2). In contrast, Abs (5344) that have pro-Treg effects show reduced / no binding to IL-2 variants. These results confirm that the designed IL-2V can be used as a powerful tool for screening anti-IL-2 Abs with CD25 mimetic epitope activity, including Abs with pro-Teff activity and Abs with pro-Treg activity.

[0226] Example 4. In vitro evaluation of the biological activity of IL-2 variants

[0227] It was evaluated whether different IL-2 variants displayed different affinities / selectivities for T cell populations expressing intermediate affinity IL-2R (present on CD4+ or CD8+ T effector cells) or high affinity IL-2R (present on Treg cells).

[0228] Therefore, CD3 positive T cells were enriched from PBMCs from healthy donors according to manufacturer's instructions (MILTENYI). The cells enriched with CD3 were cultured in RPMI culture medium (GIBCO, France) without SVF containing Proleukine (NOVARTIS) or IL-2V1, IL-2V2, IL-2V3, IL-2V4, IL-2V5 and IL-2V6 (0.0001nM to 1000nM) at 75 μL / well in 96-well plates. The phosphorylation of STAT5 (STAT5-P) in human Treg cells (IL-2Rαβγ), CD4+Teff cells (IL-2R) and CD8+T cells (IL-2Rβγ) was measured by flow cytometry as follows. After 15 minutes of stimulation, the culture was fixed with PBS / 2% paraformaldehyde in 200 μL / wells for 10 minutes at room temperature. After washing with PBS / 0.2% BSA, cells were permeabilized with 100 μL / well of ice-cold methanol on ice for 10 minutes. Then, cells were washed with PBS / 0.2% BSA and stained with anti-CD3 PE-Cy7 (clone UCHT1; 1:200; BD BIOSCIENCES), anti-CD4 PE-CF594 (clone RPA-T41:100; OZYME), anti-CD25 PE (clone M-A251; 1:5; BD BIOSCIENCES), anti-Foxp3 Alexa488 (clone 236A / E7; 1:20; EBIOSCIENCES), and anti-pSTAT5 Alexa647 (clone 47 / Stat5 (pY694); 1:20; BD BIOSCIENCES) at 4°C for 45 minutes. Cells were acquired on an LSRII flow cytometer and analyzed with FlowJo software.

[0229] The results showed that human IL-2V2 and IL-2V3 selectively induced STAT-5 phosphorylation in Tregs, but not in T effector cells, compared with commercial human IL-2 Proleukine. IL-2V1, IL-2V4, IL-2V5, and IL-2V6 did not stimulate Tregs or effector T cells at the doses evaluated ( Figure 6 ).

[0230] Example 5. In vivo evaluation of the biological activity of IL-2 variants

[0231] In order to evaluate the in vivo effect of IL-2V, a graft-versus-host disease (GVHD) model was used, in which human peripheral blood mononuclear cells were injected into immunodeficient host mice. Human T cells are activated and induce xeno-GVHD after recognizing mouse antigens. Clinically, GVHD is evaluated by weight loss in mice. It has been previously shown that Treg cells can control GVHD (Gaidot A, Blood, 2011, 117, 2975–2983). In addition, it has been reported that low-dose IL-2 administration stimulates Treg cells, and thus inhibits inflammation and alleviates disease symptoms (Shin et al., Blood, 2011, 118, 2342-2350). It is assumed that if IL-2V specifically stimulates Treg in vivo, then Treg cells should preferably expand more than CD4 and CD8+T cells and reduce disease symptoms.

[0232] To induce acute GVHD, NSG-SGM3 female mice were given iv injections containing 6 × 10 6 PBMCs of human CD3-T cells. On the same day, mice were treated with Proleukine (6ug eq100,000IU), IL-2V1 (6ug) or IL-2V2 (6ug) by ip injection. One group of mice was not treated. Blood samples were collected on days 5, 10 and 21 after treatment for immune monitoring, and body weight was monitored during the experiment to detect the development of GVHD.

[0233] Similar to Proleukin-treated mice, untreated mice began to lose weight on day 20 after PBMC injection. IL-2V2 slightly delayed, while IL-2V1 stopped weight loss at the doses used, and the clinical effect of IL-2V1 was associated with a slightly prolonged increase in mouse survival ( Figure 7 ). In addition, IL-2V1 induced a sustained increase in the frequency of circulating human Tregs as well as CD4+CD25+Tregs, which are described as Tregs with increased suppressive function ( Figure 8 These results suggest that IL-2V can control GVHD; and IL-2V1 is more effective in preventing weight loss and increasing CD25+Treg in vivo.

[0234] To evaluate the in vivo effects of IL-2V2 and IL-2V3, C57BL / 6 healthy mice were treated with Proleukine (4 μM), IL-2V2 (40 μM) or IL-2V3 (40 μM) by ip injection for 4 consecutive days. One group of mice was not treated. Blood samples were collected on the day after the last administration of Proleukine or IL-2V for FACS analysis.

[0235] IL-2V2- and IL-2V3-treated mice showed an increase in the frequency of circulating Tregs compared with untreated mice, although to a lesser extent than Proleukine-treated mice ( Fig.10 ). However, unlike Proleukine, IL-2V2 (40 μM) and IL-2V3 (both concentrations) did not increase the frequency of effector cells (CD8+ T cells and NK cells) nor induce proliferation of those cells (measured by Ki67 expression). This was emphasized by the reduction in the CD8 and NK cell to Treg ratios in mice treated with IL-2-V compared to untreated and Proleukine-treated mice. These data show that IL-2V2 and IL-2V3 also selectively stimulate Tregs in mice in vivo (a conserved function across species). Compared to Proleukine, these data enhance the potential of IL-2V2 and IL-2V3 as new drugs that selectively stimulate Treg cells in vitro and in vivo by selectively inducing STAT-5-P on Treg cells in vitro.

[0236] Example 6. IL-2V1, IL-2V4, IL-2V5 and IL-2V6 act as IL-2 antagonists

[0237] Considering the results observed in the GVHD model, it was hypothesized that IL-2-V1 could act as an IL-2 antagonist. To further investigate this hypothesis, the ability of IL-2V1 to compete with Proleukine to induce STAT5 phosphorylation in T cells expressing either a medium-affinity IL-2R (CD4+ effector cells and CD8+ T effector cells) or a high-affinity IL-2R (Treg cells) was evaluated in vitro. For this purpose, cells were stimulated with varying concentrations of Proleukine and a constant high concentration of IL-2V1 (1000 nM) using the in vitro biological assay described above. Fig. 9 It can be observed that IL-2-V1 competes with WT IL-2 for binding to IL-2Rαβγ and IL-2Rβγ; potential antagonistic function of IL-2-V1.

[0238] Binding of IL-2 variants to IL-2Rαβγ was determined in the Kit225 cell line, an IL-2-dependent human cell line that constitutively expresses IL-2Rαβγ. For ease of detection, IL-2 variants were produced with a streptavidin label that allows detection of the molecule by FACS using an anti-streptavidin tag antibody (a-Strep-Tag Ab). For this experiment, Kit225 cells were deprived of IL-2 for 2 days. Thereafter, 0.2×10 6Cells were incubated on ice for 20 min alone or with 10 μM IL-2wt, IL-2V1, V2, V3, V4, V5, and V6. After washing with PBS / 0.2% BSA buffer, cells were stained with a-Strep-Tag Ab (Strep-Tactin-A488, Iba) for 20 min at 4°C. Cells were acquired on an LSRII flow cytometer and analyzed with FlowJo software. Fig.11 The data in show that all IL-2 variants bind to IL-2Rαβγ to varying degrees and Figure 6 and 9 Those shown in together support the hypothesis that IL-2V1 and IL-2V4-6 act as IL-2 antagonists.

[0239] This hypothesis was confirmed by in vitro data showing that IL-2V antagonists compete with IL-2 for CD4+T cell activation (Tconv and Treg cells) and inhibit Treg in vitro division by removing them from wild-type IL-2 signaling (similar to blocking anti-IL-2 antibodies), leaving active IL-2 signaling in CD8+T cells. In brief, human PBMCs and CD25+T cells labeled with CellTrace Violet (CTV) (mixed in a 50:50 ratio) were incubated with anti-CD3 / anti-CD28 beads (bead: cell ratio of 1:5) + IL-2V1, 4, 6, anti-hIL-2mAb (Mab602) or PBS (endogenous IL-2 control) for 96 hours. The CTV dilution of CD4+Tconv, Treg and CD8+T cells (as a measure of T cell division) was measured by flow cytometry. Fig.12 Shows CTV 低 The percentage (%) of cells relative to PBS conditions (maximum). The data show that IL-2V1, V4, and V6 antagonize IL-2 signaling by competing with endogenous IL-2 on CD4+ T cells and leave active IL-2 signaling in CD8+ T cells by depriving them of wild-type IL-2 signaling (similar to blocking anti-IL-2 antibodies). These data are consistent with Figure 6 , 9 Together with the data shown in 11, IL-2V1 and IL-2V4-6 act as IL-2 antagonists that can be used to deprive or starve Tregs from WT IL-2 signaling and thereby inhibit Treg function, induce Treg loss of function, or eliminate Tregs while preserving effector CD8+ T cell function.

[0240] In vivo, it is expected that inhibition of Tregs with the IL-2 antagonist variants of the present invention will reduce tumor growth, as previously shown with other IL-2 antagonists (Carmenate et al., The Journal of Immunology, 2018, 200, 3475-3484). Therefore, it is expected that the IL-2V of the present invention, as an IL-2 antagonist, has an anti-tumor effect in vivo. Therefore, the inhibition of Tregs by the IL-2 antagonist variants of the present invention will promote immune responses (lymphocytes (B, NK, CD4+ or CD8+ T cells); dendritic cells (DC); macrophages, etc.) by releasing immune cells from Treg inhibition. In addition, depending on the dose of the IL-2V antagonist used, CD8+ T cell function is maintained or moderately affected ( Fig. 9 ; Fig.12 ). Therefore, the IL-2 antagonist variants of the present invention also allow direct stimulation of CD8+ T cell immune responses, for example against tumors, pathogens or vaccines. For all these reasons, the IL-2-antagonist variants of the present invention are expected to have better immune responses against cancer, infectious agents and vaccines.

[0241] In summary, the results presented in this application show that the effect of IL-2 antagonist variants in vivo can vary depending on the immune environment, because when the immune system is overactivated and produces excessive IL-2, it reduces immune activation (by neutralizing excessive endogenous IL-2), and in different immune environments, it increases immune activation (by Treg inhibition and subsequent CD8+T cell activation). For these reasons, IL-2 antagonist variants can be used to treat diseases including overactive immune systems associated with excessive IL-2 production, such as chronic or acute inflammatory diseases, graft-versus-host disease (GVHD) and transplant rejection, as well as for the treatment of cancer and infectious diseases and for increasing immune responses to vaccines.

[0242] Example 7. Crystal structure of IL-2 variant

[0243] Experimental procedures

[0244] The IL-2 variants were cloned into modified pMT / BiP plasmids (Invitrogen) and used to obtain stable transfectants of Drosophila S2 cells. Proteins were purified from the supernatant using affinity and size exclusion chromatography. Crystals of IL-2V1 were obtained in 30% (w / v) PEG8000, 0.1M imidazole pH 8.0, 0.2M NaCl, and crystals of IL-2V4 were obtained in 15% (w / v) PEG 3350, 0.1M Hepes7, 5mM NiCl2 and 10mM MgCl2 and flash frozen in liquid nitrogen. The structure of wild-type IL-2 was used as a model to solve the structure by molecular replacement.

[0245] result

[0246] The inventors have also solved the crystal structures of some variants, which provide some insights into their mechanism of action. The inventors observed that the Y31P substitution (IL2-V4) stabilized IL-2 in a conformation similar to that observed in the IL2-Rα subunit complex, hereinafter referred to as the "α-induced conformation". In fact, there is some recent evidence from mouse IL-2 that conformational changes in loop AB (where Tyr31 is located) allosterically affect the interaction with IL-2Rβ and γ subunits (De Paula et al., PNAS, doi / 10.1073; March 17, 2020 and Spangler et al., Immunity, 2015, 42, 815-825). Therefore, it is easy to speculate that the molecular mechanism behind the activity of IL2-V4 utilizes this allosteric circuit. In fact, the inventors have also obtained the structure of IL2-V1 with the same activity as IL2-V4. Although none of the mutations present in this variant are located in the AB ring, it also shows the same "α-induced conformation".

[0247] Without being bound by theory, the inventors believe that the substitutions introduced in the IL-2 antagonist variants of the invention stabilize the bound form and induce conformational changes that allosterically affect the interaction with the IL-2Rβ and γ subunits.

[0248] in conclusion

[0249] In short, these results prove that IL-2V is a new drug that selectively stimulates Treg cells or antagonizes IL-2. Therefore, IL-2V can be used for amplifying Treg in vitro or in vivo, or blocking the overactivation of the immune system mediated by IL-2, and suppressing harmful immune responses in vivo. In particular, the IL-2V that selectively stimulates Treg cells in vivo can be used to treat diseases related to immune dysfunction, such as allergic diseases and autoimmune diseases and diseases related to overactivation of the immune system, such as chronic or acute inflammatory diseases, graft-versus-host disease (GVHD) and transplant rejection. IL-2 antagonist variants can be used to treat diseases related to overactivity of the immune system, including excessive production of IL-2, such as GVHD, transplant rejection and chronic or acute inflammatory diseases.

[0250] IL-2V, as an IL-2 antagonist, can also be used to suppress Tregs and thereby increase immune responses (B, NK, CD4+ or CD8+ T cells, DCs, macrophages, etc.), especially anti-tumor immune responses in cancer treatment and immune responses to vaccines or pathogens.

Claims

1. An interleukin-2 variant for use in preventing or treating an immune disorder, which is a variant comprising substitutions K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, K49Q, E52S, R81E, D84N, N119K, T123A, T131R and L132S; The indicated positions are determined by alignment with SEQ ID NO: 1; the variant is an IL-2 antagonist.

2. The variant according to claim 1, which is a human IL-2 variant.

3. The variant according to claim 1 or 2, wherein the IL-2 antagonist inhibits T regulatory cells and thereby stimulates an immune response, in particular a CD8+ T cell response.

4. The variant according to any one of claims 1 to 3, which is bound to a target agent in the form of a molecular complex, a particle, a conjugate or a fusion protein.

5. The variant according to claim 4, which is complexed with an anti-IL-2 antibody, preferably with an anti-IL-2 antibody having T regulatory cell-promoting or T effector cell-promoting activity. The variant according to claim 4 , which is fused to an antibody against a Treg-specific surface molecule or a functional fragment thereof comprising at least an antigen binding site.

7. The variant of claim 6, wherein the antibody is selected from the group consisting of anti-CTLA-4, anti-CD25, anti-CCR8, anti-ICOS, anti-IKZF2, anti-CD70, anti-GARP, anti-IL1R1, anti-CD39, anti-CCR4, and anti-CD177 antibodies.

8. An isolated polynucleotide encoding a variant according to any one of claims 1 to 7 in an expressible form, preferably contained in an expression vector.

9. A pharmaceutical composition comprising as an active substance an IL-2 variant antagonist according to any one of claims 1 to 7, an IL-2 variant antagonist according to any one of claims 4 to 7 finally combined with a target agent, a polynucleotide encoding the variant according to claim 8 or a cell modified with the polynucleotide, and at least one pharmaceutically acceptable excipient and / or carrier.

10. The pharmaceutical composition according to claim 9, comprising the IL-2 variant antagonist according to any one of claims 1 to 7, wherein the pharmaceutical composition is used to prevent or treat a disease selected from the group consisting of cancer and infectious diseases, including treating infectious diseases and cancer and increasing the immune response to vaccines against cancer or infectious diseases.

11. A pharmaceutical composition according to claim 9, comprising an IL-2 variant antagonist according to any one of claims 1 to 7, for preventing or treating a disease comprising an overactivation of the immune system associated with overproduction of IL-2 selected from the group consisting of acute or chronic inflammatory diseases, graft-versus-host disease and transplant rejection.

12. The pharmaceutical composition for use thereof according to claim 10 or 11, which is used in combination with chimeric antigen receptor T cell therapy, immunomodulatory monoclonal antibody therapy, or therapy using anticancer agents or anti-infective agents including therapeutic agents and vaccines against cancer and infectious diseases.

13. The pharmaceutical composition for treating cancer according to claim 10, used in combination with at least one additional cancer therapy selected from the group consisting of targeted therapy, immunotherapy such as immune checkpoint therapy and immune checkpoint inhibitors, co-stimulatory antibodies, chemotherapy and / or radiotherapy.

14. Use of the variant according to any one of claims 1 to 4 for screening anti-IL-2 antibodies having T regulatory cell-promoting or T effector cell-promoting activity.

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