Interleukin-2 variants with modified biological activity

Abstract

To provide IL-2 variants for improving interleukin-2 (IL-2)-based immunotherapies to prevent or treat immune disorders.SOLUTION: The invention relates to IL-2 variants for the prevention or treatment of immune disorders, without limitation, allergic, autoimmune, chronic or acute inflammatory and infectious diseases; graft-versus-host disease; graft rejection and cancer. The invention also relates to use of the IL-2 variants for screening anti-IL-2 antibodies having pro-T-effector or pro-T-regulatory cell activity.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to interleukin-2 (IL-2) variants for the prevention or treatment of immune disorders such as, but not limited to, acute or chronic inflammatory diseases, allergic diseases, autoimmune or infectious diseases, graft-versus-host disease, graft rejection, and cancer. The present invention also relates to the use of said IL-2 variants for screening anti-IL-2 antibodies with pro-effector T cell activity or pro-regulatory T cell activity. [Background technology]

[0002] 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 alpha (IL-2RA or CD25), beta (IL-2RB or CD122), and gamma (IL-2RG or gamma c or CD132) chains. The alpha chain binds IL-2 with low affinity and is not involved in signal transduction. The combination of the beta and gamma chains forms an intermediate-affinity receptor expressed on effector T cells (conventional T cells (Tconv; CD4+Foxp3-), CD8+, and NK cells responsible for cell-mediated immune responses; all three receptor chains form a high-affinity receptor constitutively expressed on regulatory T cells (Treg; CD4+Foxp3+) with immunosuppressive functions, and short- or transiently expressed by activated effector T cells and NK cells.

[0003] Indeed, IL-2 is a cytokine that has both immunostimulatory and suppressive functions. As a result, it has been used in the clinic at high doses to stimulate immune responses against cancer and, more recently, at lower doses to block immune responses in various physiopathological conditions such as type 1 diabetes (T1D), autoimmune diseases such as 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, pp. 127-128; Grinberg-Bleyer, Y. et al., J. Exp. Med., 2010, 207, pp. 1871-1878; Tang et al., Immunity, 2008, 28, pp. 687-697; Pilon et al., Am. J. Transplant., 2014, 14, pp. 2874-2882; Saadoun et 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; Gonzalez et al., Brain. Behav. Immun., 2015, 45, 219-232; Perol et al., Immunol. Lett., 2014, 162, 173-184).

[0004] However, high doses of IL-2 in cancer therapy are highly toxic and its efficacy is suboptimal (5–20% of responders) due to the undesirable effects of IL-2 on Tregs.

[0005] In humans, low doses of IL-2 have been successfully administered to boost 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, IL-2 has a very short half-life and requires repeated administration, so improvements need to be made.

[0006] Interestingly, when IL-2 is conjugated to an anti-IL-2 antibody (Ab), the pharmacodynamics of IL-2 are improved, its toxicity is reduced, and, depending on the Ab, the conjugate redirects the action of IL-2 to effector (pro-Teff anti-IL-2 Ab) or regulatory (pro-Treg anti-IL-2 Ab) immune cells, solving a major problem associated with IL-2-based therapy (Boyman et al., Science, 2006, 311, pp. 1924-1927; Letourneau et al., Proc. Natl. Acad. Sci., 2010, 107, pp. 2171-2176). Regarding the mechanism of action, the formation of a complex with a pro-Teff anti-IL-2 antibody has been shown to directly block the interaction of IL-2 with CD25 (Levin et al., Nature, 2012, 484, 529-533; Rojas, G. in Monoclonal Antibodies (Ossipow, V. & Fischer, N. (eds.)) 1131, 447-476 (Humana Press, 2014)).

[0007] Treg-directed IL-2 / anti-IL-2 complexes have shown impressive results in different mouse models of inflammation: T1D, asthma, EAE, atherosclerosis, chronic nephropathy, and transplantation, including solid organ transplantation (Webster et al., J. Exp. Med., 2009, 206, pp. 751-760; Dinh, TN et al., Circulation, 2012, 126, pp. 1256-1266; Polhill, T. et al., J. Am. Soc. Nephrol. JASN, 2012, 23, pp. 1303-1308; Satake et al., PLoS ONE, 2014, 9, pp. e92888; Vokaer et al., Transplant. Proc., 2012, 44, pp. 2840-2844; Goldstein et al., Front. Immunol., 2013, 4, pp. 155).

[0008] Another strategy for fine-tuning the biological activity of IL-2 is to perform timed mutations in the IL-2 molecule to generate IL-2 variants or mutants. An IL-2 protein mutant (IL-2 superkine) containing five timed mutations that provides a 300-fold higher affinity for CD122 compared to wild-type IL-2 has been disclosed (Levin et al., Nature, 2012, 484, 529-533). Immunosuppressive IL-2 variants with higher affinity for IL-2RA (CD25) and / or altered signaling via IL-2RBG have also been disclosed (WO2010 / 085495 and US 2014 / 0286898). [Prior art documents] [Patent documents]

[0009] [Patent Document 1] WO2010 / 085495 [Patent Document 2] US2014 / 0286898 [Patent Document 3] WO2004004771 [Patent Document 4] WO2004056875 [Patent Document 5] WO2006121168 [Patent Document 6] WO2008156712 [Patent Document 7] WO2009014708 [Patent Document 8] WO2009114335 [Patent Document 9] WO2013043569 [Patent Document 10] WO2014047350 [Patent Document 11] U.S. Patent No. 6,984,720 [Patent Document 12] U.S. Patent No. 8,017,114 [Patent Document 13] U.S. Patent No. 7,109,003 [Patent Document 14] U.S. Patent No. 8,143,379 [Patent Document 15] WO1997020574 [Patent Document 16] WO2007123737 [Patent Document 17] U.S. Patent No. 8,491,895 [Patent Document 18] U.S. Patent Application No. 20130177557 [Patent Document 19] U.S. Patent No. 5,773,578 [Non-patent literature]

[0010] [Non-Patent Document 1] Rosenberg et al., Sci. Transl. Med., 2012, 4, 127 [Non-patent document 2] Grinberg-Bleyer, Y. et al., J. Exp. Med., 2010, 207, pp. 1871-1878 [Non-patent document 3] Tang et al., Immunity, 2008, 28, pp. 687-697 [Non-licensed Document 4] Pilon, Am. J. Transplant., 2014, 14, pp. 2874-2882 [Non-licensed Document 5] Saadounら, N. Engl. J. Med., 2011, 365, pages 2067~2077 [Non-licensed Document 6] Korethら, N. Engl. J. Med., 2011, 365, pages 2055~2066 [Non-licensed Document 7] Kennedy-Nasser, Clin. Cancer Res. Off. J. Am. Assoc. Cancer Res., 2014, 20, pages 2215~2225 [Non-licensed Document 8] Baeyensら, Diabetes, 2013, 62, pages 3120~3131 [Non-licensed Document 9] Gonzalez, Brain. Behav. Immun., 2015, 45, pp. 219-232 [Non-licensed Document 10] Perol, Immunol. Lett., 2014, pages 162, 173~184 [Non-licensed Document 11] Boyman, Science, 2006, 311, pages 1924~1927 [Non-licensed Document 12] Letourneauら, Proc. Natl. Acad. Sci., 2010, 107, pages 2171~2176 [Non-licensed Document 13] Levinら, Nature, 2012, 484, 529~533 pages [Non-licensed Document 14] Rojas, G. in Monoclonal Antibodies (Ossipow, V. & Fischer, N. (Eds.)) Pages 1131, 447~476 (Humana Press, 2014) [Non-licensed Document 15] Webster, J. Exp. Med., 2009, 206, pages 751~760 [Non-licensed Document 16] Dinh, TNら, Circulation, 2012, 126, pages 1256~1266 [Non-licensed Document 17] Polhill, T., J. Am. Soc. Nephrol. JASN, 2012, 23, pp. 1303-1308. [Non-licensed Document 18] Satakeら, PLoS ONE, 2014, 9, page e92888 [Non-licensed Document 19] Vokaerら, Transplant. Proc., 2012, 44, pages 2840~2844 [Non-licensed Document 20] Goldstein, Front. Immunol., 2013, 4, 155 pages [Non-licensed Document 21] Carmenateら, The Journal of Immunology, 2018, 200, pages 3475~3484 [Non-licensed Document 22] De Paulaら, PNAS, doi / 10.1073; March 17, 2020 [Non-licensed Document 23] Spanglerら, Immunity, 2015, 42, pages 815~825 [Non-licensed Document 24] Arenas-Ramirezら, Sci. Transl. Med., 2016, 8, 367ra166 [Non-licensed Document 25] Fraczkiewicz, J. Comp. Chem, 1998, 19, pages 319~333 [Non-licensed Document 26] Altschul, J. Mol. Biol., 1990, 215, 403 pages - [Non-licensed Document 27] Bhairavabhotlaら, Human Immunol., 2016, 77, pages 201~13 [Non-licensed Document 28] van der Veeken, Cold Spring Harb. Symp. Quant Biol., 2013, 78, pages 215~22 [Non-licensed Document 29] Pfoertner, Genome Biol., 2006, 7, R54 [Non-licensed Document 30] Sugimotoら, Int. Immunol., 2006, 18, page 1197~ [Non-licensed Document 31] Smaldini PLら, Allergy, 2018, 73, pages 885~895 [Non-licensed Document 32] Lee-S Yら, Immunology, 2012, 137, pages 305~16 [Non-licensed Document 33] Boyman Oら, Science 2006, 311, pages 1921~27 [Non-licensed Document 34] Kriegら, PNAS, 2010, 107, pages 11906~11 [Non-licensed Document 35] Caudana Tら, 2019, Cancer Immunol Res, 7, pages 443~457 [Non-licensed Document 36] Newman RGら, Blood, 2014, 123, pages 3045~55 [Non-licensed Document 37] 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 RG2 Glioma-Bearing Rats: A Comparison Between Proton Int J Radiat Oncol Biol Phys. 2019 Jun 1;104(2):266~271

Direct Entries 38

Direct Entries 39

Outdoor Track 40

[0011] To improve IL-2-based immunotherapy, IL-2 variants are needed for the prevention or treatment of immune disorders. [Means for solving the problem]

[0012] The present inventors have engineered resurfaced variants of human IL-2 and identified IL-2 variants that can selectively stimulate regulatory T cells (Tregs) or antagonize IL-2.

[0013] The examples of the present application demonstrate that resurfaced variants of human IL-2 (Treg agonist variants) selectively stimulate Tregs in vitro ( FIG. 6 ) and increase circulating Treg cells in vivo in mice ( FIG. 10 ). Treg agonist variants are useful for indirectly reducing immune activation by selectively activating Tregs and then inhibiting immune effector cells. The Treg agonist variants of the present invention are thus useful for treating diseases in which immune modulation or immune suppression is beneficial, including, but not limited to, allergic and autoimmune diseases, and particularly diseases involving immune system hyperactivity, such as chronic or acute inflammatory diseases, graft-versus-host disease (GVHD), and graft rejection.

[0014] The examples of this application also show that some resurfaced variants of human IL-2 are IL-2 antagonists (FIG. 9).

[0015] In particular, in the context of diseases involving immune system hyperactivity associated with IL-2 overproduction, including chronic or acute inflammatory diseases, graft-versus-host disease (GVHD) and graft rejection, the IL-2 antagonist variants according to the invention directly reduce immune activation by competing with endogenous IL-2, thereby blocking IL-2-mediated hyperactivation of the immune system.

[0016] In mouse models 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. The IL-2 antagonist variants tested were more effective at halting weight loss and selectively increasing circulating human Treg cells than the Treg agonist variants tested (Figures 7 and 8).

[0017] Furthermore, IL-2 variants that are IL-2 antagonists inhibit Treg division in vitro by depleting Tregs from wild-type IL-2 signaling (similar to blockade by anti-IL-2 antibodies; Figure 12). In vivo, Treg inhibition by the IL-2 antagonist variants of the present invention will reduce tumor growth, as previously shown for other IL-2 antagonists (Carmenate et al., The Journal of Immunology, 2018, 200, pp. 3475-3484). Treg inhibition by the IL-2 antagonist variants of the present invention will consequently promote immune responses (lymphocytes (B, NK, CD4+ or CD8+ T cells); dendritic cells (DCs); macrophages, etc.) by relieving immune cells from Treg suppression. Furthermore, depending on the dose of the IL-2 antagonist variant used, CD8+ T cell function is either preserved or only modestly affected (Figures 9; 12). Thus, the IL-2 antagonist variants further allow for the direct stimulation of CD8+ T cell immune responses against, for example, tumors, pathogens, or vaccines. For all these reasons, better immune responses against cancer, infectious agents, and vaccines are expected with the IL-2 antagonist variants of the invention.

[0018] The results presented in this application collectively suggest that the effect of IL-2 antagonist variants in vivo may vary depending on the immune context, as they decrease immune activation when the immune system is overactive and producing excess IL-2 (by neutralizing excess endogenous IL-2), but increase immune activation in different immune contexts (by Treg inhibition and CD8+ T cell activation). For these reasons, IL-2 antagonist variants are useful for treating diseases involving immune system hyperactivity associated with excessive production of IL-2, such as, but not limited to, chronic or acute inflammatory diseases, graft-versus-host disease (GVHD), and graft rejection, as well as for treating cancer and infectious diseases, and increasing immune responses to vaccines.

[0019] We also analyzed the crystal structures of some variants, which provided some insight into their mechanism of action. We observed that the Y31P substitution (IL2-V4) stabilizes IL-2 in a conformation similar to that observed in complex with the IL2-R alpha subunit, hereafter referred to as the "alpha-induced conformation." Indeed, there is some recent evidence from mouse IL-2 suggesting that conformational changes in loop AB, where Tyr31 is located, allosterically affect the interaction with the IL-2R beta and gamma subunits (De Paula et al., PNAS, doi / 10.1073; March 17, 2020; and Spangler et al., Immunity, 2015, 42, pp. 815-825). Therefore, it is tempting to speculate that the molecular mechanism underlying IL2-V4 activity utilizes this allosteric circuit. Indeed, we also obtained the structure of IL2-V1, which has the same activity as IL2-V4. Although none of the mutations present in this variant are located in the AB loop, it also exhibits the same "alpha-induced conformation."

[0020] Without being bound by theory, the inventors believe that the substitutions introduced into the IL-2 antagonist variants of the present invention induce conformational changes that stabilize the bound form and allosterically affect the interaction with the IL-2R beta and gamma subunits.

[0021] These results demonstrate the ability of these human IL-2 variants to treat immune disorders and to increase immune responses to vaccines. Immune disorders include, inter alia, inflammatory, allergic, infectious and autoimmune diseases, graft-versus-host disease (GVHD), transplant rejection, and cancer. Vaccines may be directed against cancer or infectious diseases.

[0022] 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.

[0023] Thus, the present invention relates to interleukin-2 (IL-2) variants capable of selectively stimulating regulatory T cells or antagonizing IL-2, which contain at least one amino acid substitution at a surface position of IL-2 outside the region that contacts the alpha receptor.

[0024] In some embodiments, the interleukin-2 variant according to the 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; - the amino acids in positions 9 and 12 are substituted by D or E, preferably E; - 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; - the amino acid in position 23 is substituted by E, Q, T, N, G, A, V, L or I, preferably by L; - the amino acid in position 31 is substituted by P or N, preferably by P; - the amino acid at position 87 is substituted by M, V, E, D, T, C, N or Q, preferably by N; and If the substitution is at position 91, the variant comprises at least another substitution at position 9, 12, 16, 19, 23, 26, 31, 49, 52, 81, 84, 87, 95, 119, 123, 127, 131 or 132, the positions indicated being determined by alignment with SEQ ID NO:1.

[0025] In some preferred embodiments, 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 further substitution at a position selected from the group consisting of 31, 49, 52, 81, 84, 119, 123, 127, 131 and 132; - the amino acid in position 31 is substituted by P or N, preferably by P; - the amino acids at positions 49, 52, 84 and 132 are substituted by another amino acid selected from 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; - the amino acid in position 81 is substituted by D or E, preferably by E; - 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 - the amino acid in position 123 is substituted by E, Q, N, M, G, A, V, L or I, more preferably by A.

[0026] In some other preferred embodiments, the variant according to the invention comprises one substitution at position 31 and no substitutions at positions 9, 12, 16, 19, 23, 26, 49, 52, 81, 84, 87, 91, 95, 119, 123, 127, 131 and 132.

[0027] In some embodiments, the variant according to the present invention preferably comprises: - variants containing K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, N119K, T123A, and S127K substitutions; - variants containing K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, Y31P, K49Q, E52S, R81E, D84N, N119K, T123A, T131R and L132S substitutions; - a variant containing a Y31P substitution that does not contain any substitutions at positions 9, 12, 16, 19, 23, 26, 49, 52, 81, 84, 87, 91, 95, 119, 123, 127, 131 and 132; - variants containing K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, Y31P, K49Q, E52S, R81E, D84N, T131R and L132S substitutions; - variants containing K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, Y31P, N119K, T123A, and S127K substitutions; - Variants containing K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, K49Q, E52S, R81E, D84N, N119K, T123A, T131R and L132S substitutions The human IL-2 variant is selected from the group consisting of:

[0028] In some embodiments, variants according to the invention have at least 70% amino acid identity with any one of SEQ ID NOs: 1 and 3-8, and preferably do not contain 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.

[0029] In some embodiments, the variants according to the invention selectively stimulate regulatory T cells.

[0030] In some other embodiments, the variant according to the invention is an IL-2 antagonist, preferably an IL-2 antagonist that inhibits regulatory T cells and thereby stimulates an immune response, in particular a CD8+ T cell response.

[0031] In some embodiments, the variants according to the invention are bound to a drug of interest, for example in the form of a molecular complex, particle, conjugate, or fusion protein. In some preferred embodiments, the IL-2 variant is complexed with an anti-IL-2 antibody, preferably an anti-IL-2 antibody having pro-regulatory T cell activity or pro-effector T cell activity. In some preferred embodiments, the IL-2 variant is fused to an antibody against a Treg-specific surface molecule or a functional fragment thereof comprising at least one antigen-binding site. The antibody against a Treg-specific surface molecule is preferably selected from the group comprising anti-CTLA-4 antibody, anti-CD25 antibody, anti-CCR8 antibody, anti-ICOS antibody, anti-IKZF2 antibody, anti-CD70 antibody, anti-GARP antibody, anti-IL1R1 antibody, anti-CD39 antibody, anti-CCR4 antibody, and anti-CD177 antibody.

[0032] The present invention relates to polynucleotides encoding said variants in an expressible form, vectors, preferably expression vectors, containing said polynucleotides, and host cells containing said polynucleotides or vectors.

[0033] The present invention relates to pharmaceutical compositions comprising as active substances an IL-2 variant, a polynucleotide, a vector and / or a cell according to the invention, ultimately combined with a drug of interest as disclosed in the present invention, and at least one pharmaceutically acceptable vehicle and / or carrier.

[0034] The present invention relates to the use of an IL-2 variant, a polynucleotide, a vector and / or a cell according to the invention for treating an immune disorder.

[0035] In some embodiments, the IL-2 variants that selectively stimulate Tregs (Treg agonist variants), polynucleotides, vectors, and / or cells according to the 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 hyperactivity of the immune system, including chronic or acute inflammatory diseases, graft-versus-host disease (GVHD), and graft rejection, among others.

[0036] In some embodiments, the IL-2 antagonist variants, polynucleotides, vectors, and / or cells according to the invention are used to block IL-2-mediated overactivation of the immune system to treat diseases involving immune system hyperactivity associated with overproduction of IL-2, in particular chronic or acute inflammatory diseases, graft-versus-host disease (GVHD), and transplant rejection.

[0037] In some embodiments, the IL-2 antagonist variants, polynucleotides, vectors, and / or cells according to the invention are used to inhibit Tregs and thereby stimulate an immune response, including B, NK, CD4+ or CD8+ T cells, DCs, macrophages, etc., particularly a CD8+ T cell immune response, to treat cancer or an infectious disease or to increase the immune response to a vaccine, particularly a vaccine for cancer or an infectious disease.

[0038] In some embodiments, the IL-2 antagonist variants, polynucleotides, vectors, and / or cells according to the invention are used in combination with chimeric antigen receptor T cell therapy, immunomodulatory monoclonal antibody therapy, or therapy using anti-cancer or anti-infective agents, including therapeutic agents and vaccines for cancer and infectious diseases. In some preferred embodiments, the IL-2 antagonist variants, polynucleotides, vectors, and / or cells according to the invention are used in combination with at least a further cancer therapy selected from the group comprising targeted therapy, immunotherapy such as immune checkpoint therapy and immune checkpoint inhibitors, costimulatory antibodies, chemotherapy, and / or radiation therapy.

[0039] The present invention also relates to the use of an IL-2 variant according to the invention for screening for anti-IL-2 antibodies with pro-effector T cell activity or pro-regulatory T cell activity. [Brief explanation of the drawings]

[0040] [Figure 1-1]Multiple sequence alignment of IL-2 from different species used in the evolutionary filter. Human (Homo sapiens) (SEQ ID NO: 1); White-fronted gibbon (Hylobates lar) (SEQ ID NO: 15); Rhesus macaque (Macaca mulatta) (SEQ ID NO: 16); Pig-tailed macaque (Macaca nemestrina) (SEQ ID NO: 17); Cynomolgus macaque (Macaca fascicularis) (SEQ ID NO: 18); Necked mangabey (Cercocebus torquatus) (SEQ ID NO: 19); Owl monkey (Aotus vociferans) (SEQ ID NO: 20); Hamadryas baboon (Papio hamadryas) (SEQ ID NO: 21); House mouse (Mus musculus) (SEQ ID NO: 22); Brown rat (Rattus norvegicus) (SEQ ID NO: 23); Mongolian gerbil (Meriones unguiculatus) (SEQ ID NO: 24); Guinea pig (Cavia porcellus) (SEQ ID NO: 25); European rabbit (Oryctolagus cuniculus) (SEQ ID NO: 26); Cattle (Bos taurus) (SEQ ID NO: 27); sheep (Ovis aries) (SEQ ID NO: 28); red deer (Cervus elaphus hippelaphus) (SEQ ID NO: 29); Himalayan goat (Capra hircus) (SEQ ID NO: 30); beluga whale (Delphinapterus leucas) (SEQ ID NO: 31); orca (Orcinus orca) (SEQ ID NO: 32); wild boar (Sus scrofa) (SEQ ID NO: 33); horse (Equus caballus) (SEQ ID NO: 34); domestic cat (Felis catus) (SEQ ID NO: 35); dog (Canis lupus familiaris) (SEQ ID NO: 36); grey seal (Halichoerus grypus) (SEQ ID NO: 37); northern elephant seal (Mirounga angustirostris) (SEQ ID NO: 38) and red jungle fowl (Gallus gallus) (SEQ ID NO: 39). [Figure 1-2]Multiple sequence alignment of IL-2 from different species used in the evolutionary filter. Human (Homo sapiens) (SEQ ID NO: 1); White-fronted gibbon (Hylobates lar) (SEQ ID NO: 15); Rhesus macaque (Macaca mulatta) (SEQ ID NO: 16); Pig-tailed macaque (Macaca nemestrina) (SEQ ID NO: 17); Cynomolgus macaque (Macaca fascicularis) (SEQ ID NO: 18); Necked mangabey (Cercocebus torquatus) (SEQ ID NO: 19); Owl monkey (Aotus vociferans) (SEQ ID NO: 20); Hamadryas baboon (Papio hamadryas) (SEQ ID NO: 21); House mouse (Mus musculus) (SEQ ID NO: 22); Brown rat (Rattus norvegicus) (SEQ ID NO: 23); Mongolian gerbil (Meriones unguiculatus) (SEQ ID NO: 24); Guinea pig (Cavia porcellus) (SEQ ID NO: 25); European rabbit (Oryctolagus cuniculus) (SEQ ID NO: 26); Cattle (Bos taurus) (SEQ ID NO: 27); sheep (Ovis aries) (SEQ ID NO: 28); red deer (Cervus elaphus hippelaphus) (SEQ ID NO: 29); Himalayan goat (Capra hircus) (SEQ ID NO: 30); beluga whale (Delphinapterus leucas) (SEQ ID NO: 31); orca (Orcinus orca) (SEQ ID NO: 32); wild boar (Sus scrofa) (SEQ ID NO: 33); horse (Equus caballus) (SEQ ID NO: 34); domestic cat (Felis catus) (SEQ ID NO: 35); dog (Canis lupus familiaris) (SEQ ID NO: 36); grey seal (Halichoerus grypus) (SEQ ID NO: 37); northern elephant seal (Mirounga angustirostris) (SEQ ID NO: 38) and red jungle fowl (Gallus gallus) (SEQ ID NO: 39). [Figure 2] Structure of the quaternary complex of IL-2 with its α (CD25), β (CD122), and γC (CD132) receptors shown. Surface reprocessing sites in IL-2-V2 are indicated. [Figure 3]Sequence alignment of the IL-2-WT construct with the resurfaced variants IL-2-V1 and IL-2-V2. IL-2-WT construct (SEQ ID NO: 2). IL-2-V1 (SEQ ID NO: 3). IL-2-V2 (SEQ ID NO: 4). [Figure 4] Size-exclusion chromatography and SDS-PAGE analysis of recombinant wild-type and resurfaced variants of IL-2. A. IL-2 wild-type. B. IL-2-V1. C. IL-2-V2. Peaks used for experimental testing are indicated by arrowheads. 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). [Figure 5] ELISA to evaluate the specific binding of different anti-IL-2 antibodies to different IL-2 variants. ELISA tests were used to evaluate the selectivity of IL-2V for antibodies (Abs) with pro-T effector or pro-Treg function. To do so, a typical ELISA test was performed by coating plates with Proleukin, IL-2V1, IL-2V2, IL-2V3, IL-2V4, IL-2V5, or IL-2V6 (all at 6 μg / mL). The Abs evaluated were MAB605 (a mouse anti-human IL-2 Ab with in vivo pro-T effector activity), 5344 (a mouse anti-human IL-2 Ab with in vivo pro-Treg activity), and NARA (an scFV antibody with in vivo pro-T effector activity derived from Arenas-Ramirez et al., Sci. Transl. Med., 2016, 8, 367ral66). To detect anti-IL-2 Ab, anti-mouse IgG-HRP Ab or anti-M13-HRP Ab for NARA was used, and absorbance was read at 450 nm. OD values ​​are shown after subtracting the absorbance of control wells (uncoated wells incubated with the corresponding anti-IL-2 Ab + anti-mouse IgG-HRP Ab). [Figure 6]Human IL-2V2 and IL-2V3, but not IL-2V1, IL-2V4, IL-2V5, or IL-2V6, selectively induce STAT-5 phosphorylation in Treg cells. CD3+ cells were purified from human PBMCs and incubated with different concentrations of Proleukin, IL-2V1, IL-2V2, IL-2V3, IL-2V4, IL-2V5, or IL-2V6 (0.0001 nM to 1000 nM). STAT5 phosphorylation (STAT5-P) in human Treg cells (IL-2Rαβγ), CD4+ Teff cells (IL-2Rβγ), and CD8+ T cells (IL-2Rβγ) was measured by flow cytometry. (A-B) Frequency of STAT5-P+ cells among different T cell populations in two different healthy donors treated with Proleukin, IL-2V1, IL-2V2, and IL-2V3. (C) Frequency of STAT5-P+ cells among different T cell populations in one healthy donor using Proleukin, IL-2V4, IL-2V5, and IL-2V6. Results from two independent experiments are shown. [Figure 7] hIL-2V delays the onset of clinical GVHD. NSG-SGM mice were administered human PBMCs containing 6x106 CD3+ T cells by the iv route and treated with 6µg / dose of Proleukin, IL-2V1, and IL-2V2 by the ip route for 5 consecutive days. (A) Mean weight change evolution expressed as a percentage (%) of initial body weight in the different treatment groups, and (B) Kaplan-Meier survival curves of transplanted mice. Untreated n=4; Proleukin n=4; IL-2V1 n=5; IL-2V2 n=5. [Figure 8-1]hIL-2V selectively increases circulating human Treg cells in a mouse model of acute GVHD. NSG-SGM mice were administered 6x106 human PBMCs containing CD3+ T cells by the intravenous route and treated with 6 μg / dose of Proleukin, IL-2V1, and IL-2V2 by the intravenous route for 5 consecutive days. Blood samples were collected 5, 10, and 21 days after PBMC injection. (A) Frequencies of human CD8+ T cells; (B) human CD4+ Tconv cells (CD4+Foxp3- effector T cells), and human Tregs (CD4+CD45RA-Foxp3High) (C) and CD25+ Tregs (CD4+Foxp3+CD25High) (D). Squares: donor 1; circles: donor 2; naive, n=4; Proleukin, n=4; IL-2V1, n=5; IL-2V2, n=5. [Figure 8-2] hIL-2V selectively increases circulating human Treg cells in a mouse model of acute GVHD. NSG-SGM mice were administered 6x106 human PBMCs containing CD3+ T cells by the intravenous route and treated with 6 μg / dose of Proleukin, IL-2V1, and IL-2V2 by the intravenous route for 5 consecutive days. Blood samples were collected 5, 10, and 21 days after PBMC injection. (A) Frequencies of human CD8+ T cells; (B) human CD4+ Tconv cells (CD4+Foxp3- effector T cells), and human Tregs (CD4+CD45RA-Foxp3High) (C) and CD25+ Tregs (CD4+Foxp3+CD25High) (D). Squares: donor 1; circles: donor 2; naive, n=4; Proleukin, n=4; IL-2V1, n=5; IL-2V2, n=5. [Figure 9]Human IL-2V1 stimulates T cells by competing with Proleukin. CD3+ cells were purified from human PBMCs and incubated with Proleukin (0.0001 nM–1000 nM; black curve), human IL-2V1 (0.0001 nM–1000 nM; blue curve), or Proleukin (0.0001 nM–1000 nM) in the presence of a fixed concentration of IL-2V1 (1000 nM, competition curve, red curve). STAT-5 phosphorylation (STAT5-P) was measured by flow cytometry. (A) Frequency of STAT5-P cells (IL-2Rαβγ) among Treg cells. (B) Frequency of STAT5-P cells (IL-2Rβγ) among CD4+Foxp3- T cells. (C) Frequency of STAT5-P cells (IL-2Rβγ) among CD8+ T cells. (D) Frequency of STAT5-P cells among Tregs, CD4+ Teffs, and CD8+ T cells induced by IL-2V1. A schematic representation of the high-affinity (top) and intermediate-affinity (center) IL-2Rs interacting with IL-2V1 is shown. [Figure 10] IL-2V2 and IL-2V3 selectively increase the frequency of Treg cells over effector cells in vivo. C57BL / 6 healthy mice were treated by i.p. injection with Proleukin (4 μM), IL-2V2 (40 μM), or IL-2V3 (40 μM). One group of mice was left untreated. n = 3 mice per group of treated or untreated mice. After the mice received four doses of Proleukin or IL-2V, blood samples were collected for FACS analysis. (A) Frequencies of Tregs (CD4+Foxp3+); effector memory CD8+ T cells (CD8+CD44high) and NK cells among CD45+ cells; proliferative (Ki67+) Tregs, effector memory CD8+ T cells (CD44highKi67+) and NK cells among CD45+ cells; (B) Ratio of effector memory CD8+ T cells (CD8+CD44high) to Tregs; ratio of NK cells to Tregs. [Figure 11]IL-2 variants bind to IL2Rαβγ. Binding assay for the IL-2-dependent human cell line Kit225, which constitutively expresses IL-2Rαβγ. Kit225 cells, depleted of IL-2 for 2 days, were incubated alone or with 10 μM of IL-2wt, IL-2V1, V2, V3, V4, V5, and V6 produced with streptavidin tags. After incubation, cells were stained with anti-Strep-Tag antibody (A-488) and analyzed by flow cytometry. [Figure 12-1] IL-2V antagonists compete with IL-2 for CD4+ T cell activation, leaving active signaling in CD8+ T cells. CTV-labeled human PBMCs and CD25+ T cells (mixed at a 50:50 ratio) were incubated with anti-CD3 / anti-CD28 beads (1:5 bead:cell ratio) plus IL-2V1, 4, 6, anti-hIL-2 mAb (Mab602), or PBS (endogenous IL-2 control) for 96 h. CTV dilution rates of CD4+ T cells, Treg cells, and CD8+ T cells were measured by flow cytometry. The percentage (%) of CTVLow cells (as a measure of dividing cells) relative to the PBS condition (max) is shown. [Figure 12-2] IL-2V antagonists compete with IL-2 for CD4+ T cell activation, leaving active signaling in CD8+ T cells. CTV-labeled human PBMCs and CD25+ T cells (mixed at a 50:50 ratio) were incubated with anti-CD3 / anti-CD28 beads (1:5 bead:cell ratio) plus IL-2V1, 4, 6, anti-hIL-2 mAb (Mab602), or PBS (endogenous IL-2 control) for 96 h. CTV dilution rates of CD4+ T cells, Treg cells, and CD8+ T cells were measured by flow cytometry. The percentage (%) of CTVLow cells (as a measure of dividing cells) relative to the PBS condition (max) is shown. [Figure 12-3]IL-2V antagonists compete with IL-2 for CD4+ T cell activation, leaving active signaling in CD8+ T cells. CTV-labeled human PBMCs and CD25+ T cells (mixed at a 50:50 ratio) were incubated with anti-CD3 / anti-CD28 beads (1:5 bead:cell ratio) plus IL-2V1, 4, 6, anti-hIL-2 mAb (Mab602), or PBS (endogenous IL-2 control) for 96 h. CTV dilution rates of CD4+ T cells, Treg cells, and CD8+ T cells were measured by flow cytometry. The percentage (%) of CTVLow cells (as a measure of dividing cells) relative to the PBS condition (max) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0041] IL-2 variants The present invention relates to interleukin-2 (IL-2) variants capable of selectively stimulating regulatory T cells or antagonizing IL-2, which contain at least one amino acid substitution at a surface position of IL-2 outside the region that contacts the alpha receptor.

[0042] 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) that is cleaved to yield the mature protein (IL-2). Representative examples of IL-2 are shown in FIG. 1 and include, but are not limited to, human (Gene ID: 3558), rat (Gene ID: 116562), feline (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 derived from positions 21 to 153 of the precursor, corresponding to SEQ ID NO: 1.

[0043] In the following description, residues are designated in standard single-letter amino acid code, and the positions shown 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 replacement residue in 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.

[0044] The terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As such, the terms "a" (or "an"), "one or more," or "at least one" can be used interchangeably herein; unless otherwise specified, "or" means "and / or."

[0045] The present invention provides interleukin-2 (IL-2) variants capable of selectively stimulating regulatory T cells (Tregs) or antagonizing IL-2, which are useful for the treatment of immune disorders.

[0046] As used herein, "immune disorder" refers to a disease involving immune dysfunction or immune dysregulation. Immune dysfunction may include inhibition, dysfunction, or overactivity of the immune system. Immune disorders include diseases that can be prevented or treated by immunomodulation using immunomodulators, immunosuppressants, and / or immunostimulators. Immunomodulation may include selectively stimulating Tregs or inhibiting Tregs. Immunosuppression may include selectively stimulating Tregs and then inhibiting immune effector cells or directly inhibiting immune effector cells. Similarly, immunostimulation may include inhibiting Tregs and then relieving immune cells from immunosuppression or directly stimulating effector immune cells. Immunomodulators and immunostimulators that inhibit Tregs are useful for preventing or treating diseases in which inhibition of Tregs and / or stimulation of an immune response is beneficial, such as infectious diseases and cancer, including, but not limited to, treatment of infectious diseases and cancer and increasing immune responses to vaccines. Vaccines are particularly directed against cancer or infectious diseases. Immunomodulatory and immunosuppressive agents that stimulate Tregs are useful for treating diseases involving immune dysfunction, such as, but not limited to, chronic or acute inflammatory diseases, graft-versus-host disease (GVHD), including acute GVHD and graft rejection, as well as allergic and autoimmune diseases and diseases associated with overactivity of the immune system.

[0047] IL-2 variants according to the invention that selectively stimulate regulatory T cells (Treg agonist variants) are useful for expanding Tregs ex vivo or in vivo. IL-2 variants according to the invention that antagonize IL-2 (IL-2 antagonist variants) are useful for blocking IL-2-mediated overactivation of the immune system or for inhibiting Tregs by depleting Tregs from IL-2 signaling and, as a result, stimulating immune responses, including B, NK, CD4+ or CD8+ T cells, DCs, macrophages, and others, particularly CD8+ T cell immune responses in vivo, such as anti-tumor immune responses or immune responses to vaccines or pathogens. As used herein, "selectively stimulating regulatory T cells" means that the IL-2 variant promotes the proliferation or activation of regulatory T cells over non-regulatory T cells. As used herein, "regulatory T 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, "effector T cells," "Teff," or "Teffs" refer to one or more of Tconv cells (CD3+CD4+Foxp3-); CD8+ T cells (CD3+CD8+), and NK cells (CD3-CD16+). As used herein, "inhibiting Tregs" means that an IL-2 variant can inhibit the proliferation, activation, or suppressive function of Treg cells by depleting them from IL-2 signaling. Inhibiting Tregs includes inhibiting Treg function or loss of Treg function, particularly Treg immunosuppressive function, and eliminating Tregs. The ability of the IL-2 variants of the present invention to selectively stimulate regulatory T cells can be measured by standard assays well known in the art and disclosed in the Examples of this application, including, but not limited to, STAT5 phosphorylation assays or flow cytometry analysis of T cell populations derived from in vivo treated subjects or in vitro treated peripheral blood samples.The ability of an IL-2 variant to antagonize IL-2 can be determined by standard assays well known in the art and disclosed in the Examples of this application, such as STAT5 phosphorylation assays competing with Proleukin on in vitro treated peripheral blood samples or quantification of proliferation using a CellTrace Violet (CTV) dilution assay.

[0048] The Treg agonist variants are useful for indirectly reducing immune activation by selectively activating Tregs and then inhibiting immune effector cells. The Treg agonist variants of the present invention are thus useful for treating diseases in which immunomodulation or immunosuppression is beneficial, such as, but not limited to, allergic and autoimmune diseases, and diseases involving hyperactivity of the immune system, such as, but not limited to, chronic or acute inflammatory diseases, graft-versus-host disease (GVHD), and graft rejection.

[0049] The IL-2 variants according to the present invention, which are IL-2 antagonists, are useful for inhibiting Tregs and, as a result, stimulating immune responses (B, NK, CD4+ or CD8+ T cells, DCs, macrophages, etc.). The IL-2 antagonist variants according to the present invention are useful for stimulating anti-tumor immune responses or immune responses to pathogens or vaccines, including vaccines against cancer or infectious diseases, in particular anti-tumor CD8+ T cell responses and anti-CD8+ T cell responses to pathogens or vaccines. Furthermore, since the results of the present application suggest that the effects of IL-2 antagonists in vivo may vary depending on the immune status, IL-2 antagonists are also useful for reducing immune activation by blocking excess IL-2 present in patients in disease situations involving immune system hyperactivity associated with IL-2 overproduction, such as, but not limited to, chronic or acute inflammatory diseases, graft-versus-host disease (GVHD), and graft rejection.

[0050] The present invention provides interleukin-2 (IL-2) variants containing at least one amino acid substitution at a surface position of IL-2 outside the region that contacts the α receptor. Residues that contact the α receptor are defined as residues of IL-2 that have at least one atom within 8 Å or less of any atom of the α receptor. Surface residues are defined as those with greater than 50% exposed side chain surface area, as determined according to standard methods well known in the art (Fraczkiewicz et al., J. Comp. Chem. 1998, 19, 319-333).

[0051] 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, the positions indicated being determined by alignment with SEQ ID NO:1.

[0052] "Containing at least one substitution" means that the IL-2 variant has one or more amino acid substitutions as shown with respect to 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 of these possible combinations are specifically contemplated.

[0053] 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.

[0054] In some embodiments, the IL-2 variant comprises 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, where the positions shown are 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 additional substitutions listed above. All of these possible combinations are specifically contemplated.

[0055] In some more preferred embodiments, the variants comprise further substitutions at 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.

[0056] In some preferred embodiments, when the substitution is at position 91, the variant includes at least another 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 include 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 includes substitutions at positions 9, 12, 16, 19, 23, 26, 87, 91, and 95, and ultimately also includes 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. The IL-2 variant may include one, two, three, four, five, six, seven, eight, nine, 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 includes additional substitutions at 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.

[0057] In some other embodiments, the IL-2 variant contains one substitution at position 31; the IL-2 variant contains only one of said substitutions, meaning that the IL-2 variant does not contain any substitutions at the other indicated positions (9, 12, 16, 19, 23, 26, 87, 91, 95, and 49, 52, 81, 84, 119, 123, 127, 131, and 132).

[0058] In some preferred embodiments, the amino acids at positions 9, 12 and 81 are substituted with D or E, preferably E.

[0059] In some preferred embodiments, the amino acids at positions 16, 19, 26, 91, 95, 119, 127, and 131 are substituted by K or R; preferably, the amino acids at positions 16, 19, and 131 are substituted by R, and the amino acids at positions 26, 91, 95, and 127 are substituted by K.

[0060] In some preferred embodiments, the amino acids at positions 23 and 123 are substituted with 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 with L and the amino acid at position 123 is substituted with A.

[0061] In some preferred embodiments, the amino acid at position 31 is substituted with N or P, preferably P.

[0062] In some preferred embodiments, the amino acids at positions 49, 52, 84, 87 and 132 are substituted by another amino acid selected from 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.

[0063] An IL-2 variant may include 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, and all of these possible combinations are specifically contemplated.

[0064] 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) substitutions selected from K9E or K9D, L12E or L12D, H16R or H16K, L19R or L19K, M23L, N26K or N26R, S87N, V91K or V91R, E95K or E95R substitutions; preferably, one or more (i.e., 1, 2, 3, 4, 5, 6, 7, 8, or 9) substitutions selected from 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 variants are 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, 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 group consisting of 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.

[0065] In some preferred embodiments, the IL-2 variant is a human IL-2 variant.

[0066] According to said preferred embodiment, said IL-2 variant, preferably a human IL-2 variant, a) K9E or K9D, L12E or L12D, H16R or H16K, L19R or L19K, M23L, N26K or N26R, S87N, V91K or V91R, E95K or E95R substitutions; preferably K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K and E95K substitutions; and a1) N119K or N119R, T123A, and S127K or S127R; preferably N119K, T123A, and S127K; a2) Y31P, N119K or N119R, T123A, and S127K or S127R; preferably Y31P, N119K, T123A, and S127K; 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; 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 a5) Y31P, K49Q, E52S, R81E or R81D, D84N, T131R or T131K and L132S; preferably Y31P, K49Q, E52S, R81E, D84N, T131R and L132S a further substitution selected from the group consisting of: b) Y31P or Y31N, preferably Y31P substitution Including, The positions shown are determined by alignment with SEQ ID NO:1.

[0067] According to the present invention, IL-2 variants having the substitution in a) and a further substitution in a3) or a5) are capable of selectively stimulating regulatory T cells (Treg agonist variants), whereas IL-2 variants having the substitution in a) and a further substitution in a1), a2) or a4), as well as IL-2 variants having the substitution in b) are capable of antagonizing IL-2 (IL-2 antagonist variants).

[0068] In some more preferred embodiments, the human IL-2 variant is - a variant comprising K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, N119K, T123A, and S127K substitutions; this variant is designated IL2-v1 (IL2V1, IL2-V1, IL-2V1, IL-2-V1, or IL-2 V1) in the examples; - a variant comprising K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, Y31P, K49Q, E52S, R81E, D84N, N119K, T123A, T131R, and L132S substitutions; this variant is designated IL2-v2 (IL2V2, IL2-V2, IL-2V2, IL-2-V2, or IL-2 V2) in the examples; - a variant comprising K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, Y31P, K49Q, E52S, R81E, D84N, T131R and L132S substitutions; this variant is designated IL2-v3 (IL2V3, IL2-V3, IL-2V3, IL-2-V3 or IL-2 V3) in the examples; - a variant comprising a Y31P substitution without any substitutions at the other indicated positions (9, 12, 16, 19, 23, 26, 87, 91, 95 and 49, 52, 81, 84, 119, 123, 127, 131 and 132); this variant is designated IL2-v4 (IL2V4, IL2-V4, IL-2V4, IL-2-V4 or IL-2 V4) in the examples; - a variant comprising K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, Y31P, N119K, T123A, and S127K substitutions; this variant is designated IL2-v5 (IL2V5, IL2-V5, IL-2V5, IL-2-V5, or IL-2 V5) in the examples; - a variant comprising the substitutions K9E, L12E, H16R, L19R, M23L, N26K, S87N, V91K, E95K, K49Q, E52S, R81E, D84N, N119K, T123A, T131R and L132S; this variant is designated IL2-v6 (IL2V6, IL2-V6, IL-2V6, IL-2-V6 or IL-2 V6) in the examples; is selected from the group consisting of:

[0069] 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 an additional amino acid mutation. 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 a position selected from S4, S5, or S6, the indicated positions being determined by alignment with SEQ ID NO: 1. The IL-2 variant may include one, two, or all of the above-listed deletions, along with the above-listed substitutions. All of these possible combinations are specifically contemplated.

[0070] 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 the 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 NOs: 3-8.

[0071] The IL-2 variants are at least 125 amino acids in size. Preferably, the IL-2 variants are at least 130 amino acids in size or greater.

[0072] In some preferred embodiments, the IL-2 variant has at least 70% amino acid identity to 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 to said 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 to said sequence, and even more preferably at least 95%, 96%, 97%, 98%, or 99% identity to said sequence.

[0073] The percent amino acid sequence identity is defined as the percentage of amino acid residues in a comparison sequence that are identical to a reference sequence after aligning the sequences and introducing gaps, if necessary, to achieve maximum sequence identity and not considering any conservative substitutions as part of the sequence identity. Sequence identity is calculated over the entire length of the reference sequence. Alignment for determining percent amino acid sequence identity can be achieved in various ways known to those skilled in the art, for example, using publicly available computer software such as BLAST (Altschul et al., J. Mol. Biol., 1990, 215, 403-). When using such software, default parameters, for example, gap penalty and extension penalty, are preferably used. The BLASTP program uses a word length (W) of 3 and an expectation (E) of 10 as default.

[0074] 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.

[0075] In some preferred embodiments, the IL-2 variant is selected from the group consisting of SEQ ID NOs: 3-8 and sequences having at least 70% amino acid identity thereto. 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 thereto. 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 with the sequence, even more preferably at least 95%, 96%, 97%, 98% or 99% identity with the sequence; more preferably, the IL-2 variant has at least 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.

[0076] In some embodiments, the IL-2 variant is capable of selectively stimulating regulatory T cells (Tregs). Non-limiting examples of such variants include the variants IL-2V2 and IL-2V3 defined above. In some preferred embodiments, the IL-2 variant is selected from the group consisting of the sequences of SEQ ID NO: 4 and SEQ ID NO: 5, and sequences having at least 70% amino acid identity therewith, as defined above; more preferably, the IL-2 variant is selected from the group consisting of 11, 13, 15, 18, 20, 22, 29, 30, 35, 37, 48, 68, 69, 71, 74, 75, 76, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 115, 118, 119, 120, 122, 129, 130, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, does not contain any substitutions at positions 5, 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 The IL-2 variants capable of selectively stimulating Tregs (Treg agonists) of the present invention are useful for treating diseases in which immunomodulation or immunosuppression is beneficial, including, but not limited to, allergic and autoimmune diseases, and diseases involving hyperactivity of the immune system, including, but not limited to, chronic or acute inflammatory diseases, graft-versus-host disease (GVHD) and graft rejection.

[0077] In some embodiments, the IL-2 variant is an IL-2 antagonist. Non-limiting examples of such variants include the variants IL-2V1, IL-2V4, IL-2V5, and IL-2V6 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 the sequences of SEQ ID NOs: 3, 6, 7, and 8, and sequences having at least 70% amino acid identity therewith, as defined above, preferably SEQ ID NO: 3 or sequences having at least 70% amino acid identity therewith; more preferably, the IL-2 variant is selected from the group consisting of 11, 13, 15, 18, 20, 22, 29, 30, 35, 37, 48, 6 and 133 or 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 to stimulate beneficial immune responses in vivo. Thus, IL-2 antagonists according to the present invention are useful for treating diseases involving immune system hyperactivity associated with IL-2 overproduction, including, but not limited to, chronic or acute inflammatory diseases, graft-versus-host disease (GVHD), and transplant rejection. IL-2 antagonists are also useful for inhibiting Tregs by depleting Tregs from endogenous wild-type IL-2 signaling, thereby stimulating immune responses (B, NK, CD4+ or CD8+ T cells, DCs, macrophages, etc.), particularly CD8+ T cell immune responses. Thus, IL-2 antagonist variants are also useful for preventing or treating immune disorders in which Treg inhibition and / or stimulation of an immune response, particularly a CD8+ T cell immune response, is beneficial, including, but not limited to, cancer, infectious diseases, and vaccination.

[0078] The present invention encompasses IL-2 variants with one or more modifications in one or more amino acid residues, peptide bonds, the N- and / or C-terminus, so long as the modified variant is functional (i.e., capable of selectively stimulating Tregs or antagonizing IL-2). Preferred modifications are those that increase the stability or bioavailability of the IL-2 variants according to the invention, in particular those that increase their half-life in vivo; reduce their immunogenicity; or facilitate their purification, detection, or targeting to specific cell types or tissues. These modifications, which can be introduced into the variants by conventional methods known to those skilled in the art, include, but are not limited to, mutations (insertion, deletion, substitution) of one or more amino acids in the amino acid sequence; fusion with an amino acid moiety of interest (protein of interest for therapeutic use or tag for purification, detection (antibody epitope or labeling) or coupling to a molecule or drug of interest; substitution of natural amino acids with non-proteinogenic amino acids (D amino acids or amino acid analogs); modification of peptide bonds, in particular by retro or retro-inverso bonds or bonds different from peptide bonds; cyclization and modification of amino acid side chains or N and / or N-terminal amino acids of the variants, in particular for coupling to a molecule or drug of interest to the variant. Modifications include the addition of chemical groups to the N-terminus or C-terminus. Modifications include, but are not limited to, esterification, glycosylation, acylation such as acetylation or linkage of 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 suitable, at amino acids other than the terminal amino acids (e.g., farnesyl coupling to a cysteine ​​side chain). Conversion of amino acid functions on the C-terminus to an aldehyde and alkylation of the thiol function of a cysteine ​​residue can be used for chemoselective ligation or reduced peptide bond formation.

[0079] In particular, the present invention encompasses IL-2 variants comprising or consisting of chains of naturally occurring amino acids (the 20 genetically encoded amino acids (A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, X, and Y) in the L and / or D configuration) linked via peptide bonds, and further includes peptidomimetics of such variants in which amino acids, peptide bonds, N- and / or C-termini are replaced by functional analogs. Such functional analogs of amino acids include all known amino acids other than the 20 genetically encoded amino acids.

[0080] In some embodiments, the IL-2 variant is provided in a form in which it is bound to at least a drug of interest, for example, in the form of a molecular complex or particle; a complex such as a conjugate or fusion protein. Drugs of interest include, but are not limited to, any therapeutic agent, including cells such as patient chimeric antigen receptor (CAR) T cells; any agent that increases the stability or bioavailability of the IL-2 variant of the present invention, particularly its half-life in vivo; any agent that reduces its immunogenicity; or any agent that facilitates its purification, detection, or targeting to specific cell types or tissues. The drug of interest may be a small or large compound, macromolecule, or particle. Particles include, but are not limited to, nanoparticles, including liposomes, micelles, and nanoparticles carrying active agents, such as liposomes, micelles, and nanocarriers. For example, the IL-2 variant, and ultimately, other drugs of interest, can be encapsulated in or grafted onto the particles by means well known in the art. In a preferred embodiment, the agent of interest is selected from the group consisting of peptides; proteins, including antibodies; bioactive substances such as drugs for the treatment of human or animal diseases; labels, tags and particles.

[0081] In some more preferred embodiments, the agent of interest is an anti-IL-2 antibody, preferably an anti-IL-2 antibody with pro-Treg or pro-T effector function or a functional fragment thereof comprising at least the antigen-binding site; more preferably a human or humanized anti-IL-2 antibody. Anti-IL-2 antibodies with pro-Treg function or fragments thereof are useful for treating diseases associated with overactivity of the immune system, including, but not limited to, chronic or acute inflammatory diseases, graft-versus-host disease (GVHD), and graft rejection. Anti-IL-2 antibodies with pro-T effector function or fragments thereof are useful for preventing or treating immune disorders in which Treg inhibition and / or stimulation of CD8+ T cell responses is beneficial, including, but not limited to, cancer, infectious diseases, and vaccination. In some embodiments, the anti-IL-2 antibody, preferably an anti-IL-2 antibody with pro-Treg or pro-T effector function, is a whole antibody molecule.

[0082] In some more preferred embodiments, the agent of interest comprises or consists of a ligand of a surface molecule specific for Tregs. Treg-specific surface molecules are known in the art and are disclosed, for example, in Bhairavabhotla et al., Human Immunol., 2016, 77, pp. 201-13; van der Veeken et al., Cold Spring Harb. Symp. Quant Biol., 2013, 78, pp. 215-22; Pfoertner et al., Genome Biol., 2006, 7, R54; Sugimoto et al., Int. Immunol., 2006, 18, pp. 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.

[0083] The agent of interest is preferably an antibody against said Treg-specific surface molecule or a functional fragment thereof comprising at least the antigen-binding site; preferably an anti-CTLA-4 antibody, anti-CD25 antibody, anti-CCR8 antibody, anti-ICOS antibody, anti-IKZF2 antibody, anti-CD70 antibody, anti-GARP antibody, anti-IL1R1 antibody, anti-CD39 antibody, anti-CCR4 antibody, or anti-CD177 antibody; more preferably a human or humanized antibody. In some embodiments, the antibody against the Treg-specific surface molecule is a whole antibody molecule, preferably a fully human or humanized antibody molecule.

[0084] In some more preferred embodiments, the agent of interest is selected from the group consisting of a whole antibody or antibody Fc region, preferably a fully human antibody or a human Fc region; a multispecific antigen-binding protein such as a bispecific antibody; ankyrin and 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 peptides in the MHC-peptide multimer may be derived from antigens capable of inducing an autoimmune or alloimmune response, such as autoantigens, or foreign antigens, such as antigens derived from pathogens or tumors, in particular vaccine antigens against pathogens or tumors.

[0085] It is within the scope of the present invention that a complex, conjugate or fusion protein may comprise more than one IL-2 variant according to the present invention, i.e., a plurality of such variants, whereby the plurality of variants may comprise a plurality of the same or different variants. Also, a complex, conjugate or fusion protein according to the present invention may comprise more than one agent of interest, whereby the plurality of agents may comprise a plurality of the same or different agents.

[0086] The desired agent is covalently or non-covalently linked to the IL-2 variant of the present invention. The desired agent can be coupled to the IL-2 variant directly or indirectly. Indirect coupling of the desired agent to the IL-2 variant can be via a linker attached to the IL-2 variant of the present invention. Linkers, also called spacers, that can be used to physically separate the IL-2 variant of the present invention from the desired agent are known in the art and include peptide bonds, amino acids, peptides of appropriate length, or different molecules that provide desired characteristics. The linker can be attached to the N-terminus, C-terminus, or, if deemed appropriate, to any amino acid other than the terminal amino acid. The IL-2 variant of the present invention can be chemically linked to the desired agent by covalent bonding using standard conjugation techniques. The desired agent can be linked to the N-terminus, C-terminus, or, if applicable, to any amino acid other than the terminal amino acid of the IL-2 variant. Functional groups, modifications, or linkers, also referred to as derivatizations, can be introduced into the IL-2 variants to conjugate them to a desired drug. Such covalent bonds are preferably formed between a suitable reactive group on the IL-2 variant and the desired drug, more preferably between the terminus of the IL-2 variant according to the present invention and the desired drug. Depending on the chemical nature of the desired drug, the moiety, group, or radical at which such a covalent bond is formed will vary, and creating such a bond is within the skill of those skilled in the art. The chemical bond may be via a disulfide bond, a thioether, a thiol-maleimide, or an amide bond. Other methods for linking the IL-2 variant to a cargo include using a C-terminal aldehyde to form an oxime, using the Click reaction, or forming a morpholino bond with a basic amino acid on a peptide.

[0087] When the agent of interest is a peptide or protein, including an antibody or functional fragment thereof, the IL-2 variant is advantageously provided as a chimeric fusion protein comprising a heterologous agent of interest (other than IL-2 or an IL-2 fragment) fused directly or via a peptide spacer as described above to the N- or C-terminus of the IL-2 variant of the invention, or inserted into its amino acid sequence. The fusion protein is expressed from a chimeric construct in which a nucleotide sequence encoding the IL-2 variant of the invention is fused in frame to a nucleotide sequence encoding the peptide / protein of interest using standard recombinant DNA techniques. The resulting fusion protein / peptide is of heterologous origin, i.e., it is different from a naturally occurring peptide or protein, such as IL-2 or other cytokines of the same family.

[0088] The IL-2 variant can also be linked to the agent of interest (molecule or particle carrying the molecule) by non-covalent bonds, such as ionic bonds, hydrogen bonds, or hydrophobic interactions, or a combination of such bonds. Non-limiting examples include antigen-antibody interactions between an IL-2 variant and an anti-IL-2 antibody; streptavidin-biotin interactions between a biotinylated IL-2 variant and an agent of interest (e.g., a nanoparticle such as a quantum dot) conjugated to streptavidin, or between a biotinylated agent and an IL-2 variant conjugated to streptavidin.

[0089] In a preferred embodiment, the IL-2 variant is provided as a chimeric fusion protein comprising a heterologous protein or peptide of interest fused directly or via a peptide spacer to the N- or C-terminus of the IL-2 variant of the invention or inserted into its amino acid sequence. The protein or peptide of interest is preferably selected from the group consisting of whole antibodies or antibody Fc regions, preferably fully human antibodies or human Fc regions; multispecific antigen-binding proteins such as bispecific antibodies; ankyrin and designed ankyrin repeat proteins (DARPins); MHC-peptide multimers such as class I or class II MHC-peptide tetramers, particularly MHC-peptide multimers in which 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 surface molecule specific for Tregs or a functional fragment thereof comprising at least the antigen-binding site, preferably an anti-CTLA-4 antibody, an anti-CD25 antibody, an anti-CCR8 antibody, an anti-ICOS antibody, an anti-IKZF2 antibody, an anti-CD70 antibody, an anti-GARP antibody, an anti-IL1R1 antibody, an anti-CD39 antibody, an anti-CCR4 antibody or an anti-CD177 antibody; more preferably, a human or humanized antibody or a whole antibody molecule, even more preferably a whole human or humanized antibody molecule.

[0090] The protein or peptide of interest is advantageously fused to the N-terminus or C-terminus of the IL-2 variant of the invention, either directly or via a peptide spacer.

[0091] In preferred embodiments, the IL-2 variants or derived fusion proteins disclosed above are conjugated to an anti-IL-2 antibody. In some more preferred embodiments, the anti-IL-2 antibody is an IL-2 antibody that has pro-Treg function, i.e., blocks the IL-2RB / IL-2 interaction and induces structural alterations on IL-2. Such antibodies are well known in the art and include, for example, clone JES6-1A12 (a rat IgG2A anti-mouse IL-2 antibody) and clone 5344.111 (a mouse IgG1 anti-human IL-2 antibody). These Abs have demonstrated the ability to expand Tregs in vivo in preclinical models of diabetes (Tang Q et al., Immunity, 2008, 28, pp. 687-97; Grinberg-Bleyer Y et al., J Exp Med, 2010, 207, pp. 1871-8), allergy (Smaldini PL et al., Allergy, 2018, 73, pp. 885-895), multiple sclerosis (Webster KE et al., J Exp Med, 2009, 206, pp. 751-60), rheumatoid arthritis (Lee-S Y et al., Immunology, 2012, 137, pp. 305-16), and transplantation (Vokaer B et al., Transplant Proc., 2012, 44, pp. 2840-4). In some other more preferred embodiments, the anti-IL-2 antibody is an IL-2 antibody with pro-Teff function. Such antibodies are well known in the art and include, for example, clone S4B6 (a rat IgG2A anti-mouse IL-2 antibody) and clone Mab602 (a mouse IgG2a anti-human IL-2 antibody). These Abs have shown better in vivo tumor growth control than IL-2 in melanoma (as single treatment or in combination) (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., Blood 2014, 123, 3045-55). The anti-IL-2 antibody is preferably a human or humanized anti-IL-2 antibody, including a humanized antibody derived from the above-mentioned mouse or rat monoclonal antibodies.

[0092] In a preferred embodiment, the above disclosed IL-2 variants or derived fusion proteins are conjugated to antibodies, preferably whole antibodies, more preferably whole human antibodies, directed against Treg-specific surface molecules as defined above.

[0093] In a preferred embodiment, the IL-2 variant is linked to a CAR T cell, in particular a patient's CAR T cell.

[0094] The term "IL-2 variant" as used herein encompasses the different forms of IL-2 variant disclosed herein, such as IL-2 variants modified or unmodified, bound or unbound with at least an agent of interest in the form of a complex, conjugate or fusion protein as disclosed above.

[0095] IL-2 variants according to the invention can be produced by routine techniques in the art, in particular by expression of recombinant DNA in suitable cell lines (eukaryotic or prokaryotic), and screened for activity (i.e., ability to selectively stimulate regulatory T cells) using the assays described herein or other similar assays.

[0096] Polynucleotides, Vectors, and Host Cells The present invention also relates to isolated polynucleotides encoding IL-2 variants in expressible form.

[0097] A polynucleotide encoding an expressible form of an IL-2 variant refers to a nucleic acid molecule that, upon expression in a cell or cell-free system, results in a functional protein.

[0098] The synthetic or recombinant polynucleotide may 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, at least one translational regulatory sequence. Preferably, the polynucleotide comprises a coding sequence optimized for the host in which the IL-2 variant is to be expressed; more preferably, the polynucleotide sequence is selected from the group consisting of SEQ ID NOs: 9 to 14, which encode the IL-2 variants of SEQ ID NOs: 3 to 8, respectively.

[0099] Polynucleotides according to the invention are prepared by conventional methods known in the art, for example, by amplification of nucleic acid sequences by PCR or RT-PCR, by screening of genomic DNA libraries by hybridization with homologous probes, or otherwise produced by total or partial chemical synthesis.

[0100] Another aspect of the present invention is a recombinant vector comprising said polynucleotide. The recombinant vector is preferably an expression vector capable of expressing said polynucleotide when delivered into a host cell, such as a prokaryotic or eukaryotic cell, e.g., a mammalian or bacterial cell. Recombinant vectors include conventional vectors used in genetic engineering and gene therapy, including, for example, plasmids and viral vectors.

[0101] Recombinant vectors are constructed and introduced into host cells by conventional recombinant DNA, genetic engineering, and gene therapy techniques that are well known in the art.

[0102] Thus, a further aspect of the present invention provides a host cell comprising the polynucleotide or recombinant vector described above.

[0103] In some embodiments, the host cells are patient cells, such as T cells, NK cells or CAR T cells, that have been modified with a polynucleotide or vector according to the invention, e.g., for use in adoptive T cell therapy.

[0104] In some embodiments, a host cell of the invention is a host cell deposited at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, FR, on November 15, 2018 under deposit number CNCM I-5377 (IL2-V1).

[0105] In some embodiments, a host cell of the invention is a host cell deposited at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, FR, on November 15, 2018 under deposit number CNCM I-5378 (IL2-V2).

[0106] In some embodiments, a host cell of the invention is a host cell deposited at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, FR, on November 15, 2018 under deposit number CNCM I-5379 (IL2-V3).

[0107] In some embodiments, a host cell of the invention is a host cell deposited at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, FR, on November 15, 2018 under deposit number CNCM I-5380 (IL2-V4).

[0108] In some embodiments, a host cell of the invention is a host cell deposited at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, FR, on November 15, 2018 under deposit number CNCM I-5381 (IL2-V5).

[0109] In some embodiments, a host cell of the invention is a host cell deposited at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, FR, on November 15, 2018 under deposit number CNCM I-5382 (IL2-V6).

[0110] The polynucleotides, vectors and cells of the invention are useful for producing the variants of the invention using well-known recombinant DNA techniques.

[0111] Pharmaceutical Compositions and Therapeutic Uses The IL-2 variants, polynucleotides, vectors and / or cells according to the invention are used to treat immune disorders.

[0112] As used herein, the term "treatment" or "treating" refers to both prophylactic or preventative treatment and curative or disease-modifying treatment, including treatment of patients at risk of or suspected of having a disease, as well as patients who are ill or have been diagnosed with a disease or medical condition, including the suppression of clinical recurrence. Treatment can be administered to a patient who has a medical disorder or who may ultimately acquire a disorder in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurrent disorder, or to prolong the patient's survival beyond that expected in the absence of such treatment.

[0113] In some embodiments, IL-2 variants, polynucleotides, vectors, and / or cells according to the invention are used to expand Tregs ex vivo or in vivo or to block IL-2-mediated overactivation of the immune system to treat diseases associated with immune dysregulation where immunomodulation or immunosuppression is beneficial, including diseases associated with immune system dysfunction or overactivity. In particular, IL-2 variants capable of selectively stimulating regulatory T cells (Treg agonists) are used to treat immune disorders associated with immune system dysfunction, such as allergic and autoimmune diseases, and immune disorders associated with immune system overactivity, such as acute or chronic inflammatory diseases, GVHD, and graft rejection. IL-2 antagonist variants are used to treat diseases associated with immune system overactivity, including overproduction of IL-2, such as GVHD, particularly acute GVHD, and other diseases such as acute or chronic inflammatory diseases and graft rejection.

[0114] For ex vivo therapy, a peripheral blood sample is collected from the patient; T cells are expanded in vitro using an IL-2 variant, polynucleotide and / or vector according to the invention, and the expanded Tregs are then re-infused into the patient. For in vivo therapy, an IL-2 variant, polynucleotide and / or vector according to the invention is administered to the patient and Tregs are expanded in vivo in the patient. Alternatively, or in addition, IL-2 already present in the patient is antagonized by the IL-2 variant.

[0115] In some embodiments, the IL-2 antagonist variants, polynucleotides, vectors and / or cells according to the invention are used to inhibit Tregs by depleting them from IL-2, thus allowing stimulation of an immune response including B, NK, CD4+ or CD8+ T cells, DCs, macrophages, etc., to treat cancer or infectious diseases or to increase the immune response to a vaccine, particularly a vaccine for cancer or infectious diseases. In some preferred embodiments, the IL-2 antagonist variants, polynucleotides, vectors and / or cells according to the invention are used to stimulate an anti-tumor CD8+ T cell response or a CD8+ T cell response to a pathogen or a vaccine, including a vaccine against cancer or infectious diseases.

[0116] For in vivo therapy, IL-2 antagonist variants, polynucleotides, vectors and / or cells according to the invention are administered to a patient and Tregs are inhibited or eliminated in vivo in the patient, relieving immune cells (B, NK, CD4+ or CD8+ T cells; DCs; macrophages, etc.) from Treg suppression.

[0117] The present invention relates to pharmaceutical compositions comprising, as active substances, an IL-2 variant, a polynucleotide, a vector and / or a cell according to the invention and at least one pharmaceutically acceptable vehicle and / or carrier.

[0118] Pharmaceutical compositions are formulated for administration by several routes, including, but not limited to, oral, parenteral, and topical. Pharmaceutical vehicles are appropriate for the intended route of administration, as is well known in the art.

[0119] The pharmaceutical composition may further comprise a carrier. Non-limiting examples of carriers suitable for use in the compositions of the present invention include unilamellar or multilamellar liposomes, ISCOMS, virosomes, viral pseudoparticles, saponin micelles, saccharide (poly(lactide-co-glycolide)) or gold microspheres, and nanoparticles.

[0120] The pharmaceutical composition comprises a therapeutically effective amount of an IL-2 variant, polynucleotide, vector, and / or cell sufficient to elicit a positive medical response in the individual to whom it is administered. A positive medical response refers to a reduction in subsequent (preventive treatment) or established (therapeutic treatment) disease symptoms. A positive medical response includes partial or total 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 increased survival. The medical response to the compositions according to the present invention can be readily verified in appropriate animal models of disease, which are well known in the art and exemplified in the Examples of this application.

[0121] The pharmaceutically effective dose will depend 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, any concomitant medications, and other factors recognized by those skilled in the medical arts.

[0122] "Therapeutic regimen" refers to a pattern of treatment for a disease, e.g., a pattern of medication used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or a portion of a therapeutic regimen) used for the initial treatment of a disease. The general goal of an induction regimen is to provide high levels of drug to the patient during the initial period of the treatment regimen. An induction regimen may also use a "loading regimen," which may involve (in part or in whole) the physician administering a higher dose of drug than used during a maintenance regimen, the physician administering the drug more frequently than the physician administers the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or a portion of a therapeutic regimen) used to maintain a patient during treatment of a disease, for example, to keep the patient in remission for an extended period of time (months or years). Maintenance regimens may use continuous therapy (e.g., administering a drug at regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., discontinued treatment, intermittent treatment, treatment upon relapse, or treatment upon achievement of certain predetermined criteria (e.g., pain, disease symptoms, etc.)).

[0123] In some embodiments, the pharmaceutical composition contains another active agent, which is a pharmaceutical or therapeutic agent capable of preventing, treating, or ameliorating a disease in a human or animal. The active agent may 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 a DNA, RNA, or peptide aptamer; a small molecule or polymeric chemical; or a mixture thereof. In particular, the active agent may be an anti-inflammatory or immunomodulatory agent, such as rapamycin or a corticoid. The active agent may also be an anti-cancer or anti-infective agent, or an antigen, such as a tumor antigen or a pathogen antigen.

[0124] The present invention also provides an IL-2 variant, polynucleotide, vector, cell or pharmaceutical composition according to the invention for use as a medicament.

[0125] The present invention also provides an IL-2 variant, polynucleotide, vector, cell or pharmaceutical composition according to the invention for use in the prevention or treatment of an immune disorder.

[0126] 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 overactivity of the immune system, such as, but not limited to, chronic or acute inflammatory diseases, graft-versus-host disease (GVHD) or graft 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 diseases such as Crohn's disease and ulcerative colitis, autoimmune thyroid diseases such as Addison's disease, Graves' disease, Sjogren's disease, alopecia areata, Hashimoto's thyroiditis, myasthenia gravis, vasculitis including HVC-associated vasculitis and systemic vasculitis, uveitis, myositis, pernicious anemia, celiac disease, Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, scleroderma, hemolytic anemia, glomerulonephritis, autoimmune encephalitis, fibromyalgia, aplastic anemia, and others. 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 nephropathy, etc. The disease may also be allograft rejection, including transplant rejection, graft-versus-host disease (GVHD), and spontaneous abortion.

[0127] In some embodiments, the disease associated with immune dysregulation or dysfunction is one in which inhibition 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 as well as increasing the immune response to vaccines, particularly vaccines for cancer or infectious diseases.

[0128] As used herein, the term "cancer" refers to any of a number of classes of diseases or disorders characterized by the ability of these cells to invade other tissues, either by direct growth in neighboring tissues through uncontrolled cell division and invasion or by transplantation to distant sites through metastasis. Metastasis is defined as the 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. Cancers are classified by the type of cell to which tumors resemble and therefore the tissue presumed to be the tumor's origin. For example, carcinomas are malignant tumors derived from epithelial cells. This group is the most common cancer, including common forms of breast, prostate, lung, and colon cancer. Lymphomas and leukemias include malignant tumors derived from blood and bone marrow cells. Sarcomas are malignant tumors derived from connective tissue or mesenchymal cells. Mesotheliomas are tumors derived from mesothelial cells, which reinforce the peritoneum and pleura. Gliomas are tumors derived from glia, the most common type of brain cell. Germinomas are tumors that originate from germ cells, usually found in the testes and ovaries. Choriocarcinomas are malignant tumors that originate from the placenta.

[0129] As used herein, the term "cancer" refers to cancer of the following tissues or organs: breast; liver; kidney; heart, mediastinum, pleura; floor of the mouth; lips; salivary glands; tongue; gums; oral cavity; palate; tonsils; larynx; trachea; bronchi, lungs; pharynx, hypopharynx, oropharynx, nasopharynx; esophagus; digestive tract such as stomach, intrahepatic bile duct, bile duct, pancreas, small intestine, colon; rectum; urinary tract such as bladder, gallbladder, ureter; rectosigmoid junction; anus, anal canal; skin; bone; joints, limbs, etc. refers to any cancer that can affect any one of the following: articular cartilage; eyes and adnexa; brain; peripheral nerves, autonomic nervous system; spinal cord, cranial nerves, meninges; and various parts of the central nervous system; connective, subcutaneous, and other soft tissues; retroperitoneum, peritoneum; adrenal glands; thyroid gland; endocrine glands and associated structures; female genitalia, such as ovaries, uterus, and cervix; uterine corpus, vagina, and vulva; male genitalia, such as penis, testes, and prostate; hematopoietic and retinal endothelial systems; blood; lymph nodes; or thymus.

[0130] The term "cancer" according to the present invention includes leukemia, seminoma, melanoma, teratoma, lymphoma, non-Hodgkin's lymphoma, neuroblastoma, glioma, adenocarcinoma, mesothelioma (pleural mesothelioma, peritoneal mesothelioma, pericardial mesothelioma and late stage mesothelioma), rectal cancer, endometrial cancer, thyroid cancer (including papillary thyroid carcinoma, follicular thyroid carcinoma, medullary thyroid carcinoma, anaplastic thyroid carcinoma, multiple endocrine adenoma type 2A, multiple endocrine adenoma type 2B, familial medullary thyroid carcinoma, pheochromocytoma and paraganglioma), skin cancer (malignant melanoma, basal cell carcinoma, squamous cell carcinoma, Kaposi's sarcoma, keratoacanthoma, leukemia ... cancer of the nervous system, brain tumors (astrocytoma, medulloblastoma, glioma, low-grade glioma, ependymoma, germ cell tumor (pinealoma), glioblastoma multiforme, oligodendroglioma, schwannoma, retinoblastoma, congenital tumor, spinal neurofibroma, glioma or sarcoma), cranial cancer (including osteoma, hemangioma, granuloma, xanthomas or osteitis deformans), meningeal cancer (including meningioma, meningeal sarcoma or gliomatosis), head and neck cancer (head and neck squamous cell carcinoma) and oral cancer (e.g. buccal cancer of the oral cavity, lip, tongue, mouth or pharynx, etc.), lymph node cancer, gastrointestinal cancer, liver cancer (including hepatoma, hepatocellular carcinoma, cholangiocarcinoma, hepatoblastoma, angiosarcoma, hepatocellular adenoma and hemangioma), colon cancer, stomach (or gastric) cancer, esophageal cancer (including squamous cell carcinoma, laryngeal, adenocarcinoma, leiomyosarcoma or lymphoma), colorectal cancer, intestinal cancer, small bowel (or small intestine) cancer (e.g., adenocarcinoma lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma or fibroma, etc.), large bowel (or large intestine) cancer (e.g., adenocarcinoma lymphoma, carcinoid tumor, Kaposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma or fibroma, etc.), large intestine (e.g., large bowel or large intestine) cancer intestine) cancer (e.g., adenocarcinoma, tubular adenoma, villous adenoma, hamartoma, or leiomyoma), pancreatic cancer (including pancreatic ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoid tumor, or vipoma), ear, nose, and throat (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, endometrial stromal sarcoma, and endometrial cancer such as malignant mixed Müllerian tumor, uterine sarcoma, leiomyosarcoma, and gestational trophoblastic disease), ovarian cancer (dysgerminoma,granulosa theca cell tumor and Sertoli-Leydig cell tumor), cervical cancer, vaginal cancer (including vaginal squamous cell carcinoma, vaginal adenocarcinoma, vaginal clear cell adenocarcinoma, vaginal germ cell tumor, vaginal botryoid sarcoma and vaginal melanoma), vulvar cancer (including vulvar squamous cell carcinoma, vulvar verrucous carcinoma, vulvar melanoma, vulvar basal cell carcinoma, Bartholin's gland carcinoma, vulvar adenocarcinoma and Kehler's erythroplasia), genitourinary tract cancer, kidney cancer (including renal clear cell carcinoma, chromophobe renal carcinoma, papillary renal cell carcinoma, adenocarcinoma, Wilms' tumor, nephroblastoma, lymphoma or leukemia), adrenal gland cancer, bladder cancer, urethral cancer (e.g., tonsillar cancer, squamous cell carcinoma, transitional cell carcinoma or adenocarcinoma, etc.), prostate cancer (e.g., adenocarcinoma or sarcoma, etc.) and testicular cancer (e.g., seminoma, teratoma, embryonal carcinoma, teratocarcinoma, choriocarcinoma, sarcoma, stromal cell carcinoma, fibroma, fibroadenoma, adenooid tumor or lipoma), lung cancer (including small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC) including squamous cell carcinoma of the lung, adenocarcinoma of the lung (LUAD), and large cell lung carcinoma, bronchogenic carcinoma, alveolar carcinoma, bronchogenic adenoma, pulmonary sarcoma, chondroitinoma and pleural mesothelioma), sarcomas (Askin tumor, botryoid sarcoma, chondrosarcoma, Ewing sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma and soft tissue sarcomas), soft tissue sarcomas (alveolar soft part sarcoma, angiosarcoma, cystosarcoma phyllodes, dermatofibrosarcoma protuberans, desmoid tumor, desmoplastic small round cell tumor, epithelioid sarcoma, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, gastrointestinal stromal tumor (GIST), hemangiopericytoma, angiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant peripheral nerve sheath tumor (MPNST), neurofibrosarcoma, reticular fibrohistiocytic tumor, rhabdomyosarcoma, synovial sarcoma and undifferentiated pleomorphic sarcoma), cardiac cancer (e.g., sarcomas such as angiosarcoma, fibrosarcoma, rhabdomyosarcoma or liposarcoma, myxoma, sarcoma ... tumor, fibroma, lipoma and teratoma), bone cancer (including osteogenic sarcoma, osteosarcoma, fibrosarcoma, malignant fibrous histiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma and retinal cell sarcoma, multiple myeloma, malignant giant cell tumor, chordoma, osteochondroma, osteochondroma, benign chondroma, 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,Includes non-Hodgkin's lymphoma and hairy cell and lymphoid disorders, and their metastases.

[0131] Infectious diseases include, for example, viral, bacterial, fungal and parasitic diseases such as HIV / AIDS, viral hepatitis such as hepatitis A, hepatitis B and hepatitis C, measles, malaria, and Chagas disease.

[0132] The present invention also provides a method for treating a disease associated with immune dysfunction or dysregulation, comprising administering to a patient a therapeutically effective amount of a pharmaceutical composition according to the present invention.

[0133] The pharmaceutical compositions of the present invention are generally administered in accordance with known procedures at dosages and for periods effective to induce a beneficial effect in an individual. Administration may be by injection or oral, sublingual, intranasal, rectal or vaginal administration, inhalation, or transdermal application. Injection may be subcutaneous, intramuscular, intravenous, intraperitoneal, intradermal, or other.

[0134] The pharmaceutical compositions of the present invention are advantageously used in combination with another therapy, particularly immunotherapy such as CAR-T cell therapy; therapy with immunomodulatory agents, particularly immunomodulatory monoclonal antibodies or functional derivatives thereof; or therapy with anti-cancer or anti-infective agents, including therapeutic agents and vaccines against cancer and infectious diseases. The combination therapy may be separate, simultaneous, and / or sequential.

[0135] In some embodiments, the pharmaceutical compositions are used for the prevention or treatment of humans.

[0136] In some embodiments, the pharmaceutical compositions are used for the treatment of animals.

[0137] In some embodiments, the IL-2 variants, polynucleotides, vectors, cells and / or pharmaceutical compositions according to the invention are administered in combination with a further cancer therapy. In particular, the IL-2 variants, polynucleotides, vectors, cells and / or pharmaceutical compositions of the invention may be administered in combination with targeted therapies, immunotherapies such as immune checkpoint therapies and immune checkpoint inhibitors, costimulatory antibodies, chemotherapy and / or radiation therapy.

[0138] Immune checkpoint therapies such as checkpoint inhibitors include, but are not limited to, programmed cell death-1 (PD-1) inhibitors, programmed cell death ligand-1 (PD-L1) inhibitors, programmed cell 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 suppressor 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 anti-PD1 antibodies, anti-PD-L1 antibodies, anti-CTLA-4 antibodies, anti-TIM-3 antibodies, and anti-LAG3 antibodies.Co-stimulatory antibodies deliver positive signals through immunoregulatory receptors, including but not limited to, ICOS, CD137, CD27, OX-40, and GITR.

[0139] Examples of anti-PD1 antibodies include, but are not limited to, nivolumab, cemiplimab (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, GTPL7335 or Opdivo), pembrolizumab (MK-3475, MK03475, lambrolizumab, SCH-900475 or Keytruda), and antibodies described in International Patent Applications WO2004004771, WO2004056875, WO2006121168, WO2008156712, WO2009014708, WO2009114335, WO2013043569, and WO2014047350.

[0140] Examples of anti-PL-L1 antibodies include, but are not limited to, LY3300054, atezolizumab, durvalumab, and avelumab.

[0141] Examples of anti-CTLA-4 antibodies include, but are not limited to, ipilimumab (see, e.g., U.S. Patent Nos. 6,984,720 and 8,017,114), tremelimumab (see, e.g., U.S. Patent Nos. 7,109,003 and 8,143,379), single-chain anti-CTLA4 antibodies (see, e.g., International Patent Applications WO1997020574 and WO2007123737), and antibodies described in U.S. Patent No. 8,491,895.

[0142] Examples of anti-VISTA antibodies are described in U.S. Patent Application No. 20130177557.

[0143] Examples of inhibitors of the LAG3 receptor are described in US Pat. No. 5,773,578.

[0144] An example of a KIR inhibitor is IPH4102, which targets KIR3DL2.

[0145] 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 to radioisotopes or toxins, allowing these cytotoxic agents to be specifically delivered to the intended cancer cell target. Small molecules can penetrate the cell membrane and interact with intracellular targets. Small molecules are usually designed to interfere with the enzymatic activity of target proteins, such as proteasome inhibitors, tyrosine kinase or cyclin-dependent kinase inhibitors, and histone deacetylase inhibitors. Targeted therapy may 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, PDGFR beta, VEGFR1 / 2 / 3), belimumab (BAFF), belinstat (HDAC), bevacizumab (VEGF), ligand), blinatumomab (CD19 / CD3), bortezomib (proteasome), brentuximab vedotin (CD30), bosutinib (ABL), brigatinib (ALK), cabozantinib (FLT3, KIT, MET, RET, VEGFR2), canakinumab (IL-1 beta), carfilzomib (proteasome), ceritinib (ALK), cetuximab (EGFR), cofimetinib (MEK), crizotinib (ALK, MET, ROS1), dabrafenib (BRAF), daratumumab (CD38), dasatinib (ABL), denosumab (RANKL), dinutuximab (B4GALNT1(GD2)), elotuzumab (SLAMF7), enasidenib (IDH2), el These include rotinib (EGFR), everolimus (mTOR), gefitinib (EGFR), ibritumomab tiuxetan (CD20), sonidegib (Smoothened), sipuleucel-T, siltuximab (IL-6), sorafenib (VEGFR, PDGFR, KIT, RAF), tocilizumab (IL-6R), temsirolimus (mTOR), tofacitinib (JAK3), trametinib (MEK), tositumomab (CD20), trastuzumab (HER2), vandetanib (EGFR), vemurafenib (BRAF), venetoclax (BCL2), vismodegib (PTCH, Smoothened), vorinostat (HDAC), and Ziv-aflibercept (PIGF, VEGFA / B).

[0146] 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 consists in administering a chemotherapeutic agent to a patient. Chemotherapeutic agents include, but are not limited to, alkylating agents such as thiotepa and cyclophosphamide; alkylsulfonic acids such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, metuledopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; acetogenins (particularly bullatacin and bullatacinone); camptothecins (including the synthetic analog topotecan); bryostatin; kallistatin; CC-1065 (including its synthetic analogs adozelesin, carzelesin, and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatins; duocarmycins (including synthetic analogs KW-2189 and CB1-TM1). eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembitine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gamma and calicheamicin omegal); dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamicin;and neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfilomycin, puromycin, querama antimetabolites such as isin, lodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, and testolactone; anti-adrenal cortical hormone synthesis inhibitors such as aminoglutethimide, mitotane, and trilostane; folinic acid Folic acid supplements such as aceglatone, aldophosphamide glycosides, aminolevulinic acid, eniluracil, amsacrine, bestravcil, bisantrene, edatrexate, defofamine, demecolcine, diaziquone, elfornithine, elliptinium acetate, epothilone, etoglucide, gallium nitrate, hydroxyurea, lentinan, lonidynin, maytansinoids such as maytansine and ansamitocin, mitoguazone, mitoxantrone, mopidammol, nitraelin, pentostatin, fenametPirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; methylhydrazine derivatives including N-methylhydrazine (MIH) and procarbazine; PSK polysaccharide complex; razoxane; rhizoxin; schizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anaguidin); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobromo Man; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, 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; novantrone; teniposide; edatrexate; daunomycin; aminopterin; Xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS2000; difluoromethylomitin (DMFO); retinoids such as retinoic acid; capecitabine; anthracyclines, nitrosoureas, antimetabolites, epipodophyllotoxins, enzymes such as L-asparaginase; anthracenediones; corticosteroid antagonists such as prednisone and equivalents, hormones and antagonists including dexamethasone and aminoglutethimide; hydroxyprogesterone caproate progestins such as medroxyprogesterone acetate and megestrol acetate; estrogenic agents such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogenic agents such as tamoxifen; androgenic agents such as testosterone propionate and fluoxymesterone equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; and nonsteroidal antiandrogens such as flutamide; and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0147] 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 radiation therapy. Suitable examples of radiation therapy include, but are not limited to, external radiation therapy (superficial 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 radiation therapy (IMRT), three-dimensional conformal radiation therapy (3D-CRT), etc.); brachytherapy; unsealed source radiation therapy; tomotherapy, etc. Gamma rays are another form of photons used in radiation therapy. Gamma rays are spontaneously produced as radiation emitted by certain elements (such as radium, uranium, and cobalt-60) when they decompose or decay. In some embodiments, the radiation therapy may be proton radiation therapy or proton minibeam radiation therapy.Proton radiotherapy is an ultra-precision form of radiotherapy 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 RG2 Glioma-Bearing Rats: A Comparison Between Proton Minibeam Therapy and Standard "Proton Therapy", Int J Radiat Oncol Biol Phys. 2019 Jun 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). The radiation therapy may also be FLASH radiotherapy (FLASH-RT) or FLASH proton irradiation.FLASH radiotherapy involves the ultrafast delivery of radiation treatments at dose rates several orders of magnitude higher (ultra-high dose rates) than those currently in 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 setup 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 2018 Jul 11).

[0148] Use for antibody screening The IL-2 variants of the present invention can be used to screen for anti-IL-2 antibodies with pro-Teff or pro-Treg activity using standard immunoassays well known in the art.

[0149] In this regard, the present invention relates to a method for screening for an anti-IL-2 antibody with pro-Teff or pro-Treg activity, comprising at least the steps of 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 more than 70% (preferably 80%, 90% or more) of a reference value, the anti-IL-2 antibody is likely to have pro-Teff activity, and if the level of bound antibody is more than 50% (preferably 40%, 30%, 20%, 10% or less) below the reference value, the anti-IL-2 antibody is likely to have pro-Treg activity.

[0150] Antibodies with pro-Teff activity are in particular antibodies with CD25 mimotope binding.

[0151] The reference value is 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 immobilized on a solid surface (plate, beads). Step b) is performed using a labeled primary or secondary antibody. Depending on the label used, the assay may be an ELISA, a fluorescent immunoassay (FIA) or a chemiluminescent immunoassay (CLIA). The assay may be, for example, a high-throughput assay using a microfluidic device.

[0152] The present invention also relates to a kit for carrying out the screening method of the present invention, which comprises an IL-2 variant according to the present invention.

[0153] In various embodiments, the kit may further include instructions for use and / or a labeled antibody.

[0154] The practice of the present invention will employ, unless otherwise indicated, conventional techniques that are within the skill of the art, such techniques being explained more fully in the literature.

[0155] The present invention will now be described in conjunction with the following non-limiting examples and with reference to the accompanying drawings. [Example]

[0156] Example 1 Computational design of resurfaced variants of IL-2 IL-2 and its α (also known as CD25), β (CD122), and γ cThe 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 α receptor but modify the surface outside this region (Figure 2). The general pipeline for resurfacing design was as follows: First, residues that contact the α receptor were defined as residues in IL-2 with at least one atom within 8 Å or less of any atom of the α receptor. After discarding amino acids that contact CD25, a set of candidate positions for mutation was identified using accessibility criteria, selecting only residues with more than 50% of their side chain surface area exposed (Fraczkiewicz et al., J. Comp. Chem. 1998, 19, 319-333).

[0157] Using this procedure, 28 candidate positions were identified on the IL-2 surface. Next, ortholog sequence analysis was combined with solubility criteria to determine the tolerance of a set of mutations at each position. To incorporate evolutionary information, a multiple sequence alignment of IL-2 sequences from 26 different species was performed (Figure 1), allowing only mutations to residues present in either ortholog at a given position. This original set of mutations was further filtered by considering solubility, discarding the presence of hydrophobic residues where possible and allowing all polar and natural residues in IL-2. Finally, Rosetta's "fixed backbone design application" was used to select low-energy sequences and maintain natural rotamers in all unexposed residues (Kuhlman et al., Science, 2003, 302, 1364-8). The lowest-energy design for a particular combination of resurfaced positions was selected for experimental testing. Using this procedure, different variants, designated IL-2-V1 through IL-2-V6, were designed (Table I). A C-terminal Strep tag was added to the construct to facilitate purification. The wild-type human IL-2 construct with the C-terminal Strep tag and the derived IL-2 variants (IL-2-V1 to IL-2-V6) has a sequence of 171 amino acids (SEQ ID NOS: 2 to 8; Figure 3).

[0158] [Table 1]

[0159] Example 2 Protein expression and purification 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, a mammalian expression vector suitable for high-yield protein production (Aricescu et al., Acta Crystallogr D Biol Crystallogr., 2006, 62, 1243-50). A top 10 Escherichia coli (E. coli) strain transformed with a different pHL-sec recombinant plasmid encoding each IL-2 variant was deposited on November 15, 2018, at the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris, FR, under accession numbers CNCM I-5377 to CNCM I-5382 (IL-2-V1 to IL-2-V6). Transformed bacteria deposited at the CNCM under accession numbers CNCM I-5377 to CNCM I-5382 (IL-2-V1 to IL-2-V6) were grown under standard conditions, such as in Luria-Bertani (LB) medium supplemented with 100 μg / mL ampicillin at 37 °C and agitation at >160 rpm. The viability of transformed bacteria can be verified by standard assays, such as plating bacterial cultures on LB agar plates supplemented with 100 μg / mL ampicillin and detecting the presence of bacterial colonies. Transformed bacteria can be stored under standard conditions, such as by freezing a mixture of 0.5 mL of bacterial culture and 0.5 mL of 50% (V / V) glycerol in a 2 mL Eppendorf tube at -80°C. Proteins were produced by transient transfection of HEK293 FreeStyle cells (Thermo Scientific) maintained in serum-free medium (FreeStyle 293 Expression Medium). Cell supernatants were harvested 3 days after transfection, and Tris pH 8.0 was added to a final concentration of 100 mM.The protein was first purified by streptactin affinity chromatography using a StrepTrap column (GE Healthcare, Piscataway, NJ), and the eluted fractions were further purified by gel filtration using a Superdex75 column (GE Healthcare, Piscataway, NJ), resulting in a profile with two peaks, one corresponding to the monomeric protein and the other to an oligomer, presumably a dimer (Figure 4). Fractions containing the monomeric peak were concentrated and used for experimental testing.

[0160] Example 3 IL-2V is a novel tool for specifically selecting anti-IL-2 Abs with pro-Teff or pro-Treg activity ELISA assays were used to evaluate the selectivity of IL-2 variants for antibodies (Abs) with pro-T effector or pro-Treg function. To do so, a typical ELISA was performed by coating plates with Proleukin, IL-2V1, IL-2V2, IL-2V3, IL-2V4, IL-2V5, or IL-2V6 (all at 6 μg / mL). The Abs evaluated were MAB605 (a mouse anti-human IL-2 Ab with in vivo pro-T effector activity), 5344 (a mouse anti-human IL-2 Ab with in vivo pro-Treg activity), and NARA (an scFV antibody with in vivo pro-T effector activity derived from Arenas-Ramirez et al., Sci. Transl. Med., 2016, 8, 367ral66). Anti-IL-2 Abs were detected using anti-mouse IgG-HRP Abs, or anti-M13-HRP Abs for NARA. Absorbance was read at 450 nm. OD values ​​are shown from which the absorbance of control wells (uncoated wells incubated with the corresponding anti-IL-2 Ab + anti-mouse IgG-HRP Ab) was subtracted.

[0161] Figure 5 shows that MAB605, a pro-Teff Ab, binds to all IL-2 forms, and NARA, a pro-Teff Ab, also binds to IL-2V1 and IL-2V2 (in these preliminary experiments, the NARA Ab was tested only against IL-2V1 and IL-2V2). In contrast, 5344, a pro-Treg Ab, shows reduced / no binding to IL-2 variants. These results confirm that engineered IL-2Vs can be used as a powerful tool to screen anti-IL-2 Abs with CD25 mimotope activity, including Abs with pro-Teff and pro-Treg activity.

[0162] Example 4 In vitro evaluation of the biological activity of IL-2 variants We assessed whether different IL-2 variants exhibit different affinities / selectivities for T cell populations expressing either the intermediate affinity IL-2R (present in CD4+ or CD8+ effector T cells) or the high affinity IL-2R (present in Treg cells).

[0163] Therefore, CD3+ T cells were enriched from PBMCs derived from healthy donors according to the manufacturer's instructions (Miltenyi). CD3-enriched cells were cultured in SVF-free RPMI medium (GIBCO, France) containing different concentrations of Proleukin (Novartis) or IL-2V1, IL-2V2, IL-2V3, IL-2V4, IL-2V5, and IL-2V6 (0.0001 nM to 1000 nM) at 75 μL / well in 96-well plates. STAT5 phosphorylation (STAT5-P) was measured in human Treg cells (IL-2Rαβγ), CD4+ Teff cells (IL-2Rβγ), and CD8+ T cells (IL-2Rβγ) by flow cytometry as follows: After 15 min of stimulation, cultures were fixed with 200 μL / well of PBS / 2% paraformaldehyde for 10 min at room temperature. After washing with PBS / 0.2% BSA, cells were permeabilized with 100 μL / well of ice-cold methanol for 10 min on ice. Cells were then 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-T4 1:100; 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) for 45 min at 4°C. Cells were acquired on an LSRII flow cytometer and analyzed using FlowJo software.

[0164] The results show that human IL-2V2 and IL-2V3 selectively induce STAT-5 phosphorylation in Treg cells but not in effector T cells compared to commercially available human IL-2 Proleukin. IL-2V1, IL-2V4, IL-2V5, and IL-2V6 do not stimulate Treg or effector T cells at the doses evaluated (Figure 6).

[0165] Example 5 In vivo evaluation of the biological activity of IL-2 variants To evaluate the in vivo effects of IL-2V, we used a graft-versus-host disease (GVHD) model in which human peripheral blood mononuclear cells were injected into immunodeficient host mice. Upon recognition of murine antigens, human T cells are activated and induce xenogeneic GVHD. Clinically, GVHD is assessed by weight loss in mice. It has previously been shown that Treg cells can control GVHD (Gaidot A, Blood, 2011, 117, 2975-2983). Furthermore, it has been reported that low-dose IL-2 administration stimulates Treg cells, thus suppressing inflammation and reducing disease symptoms (Shin et al., Blood, 2011, 118, 2342-2350). It was hypothesized that if IL-2V specifically stimulates Tregs in vivo, Treg cells should be selectively expanded over CD4+ and CD8+ T cells, resulting in reduced disease symptoms.

[0166] To induce acute GVHD, NSG-SGM3 female mice were injected with 6x10 6 PBMCs containing human CD3 T cells were administered. On the same day, mice were treated by i.p. injection with Proleukin (6 μg, equivalent to 100,000 IU), IL-2V1 (6 μg), or IL-2V2 (6 μg). One group of mice remained untreated. Blood samples were taken on days 5, 10, and 21 after treatment for immune monitoring, and body weight was monitored along the experiment to detect the progression of GVHD.

[0167] Untreated mice, like Proleukin-treated mice, began to show weight loss by day 20 after PBMC injection. At the doses used, IL-2V2 slightly delayed, and IL-2V1 stopped, weight loss, a clinical effect of IL-2V1 associated with a slightly prolonged increase in mouse survival (Figure 7). Furthermore, IL-2V1 induced a sustained increase in the frequency of circulating human Tregs and also CD4+CD25+ Tregs, described as Tregs with enhanced suppressive function (Figure 8). These results demonstrate that IL-2V can control GVHD and that IL-2V1 is more effective in stopping weight loss and increasing CD25+ Tregs in vivo.

[0168] To evaluate the in vivo effects of IL-2V2 and IL-2V3, healthy C57BL / 6 mice were treated with Proleukin (4 μM), IL-2V2 (40 μM), or IL-2V3 (40 μM) by i.p. injection for four consecutive days. One group of mice remained untreated. Blood samples were collected the day after the last dose of Proleukin or IL-2V for FACS analysis.

[0169] IL-2V2- and IL-2V3-treated mice showed increased frequencies of circulating Tregs compared with untreated mice, but to a lesser extent than Proleukin-treated mice (Figure 10). However, unlike Proleukin, IL-2V2 (40 μM) and IL-2V3 (both concentrations) did not increase the frequencies of effector cells (CD8+ T cells and NK cells) or induce their proliferation (measured by Ki67 expression). This is highlighted by the decreased ratio of CD8 and NK cells to Tregs in IL-2V-treated mice compared with untreated and Proleukin-treated mice. These data indicate that IL-2V2 and IL-2V3 also selectively stimulate Tregs in mice in vivo, a function conserved across species. These data reinforce the potential of IL-2V2 and IL-2V3 as novel drugs that selectively stimulate Treg cells both in vitro and in vivo via the selective induction of STAP-5-P in Treg cells in vitro compared with Proleukin.

[0170] Example 6 IL-2V1, IL-2V4, IL-2V5, and IL-2V6 act as IL-2 antagonists Given 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 Proleukin for inducing STAT5 phosphorylation in T cells expressing the intermediate-affinity IL-2R (CD4+ effector cells and CD8+ effector T cells) or the high-affinity IL-2R (Treg cells) was evaluated in vitro. To this end, cells were stimulated with different concentrations of Proleukin and a constant high concentration of IL-2V1 (1000 nM) using the in vitro bioassay described above. IL-2-V1 competes with WT IL-2 for binding to IL-2Rαβγ and IL-2Rβγ, underlying its antagonistic function, as can be seen in Figure 9.

[0171] Binding of IL-2 variants to IL-2Rαβγ was assayed in Kit225, an IL-2-dependent human cell line that constitutively expresses IL-2Rαβγ. To facilitate detection, IL-2 variants were produced with a streptavidin tag, which allows detection of the molecules by FACS using an anti-streptavidin-tag antibody (a-Strep-Tag Ab). For the experiment, Kit225 cells were depleted of IL-2 for 2 days. Then, 0.2x10 cells were incubated on ice for 20 min. 6 Cells were incubated 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 using FlowJo software. The data presented in Figure 11 indicate that all IL-2 variants bind to IL-2Rαβγ to different extents, which, together with those shown in Figures 6 and 9, supports the hypothesis that IL-2V1 and IL-2V4-6 act as IL-2 antagonists.

[0172] 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 division in vitro by depleting them of wild-type IL-2 signaling (similar to anti-IL-2 antibody blockade), leaving active IL-2 signaling in CD8+ T cells. Briefly, CellTrace Violet (CTV)-labeled human PBMCs and CD25+ T cells (mixed at a 50:50 ratio) were incubated with anti-CD3 / anti-CD28 beads (1:5 bead:cell ratio) plus IL-2V1, 4, 6, anti-hIL-2 mAb (Mab602), or PBS (endogenous IL-2 control) for 96 h. CTV dilution (as a measure of T cell division) of CD4+ Tconv, Treg, and CD8+ T cells was measured by flow cytometry. Figure 12 shows the CTV dilution ratio relative to the PBS condition (maximum). LowPercentages (%) of cells expressing IL-2 V1, V4, and V6 are shown. This data indicates that IL-2 V1, V4, and V6 antagonize IL-2 signaling by competing with endogenous IL-2 on CD4+ T cells, inhibit Treg division in vitro by depleting them of wild-type IL-2 signaling (similar to anti-IL-2 antibody blockade), and leave active IL-2 signaling in CD8+ T cells. These data, along with the data shown in Figures 6, 9, and 11, indicate that IL-2 V1 and IL-2 V4-6 disarm Tregs by depleting or starving them from wild-type IL-2 signaling, thereby acting as IL-2 antagonists that can be used to inhibit Treg function, induce Treg function loss, or eliminate Tregs while preserving effector CD8+ T cell function.

[0173] In vivo, Treg inhibition by the IL-2 antagonist variants of the present invention will reduce tumor growth, as previously shown for other IL-2 antagonists (Carmenate et al., The Journal of Immunology, 2018, 200, pp. 3475-3484). Therefore, the IL-2 antagonist IL-2V of the present invention is expected to have antitumor effects in vivo. Treg inhibition by the IL-2 antagonist variants of the present invention will consequently promote immune responses (lymphocytes (B, NK, CD4+ or CD8+ T cells); dendritic cells (DCs); macrophages, etc.) by relieving immune cells from Treg suppression. Furthermore, depending on the dose of the IL-2Vs antagonist used, CD8+ T cell function is either preserved or only moderately affected (Figures 9 and 12). Thus, the IL-2 antagonist variants of the present invention further enable direct stimulation of CD8+ T cell immune responses, e.g., against tumors, pathogens, or vaccines. For all these reasons, better immune responses against cancer, infectious agents and vaccines are expected for the IL-2 antagonist variants of the present invention.

[0174] The results presented in this application collectively suggest that the effect of IL-2 antagonist variants in vivo may vary depending on the immune context, as they reduce immune activation when the immune system is overactive and producing excess IL-2 (by neutralizing excess endogenous IL-2), but increase immune activation in different immune contexts (by Treg inhibition and subsequent CD8+ T cell activation). For these reasons, IL-2 antagonist variants are useful for treating diseases involving immune system hyperactivity associated with IL-2 overproduction, such as chronic or acute inflammatory diseases, graft-versus-host disease (GVHD), and graft rejection, as well as for treating cancer and infectious diseases, and increasing immune responses to vaccines.

[0175] Example 7 Crystal structure of IL-2 variant Experimental procedure IL-2 variants were cloned into modified pMT / BiP plasmids (Invitrogen) and used to generate stable transfectants of Drosophila S2 cells. Proteins were purified from the supernatant using affinity and size-exclusion chromatography. Crystals of IL-2 V1 were obtained in 30% (w / v) PEG8000, 0.1 M imidazole pH 8.0, and 0.2 M NaCl. Crystals of IL-2 V4 were obtained in 15% (w / v) PEG3350, 0.1 M Hepes 7, 5 mM NiCl2, and 10 mM MgCl2 and snap-frozen in liquid nitrogen. The structures were solved by molecular replacement using the structure of wild-type IL-2 as a model.

[0176] result We also analyzed the crystal structures of some variants, which provides some insight into their mechanism of action. We observed that the Y31P substitution (IL2-V4) stabilizes IL-2 in a conformation similar to that observed in complex with the IL2-R alpha subunit, hereafter referred to as the "alpha-induced conformation." Indeed, there is some recent evidence from mouse IL-2 suggesting that conformational changes in loop AB, where Tyr31 is located, allosterically affect the interaction with the IL-2R beta and gamma subunits (De Paula et al., PNAS, doi / 10.1073; March 17, 2020; and Spangler et al., Immunity, 2015, 42, pp. 815-825). Therefore, it is tempting to speculate that the molecular mechanism underlying IL2-V4 activity utilizes this allosteric circuit. In fact, we also obtained the structure of IL2-V1, which has the same activity as IL2-V4. It also exhibits the same "alpha-inducing conformation," even though none of the mutations present in this variant are located in the AB loop.

[0177] Without being bound by theory, the inventors believe that the substitutions introduced into the IL-2 antagonist variants of the present invention induce conformational changes that stabilize the bound form and allosterically affect the interaction with the IL-2R beta and gamma subunits.

[0178] conclusion Overall, these results demonstrate that IL-2V is a novel drug that selectively stimulates Treg cells or antagonizes IL-2. Thus, IL-2V can be used to expand Treg cells ex vivo or in vivo, or to block IL-2-mediated overactivation of the immune system and suppress harmful immune responses in vivo. In particular, IL-2V, which selectively stimulates Treg cells in vivo, is useful for treating diseases involving immune dysfunction, such as allergic and autoimmune diseases, as well as diseases associated with immune system overactivity, such as chronic or acute inflammatory diseases, graft-versus-host disease (GVHD), and graft rejection. IL-2 antagonist variants are useful for treating diseases associated with immune system overactivity, including IL-2 overproduction, such as GVHD, graft rejection, and chronic or acute inflammatory diseases.

[0179] The IL-2 antagonist IL-2V is also useful for inhibiting Tregs, thus increasing immune responses (B, NK, CD4+ or CD8+ T cells, DCs, macrophages, etc.), particularly anti-tumor immune responses in cancer treatment and immune responses to vaccines or pathogens.

Claims

[Claim 1] 1. An interleukin-2 variant for the prevention or treatment of immune disorders, comprising 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, - the amino acids at positions 9 and 12 are substituted by D or E, preferably E; - 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; - the amino acid in position 23 is substituted by E, Q, T, N, G, A, V, L or I, preferably by L; - the amino acid in position 31 is substituted by P or N, preferably by P; - the amino acid at position 87 is substituted by M, V, E, D, T, C, N or Q, preferably by N; and An Interleukin-2 variant wherein if the substitution is at position 91, the variant comprises at least another substitution at position 9, 12, 16, 19, 23, 26, 31, 49, 52, 81, 84, 87, 95, 119, 123, 127, 131 or 132, the indicated positions being determined by alignment with SEQ ID NO:1.

Images