IL2 agonists

Abstract

The present invention relates to variants of interleukin-2 (IL2). In one embodiment, the IL2 variant activates effector T cells in preference to regulatory T cells. In particular, the present invention relates to polypeptides comprising mutant proteins of human IL2 or mutant proteins of human IL2 functional variants, wherein human IL2 or its functional variants are at least replaced at positions of acidic or basic amino acid residues contacting the α subunit of the αβγIL2 receptor complex (I12Kαβγ) in wild-type human IL2. Alternatively, the mutant proteins of human IL2 or mutant proteins of human IL2 functional variants include at least (i) one or more amino acid replacements that reduce affinity for the α subunit of II_2Kαβγ, and (ii) one or more amino acid replacements that enhance affinity for II_2Kβγ. The present invention also relates to polynucleotides encoding the polypeptides of the present invention, host cells comprising the polynucleotides, pharmaceutical compositions comprising the polypeptides, polynucleotides or host cells, therapeutic or preventive methods for treatment using the polypeptides, polynucleotides, host cells or pharmaceutical compositions, and pharmaceutical preparations comprising the polypeptides, polynucleotides, host cells or pharmaceutical compositions.

Description

Technical Field

[0001] The present invention relates to variants of interleukin-2 (IL2). In particular, the present invention relates to polypeptides comprising mutant proteins of human IL2 or mutant proteins of functional variants of human IL2, wherein human IL2 or its functional variant is replaced at least at the position of the acidic or basic amino acid residue in contact with the α subunit of the αβγ IL2 receptor complex (IL2Rαβγ) in wild-type human IL2. In one embodiment, the substitution reduces the affinity for IL2Rαβγ, preferably to a greater extent than the βγIL2 receptor complex (IL2Rβγ). In one embodiment, the polypeptide activates effector T cells over regulatory T cells. The present invention also relates to polynucleotides encoding polypeptides of the present invention, host cells comprising the polynucleotides, pharmaceutical compositions comprising the polypeptides, polynucleotides or host cells, therapeutic or preventive methods for treatment using the polypeptides, polynucleotides, host cells or pharmaceutical compositions, and pharmaceutical preparations comprising the polypeptides, polynucleotides, host cells or pharmaceutical compositions. Background Art

[0002] The immune system plays an important role in cancer, autoimmunity, allergy and pathogen-related diseases. T cells and NK cells are important mediators of anti-tumor immune responses. CD8+T cells and NK cells can directly lyse tumor cells. On the other hand, CD4+T cells can mediate the influx of different immune subsets (including CD8+T cells and NK cells) into tumors. CD4+T cells can permit dendritic cells (DC) to trigger anti-tumor CD8+T cell responses, and can directly act on tumor cells through IFNγ-mediated MHC upregulation and growth inhibition. CD8+ and CD4+ tumor-specific T cell responses can be induced by vaccination or by adoptive transfer of T cells. In the case of mRNA-based vaccine platforms, mRNA can be delivered to antigen-presenting cells located in secondary lymphoid organs via liposomal formulations (RNA-lipid complexes, RNA-LPX) without any additional adjuvants for immune stimulation (Kreiter, S. et al. Nature 520, 692-696 (2015); Kranz, LM et al. Nature 534, 396-401 (2016)).

[0003] Cytokines play an important role in immunity. For example, interleukin-2 (IL2) is a potent immunostimulant that activates a variety of cells of the immune system, including T cells, B cells, monocytes and natural killer (natural killer, NK) cells. It is known that IL2 supports the differentiation, proliferation, survival and effector functions of T cells and NK cells (Blattman, JNetal.Nat.Med.9, 540-7 (2003)), and has been used in the treatment of advanced malignant melanoma for decades (Maas, RA, Dullens, HF & Den Otter, W.Cancer Immunol.Immunother.36, 141-8 (1993)). Therefore, immunotherapy such as T cell vaccines or (initial or T cell receptor transgenic or chimeric antigen receptor transgenic) T cells or NK cells adoptive transfer can benefit from the simultaneous administration of cytokines (such as IL2). However, one disadvantage of recombinant IL2 is its short plasma half-life (Lotze, MT et al. JAMA 1986, 256, 3117-24.; West, W Hetal. N. Engl. J. Med. 316, 898-905 (1987)). This requires frequent injections of large amounts of IL2, leading to serious side effects such as vascular leak syndrome (VLS) (Rosenberg, SA et al. N. Engl. J. Med. 316, 889-97 (1987)). VLS causes accumulation of intravascular fluid in the lungs and liver, leading to pulmonary edema and liver damage. Until recently, it was believed that VLS was caused by the release of proinflammatory cytokines by NK cells activated by IL2. However, recent reports indicate that direct binding of IL2 to lung endothelial cells is allegedly the cause of VLS (Krieg et al, PNAS USA 107 (26) 11906-11911 (2010)). The second disadvantage of IL2 is its inherent ability to stimulate regulatory T cells (Treg). Treg is associated with reduced survival in cancer patients because they can inhibit the function of anti-tumor effector T cells and NK cells (Nishikawa, H. & Sakaguchi. Curr. Opin. Immunol. 27, 1-7 (2014)). IL2 signals through the IL2 receptor, which exists in high-affinity and medium-affinity forms. The high-affinity IL2 receptor (IL2Rαβγ) is composed of CD25 (IL2Rα), CD122 (IL2Rβ) and CD132 (IL2Rγ) and is expressed on Treg and activated CD4+ and CD8+ T cells. Treg activation can aggravate immunosuppression, potentially impairing the expected therapeutic response.The medium affinity receptor (IL2Rβγ) lacks CD25 and is generally found on naive T cells, memory T cells, and NK cells. As a result, IL2 preferentially stimulates CD25-expressing Tregs (Todd, JA et al. PLoS Med. 13, e1002139 (2016)) and activated CD4+ and CD8+ T cells. In addition, high doses of IL2 are required to activate naive T cells, memory T cells, and NK cells. The attempt to change IL2 in such a way that it loses its preference for cells expressing CD25, thereby relatively increasing the stimulation potential of naive T cells, memory T cells, and NK cells, has shown an increase in its anti-tumor potential (Arenas-Ramirez, N. et al. Sci. Transl. Med. 8, 1-13 (2016)).

[0004] New strategies are needed to improve the effectiveness of vaccines, especially cancer vaccines, and other immunotherapies, especially cancer immunotherapies, such as adoptive transfer of (naive or T cell receptor transgenic or chimeric antigen receptor transgenic) T cells and NK cells.

[0005] We have discovered a variant of human IL2 that preferentially activates cells expressing the intermediate affinity IL2 receptor IL2Rβγ over cells expressing the high affinity IL2 receptor IL2Rαβγ.

[0006] It is assumed that the interaction of IL2 with IL2Rα is destroyed by appropriately modifying the specific binding residues on the IL2 binding surface, which will prevent effective binding (and thus activation) to cells expressing IL2Rαβγ. However, binding to cells still occurs on cells expressing IL2Rβγ. Those IL2 variants that are able to selectively activate the medium-affinity IL2 receptor on certain T cells (such as memory T cells, initial T cells and effector T cells and NK cells) in preference to the high-affinity IL2 receptor on regulatory T cells are expected to have improved therapeutic indexes (therapeutic index) and reduced toxicity characteristics relative to wild-type IL2. IL2 variants with improved therapeutic indexes will have a significantly expanded range of use in the treatment of conditions requiring immune system stimulation, such as in the treatment of cancer (as direct treatment and / or adjuvant therapy). In particular, the administration of IL2 variant RNA is a promising method for enhancing the therapeutic efficacy of a variety of T cell and NK cell-based (cancer) immunotherapies.

[0007] The present disclosure provides new IL2 variants. Specifically, variants of IL2 are described, which include mutations that affect CD25 binding ("mutCD25") and optionally also include mutations that enhance IL2Rβγ binding ("mutβγ"), thereby reducing Treg expansion and improving effector T cells and NK cell stimulation. In vitro, mRNA-encoded IL2 mutCD25 variants induce the proliferation of weaker IL2Rαβγ-positive T cells compared with wild-type IL2, and show reduced binding to CD25. Compared with wild-type IL2, the mutβγ variant without mutCD25 mutations not only strongly improves the efficacy of amplifying all T cells, but also improves the preferential amplification of Treg. Importantly, the combination of mutCD25 and mutβγ mutations leads to a strong reduction in IL2-mediated Treg amplification, while retaining the efficacy of amplifying CD8+T cells and FoxP3-CD4+T cells. In vivo, nanoparticle-formulated mRNA-encoded IL2 mutCD25 variants were targeted to the liver of mice for preferential or exclusive enhancement of systemic availability of effector T cell responses induced by RNA-LPX vaccination compared to Tregs. The combination of mutCD25 and mutβγ mutations further enhanced the potency of the mutCD25IL2 variant compared to wild-type IL2 and resulted in preferential expansion of antigen-specific T cells and significantly reduced Treg expansion and increased CD8 to Treg ratios. Summary of the invention

[0008] In one embodiment, an IL2 variant is described herein, which has an amino acid substitution at the IL2 region that contacts the alpha (α) subunit of the heterotrimeric IL2 receptor complex IL2Rαβγ, thereby reducing its ability to bind and activate the heterotrimer (also referred to herein as a "mutCD25" mutation). The IL2Rαβγ complex is constitutively expressed on regulatory T cells (Treg). Therefore, the IL2 replacement described herein reduces the affinity of IL2Rαβγ to a greater extent than that of IL2Rβγ, and the receptor complex is mainly on initial T cells and memory T cells and NK cells. In the tumor microenvironment, relative to Treg, in vivo treatment using the IL2 variant described herein is more conducive to activating T cells (e.g., CD8 T cells) to provide anti-tumor efficacy.

[0009] In one aspect, the present invention relates to a polypeptide comprising a mutant protein of human interleukin-2 (IL2) or a mutant protein of a functional variant of human IL2, wherein human IL2 or its functional variant is substituted at least at a position having an acidic or basic amino acid residue in wild-type human IL2 that contacts the α subunit of the αβγ IL2 receptor complex (IL2Rαβγ), wherein if the amino acid residue is an acidic amino acid residue in wild-type human IL2 (glutamic acid (Glu / E) or aspartic acid (Asp / D), in particular glutamic acid), the substitution is by a basic amino acid residue (lysine (Lys / K) or arginine (Arg / R), in particular lysine), and if the amino acid residue is a basic amino acid residue in wild-type human IL2 (lysine (Lys / K) or arginine (Arg / R), in particular lysine), the substitution is by an acidic amino acid residue (glutamic acid (Glu / E) or aspartic acid (Asp / D), in particular glutamic acid). In different embodiments, human IL2 or a functional variant thereof is replaced at one or more, e.g., two or more or three or more, e.g., 2, 3, 4, 5, 6, 7 or 8, positions having acidic or basic amino acid residues that in wild-type human IL2 contact the α subunit of the αβγIL2 receptor complex (IL2Rαβγ).

[0010] In one embodiment, the wild-type human IL2 has the amino acid sequence according to SEQ ID NO:17.

[0011] In one embodiment, an acidic amino acid residue in wild-type human IL2 is contacted with a basic amino acid residue in the α subunit of IL2Rαβγ. In one embodiment, a basic amino acid residue in wild-type human IL2 is contacted with an acidic amino acid residue in the α subunit of IL2Rαβγ.

[0012] In one embodiment, the substitution reduces affinity for IL2Rαβγ.In one embodiment, the substitution reduces affinity for IL2Rαβγ to a greater extent than for the βγIL2 receptor complex (IL2Rβγ).

[0013] In one embodiment, the polypeptide preferentially activates effector T cells (e.g., CD8+ T cells and / or CD4+ T cells, such as CD4+ T cells that are CD25- and / or FoxP3-) relative to regulatory T cells (e.g., CD4+CD25+FoxP3+ T cells).

[0014] In one embodiment, human IL2 or a functional variant thereof is substituted at least one of positions 35 (lysine), 43 (lysine), 61 (glutamic acid) and 62 (glutamic acid) relative to wild-type human IL2 and is numbered according to wild-type human IL2.

[0015] In various embodiments, human IL2 or a functional variant thereof is replaced at least at the following positions:

[0016] - No. 35,

[0017] - No. 43,

[0018] - No. 61,

[0019] - No. 62,

[0020] - 35th and 43rd,

[0021] - 35th and 61st,

[0022] - 35th and 62nd,

[0023] - 43rd and 61st,

[0024] - 43rd and 62nd,

[0025] - 61st and 62nd,

[0026] - 35th, 43rd and 61st,

[0027] - 35th, 43rd and 62nd,

[0028] - 35th, 61st and 62nd,

[0029] - 43rd, 61st and 62nd, or

[0030] -35th, 43rd, 61st and 62nd.

[0031] In one embodiment, position 35 is replaced with glutamic acid. In one embodiment, position 43 is replaced with glutamic acid. In one embodiment, position 61 is replaced with lysine. In one embodiment, position 62 is replaced with lysine.

[0032] In one embodiment, position 35 is substituted. In one embodiment, position 35 is substituted with glutamic acid.

[0033] In one embodiment, position 43 is substituted. In one embodiment, position 43 is substituted with glutamic acid.

[0034] In one embodiment, position 61 is substituted. In one embodiment, position 61 is substituted with lysine.

[0035] In one embodiment, position 62 is substituted. In one embodiment, position 62 is substituted with lysine.

[0036] In one embodiment, positions 43 and 61 are substituted. In one embodiment, position 43 is substituted with glutamic acid and position 61 is substituted with lysine.

[0037] In one embodiment, positions 35, 43 and 61 are substituted. In one embodiment, position 35 is substituted with glutamic acid, position 43 is substituted with glutamic acid and position 61 is substituted with lysine.

[0038] In one embodiment, position 61 and position 62 are substituted. In one embodiment, position 61 is substituted with lysine and position 62 is substituted with lysine.

[0039] In one aspect, the present invention relates to a polypeptide comprising a mutant protein of human interleukin-2 (IL2) or a mutant protein of a functional variant of human IL2, wherein human IL2 or its functional variant is substituted at least one of position 35 (lysine), position 43 (lysine), position 61 (glutamic acid) and position 62 (glutamic acid) relative to wild-type human IL2, and numbered according to wild-type human IL2. In one embodiment, if the amino acid residue is an acidic amino acid residue in wild-type human IL2, the substitution is substituted by a basic amino acid residue, and if the amino acid residue is a basic amino acid residue in wild-type human IL2, the substitution is substituted by an acidic amino acid residue.

[0040] In various embodiments, human IL2 or a functional variant thereof is replaced at least at the following positions:

[0041] - No. 35,

[0042] - No. 43,

[0043] - No. 61,

[0044] - No. 62,

[0045] - 35th and 43rd,

[0046] - 35th and 61st,

[0047] - 35th and 62nd,

[0048] - 43rd and 61st,

[0049] - 43rd and 62nd,

[0050] - 61st and 62nd,

[0051] - 35th, 43rd and 61st,

[0052] - 35th, 43rd and 62nd,

[0053] - 35th, 61st and 62nd,

[0054] - 43rd, 61st and 62nd, or

[0055] -35th, 43rd, 61st and 62nd.

[0056] In one embodiment, position 35 is replaced with glutamic acid. In one embodiment, position 43 is replaced with glutamic acid. In one embodiment, position 61 is replaced with lysine. In one embodiment, position 62 is replaced with lysine.

[0057] In one embodiment, position 35 is substituted. In one embodiment, position 35 is substituted with glutamic acid.

[0058] In one embodiment, position 43 is substituted. In one embodiment, position 43 is substituted with glutamic acid.

[0059] In one embodiment, position 61 is substituted. In one embodiment, position 61 is substituted with lysine.

[0060] In one embodiment, position 62 is substituted. In one embodiment, position 62 is substituted with lysine.

[0061] In one embodiment, positions 43 and 61 are substituted. In one embodiment, position 43 is substituted with glutamic acid and position 61 is substituted with lysine.

[0062] In one embodiment, positions 35, 43 and 61 are substituted. In one embodiment, position 35 is substituted with glutamic acid, position 43 is substituted with glutamic acid and position 61 is substituted with lysine.

[0063] In one embodiment, position 61 and position 62 are substituted. In one embodiment, position 61 is substituted with lysine and position 62 is substituted with lysine.

[0064] In one embodiment, the wild-type human IL2 has the amino acid sequence according to SEQ ID NO:17.

[0065] In one embodiment, the substitution reduces affinity for IL2Rαβγ.In one embodiment, the substitution reduces affinity for IL2Rαβγ to a greater extent than for the βγIL2 receptor complex (IL2Rβγ).

[0066] In one embodiment, the polypeptide preferentially activates effector T cells (e.g., CD8+ T cells and / or CD4+ T cells, such as CD4+ T cells that are CD25- and / or FoxP3-) relative to regulatory T cells (e.g., CD4+CD25+FoxP3+ T cells).

[0067] In one embodiment, the above-mentioned IL2 replaced or its functional variant (IL2 mutant protein) has the same amino acid sequence as wild-type IL2 at other unsubstituted residues. In one embodiment, the above-mentioned IL2 mutant protein has amino acid modifications, such as amino acid replacements, at one or more sites in or at other residues of wild-type human IL2. In one embodiment, when compared with wild-type IL2, such amino acid replacements result in a relative increase in affinity for IL2Rβγ (also referred to herein as "mutβγ" mutations). Such mutants are potent IL2 signaling agonists. In one embodiment, such amino acid replacements are at amino acid residues in contact with IL2Rβ and / or IL2Rγ.

[0068] In one embodiment, human IL2 or a functional variant thereof is replaced at least one of positions 24, 65, 74, 80, 81, 85, 86, 89, 92 and 93 relative to wild-type human IL2 and numbered according to wild-type human IL2. The replaced amino acid residues may be, but are not necessarily, conservative replacements. For example, the mutations may be: I24V, P65H, Q74R, Q74H, Q74N, Q74S, L80F, L80V, R81I, R81T, R81D, L85V, I86V, I89V, I92F, V93I.

[0069] In one embodiment, the IL2 mutant protein comprises the following amino acid substitution group: 80F / 81D / 85V / 86V / 92F. The IL2 mutant protein may also comprise the amino acid substitution 42A. The IL2 mutant protein may also comprise one or more of the following amino acid substitutions: 24V, 65H, 74R, 74H, 74N, 74S, 89V, 93I.

[0070] In some embodiments, the IL2 mutein comprises an amino acid substitution group selected from:

[0071] (i) 74N, 80F, 81D, 85V, 86V, 89V, 92F;

[0072] (ii)74H, 80F, 81D, 85V, 86V, 92F;

[0073] (iii)74S, 80F, 81D, 85V, 86V, 92F;

[0074] (iv) 74N, 80F, 81D, 85V, 86V, 92F;

[0075] (v)80F, 81D, 85V, 86V, 92F;

[0076] (vi)80F, 81D, 85V, 86V, 89V, 92F, 93I;

[0077] (vii) 18R, 22E, 80F, 81D, 85V, 86V, 89V, 92F, 93I, 126T;

[0078] (viii)18R, 22E, 74S, 80F, 81T, 85V, 86V, 89V, 92F, 93I, 126T.

[0079] In one aspect, the present invention relates to a polypeptide comprising a mutant protein of human interleukin-2 (IL2) or a mutant protein of a functional variant of human IL2, wherein human IL2 or its functional variant comprises at least (i) one or more amino acid substitutions that reduce affinity for the α subunit of IL2Rαβγ (also referred to herein as "mutCD25" mutation), and (i) one or more amino acid substitutions that enhance affinity for IL2Rβγ (also referred to herein as "mutβγ" mutation).

[0080] In one embodiment, the polypeptide preferentially activates effector T cells (e.g., CD8+ T cells and / or CD4+ T cells, such as CD4+ T cells that are CD25- and / or FoxP3-) relative to regulatory T cells (e.g., CD4+CD25+FoxP3+ T cells).

[0081] In one embodiment, the one or more amino acid substitutions that reduce affinity for the α subunit of IL2Rαβγ are at an amino acid residue of IL2 or a functional variant thereof that contacts IL2Rα. In one embodiment, the one or more amino acid substitutions that reduce affinity for the α subunit of IL2Rαβγ reduce affinity for IL2Rαβγ to a greater extent than for IL2Rβγ.

[0082] In one embodiment, the one or more amino acid substitutions that reduce affinity for the α subunit of IL2Rαβγ comprise substitutions at one or more positions of IL2 or a functional variant thereof selected from K35, T37, R38, T41, F42, K43, F44, Y45, E61, E62, K64, P65, E68, L72 and Y107.

[0083] In one embodiment, one or more amino acid substitutions that reduce affinity for the α subunit of IL2Rαβγ are at one or more positions of IL2 or a functional variant thereof, said positions having an acidic or basic amino acid residue in wild-type human IL2 that contacts the α subunit of IL2Rαβγ, wherein if the amino acid residue is an acidic amino acid residue in wild-type human IL2 (glutamic acid (Glu / E) or aspartic acid (Asp / D), in particular glutamic acid), the substitution is by a basic amino acid residue (lysine (Lys / K) or arginine (Arg / R), in particular lysine), and if the amino acid residue is a basic amino acid residue in wild-type human IL2 (lysine (Lys / K) or arginine (Arg / R), in particular lysine), the substitution is by an acidic amino acid residue (glutamic acid (Glu / E) or aspartic acid (Asp / D), in particular glutamic acid). In various embodiments, human IL2 or a functional variant thereof is replaced at one or more, such as two or more or three or more, such as 2, 3, 4, 5, 6, 7 or 8 such positions, wherein the positions have acidic or basic amino acid residues that contact the α subunit of IL2Rαβγ in wild-type human IL2.

[0084] In one embodiment, an acidic amino acid residue in wild-type human IL2 is contacted with a basic amino acid residue in the α subunit of IL2Rαβγ. In one embodiment, a basic amino acid residue in wild-type human IL2 is contacted with an acidic amino acid residue in the α subunit of IL2Rαβγ.

[0085] In one embodiment, the wild-type human IL2 has the amino acid sequence according to SEQ ID NO:17.

[0086] In one embodiment, the one or more amino acid substitutions that reduce affinity for the α subunit of IL2Rαβγ comprise substitutions at least one of position 35 (lysine), position 43 (lysine), position 61 (glutamate) and position 62 (glutamate) relative to and numbered according to wild-type human IL2.

[0087] In some different embodiments, the one or more amino acid substitutions that reduce affinity for the α subunit of IL2Rαβγ are at one or more positions of IL2 or a functional variant thereof, the substitutions being selected from:

[0088] - No. 35,

[0089] - No. 43,

[0090] - No. 61,

[0091] - No. 62,

[0092] - 35th and 43rd,

[0093] - 35th and 61st,

[0094] - 35th and 62nd,

[0095] - 43rd and 61st,

[0096] - 43rd and 62nd,

[0097] - 61st and 62nd,

[0098] - 35th, 43rd and 61st,

[0099] - 35th, 43rd and 62nd,

[0100] - 35th, 61st and 62nd,

[0101] - 43rd, 61st and 62nd, or

[0102] -35th, 43rd, 61st and 62nd.

[0103] In one embodiment, position 35 is replaced with glutamic acid. In one embodiment, position 43 is replaced with glutamic acid. In one embodiment, position 61 is replaced with lysine. In one embodiment, position 62 is replaced with lysine.

[0104] In one embodiment, position 35 is substituted. In one embodiment, position 35 is substituted with glutamic acid.

[0105] In one embodiment, position 43 is substituted. In one embodiment, position 43 is substituted with glutamic acid.

[0106] In one embodiment, position 61 is substituted. In one embodiment, position 61 is substituted with lysine.

[0107] In one embodiment, position 62 is substituted. In one embodiment, position 62 is substituted with lysine.

[0108] In one embodiment, positions 43 and 61 are substituted. In one embodiment, position 43 is substituted with glutamic acid and position 61 is substituted with lysine.

[0109] In one embodiment, positions 35, 43 and 61 are substituted. In one embodiment, position 35 is substituted with glutamic acid, position 43 is substituted with glutamic acid and position 61 is substituted with lysine.

[0110] In one embodiment, position 61 and position 62 are substituted. In one embodiment, position 61 is substituted with lysine and position 62 is substituted with lysine.

[0111] In one embodiment, the one or more amino acid substitutions that enhance affinity for IL2Rβγ are at amino acid residues of IL2 that contact IL2Rβ and / or IL2Rγ.

[0112] In one embodiment, the one or more amino acid substitutions that enhance affinity for IL2Rβγ comprise substitutions at one or more positions of IL2 selected from K9, L12, Q13, E15, H16, D20, Q74, L80, R81, D84, L85, I86, N88, I92, L94 and E95.

[0113] In one embodiment, the one or more amino acid substitutions that enhance affinity for IL2Rβγ comprise at least one of positions 24, 65, 74, 80, 81, 85, 86, 89, 92, and 93 relative to and numbered according to wild-type human IL2. The replaced amino acid residues may be, but are not necessarily, conservative substitutions. For example, the mutations may be: I24V, P65H, Q74R, Q74H, Q74N, Q74S, L80F, L80V, R81I, R81T, R81D, L85V, I86V, I89V, I92F, V93I.

[0114] In one embodiment, the IL2 mutant protein comprises the following amino acid substitution group: 80F / 81D / 85V / 86V / 92F. The IL2 mutant protein may also comprise the amino acid substitution 42A. The IL2 mutant protein may also comprise one or more of the following amino acid substitutions: 24V, 65H, 74R, 74H, 74N, 74S, 89V, 93I.

[0115] In some embodiments, the IL2 mutant protein comprises an amino acid substitution group selected from the group consisting of:

[0116] (i) 74N, 80F, 81D, 85V, 86V, 89V, 92F;

[0117] (ii)74H, 80F, 81D, 85V, 86V, 92F;

[0118] (iii)74S, 80F, 81D, 85V, 86V, 92F;

[0119] (iv) 74N, 80F, 81D, 85V, 86V, 92F;

[0120] (v)80F, 81D, 85V, 86V, 92F;

[0121] (vi)80F, 81D, 85V, 86V, 89V, 92F, 93I;

[0122] (vii) 18R, 22E, 80F, 81D, 85V, 86V, 89V, 92F, 93I, 126T;

[0123] (viii)18R, 22E, 74S, 80F, 81T, 85V, 86V, 89V, 92F, 93I, 126T.

[0124] The IL2 muteins described herein may be linked to a pharmacokinetic modifying group and, thus, may be "pharmacokinetic (PK)-prolonged IL2".

[0125] In one aspect, the present invention relates to a polypeptide as described herein, which is a pharmacokinetic (PK) prolonged IL2, further comprising an amino acid sequence heterologous to IL2 or a functional variant thereof fused to an IL2 mutein.

[0126] In one embodiment, the amino acid sequence heterologous to IL2 or a functional variant thereof is selected from serum albumin, an immunoglobulin fragment, transferrin and Fn3, or a variant thereof. In one embodiment, the serum albumin comprises mouse serum albumin or human serum albumin. In one embodiment, the immunoglobulin fragment comprises an immunoglobulin Fc domain.

[0127] In one aspect, the invention relates to a polypeptide comprising an amino acid sequence selected from SEQ ID NO: 2 to 16, e.g., SEQ ID NO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 of the sequence listing.

[0128] The above polypeptides are also referred to herein as "IL2 variant polypeptides" or simply as "IL2 variants".

[0129] In one aspect, the invention relates to polynucleotides encoding the IL2 variant polypeptides described herein. In one embodiment, the polynucleotide is RNA.

[0130] In one aspect, the invention relates to a host cell comprising a polynucleotide encoding an IL2 variant polypeptide described herein.

[0131] In one aspect, the present invention relates to a pharmaceutical composition comprising an IL2 variant polypeptide as described herein, a polynucleotide encoding an IL2 variant polypeptide as described herein, or a host cell comprising a polynucleotide encoding an IL2 variant polypeptide as described herein.

[0132] In one aspect, the present invention relates to a method of treating a subject, comprising administering to the subject an IL2 variant polypeptide as described herein, a polynucleotide encoding an IL2 variant polypeptide as described herein, a host cell comprising a polynucleotide encoding an IL2 variant polypeptide as described herein, or a pharmaceutical composition as described herein. In one embodiment, the subject suffers from cancer.

[0133] In one embodiment, the subject is further treated with one or more immunotherapies, such as vaccination or adoptive transfer of T cells, such as a T cell vaccine or adoptive transfer of (naive or T cell receptor transgenic or chimeric antigen receptor transgenic) T cells or NK cells.

[0134] The effectiveness of vaccines in which the antigen itself or as a polynucleotide, in particular as an RNA encoding the antigen (e.g., an RNA encoding a peptide or protein for vaccination (also referred to herein as "a peptide or protein comprising an epitope for inducing an immune response to the antigen")) is delivered can be improved by co-administering an IL2 variant polypeptide as described herein, wherein the IL2 variant polypeptide itself or as a polynucleotide, in particular an RNA encoding the IL2 variant polypeptide is delivered. The vaccine is particularly effective if the RNA encoding the IL2 variant polypeptide is targeted to the liver for systemic availability. Hepatocytes can be efficiently transfected and are able to produce large amounts of protein. The mRNA encoding the antigen is preferably targeted to secondary lymphoid organs. In addition, the vaccine is particularly effective if an immune checkpoint inhibitor such as an anti-PD-L1 antibody is additionally administered.

[0135] In one aspect, the invention relates to a method for inducing an immune response in a subject, comprising administering to the subject:

[0136] a. an IL2 variant polypeptide described herein, a polynucleotide encoding an IL2 variant polypeptide described herein, a host cell comprising a polynucleotide encoding an IL2 variant polypeptide described herein, or a pharmaceutical composition described herein; and

[0137] b. A peptide or protein comprising an epitope for inducing an immune response against an antigen in a subject, or a polynucleotide encoding the peptide or protein.

[0138] In one embodiment, the polynucleotide encoding the peptide or protein is RNA.

[0139] In one embodiment, the method for inducing an immune response in a subject comprises administering to the subject:

[0140] a. RNA encoding the IL2 variant polypeptide described herein; and

[0141] b. RNA encoding a peptide or protein comprising an epitope for inducing an immune response against an antigen in a subject.

[0142] In one embodiment, the polypeptide preferentially activates effector T cells (e.g., CD8+ T cells and / or CD4+ T cells, such as CD4+ T cells that are CD25- and / or FoxP3-) relative to regulatory T cells (e.g., CD4+CD25+FoxP3+ T cells).

[0143] In one embodiment, the method is a method for treating or preventing cancer in a subject, wherein the antigen is a tumor-associated antigen.

[0144] In one aspect, the invention relates to a method for treating or preventing cancer in a subject, comprising administering to the subject:

[0145] a. an IL2 variant polypeptide described herein, a polynucleotide encoding an IL2 variant polypeptide described herein, a host cell comprising a polynucleotide encoding an IL2 variant polypeptide described herein, or a pharmaceutical composition described herein; and

[0146] b. A peptide or protein comprising an epitope for inducing an immune response against a tumor-associated antigen in a subject, or a polynucleotide encoding the peptide or protein.

[0147] In one embodiment, the polynucleotide encoding the peptide or protein is RNA.

[0148] In one embodiment, the method for treating or preventing cancer in a subject comprises administering to the subject:

[0149] a. RNA encoding the IL2 variant polypeptide described herein; and

[0150] b. RNA encoding a peptide or protein comprising an epitope for inducing an immune response against a tumor-associated antigen in a subject.

[0151] In one embodiment, the cancer is selected from melanoma, leukemia, lymphoma, lung cancer, breast cancer, prostate cancer, ovarian cancer, colon cancer, mesothelioma, renal cell carcinoma, and brain cancer.

[0152] In one embodiment, the method described herein further comprises administering an immune checkpoint inhibitor to a subject. In one embodiment, the immune checkpoint inhibitor targets (i) the interaction between PD-1 and PD-L1, or (ii) the interaction between CTLA-4 and CD80 or CD86. In one embodiment, the immune checkpoint inhibitor is an antibody or antibody fragment. In one embodiment, the antibody or antibody fragment targets PD-1, PD-L1 or CTLA-4.

[0153] In one embodiment, RNA encoding an IL2 variant polypeptide described herein, RNA encoding a peptide or protein comprising an epitope for inducing an immune response to an antigen in a subject, and optionally an immune checkpoint inhibitor are administered simultaneously or sequentially.

[0154] In one aspect, the invention relates to a pharmaceutical formulation comprising:

[0155] a. An IL2 variant polypeptide described herein, a polynucleotide encoding an IL2 variant polypeptide described herein, a host cell comprising a polynucleotide encoding an IL2 variant polypeptide described herein, or a pharmaceutical composition described herein.

[0156] In one embodiment, the pharmaceutical formulation further comprises:

[0157] b. A peptide or protein comprising an epitope for inducing an immune response against an antigen in a subject, or a polynucleotide encoding the peptide or protein.

[0158] In one embodiment, the polynucleotide encoding the peptide or protein is RNA.

[0159] In one embodiment, the pharmaceutical formulation comprises:

[0160] a. RNA encoding the IL2 variant polypeptide described herein; and

[0161] b. RNA encoding a peptide or protein comprising an epitope for inducing an immune response against an antigen in a subject.

[0162] In one embodiment of the pharmaceutical formulation, the RNA is present in a form selected from a liquid form, a solid form, or a combination thereof. In one embodiment, the solid form is a frozen form or a dehydrated form. In one embodiment, the dehydrated form is a freeze-dried or spray-dried form.

[0163] In one embodiment, the pharmaceutical preparation further comprises an immune checkpoint inhibitor. In one embodiment, the immune checkpoint inhibitor targets (i) the interaction between PD-1 and PD-L1, or (ii) the interaction between CTLA-4 and CD80 or CD86. In one embodiment, the immune checkpoint inhibitor is an antibody or antibody fragment. In one embodiment, the antibody or antibody fragment targets PD-1, PD-L1 or CTLA-4.

[0164] In one embodiment, the pharmaceutical preparation is a pharmaceutical kit. In one embodiment, the pharmaceutical preparation is a pharmaceutical composition.

[0165] In one embodiment, the pharmaceutical formulation comprises each component a. and b. in separate containers.

[0166] In one embodiment, the pharmaceutical preparation is a pharmaceutical composition.In one embodiment, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0167] In one embodiment, the pharmaceutical preparation further comprises instructions for use of the pharmaceutical preparation for treating or preventing cancer, wherein the antigen is a tumor-associated antigen.

[0168] In one aspect, the invention relates to a pharmaceutical formulation as described herein for use in medicine.

[0169] In one embodiment, pharmaceutical uses include therapeutic or prophylactic treatment of a disease or condition.

[0170] In one aspect, the invention relates to a pharmaceutical formulation as described herein for use in a method of treating or preventing cancer in a subject, wherein the antigen is a tumor-associated antigen.

[0171] In one embodiment, the cancer is selected from melanoma, leukemia, lymphoma, lung cancer, breast cancer, prostate cancer, ovarian cancer, colon cancer, mesothelioma, renal cell carcinoma, and brain cancer.

[0172] In another aspect, the present invention relates to an IL2 variant polypeptide described herein, a polynucleotide encoding an IL2 variant polypeptide described herein, a host cell comprising a polynucleotide encoding an IL2 variant polypeptide described herein, or a pharmaceutical composition described herein, for use in a method of treating a subject, the method comprising administering the polypeptide, the polynucleotide, the host cell or the pharmaceutical composition to the subject.

[0173] In one embodiment, the subject has cancer.

[0174] In another aspect, the present invention relates to an IL2 variant polypeptide as described herein, a polynucleotide encoding an IL2 variant polypeptide as described herein, a host cell comprising a polynucleotide encoding an IL2 variant polypeptide as described herein, or a pharmaceutical composition as described herein, for use in a method of inducing an immune response in a subject, the method comprising administering to the subject:

[0175] a. the polypeptide, the polynucleotide, the host cell or the pharmaceutical composition; and

[0176] b. A peptide or protein comprising an epitope for inducing an immune response against an antigen in a subject, or a polynucleotide encoding the peptide or protein.

[0177] In one embodiment, the polynucleotide encoding the peptide or protein is RNA.

[0178] In one embodiment, the method for inducing an immune response in a subject comprises administering to the subject:

[0179] a. RNA encoding the IL2 variant polypeptide described herein; and

[0180] b. RNA encoding a peptide or protein comprising an epitope for inducing an immune response against an antigen in a subject.

[0181] In one embodiment, the polypeptide preferentially activates effector T cells (e.g., CD8+ T cells and / or CD4+ T cells, such as CD4+ T cells that are CD25- and / or FoxP3-) relative to regulatory T cells (e.g., CD4+CD25+FoxP3+ T cells).

[0182] In one embodiment, the method is a method for treating or preventing cancer in a subject, wherein the antigen is a tumor-associated antigen.

[0183] In another aspect, the present invention relates to an IL2 variant polypeptide as described herein, a polynucleotide encoding an IL2 variant polypeptide as described herein, a host cell comprising a polynucleotide encoding an IL2 variant polypeptide as described herein, or a pharmaceutical composition as described herein, for use in a method of treating or preventing cancer in a subject, the method comprising administering to the subject:

[0184] a. the polypeptide, the polynucleotide, the host cell or the pharmaceutical composition; and

[0185] b. A peptide or protein comprising an epitope for inducing an immune response against a tumor-associated antigen in a subject, or a polynucleotide encoding the peptide or protein.

[0186] In one embodiment, the polynucleotide encoding the peptide or protein is RNA.

[0187] In one embodiment, the method for treating or preventing cancer in a subject comprises administering to the subject:

[0188] a. RNA encoding the IL2 variant polypeptide described herein; and

[0189] b. RNA encoding a peptide or protein comprising an epitope for inducing an immune response against a tumor-associated antigen in a subject.

[0190] In one embodiment, the cancer is selected from melanoma, leukemia, lymphoma, lung cancer, breast cancer, prostate cancer, ovarian cancer, colon cancer, mesothelioma, renal cell carcinoma, and brain cancer.

[0191] In one embodiment, the method described herein further comprises administering an immune checkpoint inhibitor to a subject. In one embodiment, the immune checkpoint inhibitor targets (i) the interaction between PD-1 and PD-L1, or (ii) the interaction between CTLA-4 and CD80 or CD86. In one embodiment, the immune checkpoint inhibitor is an antibody or antibody fragment. In one embodiment, the antibody or antibody fragment targets PD-1, PD-L1 or CTLA-4.

[0192] In one embodiment, RNA encoding an IL2 variant polypeptide described herein, RNA encoding a peptide or protein comprising an epitope for inducing an immune response to an antigen in a subject, and optionally an immune checkpoint inhibitor are administered simultaneously or sequentially. BRIEF DESCRIPTION OF THE DRAWINGS

[0193] Figure 1 : In vitro expression of RNA-encoded IL2 variants and IL2Rβγ binding. 6 HEK293T / 17 cells were seeded in 6-well plates and lipofected with 3 μg mRNA (400 ng complexed mRNA / μL Lipofectamine MessengerMAX) in a total volume of 3.25 mL DMEM + 10% FBS after reaching about 80% confluence. After incubation for 20 hours at 37°C, 5% CO2, the supernatant was collected and serial dilutions thereof were incubated with the medium affinity IL2 receptor (IL2Rβγ) expressing human cell line TF-1_IL2Rβγ. Three days later, the cells were transfected with 1 μg mRNA (400 ng complexed mRNA / μL Lipofectamine MessengerMAX) in a total volume of 3.25 mL DMEM + 10% FBS. The assay measures the proliferative response by quantifying the amount of ATP in viable cells. Data shown are mean ± standard deviation (SD) of n = 2 technical replicates. RLU = relative luminescence unit.

[0194] Figure 2 : CD25 binding of IL2 mutCD25 variants. 100 ng of plate-bound recombinant human CD25 (A) or mouse CD25 (B) was incubated with 1:2 diluted supernatants containing IL2 variants from liposome transfections of HEK293T / 17, and bound proteins were detected by HRP-conjugated anti-human serum albumin antibody. Supernatants from HEK293T / 17 cells transfected with mRNA liposomes encoding only hAlb were used as negative controls. Data shown are mean ± SD of n = 2 technical replicates.

[0195] Figure 3 : Biological activity of IL2 mutCD25 variant in high affinity IL2 receptor (IL2Rαβγ) dependent cell culture. 高 Proliferative response of the mouse T cell line CTLL-2. Cells were incubated with serial dilutions of IL2 variant-containing supernatants for three days and the expression of IL2 variants was determined by using The assay measures proliferation by quantifying viable cells by ATP levels. Data shown are means ± SD of n = 2 to 3 technical replicates and were fitted with a four-parameter logistic fit to calculate EC 50 Value. RLU = relative luminescence unit.

[0196] Figure 4 :The biological activity of IL2 variants on different T cell subsets in human PBMC measured by the enhancement of IL2-mediated antigen nonspecific proliferation.CFSE-labeled PBMCs were incubated for 4 days with the anti-CD3 antibody (clone UCHT1) of suboptimal concentration and the serial dilutions containing IL2 variant supernatant.The proliferation of CD4+T cells (A and D), CD8+T cells (B and E) and CD56+NK cells (C and F) was measured by flow cytometry.The data from a representative donor are shown as the mean value of the amplification index calculated using FlowJo v10.4 software and fitted with four-parameter logarithmic fitting.Error bars (SD) indicate the variation within the experiment (three repetitions using the cells from a donor).

[0197] Figure 5 : Relative biological activity of different IL2 variants in medium affinity IL2 receptor (IL2Rβγ) and high affinity IL2 receptor (IL2Rαβγ) dependent cell culture. (A to B) The proliferation response of the medium affinity IL2 receptor (IL2Rβγ) expressing human cell line TF-1_IL2Rβγ and (C to D) the high affinity IL2 receptor (IL2Rαβγ) expressing mouse T cell line CTLL-2 is shown. Cell cultures were incubated with serial dilutions containing IL2 variant supernatants for three days and were measured by using The assay measures proliferation by quantifying viable cells by ATP levels. Data shown are means ± SD of n = 2 to 3 technical replicates and were fitted with a four-parameter logistic fit to calculate EC 50 Value. RLU = relative luminescence unit.

[0198] Figure 6 : Functional properties of IL2 variants on different T cell subsets in human PBMCs measured by IL2-mediated STAT5 phosphorylation. Dose-response curves of STAT5 phosphorylation (pSTAT5) on CD4+CD25+FoxP3+ regulatory T cells (A), CD4+CD25-T effector cells and memory cells (B), and CD8+ cytotoxic T cells (C). PBMCs were incubated with serial dilutions of IL2 variant supernatants, and STAT5 phosphorylation was subsequently analyzed in different T cell subsets by flow cytometry. The data shown are the mean ± SD of n = 2 technical replicates, and were fitted with a four-parameter logistic fit to calculate EC 50 value.

[0199] Figure 7 Effects of the IL2 mutCD25 variant on T cell vaccination in vivo

[0200] A, BALB / c mice (n=5 per group) were vaccinated intravenously (iv) with 20 μg RNA-LPX encoding CD8+T cell antigen gp70. Three days after vaccination, hAlb (negative control) or increasing doses of hAlb-hIL2, hAlb-hIL2_A4, hAlb-hIL2_A6 RNA formulated as lipid nanoparticles (LNP) were iv administered. Liver weight (B) is shown; aspartate aminotransferase (ASAT) activity in serum (C); gp70 antigen-specific CD8 positive cells (D), CD8 positive cells (E), CD45 positive cells (F) or CD4FoxP3CD25 positive cells (G) per μL of blood; and CD8 T cells to Treg ratio (H). Points represent individual mice and lines represent group averages. I, shows the fold change (mean + standard error of the mean (SEM)) of gp70-specific or nonspecific CD8-positive T cells relative to the respective mean hAlb control values ​​of treated mice. Statistical significance was determined using one-way ANOVA and Dunnett's multiple comparison test (B to H) or two-way ANOVA followed by Sidak's multiple comparison test (I). All analyses were two-tailed and performed using GraphPad Prism 6. ns: P>0.05, * P<0.05, ** P<0.01,*** P<0.001.

[0201] Figure 8 : Improving the potency of the IL2 mutCD25 variant by adding the mutβγ mutation

[0202] BALB / c mice (n=5 per group) were vaccinated intravenously (iv) with 20 μg RNA-LPX encoding CD8+T cell antigen gp70. Three days after vaccination, hAlb (negative control) or hAlb-hIL2_A4, hAlb-hIL2_A4s, hIL2_A6-hAlb and hAlb-hIL2_A6s RNA formulated as LNPs were administered iv. The frequencies of gp70 antigen-specific CD8 positive cells (A), CD4FoxP3CD25 positive cells (B) and CD4CD8 negative CD49b positive NK cells (D) are shown. Points represent individual mice and lines represent group averages. C, shows the fold changes (mean + SEM) of gp70-specific or nonspecific CD8 positive T cells relative to the respective average hAlb control values ​​of treated mice. Statistical significance was determined using one-way ANOVA and Dunnett's multiple comparison test (A, B, D) or two-way ANOVA followed by Sidak's multiple comparison test (C). All analyses were two-tailed and performed using GraphPad Prism 6. ns: P>0.05, * P<0.05, ** P<0.01, *** P<0.001.

[0203] Fig. 9 : IL2 variants with combined mutCD25 and mutβγ mutations outperform wild-type IL2 and mutβγ IL2 variants

[0204] BALB / c mice (n=5 per group) were vaccinated iv with 20 μg gp70 RNA-LPX on days 0 and 7 and with cytokine RNA-LNP (doses indicated in the figures) on days 3 and 10. Blood lymphocyte analysis was performed by flow cytometry (see Example 8) on days 7 and 14 (A). B, number of gp70 antigen-specific CD8-positive T cells per μL of blood (mean ± SEM). C, number of CD4 CD25 FoxP3-positive Tregs per μL of blood (mean ± SEM). D, number of CD8-positive T cells per μL of blood (mean ± SEM). The ratio of antigen-specific T cells (E) or CD8+T cells (F) to Tregs (mean ± SEM) is shown. G, the fold change of gp70-specific or non-specific CD8-positive T cells relative to the respective mean hAlb control values ​​of treated mice is shown (mean + SEM). H, shows the mean (± SEM) fraction of CD127-negative KLRG1 tetramer CD8-positive T cells. I, Number of CD49-positive CD4 CD8-negative NK cells per μL of blood (mean ± SEM). H, shows the fraction of KLRG1-positive NK cells per mouse (line represents group mean). Statistical significance was determined using two-way ANOVA and Dunnett's multiple comparison test (B to F, H, I) or Sidak test (G) or using one-way ANOVA followed by Tukey's multiple comparison test (J). All analyses were two-tailed and performed using GraphPad Prism6. ns: P>0.05, * P<0.05, ** P<0.01, *** P<0.001.

[0205] Fig.10 Administration of an IL2 variant with combined mutCD25 and mutβγ mutations enhances CD4+ T cell responses

[0206] C57BL / 6 mice (n=7 per group) were treated iv with 20 μg B16_M30 (Kreiter, S. et al. Nature 520, 692-696 (2015)) RNA-LPX and 3 μg hAlb, hAlb-hIL2 or hAlb-hIL2_A4s RNA-LNP on days 0, 7 and 14. Blood lymphocytes were analyzed by flow cytometry on day 19 (A). B16_M30 is a class II MHC restricted neoepitope of the B16F10 tumor cell line recognized by CD4+ T cells (Kreiter, S. et al. Nature 520, 692-696 (2015)). Only co-administration of hAlb-hIL2_A4s but not hAlb-hIL2 increased the number of CD4+ effector T cells / non-Treg (CD25-FoxP3-CD4+) and B16_M30-specific tetramer+CD4+ T cells, respectively (B, C). DETAILED DESCRIPTION

[0207] Although the present disclosure is described in detail below, it should be understood that the present disclosure is not limited to the specific methods, protocols and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing some specific embodiments, and are not intended to limit the scope of the present disclosure, which will only be limited by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those of ordinary skill in the art are generally understood.

[0208] Preferably, the terms used herein are as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", HGW Leuenberger, B. Nagel and H. ed., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0209] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology and recombinant DNA techniques, which are described in the literature in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0210] Hereinafter, the elements of the present disclosure will be described. These elements are listed together with some specific embodiments, however, it should be understood that they can be combined in any way and in any number to produce other embodiments. Some examples and embodiments of different descriptions should not be interpreted as limiting the present disclosure to only some embodiments clearly described. This specification should be understood to disclose and cover the embodiments that will clearly describe the embodiment and any number of disclosed elements combined. In addition, unless the context otherwise indicates, any arrangement and combination of all description elements should be considered to be disclosed by this specification.

[0211] The term "about" means approximately or close to, and in the context of a numerical value or range listed herein, means in one embodiment ±20%, ±10%, ±5%, or ±3% of the recited or claimed numerical value or range.

[0212] Unless otherwise noted herein or clearly contradictory to the context, otherwise the nouns modified by the use of quantifiers in the context of describing the present disclosure (especially in the context of the claims) should be interpreted as covering one / kind and / or more / kind. The recording of numerical ranges herein is only intended to be used as a shorthand method for referring to each individual value falling within the scope individually. Unless otherwise noted herein, each individual value is incorporated into this specification as it is recorded individually in this article. Unless otherwise noted herein or clearly contradictory to the context in addition, all methods described herein can be carried out in any suitable order. The use of any and all examples or exemplary language (such as "for example") provided herein is only intended to better illustrate the present disclosure, without limiting the scope of the claims. The language in this specification should not be interpreted as indicating any unclaimed elements necessary for the implementation of the present disclosure.

[0213] Unless explicitly stated otherwise, the term "includes / comprising" is used in the context of this document to indicate that other members may optionally be present in addition to the members of the list introduced by "includes / comprising". However, it is contemplated that as a specific embodiment of the present disclosure, the term "includes / comprising" encompasses the possibility that no other members are present, that is, for this purpose, the embodiment "includes / comprising" should be understood to have the meaning of "consisting of ..."

[0214] Several documents are cited throughout the text of this specification. Each document cited herein, whether above or below (including all patents, patent applications, scientific publications, manufacturer specifications, guidelines, etc.) is hereby incorporated by reference in its entirety. None of the content herein should be construed as admitting that the present disclosure is not entitled to predate such disclosure.

[0215] Definitions applicable to all aspects of the present disclosure are provided below. Unless otherwise indicated, the following terms have the following meanings. Any undefined term has its art-recognized meaning.

[0216] definition

[0217] As used herein, "reduce," "lower," or "inhibit" means an overall decrease in the level (e.g., binding level) or the ability to cause an overall decrease, preferably 5% or greater, 10% or greater, 20% or greater, more preferably 50% or greater, and most preferably 75% or greater.

[0218] Terms such as "increase" or "enhance" preferably relate to an increase or enhancement of about at least 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 100%, at least 200%, at least 500%, or even more.

[0219] According to the present disclosure, the term "peptide" includes oligopeptides and polypeptides, and refers to a substance comprising about two or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids connected to each other by peptide bonds. The term "protein" or "polypeptide" refers to large peptides, particularly peptides with at least about 50 amino acids, but the terms "peptide", "protein" and "polypeptide" are often used as synonyms herein.

[0220] A "therapeutic protein" has a positive or beneficial effect on the condition or disease state of a subject when provided to the subject in a therapeutically effective amount. In one embodiment, the therapeutic protein has curative or palliative properties and can be administered to improve, alleviate, mitigate, reverse, delay the onset of a disease or condition, or reduce the severity of one or more symptoms of a disease or condition. Therapeutic proteins may have preventive properties and may be used to delay the onset of a disease or reduce the severity of such a disease or pathological condition. The term "therapeutic protein" includes complete proteins or peptides, and may also refer to therapeutically active fragments thereof. It may also include therapeutically active variants of proteins. Some examples of therapeutically active proteins include, but are not limited to, cytokines.

[0221] For amino acid sequences (peptides or proteins), "fragment" refers to a part of the amino acid sequence, i.e., a sequence representing an amino acid sequence shortened at the N-terminus and / or the C-terminus. A fragment shortened at the C-terminus (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. A fragment shortened at the N-terminus (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains a start codon for initiating translation. A fragment of an amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues from the amino acid sequence. A fragment of an amino acid sequence preferably comprises at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50 or at least 100 consecutive amino acids from the amino acid sequence.

[0222] "Variant" or "variant protein" or "variant polypeptide" herein means a protein that differs from a wild-type protein due to at least one amino acid modification. A parent polypeptide may be a naturally occurring or wild-type (WT) polypeptide, or may be a modified form of a wild-type polypeptide. Preferably, a variant polypeptide has at least one amino acid modification compared to a parent polypeptide, for example, 1 to about 20 amino acid modifications compared to a parent polypeptide, and preferably 1 to about 10 or 1 to about 5 amino acid modifications.

[0223] As used herein, "parent polypeptide", "parent protein", "precursor polypeptide" or "precursor protein" refers to an unmodified polypeptide that is subsequently modified to produce a variant. The parent polypeptide may be a wild-type polypeptide, or a variant or engineered form of a wild-type polypeptide.

[0224] "Wild type" or "WT" or "native" herein means an amino acid sequence that occurs in nature, including allelic variations. A wild type protein or polypeptide has an amino acid sequence that has not been intentionally modified.

[0225] For the purposes of this disclosure, "variants" of an amino acid sequence (peptide, protein or polypeptide) include amino acid insertion variants, amino acid addition variants, amino acid deletion variants and / or amino acid substitution variants. The term "variant" includes all splice variants, post-translationally modified variants, conformers, isomers and species homologs, particularly those naturally expressed by cells.

[0226] Amino acid insertion variants include the insertion of a single or two or more amino acids in a specific amino acid sequence. In the case of an amino acid sequence variant with insertion, one or more amino acid residues are inserted into a specific site in the amino acid sequence, although random insertion and suitable screening of the resulting product are also possible. Amino acid addition variants include one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids of amino and / or carboxyl terminal fusions. Amino acid deletion variants are characterized by removing one or more amino acids from the sequence, for example, removing 1, 2, 3, 5, 10, 20, 30, 50 or more amino acids. The deletion can be in any position of the protein. Amino acid deletion variants comprising a deletion at the N-terminal and / or C-terminal end of the protein are also referred to as N-terminal and / or C-terminal truncated variants. Amino acid substitution variants are characterized by removing at least one residue in the sequence and inserting another residue in its position. It is preferred to consider modifications in positions in non-conserved amino acid sequences between homologous proteins or peptides and / or consider replacing amino acids with other amino acids with similar properties. Preferably, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., replacements of similar charged or uncharged amino acids. Conservative amino acid changes involve replacements of one of the amino acid families associated in their side chains. Naturally occurring amino acids are generally divided into four families: acidic amino acids (aspartic acid, glutamic acid); basic amino acids (lysine, arginine, histidine); non-polar amino acids (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar amino acids (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine, tryptophan and tyrosine are sometimes collectively classified as aromatic amino acids. In one embodiment, conservative amino acid replacements include replacements within the following groups:

[0227] Glycine, Alanine;

[0228] valine, isoleucine, leucine;

[0229] Aspartic acid, glutamic acid;

[0230] Asparagine, glutamine;

[0231] Serine, threonine;

[0232] lysine, arginine; and

[0233] Phenylalanine, tyrosine.

[0234] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence will be at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably given for an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the full length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180 or about 200 amino acids, preferably continuous amino acids. In some preferred embodiments, the degree of similarity or identity is given for the full length of the reference amino acid sequence. Comparisons for determining sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using optimal sequence alignment, for example, using Align, using standard settings, preferably EMBOSS::needle, matrix: Blosum62, gap open (Gap Open) 10.0, gap extension (Gap Extend) 0.5.

[0235] "Sequence similarity" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of identical amino acids between the sequences.

[0236] The term "percent identity" is intended to mean the percentage of identical amino acid residues between the two sequences to be compared obtained after optimal alignment, which percentage is purely statistical, and the differences between the two sequences are randomly distributed and randomly distributed over their entire length. The sequence comparison between two amino acid sequences is conventionally performed by comparing these sequences after optimal alignment thereof, the comparison being performed by segments or by a "comparison window" in order to identify and compare local areas of sequence similarity. Optimal alignment of sequences for comparison can be produced, in addition to manual work, by the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by the similarity search method of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444, or by computer programs that use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0237] The percent identity is calculated by determining the number of identical positions between the two sequences being compared, dividing that number by the number of positions being compared, and multiplying the result by 100 in order to obtain the percent identity between the two sequences.

[0238] According to the present disclosure, homologous amino acid sequences show at least 40%, particularly at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and preferably at least 95%, at least 98% or at least 99% identity of the amino acid residues.

[0239] The amino acid sequence variants described herein can be easily prepared by a skilled person, for example, by recombinant DNA manipulation. The manipulation of DNA sequences for preparing peptides or proteins with substitutions, additions, insertions or deletions is described in detail, for example, in Sambrook et al. (1989). In addition, the peptides and amino acid variants described herein can be easily prepared by means of known peptide synthesis techniques, for example, such as by solid phase synthesis and similar methods.

[0240] In one embodiment, a fragment or variant of an amino acid sequence (peptide or protein) is preferably a "functional fragment" or "functional variant". The term "functional fragment" or "functional variant" of an amino acid sequence relates to any fragment or variant that exhibits one or more functional properties that are identical or similar to the functional properties of the amino acid sequence from which the fragment or variant is derived (i.e., it is functionally equivalent). With respect to cytokines, one specific function is one or more immunomodulatory activities exhibited by the amino acid sequence from which the fragment or variant is derived and / or binding to a receptor to which the amino acid sequence from which the fragment or variant is derived binds.

[0241] An amino acid sequence (peptide, protein or polypeptide) "derived from" a specified amino acid sequence (peptide, protein or polypeptide) refers to the source of the first amino acid sequence. Preferably, the amino acid sequence derived from a specific amino acid sequence has an amino acid sequence that is identical, substantially identical or homologous to the specific sequence or a fragment thereof. The amino acid sequence derived from a specific amino acid sequence may be a variant of the specific sequence or a fragment thereof.

[0242] The term "polynucleotide" is interpreted broadly herein and includes DNA and RNA, including modified DNA and RNA.

[0243] In the present disclosure, the term "RNA" refers to a nucleic acid molecule comprising ribonucleotide residues. In some preferred embodiments, RNA comprises all or most of the ribonucleotide residues. "Ribonucleotide" as used herein refers to a nucleotide having a hydroxyl group at the 2'-position of a β-D-ribofuranosyl group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA (e.g., partially purified RNA), substantially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that is different from naturally occurring RNA by adding, deleting, replacing, and / or changing one or more nucleotides. Such a change may refer to adding a non-nucleotide substance to an internal RNA nucleotide or an RNA end. It is also contemplated herein that the nucleotides in the RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the present disclosure, these altered RNAs are considered to be analogs of naturally occurring RNA.

[0244] In certain embodiments of the present disclosure, RNA is a messenger RNA (mRNA) associated with an RNA transcript encoding a peptide or protein. As recognized in the art, mRNA generally comprises a 5' untranslated region (5'-UTR), a peptide coding region, and a 3' untranslated region (3'-UTR). In some embodiments, RNA is produced by in vitro transcription or chemical synthesis. In one embodiment, mRNA is produced by in vitro transcription using a DNA template, wherein DNA refers to a nucleic acid comprising deoxyribonucleotides.

[0245] In one embodiment, the RNA is an in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of a suitable DNA template. The promoter used to control transcription can be any promoter of any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning nucleic acid, particularly cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0246] In one embodiment, the RNA may have modified ribonucleotides. Some examples of modified ribonucleotides include, but are not limited to, 5-methylcytidine, pseudouridine, and / or 1-methyl-pseudouridine.

[0247] In some embodiments, the RNA according to the present disclosure comprises a 5'-cap. In one embodiment, the RNA of the present disclosure does not have an uncapped 5'-triphosphate. In one embodiment, the RNA may be modified by a 5'-cap analog. The term "5'-cap" refers to a structure found at the 5'-end of an mRNA molecule, and is typically composed of a guanosine nucleotide linked to the mRNA via a 5' to 5' triphosphate linkage. In one embodiment, the guanosine is methylated at position 7. Providing RNA with a 5'-cap or a 5'-cap analog can be achieved by in vitro transcription, wherein the 5'-cap is co-transcribed into the RNA chain, or can be linked to the RNA post-transcriptionally using a capping enzyme.

[0248] In some embodiments, the RNA according to the present disclosure comprises a 5'-UTR and / or a 3'-UTR. The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or to a corresponding region in an RNA molecule (e.g., an mRNA molecule). An untranslated region (UTR) may be present at the 5' (upstream) (5'-UTR) and / or the 3' (downstream) (3'-UTR) of an open reading frame. The 5'-UTR, if present, is located at the 5' end, upstream of the start codon of the protein coding region. The 5'-UTR is located downstream of the 5'-cap (if present), for example, directly adjacent to the 5' cap. The 3'-UTR, if present, is located at the 3' end, downstream of the stop codon of the protein coding region, but the term "3'-UTR" preferably does not include a poly (A) tail. Therefore, the 3'-UTR is located upstream of the poly (A) sequence (if present), for example, directly adjacent to the poly (A) sequence.

[0249] In some embodiments, the RNA according to the present disclosure comprises a 3'-poly (A) sequence. The term "poly (A) sequence" refers to a sequence of adenylic acid (A) residues that are typically located at the 3' end of an RNA molecule. According to the present disclosure, in one embodiment, the poly (A) sequence comprises at least about 20, at least about 40, at least about 80, or at least about 100, and up to about 500, up to about 400, up to about 300, up to about 200, or up to about 150 A nucleotides, and in particular about 120 A nucleotides.

[0250] In the context of the present disclosure, the term "transcription" refers to the process in which the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA can be translated into a peptide or protein.

[0251] With respect to RNA, the term "expression" or "translation" refers to the process in the ribosomes of a cell that directs the assembly of an amino acid sequence through its strand of mRNA to produce a peptide or protein.

[0252] According to the present disclosure, the term "RNA encoding" means that, if present in a suitable environment, such as within a cell of a target tissue, the RNA can guide the assembly of amino acids during translation to produce a peptide or protein encoded by it. In one embodiment, the RNA is capable of interacting with the cell translation machinery to allow translation of the peptide or protein. The cell can produce the encoded peptide or protein within the cell (e.g., in the cytoplasm and / or in the nucleus), can secrete the encoded peptide or protein, or can cause it to be produced on the surface.

[0253] As used herein, the terms "linked", "fused" or "fusion / fusion" are used interchangeably. These terms refer to the joining of two or more elements or components or domains.

[0254] "Half-life" as used herein refers to the time it takes for the serum or plasma concentration of a compound (e.g., a peptide or protein) to be reduced by 50% in vivo, for example, due to degradation and / or clearance or chelation by natural mechanisms. The interleukins (ILs) suitable for PK extension herein are stable in vivo, and their half-life is increased by, for example, fusion with serum albumin (e.g., HSA or MSA) that resists degradation and / or clearance or chelation. The half-life can be determined in any manner known per se, for example, by pharmacokinetic analysis. Suitable techniques will be obvious to those skilled in the art, and may, for example, generally include the following steps: appropriately administering a suitable dose of an amino acid sequence or compound to a subject; regularly collecting blood samples or other samples from the subject; determining the level or concentration of the amino acid sequence or compound in the blood sample; and calculating the time until the level or concentration of the amino acid sequence or compound is reduced by 50% compared to the initial level at the time of administration, based on the data (graphs) thus obtained. Further details are provided, for example, in standard handbooks, such as Kenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and in Peters et al., Pharmacokinetic Analysis: A Practical Approach (1996). Reference is also made to Gibaldi, M. et al., Pharmacokinetics, 2nd Rev. Edition, Marcel Dekker (1982).

[0255] Cytokines are a class of small proteins (about 5 to 20 kDa) that are important in cell signaling. Their release has an effect on the behavior of cells around them. Cytokines participate in autocrine signaling, paracrine signaling, and endocrine signaling as immunomodulators. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, but usually do not include hormones or growth factors (although the terms are somewhat overlapping). Cytokines are produced by a wide variety of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes, and mast cells, as well as endothelial cells, fibroblasts, and a variety of stromal cells. A given cytokine can be produced by more than one cell type. Cytokines act through receptors and are particularly important in the immune system; cytokines regulate the balance between humoral immune responses and cell-based immune responses, and they regulate the maturation, growth, and reactivity of specific cell populations. Some cytokines enhance or inhibit the effects of other cytokines in a complex manner.

[0256] Interleukin-2 (IL2) is a cytokine that induces the proliferation of antigen-activated T cells and stimulates natural killer (NK) cells. The biological activity of IL2 is mediated by the multi-subunit IL2 receptor complex (IL2R) of the following three polypeptide subunits across the cell membrane: p55 (IL2Rα, α subunit, also known as CD25 in humans), p75 (IL2Rβ, β subunit, also known as CD122 in humans), and p64 ​​(IL2Rγ, γ subunit, also known as CD132 in humans). The response of T cells to IL2 depends on multiple factors, including: (1) the concentration of IL2; (2) the number of IL2R molecules on the cell surface; and (3) the number of IL2Rs occupied by IL2 (i.e., the affinity of the binding interaction between IL2 and IL2R (Smith, "Cell Growth Signal Transduction is Quantal" In Receptor Activation by Antigens, Cytokines, Hormones, and Growth Factors 766:263-271, 1995)). The IL2:IL2R complex is internalized after ligand binding, and the different components are differentially sorted. When administered as an intravenous (iv) bolus, IL2 has rapid systemic clearance (an initial clearance phase with a half-life of 12.9 minutes, followed by a slower clearance phase with a half-life of 85 minutes) (Konrad et al., Cancer Res. 50:2009-2017, 1990).

[0257] In eukaryotic cells, human IL2 is synthesized as a 153 amino acid precursor polypeptide from which 20 amino acids are removed to generate mature secreted IL2. Recombinant human IL2 has been produced in Escherichia coli (E. coli), insect cells, and mammalian COS cells.

[0258] The results of systemic IL2 administration in cancer patients are far from ideal. Although 15% to 20% of patients objectively respond to high-dose IL2, most patients do not respond, and many patients suffer from serious, life-threatening side effects, including nausea, confusion, hypotension, and septic shock. Attempts have been made to reduce serum concentrations by reducing the dose and adjusting the dosing regimen, and although the toxicity is low, the effect of such treatment is also minimal.

[0259] According to the present disclosure, in certain embodiments, the IL2 variant polypeptide described herein comprises a pharmacokinetic modifying group. In one embodiment, the IL2 variant part or mutant protein described herein is connected to a pharmacokinetic modifying group. Relative to free IL2, the resulting molecule (hereinafter referred to as "IL2 with extended pharmacokinetic (PK)") has an extended circulation half-life. The extended circulation half-life of the IL2 extended by PK maintains the concentration of serum IL2 in the body within the therapeutic range, thereby likely causing the activation of many types of immune cells (including T cells) to be enhanced. Due to the favorable pharmacokinetic profile of the IL2 extended by PK, it can be administered with a lower frequency and last longer than unmodified IL2.

[0260] As used herein, "human IL2" or "wild-type human IL2" means IL2 (whether native or recombinant) having the normally occurring 133 amino acid sequence of native human IL2 (minus the signal peptide, which consists of an additional 20 N-terminal amino acids), the amino acid sequence of which is described in Fujita, et.al, PNAS USA, 80, 7437-7441 (1983), with or without an additional N-terminal methionine, which must be included when the protein is expressed as an intracellular part in E. coli. In one embodiment, human IL2 comprises the amino acid sequence of SEQ ID NO: 17. In one embodiment, the functional variant of human IL2 comprises an amino acid sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 17. In one embodiment, the functional variant of human IL2 binds to the IL2 receptor, in particular the alpha subunit of the IL2 receptor.

[0261] In certain embodiments described herein, the IL2 variant portion or mutant protein is fused to a heterologous polypeptide (i.e., a polypeptide that is not IL2 and preferably is not a variant of IL2). The heterologous polypeptide can increase the circulating half-life of IL2. As discussed in further detail below, the polypeptide that increases the circulating half-life can be serum albumin, such as human or mouse serum albumin.

[0262] As used herein, "IL2 mutant protein" means a variant of IL2 (including its functional variant), in particular a polypeptide in which a specific replacement has been made to the IL2 protein. In one embodiment, the human IL2 protein is replaced at least at a position in contact with the α subunit of the αβγ IL2 receptor complex (IL2Rαβγ). In one embodiment, such a position has an acidic or basic amino acid residue in wild-type human IL2, wherein if the amino acid residue is an acidic amino acid residue in wild-type human IL2, the replacement is replaced by a basic amino acid residue, and if the amino acid residue is a basic amino acid residue in wild-type human IL2, the replacement is replaced by an acidic amino acid residue. Some particularly preferred embodiments include the following: a lysine (Lys) residue at position 35, a lysine (Lys) residue at position 43, a glutamic acid (Glu) residue at position 61, and a glutamic acid (Glu) residue at position 62, or any combination thereof, relative to wild-type human IL2 and numbered according to wild-type human IL2.

[0263] The IL2 mutant protein may have the same amino acid sequence as wild-type IL2 at other unsubstituted residues (i.e., the IL2 mutant protein comprises a "mutCD25" mutation, such as those wherein the sequence of any one of SEQ ID NOs: 2 to 6 differs from the sequence of SEQ ID NO: 17). However, the IL2 mutant protein may also be characterized by amino acid insertions, deletions, substitutions and modifications at one or more sites in or at other residues of the native IL2 polypeptide chain. According to the present invention, any such insertions, deletions, substitutions and modifications may produce an IL2 mutant protein that retains affinity for IL2Rβγ while having reduced affinity for IL2Rαβγ.

[0264] For example, the IL2 mutant protein may also be characterized by amino acid substitutions at one or more sites in or at other residues of the native IL2 polypeptide chain, such amino acid substitutions result in, for example, a relatively increased affinity for IL2Rβγ compared to wild-type IL2, such that IL2-mediated stimulation no longer requires the participation of IL2Rα (i.e., the IL2 mutant protein comprises a "mutβγ" mutation in addition to the mutCD25 mutation, e.g., those mutations in which the sequence of SEQ ID NO: 18 differs from the sequence of SEQ ID NO: 17). Such mutants are potent IL2 signaling agonists. These mutations may be at amino acid residues that contact IL2Rβ and / or IL2Rγ.

[0265] In various embodiments, the IL2 muteins described herein may differ from wild-type IL2 by replacing one or more of the residues at positions 24, 65, 74, 80, 81, 85, 86, 89, 92, and 93 of wild-type IL2. The replaced amino acid residues may be, but are not necessarily, conservative replacements.

[0266] For example, the mutations may be: I24V, P65H, Q74R, Q74H, Q74N, Q74S, L80F, L80V, R81I, R81T, R81D, L85V, I86V, I89V, I92F, V93I.

[0267] In one embodiment, an IL2 mutant protein is provided, wherein the mutant protein comprises the following amino acid substitution group: 80F / 81D / 85V / 86V / 92F. The mutant protein may also comprise the amino acid substitution 42A. The mutant protein may also comprise one or more of the following amino acid substitutions: 24V, 65H, 74R, 74H, 74N, 74S, 89V, 93I.

[0268] In some embodiments, an IL2 mutant protein is provided, wherein the mutant protein comprises an amino acid substitution group selected from the following:

[0269] (i) 74N, 80F, 81D, 85V, 86V, 89V, 92F;

[0270] (ii)74H, 80F, 81D, 85V, 86V, 92F;

[0271] (iii)74S, 80F, 81D, 85V, 86V, 92F;

[0272] (iv) 74N, 80F, 81D, 85V, 86V, 92F;

[0273] (v)80F, 81D, 85V, 86V, 92F;

[0274] (vi)80F, 81D, 85V, 86V, 89V, 92F, 93I;

[0275] (vii) 18R, 22E, 80F, 81D, 85V, 86V, 89V, 92F, 93I, 126T;

[0276] (viii)18R, 22E, 74S, 80F, 81T, 85V, 86V, 89V, 92F, 93I, 126T.

[0277] By "numbering according to wild-type IL2" we mean that the selected amino acid is identified with reference to the position in which the amino acid normally occurs in the mature sequence of wild-type IL2. In the case of insertions or deletions made to an IL2 mutant protein, those skilled in the art will appreciate that an amino acid normally present at a particular position may shift position in the mutant protein. However, the position of the shifted amino acid can be readily determined by inspection and correlation of the flanking amino acids with those flanking the amino acid in wild-type IL2.

[0278] The IL2 variant polypeptides described herein and the polynucleotides encoding them can be produced by any suitable method known in the art. Such methods include introducing appropriate nucleotide changes into the nucleic acid encoding IL2 or by in vitro synthesis of IL2 polynucleotides or proteins. For example, a DNA sequence encoding the IL2 variant polypeptides described herein can be constructed, and those sequences can be expressed in a suitably transformed host or in any other suitable expression system. The method will produce the IL2 variant polypeptides described herein and / or the RNA encoding them. However, the IL2 variant polypeptides described herein and the polynucleotides encoding them can also be produced by chemical synthesis, although less preferred.

[0279] The IL2 variant polypeptides described herein may bind to IL2Rαβγ with a lower affinity than the affinity with which wild-type IL2 binds to IL2Rαβγ. In one embodiment, the IL2 variant polypeptides described herein may bind to IL2Rβγ with a greater affinity than the affinity with which wild-type IL2 binds to IL2Rβγ.

[0280] The affinity of the IL2 variant polypeptides described herein for IL2Rαβγ may be at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold lower than the affinity of wild-type IL2 for binding to IL2Rαβγ. In addition, the affinity of the IL2 variant polypeptides described herein for IL2Rβγ may be at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold greater than the affinity of wild-type IL2 for binding to IL2Rβγ.

[0281] The IL2 variant polypeptides described herein may have an affinity for IL2Rβγ that is at least 2-fold, at least 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold greater than their affinity for IL2Rαβγ.

[0282] The IL2 variant polypeptides described herein may have reduced ability to stimulate regulatory T cells compared to wild-type IL2, particularly when compared to the ability to stimulate effector T cells and / or NK cells.

[0283] The IL2 variant polypeptides described herein may have mutations (e.g., deletions, additions or substitutions) of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid residues relative to wild-type IL2.

[0284] The IL2 variant polypeptides described herein may comprise an amino acid sequence that is at least about 50%, at least about 65%, at least about 70%, at least about 80%, at least about 85%, at least about 87%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or at least about 99% identical to wild-type IL2.

[0285] In one embodiment, the IL2 variant polypeptides described herein have one or more, preferably all, of the following properties:

[0286] 1) Agonist effect on IL2Rβγ. This property can be directly assessed in in vitro proliferation assays of IL2-dependent cell lines.

[0287] 2) Loss of the ability to stimulate regulatory T cell populations in vitro and / or in vivo compared to wild-type IL2. This property can be assessed, for example, by studying the ability of the mutant protein to induce regulatory T cell expansion compared to a mutant protein of wild-type IL2.

[0288] 3) Improved therapeutic effects relative to native IL2 in animal models. This property can be evaluated, for example, by comparing the anti-tumor or anti-metastatic effects of the IL2 variant polypeptides described herein with wild-type IL2 as monotherapy in transplantable tumor models (e.g., B16 melanoma). It can also be evaluated by the enhancement of the cellular and / or humoral responses to the target vaccine.

[0289] When mounting an immune response (including eliciting CD8 T cells), many immune cells transiently upregulate IL2Rαβγ after activation to increase IL2 sensitivity. Since some IL2Rαβγ may be necessary for binding by IL2, the present invention envisions the use of a mixture of IL2Rβγ selective IL2 variant polypeptides described herein in combination with IL2 (including functional variants thereof) (e.g., wild-type IL2) that does not exhibit a preferential affinity for IL2Rβγ. In certain embodiments, the molar ratio of the IL2Rβγ-selective IL2 variant polypeptides described herein to the IL2 that does not exhibit a preferential affinity for IL2Rβγ is 50:1 to 1:1, 20:1 to 2:1, 10:1 to 5:1, or 5:1 to 3:1.

[0290] The IL2 variant polypeptides described herein can be prepared as fusion polypeptides or chimeric polypeptides comprising an IL2 variant portion and a heterologous polypeptide (i.e., a polypeptide that is not IL2 or a variant thereof). The IL2 variant can be fused to a PK-extended group, which increases the circulating half-life. Some non-limiting examples of PK-extended groups are described below. It should be understood that other PK groups that increase the circulating half-life of cytokines or variants thereof are also applicable to the present disclosure. In certain embodiments, the PK-extended group is a serum albumin domain (e.g., mouse serum albumin, human serum albumin).

[0291] The term "PK" as used herein is an acronym for "pharmacokinetics" and encompasses the properties of a compound including, for example, absorption, distribution, metabolism, and clearance by a subject. As used herein, a "PK-extended group" refers to a protein, peptide, or moiety that increases the circulating half-life of a biologically active molecule when fused to or administered together with a biologically active molecule. Some examples of PK-extended groups include serum albumin (e.g., HSA), Fc or Fc fragments and variants thereof, transferrin and variants thereof, and human serum albumin (HSA) binders (as disclosed in U.S. Publication Nos. 2005 / 0287153 and 2007 / 0003549). Other exemplary PK-extended groups are disclosed in Kontermann, Current Opinion in Biotechnology 2011; 22: 868-876 and Kontermann, Expert Opin Biol Ther. 2016; 16: 903-15, which are incorporated herein by reference in their entirety. As used herein, "PK-extended IL" refers to an interleukin (IL) portion (including an IL variant portion) combined with a PK-extended group. In one embodiment, the PK-extended IL is a fusion protein in which the IL portion is linked or fused to the PK-extended group. An exemplary fusion protein is an HSA / IL2 fusion in which the IL2 portion is fused to HSA.

[0292] In certain embodiments, relative to the serum half-life of a single IL (i.e., an IL not fused to a PK-extended group), the serum half-life of the IL extended by PK is improved. In certain embodiments, relative to the serum half-life of a single IL, the serum half-life of the IL extended by PK is at least 20%, 40%, 60%, 80%, 100%, 120%, 150%, 180%, 200%, 400%, 600%, 800% or 1000% longer. In certain embodiments, the serum half-life of the IL extended by PK is at least 1.5 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 6 times, 7 times, 8 times, 10 times, 12 times, 13 times, 15 times, 17 times, 20 times, 22 times, 25 times, 27 times, 30 times, 35 times, 40 times or 50 times longer than the serum half-life of a single IL. In certain embodiments, the serum half-life of the PK-extended IL is at least 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200 hours.

[0293] In certain embodiments, the PK extended group includes serum albumin or a fragment thereof or a variant of serum albumin or a fragment thereof (all of which are included by the term "albumin" for the purposes of this disclosure). The polypeptides described herein can be fused with albumin (or a fragment or variant thereof) to form an albumin fusion protein. Such albumin fusion proteins are described in U.S. Publication No. 20070048282.

[0294] As used herein, "albumin fusion protein" refers to a protein formed by fusing at least one albumin molecule (or a fragment or variant thereof) to at least one protein molecule, such as a therapeutic protein, particularly IL2 (or a variant thereof). The albumin fusion protein can be produced by translating a nucleic acid in which a polynucleotide encoding a therapeutic protein is linked in frame to a polynucleotide encoding an albumin. The therapeutic protein and the albumin (which was once part of the albumin fusion protein) can each be referred to as a "portion", "region" or "moiety" of the albumin fusion protein (e.g., a "therapeutic protein portion" or "albumin protein portion"). In a highly preferred embodiment, the albumin fusion protein comprises at least one therapeutic protein molecule (including but not limited to a mature form of a therapeutic protein) and at least one albumin molecule (including but not limited to a mature form of albumin). In one embodiment, the albumin fusion protein is processed by a host cell (e.g., a cell of a target organ (e.g., a hepatocyte)) for the administered RNA and secreted into the circulation. Processing of the nascent albumin fusion protein that occurs in the secretory pathway of the host cell used to express the RNA may include, but is not limited to: signal peptide cleavage; disulfide bond formation; proper folding; addition and processing of carbohydrates (e.g., such as N- and O-linked glycosylation); specific proteolytic cleavages; and / or assembly into multimeric proteins. The albumin fusion protein is preferably encoded by the RNA in a non-processed form, particularly with a signal peptide at its N-terminus, and after secretion from the cell is preferably present in a processed form, wherein particularly the signal peptide has been cleaved off. In a most preferred embodiment, a "processed form of the albumin fusion protein" refers to an albumin fusion protein product that has undergone cleavage of the N-terminal signal peptide, also referred to herein as a "mature albumin fusion protein".

[0295] In some preferred embodiments, the albumin fusion protein comprising a therapeutic protein has a higher plasma stability compared to the plasma stability of the same therapeutic protein not fused to albumin. Plasma stability generally refers to the period of time between when a therapeutic protein is administered in vivo and carried into the bloodstream and when the therapeutic protein is degraded and cleared from the bloodstream to an organ (e.g., kidney or liver) that ultimately clears the therapeutic protein from the body. Plasma stability is calculated based on the half-life of the therapeutic protein in the bloodstream. The half-life of a therapeutic protein in the bloodstream can be readily determined by conventional assays known in the art.

[0296] As used herein, "albumin" refers collectively to an albumin protein or amino acid sequence having one or more functional activities (e.g., biological activity) of albumin, or an albumin fragment or variant. In particular, "albumin" refers to human albumin or a fragment or variant thereof, in particular the mature form of human albumin, or albumin or a fragment thereof from other vertebrates, or a variant of these molecules. Albumin may be derived from any vertebrate, in particular any mammal, such as humans, cattle, sheep or pigs. Non-mammalian albumins include, but are not limited to, hens and salmon. The albumin portion of the albumin fusion protein may be from an animal different from the therapeutic protein portion.

[0297] In certain embodiments, the albumin is human serum albumin (HSA) or a fragment or variant thereof, such as those disclosed in US 5,876,969, WO 2011 / 124718, WO 2013 / 075066 and WO 2011 / 0514789.

[0298] The terms human serum albumin (HSA) and human albumin (HA) are used interchangeably herein. The terms "albumin" and "serum albumin" are broader and encompass human serum albumin (and fragments and variants thereof) as well as albumins (and fragments and variants thereof) from other species.

[0299] As used herein, an albumin fragment sufficient to prolong the therapeutic activity or plasma stability of a therapeutic protein refers to an albumin fragment whose length or structure is sufficient to stabilize or prolong the therapeutic activity or plasma stability of the protein, such that the plasma stability of the therapeutic protein portion of the albumin fusion protein is prolonged or extended compared to the plasma stability in the non-fused state.

[0300] The albumin portion of the albumin fusion protein may comprise the full length of the albumin sequence, or may comprise one or more fragments thereof that are capable of stabilizing or prolonging therapeutic activity or plasma stability. Such fragments may be 10 or more amino acids in length, or may comprise about 15, 20, 25, 30, 50 or more consecutive amino acids from the albumin sequence, or may comprise part or all of a particular domain of albumin. For example, one or more fragments of HSA that span the first two immunoglobulin-like domains may be used. In a preferred embodiment, the HSA fragment is a mature form of HSA.

[0301] Generally, an albumin fragment or variant will be at least 100 amino acids long, preferably at least 150 amino acids long.

[0302] According to the present disclosure, the albumin may be a naturally occurring albumin or a fragment or variant thereof. The albumin may be human albumin and may be derived from any vertebrate, in particular any mammal. In one embodiment, the albumin comprises the amino acid sequence of SEQ ID NO: 21 or an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 21.

[0303] Preferably, the albumin fusion protein comprises albumin as the N-terminal portion and comprises a therapeutic protein as the C-terminal portion. Alternatively, an albumin fusion protein comprising albumin as the C-terminal portion and comprising a therapeutic protein as the N-terminal portion may also be used. In other embodiments, the albumin fusion protein has a therapeutic protein fused to both the N-terminus and the C-terminus of the albumin. In a preferred embodiment, the therapeutic protein fused at the N-terminus and the C-terminus is the same therapeutic protein. In another preferred embodiment, the therapeutic proteins fused at the N-terminus and the C-terminus are different therapeutic proteins. In one embodiment, the different therapeutic proteins can be used to treat or prevent the same or related diseases, disorders or conditions. In one embodiment, the different therapeutic proteins are all cytokines, wherein one of the different therapeutic proteins is an IL2 variant, and the other is preferably an interferon, such as IFNβ. In one embodiment, the albumin fusion protein has IFNβ fused to the N-terminus of the albumin and an IL2 variant fused to the C-terminus of the albumin.

[0304] In one embodiment, the therapeutic protein is linked to the albumin via a peptide linker. The linker peptide between the fused parts can provide greater physical separation between the parts and thus maximize the accessibility of the therapeutic protein parts, for example, for binding to its binding receptor. The linker peptide can be composed of amino acids so that it is flexible or more rigid. The linker sequence can be cleaved by proteases or chemically.

[0305] The term "Fc region" as used herein refers to a portion of a natural immunoglobulin formed by the respective Fc domains (or Fc parts) of its two heavy chains. The term "Fc domain" as used herein refers to a portion or fragment of a single immunoglobulin (Ig) heavy chain, wherein the Fc domain does not include an Fv domain. In certain embodiments, the Fc domain begins in the hinge region just upstream of the papain cleavage site and terminates at the C-terminus of the antibody. Therefore, a complete Fc domain includes at least a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments, the Fc domain includes at least one of the following: a hinge (e.g., an upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, a CH4 domain, or a variant, part, or fragment thereof. In certain embodiments, the Fc domain includes a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In certain embodiments, the Fc domain includes a hinge domain (or a part thereof) fused to a CH3 domain (or a part thereof). In certain embodiments, the Fc domain comprises a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof). In certain embodiments, the Fc domain consists of a CH3 domain or a portion thereof. In certain embodiments, the Fc domain consists of a hinge domain (or a portion thereof) and a CH3 domain (or a portion thereof). In certain embodiments, the Fc domain consists of a CH2 domain (or a portion thereof) and a CH3 domain (or a portion thereof). In certain embodiments, the Fc domain consists of a hinge domain (or a portion thereof) and a CH2 domain (or a portion thereof). In certain embodiments, the Fc domain lacks at least a portion of the CH2 domain (e.g., all or part of the CH2 domain). The Fc domain herein generally refers to a polypeptide comprising all or part of the Fc domain of an immunoglobulin heavy chain. This includes, but is not limited to, polypeptides comprising complete CH1, hinge, CH2 and / or CH3 domains, and fragments of such peptides comprising only, for example, hinge, CH2 and CH3 domains. The Fc domain can be derived from any species and / or any subtype of immunoglobulin, including but not limited to: human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE or IgM antibodies. The Fc domain covers natural Fc and Fc variant molecules. As described herein, it will be understood by those of ordinary skill in the art that any Fc domain can be modified so that its amino acid sequence is different from the natural Fc domain of the naturally occurring immunoglobulin molecule. In certain embodiments, the Fc domain has reduced effector function (e.g., FcγR binding).

[0306] The Fc domain of the polypeptide described herein may be derived from different immunoglobulin molecules. For example, the Fc domain of the polypeptide may include a CH2 and / or CH3 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, the Fc domain may include a chimeric hinge region derived in part from an IgG1 molecule and in part from an IgG3 molecule. In another example, the Fc domain may include a chimeric hinge region derived in part from an IgG1 molecule and in part from an IgG4 molecule.

[0307] In certain embodiments, the PK extended group comprises an Fc domain or a fragment thereof or a variant of an Fc domain or a fragment thereof (all of which are included by the term "Fc domain" for the purposes of this disclosure). The Fc domain does not comprise a variable region that binds to an antigen. Fc domains suitable for use in this disclosure can be obtained from many different sources. In certain embodiments, the Fc domain is derived from human immunoglobulin. In certain embodiments, the Fc domain is from a human IgG1 constant region. However, it should be understood that the Fc domain can be derived from immunoglobulins of other mammalian species, including, for example, rodents (e.g., mice, rats, rabbits, guinea pigs) or non-human primates (e.g., chimpanzees, macaques) species.

[0308] Furthermore, the Fc domain (or fragment or variant thereof) may be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and may be derived from any immunoglobulin isotype, including IgGl, IgG2, IgG3, and IgG4.

[0309] A variety of Fc domain gene sequences (e.g., mouse and human constant region gene sequences) are all available in the form of publicly available deposits. Constant region domains comprising Fc domain sequences lacking specific effector functions and / or having specific modifications to reduce immunogenicity can be selected. The sequences of many antibodies and antibody encoding genes have been published, and suitable Fc domain sequences (e.g., hinge, CH2 and / or CH3 sequences or fragments or variants thereof) can be obtained from these sequences using techniques recognized in the art.

[0310] In certain embodiments, the PK extended group is a serum albumin binding protein, such as those described in US2005 / 0287153, US2007 / 0003549, US2007 / 0178082, US2007 / 0269422, US2010 / 0113339, WO2009 / 083804 and WO2009 / 133208, which are incorporated herein by reference in their entirety. In certain embodiments, the PK extended group is transferrin, such as disclosed in US 7,176,278 and US 8,158,579, which are incorporated herein by reference in their entirety. In certain embodiments, the PK extended group is a serum immunoglobulin binding protein, such as those disclosed in US2007 / 0178082, which are incorporated herein by reference in their entirety. In certain embodiments, the PK extended group is a fibronectin (Fn) based scaffold domain protein that binds to serum albumin, such as those disclosed in US2012 / 0094909, which is incorporated herein by reference in its entirety. Methods for preparing fibronectin based scaffold domain proteins are also disclosed in US2012 / 0094909. A non-limiting example of a PK extended group based on Fn3 is Fn3 (HSA), i.e., an Fn3 protein that binds to human serum albumin.

[0311] In certain aspects, PK-extended ILs suitable for use according to the present disclosure may employ one or more peptide linkers. As used herein, the term "peptide linker" refers to a peptide or polypeptide sequence that connects two or more domains (e.g., a PK-extended portion and an IL portion, such as IL2) in a linear amino acid sequence of a polypeptide chain. For example, a peptide linker can be used to connect an IL2 portion to an HSA domain.

[0312] The linker that is suitable for the group that prolongs PK and for example IL2 is fused is well known in the art. Some exemplary linkers include glycine-serine-peptide linkers, glycine-proline-peptide linkers and proline-alanine peptide linkers. In certain embodiments, the linker is a glycine-serine-peptide linker, i.e. a peptide consisting of glycine and serine residues.

[0313] In addition to or in place of the above-mentioned heterologous polypeptides, the IL2 variant polypeptides described herein may comprise a sequence encoding a "marker" or "reporter". Some examples of marker or reporter genes include β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase, dihydrofolate reductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidine kinase (TK), β-galactosidase, and xanthine guanine phosphoribosyltransferase (XGPRT).

[0314] Peptide and protein antigens suitable for use according to the present disclosure generally include peptides or proteins comprising epitopes for inducing an immune response. The peptide or protein or epitope may be derived from a target antigen, i.e., an antigen against which an immune response is elicited. For example, a peptide or protein antigen or an epitope contained in the peptide or protein antigen may be a target antigen or a fragment or variant of the target antigen.

[0315] The peptide and protein antigens (either by themselves or as RNA encoding the peptide and protein antigens), i.e., vaccine antigens, administered according to the present disclosure preferably cause stimulation, priming and / or amplification of T cells in the subject to which the antigen is administered. The T cells stimulated, primed and / or amplified are preferably directed against a target antigen, in particular a target antigen expressed by a diseased cell, tissue and / or organ, i.e., a disease-associated antigen. Therefore, the vaccine antigen may comprise a disease-associated antigen or a fragment or variant thereof. In one embodiment, such a fragment or variant is immunologically equivalent to a disease-associated antigen. In the context of the present disclosure, the term "fragment of an antigen" or "variant of an antigen" means an agent that causes stimulation, priming and / or amplification of T cells, which stimulate, prime and / or amplify T cells targeting antigens, i.e., disease-associated antigens, in particular when presented by diseased cells, tissues and / or organs. Therefore, the vaccine antigen administered according to the present disclosure may correspond to or may comprise a disease-associated antigen, may correspond to or may comprise a fragment of a disease-associated antigen, or may correspond to or may comprise an antigen homologous to a disease-associated antigen or a fragment thereof. If the vaccine antigen administered according to the present disclosure comprises a fragment of a disease-associated antigen or an amino acid sequence homologous to a fragment of a disease-associated antigen, the fragment or amino acid sequence may comprise an epitope (e.g., a T cell epitope of a disease-associated antigen) or a sequence homologous to an epitope (e.g., a T cell epitope of a disease-associated antigen). Therefore, the antigen administered according to the present disclosure may comprise an immunogenic fragment of a disease-associated antigen or an amino acid sequence homologous to an immunogenic fragment of a disease-associated antigen. An "immunogenic fragment of an antigen" according to the present disclosure preferably relates to a fragment of an antigen that can stimulate, elicit and / or amplify T cells when present in the presence of an MHC molecule. It is preferred that the vaccine antigen (similar to the disease-associated antigen) can be presented by a cell, such as an antigen presenting cell, thereby providing the relevant epitope for being bound by a T cell. The vaccine antigen administered according to the present disclosure may be a recombinant antigen.

[0316] The term "immunologically equivalent" means, for example, immunologically equivalent molecules, such as immunologically equivalent amino acid sequences, that exhibit the same or substantially the same immunological properties and / or exert the same or substantially the same immunological effects, for example with respect to the type of immune effector. In the context of the present disclosure, the term "immunologically equivalent" is preferably used with respect to the immunological effects or properties of an antigen or antigenic variant for immunization. For example, if an amino acid sequence induces an immune response with specificity that reacts with a reference amino acid sequence when exposed to the immune system of a subject (e.g., a T cell that binds to a reference amino acid sequence or a cell that expresses the reference amino acid sequence), the amino acid sequence is immunologically equivalent to the reference amino acid sequence. Therefore, a molecule that is immunologically equivalent to an antigen exhibits the same or substantially the same properties and / or exerts the same or substantially the same effect as the antigen targeted by the T cell in terms of stimulation, priming and / or amplification of T cells.

[0317] The term "priming" refers to the process in which a T cell first comes into contact with its specific antigen and results in differentiation into an effector T cell.

[0318] The term "clonal expansion" or "amplification" refers to a process in which a specific entity is amplified. In the context of the present disclosure, the term is preferably used in the context of an immune response in which lymphocytes are stimulated by an antigen, proliferate, and amplify specific lymphocytes that recognize the antigen. Preferably, clonal expansion results in lymphocyte differentiation.

[0319] The term "antigen" refers to a substance comprising an epitope to which an immune response can be generated. In particular, the term "antigen" includes proteins and peptides. In one embodiment, the antigen is presented by cells of the immune system (e.g., antigen presenting cells such as dendritic cells or macrophages). In one embodiment, an antigen or its processed product, such as a T cell epitope, binds to an antibody through a T or B cell receptor or through an immunoglobulin molecule such as an antibody. Therefore, an antigen or its processed product can react specifically with an antibody or a T lymphocyte (T cell). In one embodiment, the antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, and the epitope is derived from such an antigen.

[0320] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule that contains an epitope that stimulates the host's immune system to produce a cellular antigen-specific immune response and / or a humoral antibody response to the disease. Thus, a disease-associated antigen or an epitope thereof may be used for therapeutic purposes. A disease-associated antigen may be associated with infection with a microorganism (usually a microbial antigen) or may be associated with a cancer (usually a tumor).

[0321] The term "tumor antigen" refers to a component of a cancer cell, which may be derived from the cytoplasm, cell surface, and nucleus. In particular, it refers to those antigens produced in cells or produced as surface antigens on tumor cells. Tumor antigens are usually preferentially expressed by cancer cells (e.g., compared to non-cancerous cells, they are expressed at a higher level in cancer cells), and in some cases, they are only expressed by cancer cells. Some examples of tumor antigens include, but are not limited to: p53, ART-4, BAGE, β-catenin / m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, cell surface proteins of the claudin family such as claudin-6, claudin-18.2, and claudin-12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap 100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE- A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11 or MAGE-A12, MAGE-B, MAGE-C, MART-1 / Melan-A, MC1R, Myosphere MUC1, MUM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 small BCR-abL, Pml / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, survivin, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, WT, and WT-1.

[0322] The term "viral antigen" refers to any viral component that has antigenic properties, ie, is capable of eliciting an immune response in an individual. A viral antigen may be a viral ribonucleoprotein or an envelope protein.

[0323] The term "bacterial antigen" refers to any bacterial component that has antigenic properties, ie, is capable of eliciting an immune response in an individual. Bacterial antigens may be derived from the bacterial cell wall or cytoplasmic membrane.

[0324] The term "epitope" refers to a part or fragment of a molecule (e.g., an antigen) that is recognized by the immune system. For example, an epitope can be recognized by a T cell, a B cell, or an antibody. The epitope of an antigen can comprise a continuous or discontinuous portion of the antigen, and the length can be from about 5 to about 100, for example, from about 5 to about 50, more preferably from about 8 to about 30, most preferably from about 10 to about 25 amino acids, for example, the length of the epitope can be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids. In one embodiment, the length of the epitope is from about 10 to about 25 amino acids. The term "epitope" comprises a T cell epitope.

[0325] The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by a T cell when present in the context of an MHC molecule. The term "major histocompatibility complex" and the abbreviation "MHC" include class I MHC and class II MHC molecules, and relate to a gene complex present in all vertebrates. MHC proteins or molecules are important for signal transduction between lymphocytes and antigen presenting cells or diseased cells in an immune response, wherein MHC proteins or molecules bind peptide epitopes and present them to be recognized by T cell receptors on T cells. Proteins encoded by MHC are expressed on the cell surface, and both autoantigens (peptide fragments from the cell itself) and non-autoantigens (e.g., fragments of invading microorganisms) are displayed to T cells. In the context of class I MHC / peptide complexes, the binding peptide is typically about 8 to about 10 amino acids long, although longer or shorter peptides may be effective. In the case of a class II MHC / peptide complex, the binding peptide is typically about 10 to about 25 amino acids in length, and particularly about 13 to about 18 amino acids in length, although longer or shorter peptides may be effective.

[0326] The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+T cells) and cytotoxic T cells (CTL, CD8+T cells) including cell lytic T cells. The term "antigen-specific T cell" or similar terms relate to T cells that recognize antigens targeted by T cells, particularly when presented on the surface of antigen presenting cells or diseased cells (e.g., cancer cells) in the case of MHC molecules, and preferably exert the effector function of T cells. If T cells kill target cells expressing antigens, it is believed that T cells have specificity for antigens. Any of a variety of standard techniques can be used to evaluate T cell specificity, for example, in chromium release analysis or proliferation assays. Alternatively, the synthesis of lymphokines (e.g., interferon-γ) can be measured.

[0327] "Regulatory T cells" or "Tregs" are T cell subsets that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune diseases. Tregs are immunosuppressive and typically inhibit or downregulate the induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FoxP3, and CD25.

[0328] The term "naive T cell" as used herein refers to a mature T cell that has not been exposed to its cognate antigen in the periphery, as opposed to an activated T cell or a memory T cell. Naive T cells are typically characterized by surface expression of L-selectin (CD62L), the absence of activation markers CD25, CD44 or CD69, and the absence of the memory CD45RO isoform.

[0329] The term "memory T cells" as used herein refers to a subset or subpopulation of T cells that have previously been exposed to and responded to their cognate antigen. Upon a second exposure to an antigen, memory T cells can replicate to produce an immune response that is faster and stronger than the first time the immune system responded to the antigen. Memory T cells can be CD4 + or CD8 + And usually express CD45RO.

[0330] As used herein, the term "NK cell" or "natural killer cell" refers to a subset of peripheral blood lymphocytes defined by the expression of CD56 or CD16 and the absence of the T cell receptor (CD3). As provided herein, NK cells can also be differentiated from stem cells or progenitor cells.

[0331] In one embodiment, the target antigen is a tumor antigen, and the peptide or protein comprising an epitope or a fragment thereof (e.g., an epitope) is derived from a tumor antigen. Tumor antigens can be "standard" antigens, which are generally known to be expressed in a variety of cancers. Tumor antigens can also be "neoantigens," which are specific to individual tumors and have not been previously recognized by the immune system. New antigens or new epitopes can be caused by one or more cancer-specific mutations that cause amino acid changes in the genome of cancer cells. If the tumor antigen is a new antigen, the peptide or protein comprising the epitope preferably comprises an epitope or fragment of the new antigen containing one or more amino acid changes.

[0332] Cancer mutations vary from individual to individual. Therefore, cancer mutations of encoded new epitopes (neo-epitopes) represent attractive targets in the development of vaccine compositions and immunotherapy. The effectiveness of tumor immunotherapy depends on the selection of cancer-specific antigens and epitopes that can induce a potent immune response in the host. RNA can be used to deliver patient-specific tumor epitopes to patients. Dendritic cells (DCs) present in the spleen represent antigen-presenting cells that are particularly interested in RNA expression of immunogenic epitopes or antigens (e.g., tumor epitopes). The use of multiple epitopes has been shown to promote the therapeutic efficacy in tumor vaccine compositions. Rapid sequencing of tumor mutation groups can provide multiple epitopes for personalized vaccines, which can be encoded by RNA as described herein, for example, as a single polypeptide, in which epitopes are optionally separated by joints. In certain embodiments of the present disclosure, RNA encodes at least one epitope, at least two epitopes, at least three epitopes, at least four epitopes, at least five epitopes, at least six epitopes, at least seven epitopes, at least eight epitopes, at least nine epitopes, or at least ten epitopes. Some exemplary embodiments include RNAs encoding at least five epitopes (referred to as "pentatopes") and RNAs encoding at least ten epitopes (referred to as "decatopes").

[0333] Peptide and protein antigens can be 2 to 100 amino acids, including, for example, 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids in length. In some embodiments, the peptide can be greater than 50 amino acids. In some embodiments, the peptide can be greater than 100 amino acids.

[0334] The peptide or protein antigen may be any peptide or protein that can induce or enhance the ability of the immune system to produce antibodies and T cells in response to the peptide or protein.

[0335] In certain embodiments, immune checkpoint inhibitors are used in combination with other therapeutic agents described herein (e.g., RNA encoding an interleukin (IL)-2 variant polypeptide and, optionally, RNA encoding a peptide or protein comprising an epitope).

[0336] As used herein, "immune checkpoints" refer to costimulatory and inhibitory signals that regulate the magnitude and quality of T cell receptor recognition of antigens. In certain embodiments, immune checkpoints are inhibitory signals. In certain embodiments, the inhibitory signal is the interaction between PD-1 and PD-L1. In certain embodiments, the inhibitory signal is the interaction between CTLA-4 and CD80 or CD86 to replace CD28 binding. In certain embodiments, the inhibitory signal is the interaction between LAG3 and class II MHC molecules. In certain embodiments, the inhibitory signal is the interaction between TIM3 and galectin 9.

[0337] "Immune checkpoint inhibitors" used herein refer to molecules that completely or partially reduce, inhibit, interfere with or regulate one or more checkpoint proteins. In certain embodiments, immune checkpoint inhibitors prevent inhibitory signals associated with immune checkpoints. In certain embodiments, immune checkpoint inhibitors are antibodies or fragments thereof that destroy inhibitory signaling associated with immune checkpoints. In certain embodiments, immune checkpoint inhibitors are small molecules that destroy inhibitory signaling. In certain embodiments, immune checkpoint inhibitors are antibodies, fragments thereof, or antibody mimetics that prevent the interaction between checkpoint blocker proteins, for example, antibodies or fragments thereof that prevent the interaction between PD-1 and PD-L1. In certain embodiments, immune checkpoint inhibitors are antibodies or fragments thereof that prevent the interaction between CTLA-4 and CD80 or CD86. In certain embodiments, immune checkpoint inhibitors are antibodies or fragments thereof that prevent the interaction between LAG3 and its ligand or TIM-3 and its ligand. Checkpoint inhibitors can also be in the form of soluble forms of molecules (or their variants) themselves, such as soluble PD-L1 or PD-L1 fusions.

[0338] The "Programmed Death-1 (PD-1)" receptor refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is mainly expressed on previously activated T cells in vivo and binds to two ligands (PD-L1 and PD-L2). The term "PD-1" as used herein includes human PD-1 (hPD-1), variants, isoforms and species homologs of hPD-1, and analogs having at least one common epitope with hPD-1.

[0339] "Programmed Death Ligand-1 (PD-L1)" is one of the two cell surface glycoprotein ligands of PD-1 (the other is PD-L2), which downregulates T cell activation and cytokine secretion when bound to PD-1. The term "PD-L1" as used herein includes human PD-L1 (hPD-L1), variants, isoforms and species homologs of hPD-L1, and analogs that have at least one common epitope with hPD-L1.

[0340] "Cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4)" is a T-cell surface molecule and a member of the immunoglobulin superfamily. The protein downregulates the immune system by binding to CD80 and CD86. The term "CTLA-4" as used herein includes human CTLA-4 (hCTLA-4), variants, isoforms and species homologs of hCTLA-4, and analogs that have at least one common epitope with hCTLA-4.

[0341] "Lymphocyte Activation Gene-3 (LAG3)" is an inhibitory receptor associated with the inhibition of lymphocyte activity by binding to class II MHC molecules. This receptor enhances the function of Treg cells and inhibits CD8+ effector T cell function. The term "LAG3" as used herein includes human LAG3 (hLAG3), variants, isoforms and species homologs of hLAG3, and analogs having at least one common epitope.

[0342] "T cell membrane protein-3 (TIM3)" is an inhibitory receptor involved in the inhibition of lymphocyte activity by inhibiting TH1 cell responses. Its ligand is galectin 9, which is upregulated in many types of cancer. The term "TIM3" as used herein includes human TIM3 (hTIM3), variants, isoforms and species homologs of hTIM3, and analogs having at least one common epitope.

[0343] The "B7 family" refers to inhibitory ligands with undefined receptors. The B7 family encompasses B7-H3 and B7-H4, both of which are upregulated on tumor cells and tumor-infiltrating cells.

[0344] In certain embodiments, immune checkpoint inhibitors suitable for use in the methods disclosed herein are antagonists of inhibitory signals, such as antibodies targeting, for example, PD-1, PD-L1, CTLA-4, LAG3, B7-H3, B7-H4, or TIM3. These ligands and receptors are reviewed in Pardoll, D., Nature. 12: 252-264, 2012.

[0345] In certain embodiments, an immune checkpoint inhibitor is an antibody or antigen binding portion thereof that disrupts or inhibits signaling from an inhibitory immunomodulator. In certain embodiments, an immune checkpoint inhibitor is a small molecule that disrupts or inhibits signaling from an inhibitory immunomodulator.

[0346] In certain embodiments, the inhibitory immunomodulator is a component of the PD-1 / PD-L1 signaling pathway. Therefore, certain embodiments of the present disclosure provide for administering to a subject an antibody or an antigen binding portion thereof that disrupts the interaction between the PD-1 receptor and its ligand PD-L1. Antibodies that bind to PD-1 and disrupt the interaction between PD-1 and its ligand PD-L1 are known in the art. In certain embodiments, the antibody or its antigen binding portion specifically binds to PD-1. In certain embodiments, the antibody or its antigen binding portion specifically binds to PD-L1 and inhibits its interaction with PD-1, thereby increasing immune activity.

[0347] In certain embodiments, the inhibitory immunomodulator is a component of the CTLA4 signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administering to a subject an antibody, or antigen-binding portion thereof, that targets CTLA4 and disrupts its interaction with CD80 and CD86.

[0348] In certain embodiments, the inhibitory immunomodulator is a component of the LAG3 (lymphocyte activation gene 3) signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administering to a subject an antibody, or antigen-binding portion thereof, that targets LAG3 and disrupts its interaction with class II MHC molecules.

[0349] In certain embodiments, the inhibitory immunomodulator is a component of the B7 family signaling pathway. In certain embodiments, the B7 family members are B7-H3 and B7-H4. Therefore, certain embodiments of the present disclosure provide for administering to a subject an antibody or antigen binding portion thereof that targets B7-H3 or H4. The B7 family does not have any defined receptors, but these ligands are upregulated on tumor cells or tumor infiltrating cells. Preclinical mouse models have shown that blocking these ligands can enhance anti-tumor immunity.

[0350] In certain embodiments, the inhibitory immunomodulator is a component of the TIM3 (T cell membrane protein 3) signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administering to a subject an antibody or antigen binding portion thereof that targets TIM3 and disrupts its interaction with galectin 9.

[0351] One of ordinary skill in the art will appreciate that other immune checkpoint targets may also be targeted by antagonists or antibodies, provided that such targeting results in stimulation of an immune response, such as an anti-tumor immune response, as reflected in, for example, increased T cell proliferation, enhanced T cell activation, and / or increased cytokine (e.g., IFN-γ, IL2) production.

[0352] According to the present disclosure, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies and chimeric antibodies. Each heavy chain consists of a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated as VL herein) and a light chain constant region. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conservative regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (CIq) of the classical complement system.

[0353] Antibodies can be derived from different species including, but not limited to, mouse, rat, rabbit, guinea pig, and human.

[0354] The antibodies described herein include IgA (e.g., IgA1 or IgA2), IgG1, IgG2, IgG3, IgG4, IgE, IgM, and IgD antibodies. In various embodiments, the antibody is an IgG1 antibody, more particularly an IgG1, κ or IgG1, λ isotype (i.e., IgG1, κ, λ); an IgG2a antibody (e.g., IgG2a, κ, λ); an IgG2b antibody (e.g., IgG2b, κ, λ); an IgG3 antibody (e.g., IgG3, κ, λ) or an IgG4 antibody (e.g., IgG4, κ, λ).

[0355] The term "antigen-binding portion" of an antibody (or simply "binding portion") or "antigen-binding fragment" of an antibody (or simply "binding fragment") or similar terms refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Some examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of the VH domain; (vi) isolated complementarity determining regions (CDRs) and (vii) a combination of two or more isolated CDRs, which may optionally be linked by a synthetic linker. In addition, although the two domains VL and VH of the Fv fragment are encoded by independent genes, they can be connected by synthetic linkers using recombinant methods so that they can be made as a single protein chain, in which the VL and VH regions are paired to form a monovalent molecule (called single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. Another example is a binding domain immunoglobulin fusion protein comprising (i) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to the hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to the CH2 constant region. The binding domain polypeptide can be a heavy chain variable region or a light chain variable region. Binding domain immunoglobulin fusion proteins are further disclosed in US 2003 / 0118592 and US 2003 / 0133939. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for use in the same manner as complete antibodies.

[0356] According to the present invention, it is particularly preferred that the peptides, proteins or polypeptides described herein, in particular IL2 variant polypeptides and / or antigens, are administered in the form of RNA encoding the peptides, proteins or polypeptides described herein. In one embodiment, different peptides, proteins or polypeptides described herein are encoded by different RNA molecules.

[0357] According to the present disclosure, after administering the RNA described herein, at least a portion of the RNA is delivered to the target cell. In one embodiment, at least a portion of the RNA is delivered to the cytosol of the target cell. In one embodiment, the RNA is translated by the target cell to produce the encoded peptide or protein.

[0358] Some aspects of the present disclosure relate to targeted delivery of the RNA disclosed herein (RNA encoding an IL-2 variant polypeptide and optionally RNA encoding a peptide or protein comprising an epitope) to certain tissues.

[0359] In one embodiment, the disclosure relates to targeting the lymphatic system, in particular secondary lymphoid organs, more particularly the spleen. If the administered RNA is an RNA encoding a peptide or protein comprising an epitope, targeting the lymphatic system, in particular secondary lymphoid organs, more particularly the spleen is particularly preferred.

[0360] In one embodiment, the target cell is a spleen cell. In one embodiment, the target cell is an antigen presenting cell, such as a professional antigen presenting cell in the spleen. In one embodiment, the target cell is a dendritic cell in the spleen.

[0361] The "lymphatic system" is part of the circulatory system and is an important part of the immune system, containing a network of lymphatic vessels that transport lymph. The lymphatic system consists of lymphatic organs, a conducting network of lymphatic vessels, and circulating lymph. Primary or central lymphoid organs produce lymphocytes from immature progenitor cells. The thymus and bone marrow constitute the primary lymphoid organs. Secondary or peripheral lymphoid organs (including lymph nodes and spleen) maintain mature initial lymphocytes and initiate adaptive immune responses.

[0362] RNA can be delivered to the spleen by so-called lipoplex formulations, in which RNA is combined with liposomes comprising cationic lipids and optionally additional or helper lipids to form injectable nanoparticle formulations. Liposomes can be obtained by injecting a lipid solution in ethanol into water or a suitable aqueous phase. RNA lipoplex particles can be prepared by mixing liposomes with RNA. Spleen targeting RNA lipoplex particles is described in WO 2013 / 143683, which is incorporated herein by reference. It has been found that RNA lipoplex particles with a net negative charge can be used to preferentially target spleen tissue or spleen cells, such as antigen presenting cells, particularly dendritic cells. Therefore, after administration of RNA lipoplex particles, RNA accumulation and / or RNA expression occur in the spleen. Therefore, the RNA lipoplex particles of the present disclosure can be used to express RNA in the spleen. In one embodiment, after administration of RNA lipoplex particles, there is no or substantially no RNA accumulation and / or RNA expression in the lungs and / or liver. In one embodiment, RNA accumulation and / or RNA expression occurs in antigen presenting cells (e.g., professional antigen presenting cells) in the spleen following administration of the RNA lipid complex particles. Thus, the RNA lipid complex particles of the present disclosure can be used to express RNA in such antigen presenting cells. In one embodiment, the antigen presenting cells are dendritic cells and / or macrophages.

[0363] In the context of the present disclosure, the term "RNA lipid complex particle" relates to a particle comprising a lipid (particularly a cationic lipid) and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA lead to complexation and spontaneous formation of RNA lipid complex particles. Positively charged liposomes can typically be synthesized using a cationic lipid (e.g., DOTMA) and an additional lipid (e.g., DOPE). In one embodiment, the RNA lipid complex particle is a nanoparticle.

[0364] "Cationic lipid" as used herein refers to a lipid with a net positive charge. Cationic lipids are bound to negatively charged RNA via electrostatic interactions with lipid matrices. Generally speaking, cationic lipids have a lipophilic portion, such as a sterol, an acyl group, or a diacyl chain, and the head group of the lipid generally carries a positive charge. Some examples of cationic lipids include, but are not limited to: 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), dimethylbis(dioctadecyl)ammonium (DDAB), 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 1,2-diacyloxy-3-dimethylammonium propane, 1,2-dialkoxy-3-dimethylammonium propane, dioctadecyl dimethyl ammonium chloride (DODAC), 2,3 -di-tetradecyloxypropyl-(2-hydroxyethyl)-dimethylammonium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-trifluoroacetic acid propylammonium (DOSPA). Preferred are DOTMA, DOTAP, DODAC and DOSPA. In some specific embodiments, the cationic lipid is DOTMA and / or DOTAP.

[0365] Additional lipids may be incorporated to adjust the overall positive-negative charge ratio and the physical stability of the RNA lipid complex particles. In certain embodiments, the additional lipids are neutral lipids. "Neutral lipids" as used herein refer to lipids with a net charge of zero. Some examples of neutral lipids include, but are not limited to, 1,2-di-(9Z-octadecenoyl)-sn-glyceryl-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glyceryl-3-phosphocholine (DOPC), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, and cerebrosides. In some specific embodiments, additional lipids are DOPE, cholesterol, and / or DOPC.

[0366] In certain embodiments, the RNA lipid complex particle comprises both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

[0367] In some embodiments, the molar ratio of at least one cationic lipid to at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In some specific embodiments, the molar ratio can be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of at least one cationic lipid to at least one additional lipid is about 2:1.

[0368] In one embodiment, the RNA lipid complex particles described herein have an average diameter of about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 250 to about 700 nm, about 400 to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm. In some specific embodiments, the average diameter of the RNA lipid complex particles is about 200nm, about 225nm, about 250nm, about 275nm, about 300nm, about 325nm, about 350nm, about 375nm, about 400nm, about 425nm, about 450nm, about 475nm, about 500nm, about 525nm, about 550nm, about 575nm, about 600nm, about 625nm, about 650nm, about 700nm, about 725nm, about 750nm, about 775nm, about 800nm, about 825nm, about 850nm, about 875nm, about 900nm, about 925nm, about 950nm, about 975nm, or about 1000nm. In one embodiment, the average diameter of the RNA lipid complex particles is about 250nm to about 700nm. In another embodiment, the average diameter of the RNA lipid complex particles is about 300 nm to about 500 nm. In an exemplary embodiment, the average diameter of the RNA lipid complex particles is about 400 nm.

[0369] The charge of the RNA lipid complex particle of the present disclosure is the sum of the charge present in the at least one cationic lipid and the charge present in the RNA. The charge ratio is the ratio of the positive charge present in the at least one cationic lipid to the negative charge present in the RNA. The charge ratio of the positive charge present in the at least one cationic lipid to the negative charge present in the RNA is calculated by the following equation: Charge ratio = [(cationic lipid concentration (mol)) * (total number of positive charges in cationic lipid)] / [(RNA concentration (mol)) * (total number of negative charges in RNA)].

[0370] The spleen-targeted RNA lipid complex particles described herein preferably have a net negative charge at physiological pH, for example, a charge ratio of positive charge to negative charge of about 1.9: 2 to about 1: 2. In some specific embodiments, the charge ratio of positive charge to negative charge in the RNA lipid complex particles at physiological pH is about 1.9: 2.0, about 1.8: 2.0, about 1.7: 2.0, about 1.6: 2.0, about 1.5: 2.0, about 1.4: 2.0, about 1.3: 2.0, about 1.2: 2.0, about 1.1: 2.0, or about 1: 2.0.

[0371] RNA delivery systems have an inherent preference for the liver. This involves lipid-based particles, cationic and neutral nanoparticles, especially lipid nanoparticles in bioconjugates, such as liposomes, nanomicelles, and lipophilic ligands. Liver accumulation is caused by the discontinuous nature of the liver vasculature or lipid metabolism (liposomes and lipid or cholesterol conjugates).

[0372] In one embodiment of the targeted delivery of IL2 variant polypeptides described herein, the target organ is the liver and the target tissue is liver tissue. In particular, if it is desired that the IL2 variant polypeptides be present in the organ or tissue and / or if it is desired to express a large amount of IL2 variant polypeptides and / or if it is desired or required that the IL2 variant polypeptides be present systemically, particularly in significant amounts, then such target tissues are preferably delivered.

[0373] In one embodiment, RNA encoding IL2 variant polypeptides is administered in a formulation for targeting the liver. Such formulations are described above.

[0374] In order to deliver RNA to the liver in vivo, a drug delivery system can be used to transport RNA to the liver by preventing it from being degraded. For example, the composite nano micelle composed of a surface coated with poly (ethylene glycol) (PEG) and a core comprising mRNA is a useful system because the nano micelle provides excellent RNA stability in vivo under physiological conditions. In addition, the stealth characteristics provided by the composite nano nano micelle surface consisting of dense PEG fences effectively circumvents host immune defense.

[0375] The peptides, proteins, polypeptides, RNA, RNA particles, and other agents described herein (e.g., immune checkpoint inhibitors) can be administered as pharmaceutical compositions or medicaments for therapeutic or prophylactic treatment, and can be administered in the form of any suitable pharmaceutical composition.

[0376] The term "pharmaceutical composition" refers to a formulation comprising a therapeutically effective agent, preferably a therapeutically effective agent together with a pharmaceutically acceptable carrier, diluent and / or excipient. The pharmaceutical composition can be used to treat, prevent or alleviate the severity of a disease or condition by administering the pharmaceutical composition to a subject. Pharmaceutical compositions are also referred to in the art as pharmaceutical preparations. In the context of the present disclosure, a pharmaceutical composition comprises a peptide, protein, polypeptide, RNA, RNA particle and / or other agents described herein.

[0377] The pharmaceutical compositions of the present disclosure may comprise one or more adjuvants or may be administered with one or more adjuvants. The term "adjuvant" relates to a compound that prolongs, enhances or accelerates an immune response. Adjuvants include heterogeneous groups of compounds such as oil emulsions (e.g., Freund's adjuvant), mineral compounds (e.g., alum), bacterial products (e.g., Bordetella pertussis toxin), or immunostimulatory complexes. Some examples of adjuvants include, but are not limited to: LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, chemokines. Chemokines can be IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IFNα, IFNγ, GM-CSF, LT-a. Other known adjuvants are aluminum hydroxide, Freund's adjuvant, or oils, such as ISA 51. Other suitable adjuvants for use in the present disclosure include lipopeptides, such as Pam3Cys.

[0378] The pharmaceutical compositions according to the present disclosure are generally administered in a "pharmaceutically effective amount" and a "pharmaceutically acceptable formulation."

[0379] The term "pharmaceutically acceptable" refers to the non-toxicity of a substance that does not interact with the effects of the active ingredients of the pharmaceutical composition.

[0380] The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount that achieves a desired response or desired effect alone or in combination with another dose. In the case of treating a specific disease, the desired response preferably involves inhibition of the disease process. This includes slowing the progression of the disease, and in particular interrupting or reversing the progression of the disease. The desired response in the treatment of a disease may also be to delay the occurrence of the disease or the condition or to prevent its occurrence. The effective amount of the composition described herein will depend on: the condition to be treated, the severity of the disease, the individual parameters of the patient including age, physiological condition, body size and weight, the duration of treatment, the type of concomitant treatment (if any), the specific route of administration and similar factors. Therefore, the dosage of the composition described herein may depend on a variety of such parameters. In the case of an insufficient response of the patient with the initial dose, a higher dose may be used (or an effectively higher dose may be achieved by a different, more localized route of administration).

[0381] The pharmaceutical compositions of the present disclosure may comprise salts, buffers, preservatives, and optionally other therapeutic agents.In one embodiment, the pharmaceutical compositions of the present disclosure comprise one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0382] Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0383] The term "excipient" as used herein refers to a substance that may be present in a pharmaceutical composition of the present disclosure but is not an active ingredient. Some examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

[0384] The term "diluent" refers to a diluting agent and / or a thinning agent. In addition, the term "diluent" includes any one or more of a fluid, a liquid or solid suspension and / or a mixed medium. Some examples of suitable diluents include ethanol, glycerol and water.

[0385] The term "carrier" refers to a component that can be natural, synthetic, organic, inorganic, in which the active ingredients are combined to promote, enhance or achieve the use of the pharmaceutical composition. The carrier used herein can be one or more compatible solid or liquid fillers, diluents or encapsulating materials suitable for application to the object. Suitable carriers include, but are not limited to: sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes and particularly biocompatible lactide polymers, lactide / glycolide copolymers or polyoxyethylene / polyoxypropylene copolymers. In one embodiment, the pharmaceutical composition of the present disclosure comprises isotonic saline.

[0386] Pharmaceutically acceptable carriers, excipients or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro edit. 1985).

[0387] The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0388] In one embodiment, the pharmaceutical composition described herein can be administered intravenously, intra-arterially, subcutaneously, intradermally or intramuscularly. In certain embodiments, the pharmaceutical composition is formulated for topical or systemic administration. Systemic administration may include enteral administration involving absorption through the gastrointestinal tract, or parenteral administration. "Parenteral administration" used herein refers to administration in any manner except through the gastrointestinal tract, such as by intravenous injection. In a preferred embodiment, the pharmaceutical composition is formulated for systemic administration. In another preferred embodiment, systemic administration is administered intravenously.

[0389] The term "co-administration" used herein means a process in which different compounds or compositions (e.g., RNA encoding IL2 variant polypeptides, RNA encoding peptides or proteins comprising epitopes, and optionally immune checkpoint inhibitors) are applied to the same patient. RNA encoding IL2 variant polypeptides and RNA encoding peptides or proteins comprising epitopes can be applied simultaneously, substantially simultaneously or sequentially. If the application is performed sequentially, the RNA encoding IL2 variant polypeptides can be applied before or after the RNA encoding the peptide or protein comprising the epitope is applied. If the application is performed simultaneously, it is not necessary to apply the RNA encoding IL2 variant polypeptides and the RNA encoding the peptide or protein comprising the epitope in the same composition. The RNA encoding IL2 variant polypeptides and the RNA encoding the peptide or protein comprising the epitope can be applied once or more times, and the number of applications of each component can be the same or different. In addition, it is not necessary to apply the RNA encoding IL2 variant polypeptides and the RNA encoding the peptide or protein comprising the epitope at the same site.

[0390] The IL2 variant polypeptides, polynucleotides encoding IL2 variant polypeptides, host cells comprising polynucleotides encoding IL2 variant polypeptides, pharmaceutical compositions and methods of treatment described herein can be used for a variety of diseases, particularly therapeutic or prophylactic treatments of diseases (e.g., cancer, autoimmune diseases, infectious diseases) in which IL2, particularly IL2 variant polypeptides described herein, are provided to subjects, particularly for therapeutic or prophylactic treatments of diseases (e.g., cancer, autoimmune diseases, infectious diseases), vaccine adjuvants in cancer vaccines and conventional vaccine treatments for immune stimulation in elderly individuals or other immunocompromised individuals and HIV or human SCID patients, or other therapeutic applications requiring general stimulation of the immune system in any suitable animal (preferably mammals, most preferably humans). IL2 has many effects. Some of these are stimulating T cells, particularly memory T cells, naive T cells and / or effector T cells and / or NK cells. The IL2 variant polypeptides described herein will be active against cell types (e.g., memory T cells, naive T cells and / or effector T cells) that only express medium affinity IL2 receptors, rather than cell types (e.g., regulatory T cells) that express high affinity IL2 receptors. Thus, the IL2 variant polypeptides, polynucleotides encoding IL2 variant polypeptides, host cells comprising polynucleotides encoding IL2 variant polypeptides, pharmaceutical compositions, and methods of treatment described herein are contemplated for use in treating those diseases in which IL2 is expected to provide effective treatment due to its T cell activity.

[0391] Alternatively, or in addition to the method directly applied to the patient, in some embodiments, the IL2 variant polypeptide can be used in an ex vivo method. For example, cells can be cultured in vitro in a culture medium (e.g., peripheral blood lymphocytes or purified lymphocyte groups separated and placed or maintained in a culture from a patient), and the contact step can be affected by adding IL2 variant polypeptides and / or polynucleotides encoding them to the culture medium. The culture step may include other steps, wherein the cells are stimulated or treated with other reagents, such as to stimulate proliferation or amplify a cell group that is reactive to a target antigen (e.g., a cancer antigen or a viral antigen). The cells are then applied to the patient after being treated.

[0392] The term "disease" refers to an abnormal condition that affects an individual's body. Disease is generally interpreted as a medical condition associated with specific symptoms and signs. Disease may be caused by factors that are initially from an external source, such as an infectious disease, or the disease may be caused by an internal dysfunction, such as an autoimmune disease. In humans, "disease" is generally used more broadly to refer to any condition that causes pain, dysfunction, suffering, social problems or death in the sick individual or similar problems in those who come into contact with the individual. In this broader sense, disease sometimes includes injury, disability, disorder, syndrome, infection, isolated symptoms, abnormal behavior, and atypical changes in structure and function, while in other cases and for other purposes, these may be considered as distinguishable categories. Diseases generally affect individuals not only physically but also emotionally, because infection and experience with many diseases can change an individual's perspective on life and the individual's personality.

[0393] In the context of the present invention, the term "treatment" or "therapeutic intervention" relates to the management and care of a subject for the purpose of combating a condition, such as a disease or disorder. The term is intended to include the full spectrum of treatments for a given condition suffered by a subject, such as the administration of a therapeutically effective compound to alleviate symptoms or complications, delay the progression of a disease, disorder or condition, alleviate or relieve symptoms and complications, and / or cure or eliminate a disease, disorder or condition as well as prevention of a condition, wherein prevention is understood as the management and care of an individual for the purpose of combating a disease, disorder or condition, and includes the administration of an active compound to prevent the onset of symptoms or complications.

[0394] The term "therapeutic treatment" refers to any treatment that improves health and / or prolongs (increases) the life span of an individual. The treatment may eliminate the disease in an individual, prevent or slow the onset of the disease in an individual, inhibit or slow the onset of the disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce recurrence in an individual currently suffering from or previously suffering from the disease.

[0395] The term "prophylactic treatment" or "preventive therapy" relates to any treatment intended to prevent the occurrence of a disease in an individual. The terms "prophylactic treatment" or "preventive treatment" are used interchangeably herein.

[0396] The terms "individual" and "subject" are used interchangeably herein. They refer to humans or other mammals (e.g., mice, rats, rabbits, dogs, cats, cattle, pigs, sheep, horses, or primates) that may be afflicted with a disease or condition (e.g., cancer) or are susceptible to a disease or condition (e.g., cancer) but may or may not be suffering from a disease or condition. In many embodiments, the individual is a human. Unless otherwise indicated, the terms "individual" and "subject" do not indicate a specific age, and therefore encompass adults, elderly people, children, and newborns. In some embodiments of the present disclosure, an "individual" or "subject" is a "patient".

[0397] The term "patient" refers to an individual or subject being treated, particularly an individual or subject who is ill.

[0398] In one embodiment of the present disclosure, the goal is to provide an immune response against diseased cells expressing antigens, such as cancer cells expressing tumor antigens, and treat diseases, such as cancer diseases, involving cells expressing antigens, such as tumor antigens.

[0399] A pharmaceutical composition comprising RNA encoding a peptide or protein comprising an epitope can be administered to a subject to induce an immune response in the subject to an antigen comprising the epitope, which can be therapeutic or partially or completely protective. Those skilled in the art will appreciate that one of the principles of immunotherapy and vaccination is based on the fact that an immunoprotective response to a disease is produced by immunizing a subject with an antigen or epitope that is immunologically associated with the disease to be treated. Therefore, the pharmaceutical composition described herein can be applied to induce or enhance an immune response. Therefore, the pharmaceutical composition described herein can be used for preventive and / or therapeutic treatment of diseases involving antigens or epitopes.

[0400] As used herein, "immune response" refers to an integrated body response to an antigen or a cell expressing an antigen, and refers to a cellular immune response and / or a humoral immune response. A cellular immune response includes, but is not limited to, a cellular response to cells expressing an antigen and characterized by presenting the antigen with a class I or class II MHC molecule. Cellular responses are associated with T lymphocytes, which can be classified as helper T cells (also known as CD4+T cells), which play a major role in inducing apoptosis in infected cells or cancer cells by regulating immune responses or killing cells (also known as cytotoxic T cells, CD8+T cells or CTLs). In one embodiment, administering a pharmaceutical composition of the present disclosure involves stimulating an anti-tumor CD8+T cell response against cancer cells expressing one or more tumor antigens. In a specific embodiment, the tumor antigen is presented together with a class I MHC molecule.

[0401] The present disclosure contemplates an immune response that may be protective, preventative, prophylactic and / or therapeutic. As used herein, "inducing an immune response" may indicate that there was no immune response to a particular antigen prior to induction, or may indicate that there was a basal level of immune response to a particular antigen prior to induction that was enhanced after induction. Thus, "inducing an immune response" includes "enhancing an immune response."

[0402] The term "immunotherapy" relates to the treatment of a disease or condition by inducing or enhancing an immune response. The term "immunotherapy" includes antigen immunization or antigen vaccination.

[0403] The terms "immunization" or "vaccination" describe the process of administering an antigen to an individual with the purpose of inducing an immune response, for example for therapeutic or prophylactic reasons.

[0404] The peptides, proteins, polypeptides, RNA, RNA particles and other agents described herein (e.g., immune checkpoint inhibitors) can be used for therapeutic or prophylactic treatment of such diseases, wherein providing a subject with a peptide or protein comprising an epitope for inducing an immune response to an antigen in the subject will produce a therapeutic or prophylactic effect. For example, providing an antigen or epitope derived from a virus can be used to treat a viral disease caused by the virus. Providing a tumor antigen or epitope can be used to treat a cancer disease in which a cancer cell expresses the tumor antigen.

[0405] In one embodiment, the present disclosure contemplates an embodiment in which an RNA formulation (e.g., RNA lipid complex particles) described herein that targets spleen tissue is administered. The RNA encodes, for example, a peptide or protein comprising an epitope described herein. The RNA is taken up by antigen presenting cells (e.g., dendritic cells) in the spleen to express the peptide or protein. After optional processing and presentation by the antigen presenting cells, an immune response to the epitope can be generated, resulting in preventive and / or therapeutic treatment of a disease involving the epitope or an antigen comprising the epitope. In one embodiment, the immune response induced by the RNA described herein includes presentation of an antigen or fragment thereof, such as an epitope, by antigen presenting cells, such as dendritic cells and / or macrophages, and activation of cytotoxic T cells caused by the presentation. For example, a peptide or protein encoded by RNA or its processed product can be presented by a major histocompatibility complex (MHC) protein expressed on an antigen presenting cell. The MHC peptide complex can then be recognized by an immune cell (e.g., a T cell or a B cell), resulting in its activation.

[0406] Therefore, the present disclosure relates to the RNA described herein for use in the prophylactic and / or therapeutic treatment of a disease involving an antigen, preferably a cancer disease.

[0407] The term "macrophage" refers to a subgroup of phagocytes produced by differentiation of monocytes. Macrophages activated by inflammation, immune cytokines or microbial products nonspecifically engulf and kill foreign pathogens by hydrolysis and oxidation attacks in macrophages, thereby causing degradation of pathogens. Peptides from degraded proteins are displayed on the macrophage surface, where they can be recognized by T cells, and they can directly interact with antibodies on the B cell surface, thereby causing T cells and B cells to activate and further stimulate an immune response. Macrophages belong to the category of antigen presenting cells. In one embodiment, the macrophage is a spleen macrophage.

[0408] The term "dendritic cell" (DC) refers to another subtype of phagocytes, which belongs to the category of antigen presenting cells. In one embodiment, dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells are initially converted into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation potential. Immature dendritic cells constantly sample pathogens in the surrounding environment, such as viruses and bacteria. Once it contacts with presentable antigens, it is activated to become a mature dendritic cell and begins to migrate to the spleen or lymph nodes. Immature dendritic cells engulf pathogens and degrade their proteins into small pieces (piece), and after maturation, these fragments are presented at their cell surfaces using MHC molecules. At the same time, it has raised cell surface receptors that act as co-receptors in T cell activation, such as CD80, CD86 and CD40, which greatly enhances its ability to activate T cells. It also raises CCR7, which is a chemotactic receptor that induces dendritic cells to reach the spleen through the bloodstream, or to reach lymph nodes through the lymphatic system. Here it acts as an antigen presenting cell and activates helper T cells and killer T cells and B cells by presenting its antigen together with non-antigen specific co-stimulatory signals. Therefore, dendritic cells can actively induce T cell or B cell related immune responses. In one embodiment, the dendritic cell is a spleen dendritic cell.

[0409] The term "antigen presenting cell" (APC) is a cell among a variety of cells that is capable of displaying, acquiring and / or presenting at least one antigen or antigenic fragment on its cell surface (or at its surface). Antigen presenting cells can be divided into professional antigen presenting cells and non-professional antigen presenting cells.

[0410] The term "professional antigen presenting cells" refers to antigen presenting cells that constitutively express major histocompatibility complex class II (class II MHC) molecules required for interaction with naive T cells. If T cells interact with class II MHC molecule complexes on the antigen presenting cell membrane, the antigen presenting cells produce co-stimulatory molecules that induce T cell activation. Professional antigen presenting cells include dendritic cells and macrophages.

[0411] The term "non-professional antigen presenting cells" refers to antigen presenting cells that do not constitutively express class II MHC molecules, but constitutively express class II MHC molecules after being stimulated by certain cytokines such as interferon-γ. Some exemplary non-professional antigen presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic β cells or vascular endothelial cells.

[0412] "Antigen processing" refers to the degradation of an antigen into processing products that are fragments of the antigen (e.g., degradation of a protein into peptides), and to the association (e.g., by binding) of one or more of these fragments with an MHC molecule for presentation by a cell such as an antigen presenting cell to a specific T cell.

[0413] The term "antigen-related disease" or "epitope-related disease" refers to any disease associated with an antigen or epitope, such as a disease characterized by the presence of an antigen or epitope. The antigen- or epitope-related disease may be an infectious disease, or a cancer disease or simply cancer. As described above, the antigen may be a disease-associated antigen, such as a tumor-associated antigen, a viral antigen, or a bacterial antigen, and the epitope may be derived from such an antigen.

[0414] The term "infectious disease" refers to any disease (e.g., the common cold) that can be transmitted between individuals or between organisms and caused by a microbial agent. Infectious diseases are known in the art, and include, for example, viral diseases, bacterial diseases or parasitic diseases, which are caused by viruses, bacteria and parasites, respectively. In this regard, infectious diseases can be, for example, hepatitis, sexually transmitted diseases (e.g., chlamydia or gonorrhea), tuberculosis, HIV / acquired immune deficiency syndrome (AIDS), diphtheria, hepatitis B, hepatitis C, cholera, severe acute respiratory syndrome (SARS), avian influenza and influenza.

[0415] The term "cancer disease" or "cancer" refers to or describes a physiological condition in an individual that is generally characterized by unregulated cell growth. Some examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More particularly, some examples of such cancers include bone cancer, blood cancer lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, sex and reproductive organ cancer, Hodgkin's Disease, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, central nervous system (CNS) neoplasms, neuroectodermal cancer, spinal axis tumors, gliomas, meningiomas, and pituitary adenomas. The term "cancer" according to the present disclosure also includes cancer metastasis.

[0416] Due to the synergistic effects produced, combination strategies in cancer treatment can be ideal, which can be much stronger than the effects of a single treatment method. In one embodiment, the pharmaceutical composition is administered together with an immunotherapeutic agent. "Immunotherapeutic agent" as used herein refers to any agent that can participate in activating a specific immune response and / or immune effector function. The present disclosure contemplates the use of antibodies as immunotherapeutic agents. Without wishing to be bound by theory, antibodies can achieve therapeutic effects against cancer cells through a variety of mechanisms, including inducing apoptosis, blocking components of signal transduction pathways, or inhibiting the proliferation of tumor cells. In certain embodiments, the antibody is a monoclonal antibody. Monoclonal antibodies can induce cell death through antibody-dependent cell-mediated cytotoxicity (ADCC), or bind to complement proteins, resulting in direct cytotoxicity, known as complement-dependent cytotoxicity (CDC). Some non-limiting examples of anti-cancer antibodies and potential antibody targets (in parentheses) that can be used in combination with the present disclosure include: Abavolumab (CA-125), Abciximab (CD41), Adalimumab (EpCAM), Afutuzumab (CD20), Alacizumab pegol (VEGFR2), Atumotuzumab Pentetate (CEA), Amatuximab (MORAb-009), Maanatumomab (TAG-72), Apolizumab (HLA-DR), Acitumomab (CEA), Atezolizumab (PD-L1), Baviximab (phosphatidylserine), Betumumab (CD22), Belimumab (BAFF), Bevacizumab (VEGF-A), Mobivac (Bivatuzumab mertansine)(CD44v6), Blinatumomab(CD 19), Brentuximab(CD30TNFRSF8), Mocantuzumab(Mucin CanAg), Lecantuzumab(MUC1), Capromab Pendetide(Prostate Cancer Cells), Carlumab(CNT0888), Catumaxomab(EpCAM, CD3), Cetuximab(EGFR), Citatuzumab bogatox(EpCAM), Ciliatuzumab(IGF-1 Receptor), Claudiximab(Claudin), Clivatuzumab(Celiac),tetraxetan (MUC1), situzumab (TRAIL-R2), daclizumab (CD40), dalotuzumab (insulin-like growth factor I receptor), denosumab (RANKL), demuximab (B-lymphoma cells), drozitumab (DR5), emeticholic acid (GD3 ganglioside), edrecolomab (EpCAM), elotuzumab (SLAMF7), enatuzumab (PDL192), ensituximab (NPC-1C), epratuzumab (CD22), ertuinomab (HER2 / neu, CD3), irrituzumab (integrin αvβ3), farletuzumab (folate receptor 1), FBTA05 (CD20), fenclatuzumab (SCH 900105), Figitumumab (IGF-1 receptor), Flanvotumab (glycoprotein 75), Fresolimumab (TGF-β), Galiximab (CD80), Ganitatumab (IGF-I), Gemtuzumab ozogamicin (CD33), Gevokizumab (ILIβ), Girentuximab (Carbonic anhydrase 9 (CA-IX)), Glembatumumab vedotin (GPNMB), Ibritumomab tiuxetan (CD20), Icrucumab (VEGFR-1), Igovotumab (CA-125), Indatuximab ravtansine)(SDC1), Intetumumab(CD51), Intuzumab(CD22), Ipilimumab(CD 152), Iratumumab(CD30), Labetuzumab(CEA), Lexatumumab(TRAIL-R2), Rivimab(HBsAg), Lintuzumab(CD33), Lorvotuzumab mertansine(CD56), Rucamumab(CD40), Ruximumab(CD23), Mapatumumab(TRAIL-R1), Matuzumab(EGFR), Mepolizumab(IL5), Milatuzumab(CD74), Mitumumab(GD3 ganglioside), Mogamulizumab(CCR4), Moxetumomabpasudotox)(CD22), tadalafil (C242 antigen), tadalafil (5T4), natumomab (RON), necitumumab (EGFR), nimotuzumab (EGFR), nivolumab (IgG4), ofatumumab (CD20), olaratumab (PDGF-R a), Onartuzumab (human scatter factor receptor kinase), Oportuzumab monatox (EpCAM), Ogovolumab (CA-125), Oxelumab (OX-40), Panitumumab (EGFR), Patritumab (HER3), Pemtumomab (MUC1), Pertuzumab (HER2 / neu), Pintumomab (adenocarcinoma antigen), Protumumab (vimentin), Racotumomab (N-hydroxy Acetylneuraminic acid), Radretumab (fibronectin extra domain-B), Revizumab (rabies virus glycoprotein), Ramucirumab (VEGFR2), Rilotumumab (HGF), Rituximab (CD20), Robatumumab (IGF-1 receptor), Samalizumab (CD200), Ciroizumab (FAP), Siltuximab (IL6), Tabalumab (BAFF), Talizumab (alpha-fetoprotein), Pertapumomab (CD 19), tenatumomab (tenascin C), teprotumumab (CD221), simumab (CTLA-4), tegazumab (TRAIL-R2), TNX-650 (IL13), tositumomab (CD20), trastuzumab (HER2 / neu), TRBS07 (GD2), tesitumomab (CTLA-4), tucotuzumab celmoleukin (EpCAM), ubliximab (MS4A1), urelumab (4-1 BB), voroxizumab (integrin α5β1), voruximab (tumor antigen CTAA 16.88), zatutumumab (EGFR), and zatutumumab (CD4).

[0417] Citation of documents and studies cited herein is not intended to admit that any of the foregoing is relevant prior art. All statements regarding the contents of these documents are based on the information available to the applicant and do not constitute any admission that the contents of these documents are correct.

[0418] The following description is presented to enable one of ordinary skill in the art to make and use the various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications to the embodiments described herein will be apparent to one of ordinary skill in the art, and the general principles defined herein may be applied to other embodiments and applications without departing from the spirit and scope of the various embodiments. Therefore, the various embodiments are not intended to be limited to the embodiments described and illustrated herein, but are consistent with the scope of the claims.

[0419] Example

[0420] Example 1: Construct design and mRNA production

[0421] In vitro transcription of cytokine encoding mRNA is based on the pST1-T7-AGA-dEarI-hAg-MCS-FI-A30LA70 plasmid-backbone and derived DNA-constructs. These plasmid constructs contain a 5'UTR (untranslated region, a derivative of the 5'-UTR of the Homo sapiens hemoglobin subunit alpha 1 (hAg)), a 3'FI element (where F is a 136 nucleotide long 3'-UTR fragment of the split amino-terminal enhancer, mRNA and I is a 142 nucleotide long fragment of the mitochondrial encoded 12S RNA, both identified in Homo sapiens; WO 2017 / 060314) and a 100 nucleotide poly (A) tail with a linker after 70 nucleotides. Cytokine and serum albumin (hAlb) coding sequences are derived from Homo sapiens, and no changes were introduced in the resulting amino acid sequences except for the expected mutations in the mutIL2 variants described below (hIL2: NP_000577.2; NCBI protein resources; https: / / www.ncbi.nlm.nih.gov / protein / ). hAlb was added to the N-terminus or C-terminus of the IL2 variants, where both orientations were equally effective in vitro and in vivo (data not shown). The encoded protein is equipped with an N-terminal signal peptide (SP), i.e., the natural SP of the corresponding protein. In the case of fusion proteins, only the SP of the N-terminal part is maintained, and for the other parts, only the mature part (protein without SP) is encoded. Stop codons are introduced only for most C-terminal parts. The different protein parts in the cytokine and hAlb fusion constructs are separated by a 30-nucleotide long linker sequence encoding glycine and serine residues. Mutations in the hIL2 sequence were introduced to alter CD25 binding (referred to as "mutCD25" mutations, the corresponding constructs were referred to as hIL2_Ax, where x is defined below) and binding to IL2Rβγ (referred to as "mutβγ" mutations, the corresponding constructs were marked with an additional "s", e.g., hIL2s or hIL2_A4s). In the case of hIL2_A1, four amino acid residue substitutions were introduced in the mature domain, changing the arginine at position 38 to alanine (R38A), the phenylalanine at position 42 to alanine (F42A), the tyrosine at position 45 to alanine (Y45A), and the glutamic acid at position 62 to alanine (E62A) (Carmenate, T. et al. J. Immunol. 190, 6230-6238 (2013).). For hIL2_A2, the amino acid residues of the mature domain were replaced with K35A, K43A, and E61A.For hIL2_A3, the substitutions K35E, K43E and E61K were introduced; for hIL2_A4, K43E and E61K were introduced; for hIL2_A5, E61K was introduced; and for hIL2_A6, the E62K mutation was introduced. To generate the hIL2 mutβγ variant, the amino acid residues of the mature domain of the corresponding hIL2 variant were replaced in the manner of L80F, R81D, L85V, I86V and I92F (Levin, AMet al. Nature 484, 529-533 (2012)). mRNA was generated by in vitro transcription as described by Kreiter et al. (Kreiter, S.et al. Cancer Immunol. Immunother. 56, 1577-87 (2007)), in which the normal nucleoside uridine was replaced by 1-methyl-pseudouridine. The resulting mRNA was equipped with a Cap1 structure and was depleted of double-stranded (dsRNA) molecules. Purified mRNA was eluted in H2O and stored at -80°C until further use. In vitro transcription of all described mRNA constructs was performed at BioNTech RNA Pharmaceuticals GmbH. A list of all constructs used for subsequent experiments is shown in Table 1.

[0422] Table 1: Amino acid sequences of mRNA-encoded and expressed proteins.

[0423]

[0424]

[0425]

[0426]

[0427]

[0428] Example 2: In vitro expression of RNA-encoded IL2 variants and IL2Rβγ binding

[0429] The in vitro expression of the generated mRNA was analyzed by transfection of mRNA liposomes into HEK293T / 17 cells and subsequent analysis of CD25-dependent activation of IL2Rβγ-expressing reporter cells by the IL2mutCD25 variant ( Figure 1 ). One day before liposome transfection, 1.2×10 6HEK293T / 17 cells were seeded in 3 mL DMEM (Life Technologies GmbH, catalog number 31966-021) + 10% fetal bovine serum (FBS, Biochrom GmbH, catalog number S0115) in 6-well plates. For liposome transfection, 3 μg of mRNA was prepared using 400 ng mRNA / μL Lipofectamine MessengerMax (Thermo Fisher Scientific, catalog number LMRNA015) under sterile and RNase-free conditions and was plated per 10 cm 2 The culture dishes were applied to HEK293T / 17 cells at about 80% confluence. After 20 hours of expression, the supernatant was collected under sterile conditions and stored at -20°C until further use. The IL2Rβγ-dependent bioactivity of the IL2 mutCD25 variant was evaluated by measuring the specific proliferation response of the medium-affinity IL2 receptor (IL2Rβγ) expressing TF-1_IL2Rβγ cells. This cell line was generated by transduction of the TF-1 cell line (ATCC CRL-2003), a human erythroleukemia cell line that naturally expresses the IL2R common γ chain, with a retroviral vector encoding a sequence similar to that of the human IL2Rβ chain of Farner, NL et al. Blood 86, 4568-4578 (1995) (Gene ID: 3560). Briefly, TF-1_IL2Rβγ cells were washed twice with D-PBS and resuspended in RPMI 1640 (Life Technologies GmbH, catalog number 61870010) supplemented with 10% fetal bovine serum (FBS; Biochrom GmbH, catalog number S0115) and 1 mM sodium pyruvate (Life Technologies GmbH, catalog number 11360070). A total of 5,000 cells / well were seeded in a white 96-well flat-bottom plate (Fisher Scientific GmbH, catalog number 10072151) and incubated with four-fold serial dilutions of the supernatant containing IL2 variants. After three days of culture, the cells were plated using The assay (Promega, catalog number G9242) measures proliferation by quantifying viable cells by ATP levels. Luminescence was recorded on a F200 PRO reader (Tecan Deutschland GmbH) and dose-response curves were plotted in GraphPad Prism version 6.04 (GraphPad Software, Inc.).

[0430] Example 3: Binding of RNA-encoded IL2 mutCD25 variants to recombinant CD25 (IL2Rα)

[0431] Binding of the mRNA-encoded IL2 mutCD25 variant to recombinant CD25 was analyzed by ELISA ( Figure 2 Here, 1 μg / mL recombinant human or mouse CD25 (C-Fc, Novoprotein catalog number CJ78, CK32) was coated in 100 μL DPBS onto a high protein binding 96-well plate (Nunc MaxiSorp TM , Thermo Fisher Scientific, catalog number 439454). The IL2 variant-containing supernatant produced by liposome transfection of HEK-293-T-17 (as described in Example 2) was applied to the coated CD25, and the bound protein was detected by HRP-conjugated anti-human serum albumin antibody (Abcam, catalog number ab8941). Conventional ELISA reagents and procedures were applied according to the protocol of DuoSet ELISA Auxiliary Kit 2 (R&D Systems, catalog number DY008).

[0432] like Figure 2 As shown in , wild-type hAlb-hIL2 binds strongly to both human and mouse CD25. With respect to human CD25, binding to mutCD25 variants A1, A3, A4, and A6 was completely lost, while variants A2 and A5 showed some remaining binding to human CD25 ( Figure 2 A). In contrast, all mutCD25 variants lost the ability to bind to mouse CD25 ( Figure 2 B).

[0433] Example 4: Biological activity of RNA-encoded IL2 mutCD25 variant on CD25-dependent CTLL-2 proliferation.

[0434] The biological activity of the IL2 mutCD25 variants was evaluated by analyzing the cytokine-dependent proliferation of murine CTLL-2 cells (mouse C57BL / 6 T cell line, ATCC TIB-214) that highly express CD25 ( Figure 3Briefly, CTLL-2 cells were harvested, washed twice with DPBS to remove any remaining IL2 and resuspended in RPMI 1640 (Life Technologies GmbH, catalog number 61870010) supplemented with 10% fetal bovine serum (FBS; Biochrom GmbH, catalog number S0115) and 1 mM sodium pyruvate (Life Technologies GmbH, catalog number 11360070). A total of 5,000 cells / well were seeded in a white 96-well flat-bottom plate (Fisher Scientific GmbH, catalog number 10072151) and incubated with four-fold serial dilutions of the IL2 variant-containing supernatant (as described in Example 2). After three days of culture, the cells were plated using The assay (Promega, catalog number G9242) measures proliferation by quantifying viable cells by ATP levels. Luminescence was recorded on a F200PRO reader (Tecan Deutschland GmbH) and dose-response curves were plotted in GraphPad Prism version 6.04 (GraphPad Software, Inc.).

[0435] Wild-type hALb-hIL2 induced the proliferation of CTLL-2 cells in a dose-dependent manner, with a half-maximal effective concentration (EC50) of 0.2144%-supernatant ( Figure 3 ). The mutCD25 variants A2 and A5 performed comparably to wild-type hAlb-hIL2, which was also reflected by EC50 values ​​of 0.1933%-supernatant to 0.2127%-supernatant, respectively. In contrast, the IL2 variant A1 showed an approximately 1000-fold decrease in EC50 value, resulting in almost no bioactivity against CTLL-2 cells. However, variants A3, A4 and A6 induced proliferation of CTLL-2 cells, with EC50 values ​​of 15.10%-supernatant for A3, 11.43%-supernatant for A4 and 7.785%-supernatant for A6, characterized by a moderate bioactivity that was approximately 50-fold lower than that of wild-type hAlb-hIL2 ( Figure 3 ).

[0436] Example 5: Biological activity of RNA-encoded IL2 variants on the proliferation of human PBMCs.

[0437] To measure peripheral blood mononuclear cell (PBMC) proliferation, PBMC were incubated with suboptimal concentrations of anti-CD3 antibody (clone UCHT1) and supernatant or control supernatant derived from liposome transfection of HEK293T / 17 with mRNA encoding IL2 variants. Briefly, PBMC were obtained from buffy coats of healthy donors by Ficoll-Paque (VWRinternational, catalog number 17-1440-03) density gradient separation. PBMC were labeled with 1.6 μM carboxyfluorescein succinimidyl ester (CFSE; Thermo Fisher, catalog number C34564). 75,000 CFSE-labeled PBMCs were seeded per well in 96-well round-bottom plates (Costar, catalog number 734-1797) in lscove's Modified Dulbecco's Medium (IMDM; Life Technologies GmbH, catalog number 12440-053) supplemented with 5% plasma-derived human serum (PHS; One Lambda lnc., catalog number A25761) and incubated with anti-CD3 antibodies (clone UCHT1; R&D Systems, catalog number MAB100; 0.03 to 0.09 μg / mL final concentration) at a pre-determined suboptimal concentration for each donor. In parallel, four-fold serial dilutions of IL2 variant supernatants were generated in IMDM supplemented with 5% PHS (see Example 2). The seeded cells were mixed with IL2 variant supernatants 1:1 (referring to the volume of the medium in which the cells were seeded) and stimulated for four days at 37° C., 5% CO2. PBMCs were harvested and analyzed by flow cytometry. The cells were stained with the following reagents diluted 1:100 in FACS buffer (D-PBS containing 5% FBS and 5mM EDTA): anti-human CD4-PE (TONBO Bioscience, catalog 50-0049), anti-human CD8-PE (TONBO Bioscience, catalog 50-0088), anti-human CD56-APC (eBioscience, catalog 17-0567-42) and 7-AAD (Beckman Coulter, catalog A07704). The BD FACSCanto TMFlow cytometry analysis was performed on a FlowCytometer II flow cytometer (Becton Dickinson) with CFSE dilution as proliferation readout. The acquired proliferation data were analyzed using FlowJo 10.4 software, and dose-response curves were plotted in GraphPad Prism version 6.04 (GraphPad Software, Inc.) using the derived expansion index values.

[0438] Except for the variants hAlb-hIL2_A2 and hAlb-hIL2_A5, which performed identically to wild-type IL2 in the high-affinity IL2Rαβγ-dependent CTLL-2 proliferation assay, all other mutants were analyzed in an antigen-nonspecific proliferation assay using native human PBMCs. Compared to all other IL2 variants, wild-type hAlb-hIL2 strongly enhanced CD3-induced CD4+ T cells ( Figure 4 A) CD8+T cells ( Figure 4 B) and CD56+ NK cells ( Figure 4 C) proliferation and show superior biological activity.All tested variants with reduced CD25 binding capacity all show intermediate phenotype.Variant hAlb-hIL2_A4 and hAlb-hIL2_A6 performance is suitable, compared with wild-type hAlb-hIL2, the effect is reduced by about 50 times, and hAlb-hIL2_A1 and hAlb-hIL2_A3 show superimposable dose-response curve and even more strongly turn to reduce biological activity, i.e., about 75 times lower than the biological activity of hAlb-hIL2.Between all three lymphocyte subsets evaluated, the difference observed for the variant is suitable, but NK cell proliferation is usually not as obvious as T cell proliferation.

[0439] hAlb-hIL2_A4 and hAlb-hIL2_A6 with intermediate phenotype are further compared with variants comprising mutβγ mutations in addition, which are described as improving the combination with IL2Rβγ complexes (see also Example 1). On both CD4+ and CD8+ T cells, hAlb-hIL2 shows excellent biological activity, which can not be enhanced by adding mutβγ mutations, which is also reflected in the calculated EC50 values ​​(Table 2). Compared with hAlb-hIL2, mutCD25 variant hAlb-hIL2_A4 shows that biological activity on CD4+ T cells is reduced by about 50 times, and on CD8+ T cells, biological activity is reduced by 100 times. Compared with hAlb-hIL2_A4, the effectiveness of other mutCD25 variants hAlb-hIL2_A6 is even further reduced by 2 times on two T cell subsets. However, the addition of the mutβγ mutation enhanced the bioactivity of both mutCD25 variants (i.e., hAlb-hIL2_A4 and hAlb-hIL2_A6) on CD4+ and CD8+ T cells, resulting in only a 4- to 7-fold decrease in the bioactivity of hAlb-hIL2_A4s and a 9- to 16-fold decrease in the bioactivity of hAlb-hIL2_A6s compared to hAlb-hIL2 and hAlb-hIL2s ( Figure 4 D and E, Table 2). On CD56+NK cells, the mutβγ variant hAlb-hIL2s showed the highest bioactivity, slightly exceeding the bioactivity of wild-type hAlb-hIL2 (EC50 values ​​were 0.6084% and 1.662%-supernatant, see Table 2). In contrast to CD4+ and CD8+T cells, on CD56+NK cells, the mutCD25 variants hAlb-hIL2_A4 and hAlb-hIL2_A6 were only 3 to 8 times less potent than hAlb-hIL2. Adding mutβγ mutations to hAlb-hIL2_A4 and hAlb-hIL2_A6 restored the bioactivity of the variants hAlb-hIL2_A4s and hAlb-hIL2_A6s to the level of wild-type hAlb-hIL2 ( Figure 4 F).

[0440] Table 2: EC50 values ​​of hAlb-hIL2 variants in whole PBMC lymphocyte subsets derived from human PBMC proliferation dose-response [% - supernatant].

[0441] IL2 variants CD4+ T cells CD8+ T cells CD56+NK cells hAlb-hIL2 0.5455 0.1524 1.662 hAlb-hIL2s 0.5414 0.4578 0.6084 hAlb-hIL2_A4 nd 14.06 5.472 hAlb-hIL2_A4s 3.801 1.701 0.5326 hAlb-hIL2_A6 nd nd 13.64 hAlb-hIL2_A6s 8.883 4.317 1.617

[0442] Example 6: Comparison of the relative bioactivity of different IL2 variants in medium affinity IL2 receptor (IL2Rβγ) and high affinity IL2 receptor (IL2Rαβγ) expressing IL2-dependent reporter cell lines.

[0443] To dissect the role of the IL2Rα chain (CD25) as a determinant of the bioactivity of different IL2 variants, the specific proliferation response of the high affinity IL2 receptor (IL2Rαβγ) expressing mouse T cell line CTLL-2 was compared with the specific proliferation response of the medium affinity IL2 receptor (IL2Rβγ) expressing TF-1_IL2Rβγ cells, which is a human erythroleukemia cell line that naturally expresses the IL2R common γ chain and also expresses the IL2Rβ chain when transduced (see Example 2). Briefly, CTLL-2 and TF-1_IL2Rβγ cells were washed twice with D-PBS and resuspended in RPMI 1640 (Life Technologies GmbH, catalog number 61870010) supplemented with 10% fetal bovine serum (FBS; Biochrom GmbH, catalog number S0115) and 1 mM sodium pyruvate (Life Technologies GmbH, catalog number 11360070). A total of 5,000 cells / well were seeded in white 96-well flat-bottom plates (Fisher Scientific GmbH, catalog number 10072151) and incubated with five four-fold serial dilutions of IL2 variant-containing supernatants (as described in Example 2). The assay (Promega, catalog number G9242) measures proliferation by quantifying viable cells by ATP levels. Luminescence was recorded on a PRO reader (Tecan Deutschland GmbH), and dose-response curves were plotted and EC50 values ​​were calculated in GraphPad Prism version 6.04 (GraphPad Software, Inc.).

[0444] hAlb-hIL2 and hAlb-hIL2 mutCD25 variants with reduced CD25 binding affinity (i.e., hAlb-hIL2_A4 and _A6) performed comparably to CD25-independent IL2Rβγ-expressing TF-1_IL2Rβγ cells, with nearly superimposable dose-response curves ( Figure 5A, B). This is also reflected in the calculated EC50 values, which range from 2.352%-supernatant for hAlb-hIL2 to 3.657%-supernatant for hAlb-hIL2_A6 (Table 3). In addition, as expected, the mutβγ variants with enhanced binding affinity for IL2Rβ (i.e., hAlb-hIL2s, hAlb-hIL2_A4s, hAlb-hIL2_A6s) showed a 10- to 30-fold shift toward increased biological activity, with comparable EC50 values ​​for hAlb-hIL2s and hAlb-hIL2_A4s (0.080% and 0.107%-supernatant, respectively), and slightly lower activity for hAlb-hIL2_A6s (EC50 = 0.338%-supernatant).

[0445] In stark contrast, strong differences between hAlb-hIL2 and hAlb-hIL2 variants were seen in CD25-dependent IL2Rαβγ-expressing CTLL-2 cultures ( Figure 5 C, D). Here, hAlb-hIL2 exhibited the highest biological activity that could not be further enhanced by adding mutβγ mutations (Table 4; EC50 of hAlb-hIL2 and hAlb-hIL2s was 0.115% vs. 0.240%-supernatant). The activity of hAlb-hIL2_A4 was reduced 19-fold compared to hAlb-hIL2 (EC50 = 2.19% supernatant), while for hAlb-hIL2_A6, the activity was reduced by about 300-fold (EC50 = 33.72% supernatant). The corresponding mutβγ variants hAlb-hIL2_A4s and hAlb-hIL2_A6s showed enhanced biological activity, but were still less active than hAlb-hIL2, with EC50 values ​​of 0.517 and 1.575, respectively.

[0446] Table 3: EC50 values ​​of hAlb-hIL2 variants in medium affinity IL2 receptor (IL2Rβγ) dependent cell culture derived from human TF-1_IL2Rβγ proliferation dose-response [%-supernatant] Ratios of EC50 fold reduction and fold improvement data compared to hAlb-hIL2 were calculated.

[0447]

[0448] Table 4: EC50 values ​​of hAlb-hIL2 variants in high affinity IL2 receptor (IL2Rαβγ) dependent cell culture derived from mouse CTLL-2 proliferation dose-response [%-supernatant]. Ratios of EC50 fold reduction and fold improvement data compared to hAlb-hIL2 were calculated.

[0449]

[0450] Example 7: Comparison of the activity of hAlb-hIL2 variants on different T cell subsets in human PBMCs using STAT5 phosphorylation as readout.

[0451] In order to evaluate the activity of hAlb-hIL2 variants on different T cell subsets lacking or expressing CD25, all human PBMCs were stimulated with hAlb-hIL2 variants, and STAT5 phosphorylation was determined under a series of cytokine dilutions. In brief, PBMCs were obtained from the buffy coat of healthy donors by Ficoll-Paque (VWR international, catalog number 17-1440-03) density gradient separation. PBMCs were washed twice with D-PBS (Life Technologies GmbH, catalog number 14190250) and collected by centrifugation at room temperature for 5 minutes at 300 × g. PBMCs were resuspended in lscove modified Dulbecco's medium (IMDM; Life Technologies GmbH, catalog number 12440-053) supplemented with 10% fetal bovine serum (FBS; Biochrom GmbH, catalog number S0115), and left to stand for 1 hour at 37°C and 5% CO2. Next, 100,000 PBMCs / well of a 96-well V-bottom plate (Greiner Bio-One GmbH, catalog number 651101) were seeded in PBS supplemented with 10% FBS and 2x PhosSTOP TM In parallel, five four-fold serial dilutions of the hAlb-hIL2 variant supernatant were made in IMDM supplemented with 10% FBS (as described in Example 2). The seeded cells were mixed with the hAlb-hIL2 variant supernatant 1:1 (referring to the volume of the medium in which the cells were seeded) and stimulated for 10 minutes at 37°C and 5% CO2. Next, 1:1,000 of the fixable viability dye eFluor TM 780, and the cells were stimulated for another 5 minutes at 37°C and 5% CO2. The cells were fixed by adding formaldehyde (Carl Roth GmbH + Co. KG, catalog number P087.4) to 2% and incubated on ice for 10 minutes. The fixed PBMCs were washed with ice-cold D-PBS and permeabilized with 100% ice-cold methanol on ice for 30 minutes. The permeabilized PBMCs were washed twice with 1x permeabilization buffer (eBioscience Inc., catalog number 00-8333-56) and then incubated in 1x permeabilization buffer at 2 to 8°C in the dark with 1:5 Anti-Stat5(pY694) (Becton Dickinson GmbH, accession number 612598), 1:25PerCP-Cy TM 5.5 mouse anti-human CD25 (Becton Dickinson GmbH, catalog number 560503), 1:25 APC rat anti-FOXP3 (eBioscience Inc., 17-4776-41), 1:25 BV421 mouse anti-human CD4 (Becton Dickinson GmbH, catalog number 565997) and 1:25 BV510 mouse anti-human CD8 (Becton Dickinson GmbH, catalog number 563256) were stained for 30 minutes. The stained PBMCs were washed twice with ice-cold 1x permeabilization buffer and finally resuspended in D-PBS supplemented with 2% FBS and 2 mM EDTA (Sigma-Aldrich, catalog number 03690-100ML). The BD FACSCanto TM Flow cytometry analysis was performed on a Flowcytometer II (Becton Dickinson GmbH), and the acquired data were analyzed using FlowJo software version 10.3. Dose-response curves and EC50 values ​​were calculated in GraphPad Prism version 6.04 (GraphPad Software, Inc.).

[0452] On CD4+CD25+FoxP3+ regulatory T cells, hAlb-hIL2 showed superior potency relative to all IL2 mutCD25 variants with reduced CD25 binding affinity (hAlb-hIL2_A4 / _A6) and their respective mutβγ variants (hAlb-hIL2_A4s, hAlb-hIL2_A6s) ( Figure 6 and Tables 5 and 6). Specifically, the biological activity of hAlb-hIL2_A6 was strongly reduced by about 320 times compared to hAlb-hIL2, while hAlb-hIL2_A4 exhibited an intermediate phenotype (about 170 times reduced activity compared to hAlb-hIL2). The biological activity of the two mutCD25 variants can be enhanced by adding mutβγ mutations, where hAlb-hIL2_A4s is superior to hAlb-hIL2_A6s, showing about 6-fold and about 21-fold reductions in activity compared to hAlb-hIL2, respectively. The most active variant is hAlb-hIL2s, which has an even about 15-fold increase in biological activity compared to hAlb-hIL2. In the absence of CD25 expression, hAlb-hIL2 potency is strongly reduced by >40 times (see Figure 6 , Table 5, and CD4+CD25+FoxP3+Treg The same is true for the corresponding variants containing mutβγ mutations: hAlb-hIL2s showed the highest bioactivity in both CD25-negative CD4+ and CD8+ T cell subsets (see Tables 7 to 8; the potency was increased by about 5 to 10 times compared with hAlb-hIL2), followed by hAlb-hIL2_A4s, which performed comparable to hAlb-hIL2 in CD4+CD25-T helper cells and was even superior to hAlb-hIL2 in CD8+ cytotoxic T cells (see Tables 7 to 8; the bioactivity was increased by about 2 times compared with hAlb-hIL2). When compared to hAlb-hIL2_A4s, hAlb-hIL2_A6s performed almost as well as hAlb-hIL2 in CD8+ cytotoxic T cells, but was less active in CD4+CD25- T helper cells. Most importantly, the EC values ​​determined for individual hAlb-hIL2 variants on CD4+CD25- T helper cells or CD8+ cytotoxic T cells were not significantly different from those determined for hAlb-hIL2_A6s. 50 Values ​​were compared with EC values ​​determined on CD4+CD25+FoxP3+ regulatory T cells 50 The ratio of the values ​​(Table 5) allows to calculate the 'regulatory T cell bias' (Table 9) of each hAlb-hIL2 variant. Compared with its effectiveness on CD4+CD25-T helper cells or CD8+ cytotoxic T cells, hAlb-hIL2 shows 40 to 60 times of bias to regulatory CD4+CD25+FoxP3+ regulatory T cells. In sharp contrast, when focusing on mutCD25 variants hAlb-hIL2_A4 and hAlb-hIL2_A6, this regulatory T cell bias is reduced to only 1.2 to 1.9 times. The corresponding mutβγ variants hAlb-hIL2_A4s and hAlb-hIL2_A6s are accompanied by slightly improved regulatory T cell biases, which are 4.4 to 7.1 times. When compared to its potency on CD4+CD25- T helper cells or CD8+ cytotoxic T cells, hAlb-hIL2s showed the strongest bias towards CD4+CD25+FoxP3+ regulatory T cells, ranging from 63 to 182-fold.

[0453] Table 5: EC50 values ​​calculated based on the dose-response for STAT5 phosphorylation of hAlb-hIL2 variants in different human T cell subsets [% - supernatant].

[0454]

[0455] Table 6: EC50 values ​​calculated based on dose-response for STAT5 phosphorylation of hAlb-hIL2 variants in human CD4+CD25+FoxP3+ regulatory T cells [%-supernatant]. Ratios of EC50 fold reduction and fold improvement data compared to hAlb-hIL2 were calculated.

[0456]

[0457] Table 7: EC50 values ​​calculated based on dose-response for STAT5 phosphorylation of hAlb-hIL2 variants in human CD4+CD25- T helper cells [%-supernatant]. Ratios of EC50 fold reduction and fold improvement data compared to hAlb-hIL2 were calculated.

[0458]

[0459] Table 8: EC50 values ​​calculated based on dose-response for STAT5 phosphorylation of hAlb-hlL2 variants in human CD8+ cytotoxic T cells [% - supernatant]. Ratios of EC50 fold reduction and fold improvement data compared to hAlb-hlL2 were calculated.

[0460]

[0461] Table 9: The 'regulatory T cell bias' of each hAlb-hIL2 variant is given as the fold-reduced potency on CD4+CD25-T helper cells or CD8+ cytotoxic T cells compared to CD4+CD25+FoxP3+ regulatory T cells. The fold-reduced potency is calculated as the ratio of the individual EC50 values ​​determined on CD4+CD25-T helper cells or CD8+ cytotoxic T cells for each hAlb-hIL2 variant to the EC50 value determined on CD4+CD25+FoxP3+ regulatory T cells.

[0462]

[0463]

[0464] Example 8: Effect of IL2 mutCD25 variant on T cell vaccination in vivo

[0465] Subsequently, we characterized the effects of the IL2 mutCD25 variants hIL2_A4 and hIL2_A6 on RNA vaccine-induced T cell responses in vivo. BALB / c mice (n=5 per group) were vaccinated iv with 20 μg of RNA-LPX encoding the CD8+ T cell antigen gp70 (SPSYAYHQF) as described in Kranz, LMet al.Nature 534, 396-401 (2016). gp70 is a tumor antigen that can be found, for example, in the colon cancer cell line CT26. The anti-tumor efficacy of gp70-targeted vaccines increases with the increase in the number of induced gp70-specific T cells (Kranz, LMet al.Nature 534, 396-401 (2016) and unpublished). Three days after vaccination, as described in Figure 7 hAlb (negative control) or increasing doses of hAlb-hIL2, hAlb-hIL2_A4, hAlb-hIL2_A6 RNA formulated as LNPs were administered iv as shown in. Analysis was performed on day 7, including immunophenotyping of blood lymphocyte subsets and gp70-specific T cells (MHC tetramers, MBL) by flow cytometry (BD FACSCelesta) (stained as described in Kranz, LMet al. Nature 534, 396-401 (2016)).

[0466] As described in Examples 4 and 6, the mutCD25 variants have reduced potency in stimulating IL2Rαβγ-positive CTLL-2 cells. For this reason, we tested mutCD25 variants at doses approximately three times higher than wild-type hAlb-hIL2 in order to be able to induce an effect on gp70-specific effector T cells comparable to hAlb-hIL2, while increasing the CD8 to Treg ratio. Despite the higher dose, only hAlb-hIL2, but not hAlb-hIL2 mutCD25 variants, showed a significant increase in liver weight as an indicator of IL2-mediated toxicity ( Figure 7 B). Similarly, no serum aspartate aminotransferase (ASAT) ( Figure 7 C), alanine aminotransferase, lactate dehydrogenase, amylase or lipase activity (Indiko TM, Thermo Fischer Scientific) and an increase in lung weight (data not shown). Wild-type hAlb-hIL2 and both hAlb-hIL2mutCD25 variants resulted in a dose-dependent increase in gp70-specific T cells in the blood, as determined by flow cytometry, with the strongest increase observed with the highest dose of hAlb-hIL2_A4 and the weakest increase observed with hAlb-hIL2_A6 ( Figure 7 D). Interestingly, hAlb-hIL2_A4 in particular led to a very strong increase in CD8+ T cells as well as CD45+ leukocytes ( Figure 7 E, F). Compared with hAlb-hIL2 and hAlb-hIL2_A4, the administration of hAlb-hIL2_A6 did not lead to an increase in Treg ( Figure 7 G). Importantly, both hAlb-hIL2 variants mediated an increase in the ratio of CD8+ T cells to Tregs, suggesting a preferential expansion of CD8+ T cells relative to tumor-promoting Tregs ( Figure 7 H). In contrast, hAlb-hIL2 reduced the CD8+T cell to Treg ratio in a dose-dependent manner. When comparing the fold change of gp70-specific or nonspecific CD8+T cells relative to the hAlb control, hAlb-hIL2 and hAlb-hIL2_A4, but not hAlb-hIL2_A6, led to preferential expansion of gp70-specific T cells ( Figure 7 I). Taken together, these results indicate that hAlb-hIL2_A6 and hAlb-hIL2_A4 variants have no or reduced CD25 binding capacity, leading to beneficial effects on cancer immunotherapy, such as increasing the CD8+ T cell to Treg ratio.

[0467] Example 9: Improving the potency of IL2 mutCD25 variants by adding mutβγ mutations

[0468] Although the variants hAlb-hIL2_A4 and hAlb-hIL2_A6 strongly increased the CD8+T cell to Treg ratio, due to reduced binding to the high affinity IL2Rαβγ on activated T cells, a much higher dose was required for the strong expansion of antigen-specific T cells compared to hAlb-hIL2. Therefore, we tested whether the efficacy of hAlb-hIL2_A4 and hAlb-hIL2_A6 in vivo can be further improved by introducing mutβγ mutations, which have been shown to increase binding to all IL2Rβγ-positive cells in vitro (Examples 5 to 7; Levin, AMet al.Nature 484, 529-533 (2012)). For Example 8, BALB / c mice (n=5 per group) were vaccinated iv with 20 μg gp70 RNA-LPX, followed by cytokine RNA-LNP injections three days later ( Figure 7 A). In this experiment, we drastically reduced the cytokine RNA-LNP dose to ensure good coverage of the increase caused by the mutβγ mutation and to test the lower limit of the treatment dose. gp70-specific T cells, NK cells, and CD25 FoxP3-positive Tregs were analyzed 7 days after vaccination ( Figure 8 ).

[0469] As shown in Example 8, the frequencies of antigen-specific T cells and Tregs were increased by administration of hAlb-hIL2_A4 but not hAlb-hIL2_A6 ( Figure 8 A, B). Strikingly, both mutβγ mutations, including variants hAlb-hIL2_A4s and hAlb-hIL2_A6s, further increased antigen-specific T cell frequency without any detectable increase in Treg frequency (or cell count, data not shown). hAlb-hIL2_A4s and hAlb-hIL2_A6s resulted in a preferential increase in antigen-specific T cells relative to nonspecific CD8 T cells, which exceeded the hAlb-hIL2_A4 and hAlb-hIL2_A6 ( Figure 8 C). In addition, all constructs strongly expanded NK cell frequency (and count, data not shown) to almost 50% of peripheral lymphocytes ( Figure 8 D). Here, hAlb-hIL2_A4s and hAlb-hIL2_A6s showed similar potency to hAlb-hIL2_A4 at high doses, but showed superiority at low doses. In summary, this suggests that the mutβγ mutation can further improve the potency of the hAlb-hIL2mutCD25 variant without increasing Treg frequency.

[0470] Example 10: hAlb-hIL2 variants containing both mutCD25 and mutβγ mutations are superior to mutβγ mutations containing IL2 and wild-type hAlb-hIL2

[0471] In Example 9, we showed that the introduction of mutβγ mutations into mutCD25 variants increased their potency to stimulate antigen-specific T cells without increasing Treg frequency. Next, we wanted to test whether IL2 variants containing both mutCD25 and mutβγ mutations were superior to mutβγ (hAlb-hIL2s) containing IL2 variants. BALB / c mice (n=5 per group) were vaccinated iv on days 0 and 7 with 20 μg gp70 RNA-LPX and on days 3 and 10 with cytokine RNA-LNPs (doses indicated in the figures). Blood lymphocytes were analyzed by flow cytometry (see Example 8) on days 7 and 14 ( Fig. 9 A). All IL2 variants showed an increase in the number of antigen-specific T cells compared to the hAlb control ( Fig. 9 B). Importantly, although hAlb-hIL2 and, to a lesser extent, hAlb-hIL2s expanded Tregs, Treg counts were not increased in the hAlb-hIL2_A4s and hAlb-hIL2_A6s treated groups ( Fig. 9 C). The lack of Treg expansion in hAlb-hIL2_A4s and hAlb-hIL2_A6s may explain the strong enhancement of antigen-specific T cells after the second treatment that exceeds the effect of hAlb-hIL2 and hAlb-hIL2s variants ( Fig. 9 B). In contrast, for hAlb-hIL2 and hAlb-hIL2s, the expansion of antigen-specific T cells slowed down after the second treatment, probably due to the suppression of effector T cells by induced Tregs ( Fig. 9 Similarly, CD8+ T cell numbers were most strongly increased by mutCD25 and mutβγ mutations comprising hAlb-hIL2_A4s and hAlb-hIL2_A6s variants ( Fig. 9 D). Thus, the ratio of antigen-specific T cells to Tregs ( Fig. 9 E) and CD8+T cell to Treg ratios were significantly increased. All hAlb-hIL2 variants and especially hAlb-hIL2_A4s and hAlb-hIL2_A6s preferentially expanded antigen-specific T cells (tetramer+ / CD8+) ( Fig. 9G). Interestingly, hAlb-hIL2_A4s and hAlb-hIL2_A6s induced lower frequencies of KLRG1+CD127- short-lived effector T cells (SLECs) than hAlb-hIL2 and hAlb-hIL2s ( Fig. 9 H). High SLEC frequencies negatively affect the longevity of T cell responses due to a reduced likelihood of memory formation (Joshi, NS et al. Immunity 27, 281-295 (2007)). In addition, the hAlb-hIL2_A4s and hAlb-hIL2_A6s variants expanded NK cells much more strongly than hAlb-hIL2 and slightly more strongly than hAlb-hIL2s ( Fig. 9 I). All mutβγ mutation-containing variants resulted in a significantly higher fraction of KLRG1-expressing NK cells compared to hAlb-hIL2, indicating an activated effector phenotype ( Fig. 9 J). KLRG1-positive NK cells have been shown to protect against lung metastatic disease (Renner, P. et al. Oncoimmunology 3, e28328 (2014)). Due to the short-lived nature of activated NK cells, NK cell counts decreased in all mutβγ mutants containing variants after the first treatment, but remained higher than the hAlb control group ( Fig. 9 I).

[0472] In conclusion, the combination of mutCD25 and mutβγ mutations significantly enhanced the potency of hAlb-hIL2 to increase antigen-specific T cells and NK cells without expanding Tregs, resulting in a strongly increased ratio of (antigen-specific) CD8+ T cells to Tregs.

[0473] Example 11: hAlb-hIL2_A4s administration enhances CD4+ T cell responses

[0474] In previous experiments, we have demonstrated that administration of IL2 variants containing both mutCD25 and mutβγ mutations enhances the induction of antigen-specific CD8+T cells. To further test whether CD4+T cells will benefit from these IL2 variants, C57BL / 6 mice (n=7 per group) were treated iv with 20 μg B16_M30 (Kreiter, S. et al. Nature 520, 692-696 (2015)) RNA-LPX and 3 μg hAlb, hAlb-hIL2 or hAlb-hIL2_A4s RNA-LNPs on days 0, 7 and 14. Blood lymphocyte analysis was performed by flow cytometry (see Example 8) on day 19 ( Fig.10A). B16_M30 is a class II MHC restricted neoepitope of the B16F10 tumor cell line recognized by CD4+ T cells (Kreiter, S. et al. Nature 520, 692-696 (2015)).

[0475] like Fig.10 As shown in B and 10C, only co-administration of hAlb-hIL2_A4, but not hAlb-hIL2, increased the number of CD4+ effector T cells / non-Treg (CD25-FoxP3-CD4+) and B16_M30-specific tetramer+CD4+ T cells, respectively.

[0476] Example 12: IL2 variants with combined mutCD25 and mutβγ mutations enhance the anti-tumor efficacy of cancer vaccination

[0477] Tumor antigen-specific T cells induced by vaccination can control tumor growth, while Tregs inhibit the action of effector T cells (Kreiter, S. et al. Nature 520, 692-696 (2015).). We have previously shown that hAlb-hIL2 can significantly improve the anti-tumor efficacy of gp70 RNA vaccination in CT26 tumor-bearing mice. Therefore, it is expected that improved variants of hAlb-hIL2 that do not increase Tregs, such as hAlb-hIL2_A4s and hAlb-hIL2_A6s, are even more effective in treatment.

Claims

1. A polypeptide comprising a mutant protein of human interleukin-2 (IL2), wherein the human IL2 is replaced at the following positions: (i) at positions K43 and E61 of the human IL2 amino acid sequence shown in SEQ ID NO: 17; or (ii) at positions K43 and E61 of the human IL2 amino acid sequence, each as shown in SEQ ID NO: 17, and at one or more positions that enhance affinity for IL2Rβγ selected from Q74, L80, R81, L85, I86 and I92, and wherein the substitution at position 43 is K43E and the substitution at position 61 is E61K.

2. The polypeptide of claim 1, wherein the polypeptide activates effector T cells relative to regulatory T cells.

3. The polypeptide of claim 1, wherein the one or more amino acid substitutions that enhance affinity for IL2Rβγ are selected from 74R, 74H, 74N, 74S, 80F, 81D, 85V, 86V and 92F. 4 . The polypeptide of claim 1 , wherein the one or more amino acid substitutions that enhance affinity for IL2Rβγ comprise the following substitution group: 80F, 81D, 85V, 86V, 92F.

5. The polypeptide of claim 1, which is a pharmacokinetic (PK)-extended IL2, further comprising an amino acid sequence heterologous to the IL2 fused to the substituted IL2.

6. The polypeptide of claim 5, wherein the amino acid sequence heterologous to the IL2 is selected from the group consisting of serum albumin, immunoglobulin fragments, transferrin and Fn3.

7. The polypeptide according to claim 6, wherein (i) the serum albumin comprises mouse serum albumin or human serum albumin; or (ii) The immunoglobulin fragment comprises an immunoglobulin Fc domain.

8. A polynucleotide encoding the polypeptide according to any one of claims 1 to 7.

9. The polynucleotide of claim 8, which is RNA.

10. A host cell comprising the polynucleotide of claim 8 or 9.

11. A pharmaceutical composition comprising the polypeptide of any one of claims 1 to 7, the polynucleotide of claim 8 or 9, or the host cell of claim 10.

12. Use of the polypeptide of any one of claims 1 to 7, the polynucleotide of claim 8 or 9, the host cell of claim 10 or the pharmaceutical composition of claim 11 in the preparation of a medicament for treating cancer in a subject.

13. Use of: in the preparation of a medicament for inducing an immune response in a subject: a. The polypeptide according to any one of claims 1 to 7, the polynucleotide according to claim 8 or 9, the host cell according to claim 10, or the pharmaceutical composition according to claim 11; and b. a peptide or polypeptide comprising an epitope for inducing an immune response against an antigen in the subject, or a polynucleotide encoding the peptide or polypeptide.

14. The use of claim 13, wherein the polynucleotide encoding the peptide or polypeptide is RNA.

15. The use of claim 12 or 13, wherein the treatment activates effector T cells preferentially over regulatory T cells.

16. The use of claim 12, wherein the cancer is a cancer expressing a tumor-associated antigen.

17. Use of: in the preparation of a medicament for treating or preventing cancer in a subject: a The polypeptide according to any one of claims 1 to 7, the polynucleotide according to claim 8 or 9, the host cell according to claim 10, or the pharmaceutical composition according to claim 11; and b. a peptide or polypeptide comprising an epitope for inducing an immune response against a tumor-associated antigen in the subject, or a polynucleotide encoding the peptide or polypeptide.

18. The use of claim 12 or 17, wherein the cancer is selected from melanoma, leukemia, lymphoma, lung cancer, breast cancer, prostate cancer, ovarian cancer, colon cancer, mesothelioma, renal cell carcinoma and brain cancer.

19. A pharmaceutical preparation comprising: - the polypeptide according to any one of claims 1 to 7, the polynucleotide according to claim 8 or 9, the host cell according to claim 10 or the pharmaceutical composition according to claim 11; or - A polypeptide according to any one of claims 1 to 7, a polynucleotide according to claim 8 or 9, a host cell according to claim 10 or a pharmaceutical composition according to claim 11, and a peptide or polypeptide comprising an epitope for inducing an immune response against an antigen in a subject, or a polynucleotide encoding the peptide or polypeptide.

20. A kit comprising: a. The polypeptide according to any one of claims 1 to 7, the polynucleotide according to claim 8 or 9, the host cell according to claim 10 or the pharmaceutical composition according to claim 11, and b. a peptide or polypeptide comprising an epitope for inducing an immune response against an antigen in a subject, or a polynucleotide encoding the peptide or polypeptide, Wherein each component a. and b. is provided in a separate container.

21. Use of the pharmaceutical preparation according to claim 19 or the kit according to claim 20 in the preparation of a medicament for medical use.

22. Use of the pharmaceutical formulation of claim 19 or the kit of claim 20 in the preparation of a medicament for treating or preventing cancer in a subject, wherein the antigen is a tumor-associated antigen.

23. The use of claim 22, wherein the cancer is selected from melanoma, leukemia, lymphoma, lung cancer, breast cancer, prostate cancer, ovarian cancer, colon cancer, mesothelioma, renal cell carcinoma and brain cancer.

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