Il2 agonists

Abstract

Variants of interleukin-2 (IL2) are provided.SOLUTION: The present invention relates to a polypeptide comprising a mutein of human IL2 or a functional variant of human IL2, wherein the human IL2 or the functional variant thereof is substituted such that the affinity to β γ IL2 receptor complexes (IL2R β γ) is enhanced. In one embodiment, the human IL2 or functional variant thereof is further substituted to have reduced affinity for α β γ IL2 receptor complexes (IL2R α β γ). In one embodiment, the polypeptide activates effector T cells more than regulatory T cells. The invention also relates to a polynucleotide encoding a polypeptide of the invention, a host cell comprising said polynucleotide, a pharmaceutical composition comprising said polypeptide, polynucleotide or host cell.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to variants of interleukin 2 (IL2). In particular, the present invention relates to a polypeptide comprising a mutein of human IL2 or a functional variant of human IL2, wherein the human IL2 or functional variant thereof has been substituted to enhance its affinity for the βγ IL2 receptor complex (IL2Rβγ). In one embodiment, the human IL2 or functional variant thereof has been further substituted to decrease its affinity for the αβγ IL2 receptor complex (IL2Rαβγ). In one embodiment, the polypeptide activates effector T cells rather than regulatory T cells. The present invention also relates to polynucleotides encoding the polypeptides of the present invention, host cells containing the polynucleotides, pharmaceutical compositions comprising the polypeptides, polynucleotides, or host cells, methods of therapeutic or prophylactic treatment using the polypeptides, polynucleotides, host cells, or pharmaceutical compositions, and pharmaceutical preparations comprising the polypeptides, polynucleotides, host cells, or pharmaceutical compositions. [Background technology]

[0002] The immune system plays a key role in pathogen-related diseases as well as cancer, autoimmunity, and allergies. T cells and natural killer (NK) cells are important mediators of antitumor immune responses. CD8 + T cells and NK cells can directly lyse tumor cells, while CD4 + T cells are CD8 + It can mediate the influx of various immune subsets, including T cells and NK cells, into tumors. + T cells express anti-tumor CD8 + It can prime T cell responses and act directly on tumor cells by upregulating MHC and inhibiting growth via IFNγ. + and CD4 + Tumor-specific T cell responses can be induced by vaccination or adoptive transfer of T cells.

[0003] Cytokines play an important role in immunity. For example, interleukin 2 (IL2) is a potent immunostimulator that activates various cells of the immune system, including T cells, B cells, monocytes, and NK cells. IL2 is known to support the differentiation, proliferation, survival, and effector function of T cells and NK cells (Blattman, J. Net al. Nat. Med. 9, 540-7 (2003)). IL2 has been used for decades in the treatment of late-stage malignant melanoma (Maas, RA, Dullens, HF, & Den Otter, W. Cancer Immunol. Immunother. 36, 141-8 (1993)). Therefore, immunotherapies such as T cell vaccines or adoptive transfer of (naive, T cell receptor transgenic, or chimeric antigen receptor transgenic) T cells or NK cells can benefit from the coadministration of cytokines such as IL2. However, one drawback of recombinant IL2 is its short plasma half-life, which necessitates frequent injection of large amounts of the cytokine. This can cause serious side effects such as vascular leak syndrome (VLS) (Rosenberg, SA et al. N. Engl. J. Med. 316, 889-97 (1987)). The second drawback of IL-2 is that it inhibits regulatory T cells (T reg T reg T cells can suppress the function of anti-tumor effector T cells and NK cells, which correlates with decreased survival in cancer patients (Nishikawa, H. & Sakaguchi. Curr. Opin. Immunol. 27, 1-7 (2014)). reg Activation of these cells can exacerbate immunosuppression and potentially impair the intended therapeutic response. IL2 signals through the IL2 receptor, which exists as a high-affinity and intermediate-affinity form. The high-affinity IL2 receptor (IL2Rαβγ) is composed of CD25 (IL2Rα), CD122 (IL2Rβ), and CD132 (IL2Rγ) and binds to T cells. reg Cellular and activated CD4 + and CD8 +It is expressed on T cells. The intermediate affinity receptor (IL2Rβγ) lacks CD25 and is found predominantly on naive and memory T cells and NK cells. Consequently, IL2 binds to CD25-expressing T cells. reg cells (Todd, JA et al. PLoS Med. 13, e1002139 (2016)) and activated CD4 + and CD8 + High doses of IL2 are required to selectively stimulate T cells and activate naive and memory T cells as well as NK cells. Attempts to modify IL2 in a way that loses its selectivity for CD25-expressing cells, thereby relatively increasing its ability to stimulate naive and memory T cells as well as NK cells, have been shown to improve its antitumor activity (Arenas-Ramirez, N. et al. Sci. Transl. Med. 8, 1-13 (2016)).

[0004] In particular in the context of immunotherapy, particularly cancer immunotherapy, new strategies are needed to enhance the effectiveness of IL2 when used in combination with other immunotherapies such as, for example, vaccines, particularly cancer vaccines, and adoptive transfer of (naive or T cell receptor transgenic or chimeric antigen receptor transgenic) T cells and NK cells, and / or checkpoint inhibition.

[0005] Described herein are variants of human IL2 that selectively activate cells expressing the intermediate-affinity IL2 receptor IL2Rβγ compared to cells expressing the high-affinity IL2 receptor IL2Rαβγ. In particular, suitable modifications of IL2 that enhance binding to (and thus activation of) cells expressing IL2Rβγ are described herein. These modifications may be used in combination with modifications that prevent effective binding to (and thus activation of) cells expressing IL2Rαβγ. IL2 variants that can selectively activate the intermediate-affinity IL2 receptor on certain T cells, such as memory T cells, naive T cells, and effector T cells, as well as NK cells, over the high-affinity IL2 receptor on regulatory T cells, are expected to have an improved therapeutic index and a reduced toxicity profile compared to wild-type IL2. IL2 variants with an improved therapeutic index could significantly expand their use in the treatment of disorders requiring immune system stimulation, such as cancer (direct and / or adjunctive therapy). In particular, administration of IL2 variant RNA is a promising approach to enhance the therapeutic efficacy of multiple T cell- and NK cell-based (cancer) immunotherapies. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Blattman, JNet al. Nat. Med. 9, 540-7 (2003) [Non-patent document 2] Maas, RA, Dullens, HF & Den Otter, W. Cancer Immunol. Immunother. 36, 141-8 (1993) [Non-patent document 3] Rosenberg, SA et al. N. Engl. J. Med. 316, 889-97 (1987) [Non-patent document 4] Nishikawa, H. & Sakaguchi. Curr. Opin. Immunol. 27, 1-7 (2014) [Non-Patent Document 5] Todd,JAet al.PLoS Med.13,e1002139(2016) [Non-patent document 6] Arenas-Ramirez,N.et al.Sci.Transl.Med.8,1-13(2016) Summary of the Invention [Problem to be solved by the invention]

[0007] The present disclosure provides novel IL2 variants. Specifically, variants of IL2 are described that contain mutations that enhance IL2Rβγ binding, particularly CD122 binding ("mutβγ"), and optionally further contain mutations that affect IL2Rαβγ binding, particularly CD25 binding ("mutα"). In particular, T reg Reduces cell proliferation and stimulates effector T cells and NK cells, preferably IL2Rβγ + Described herein are variants of IL2 that increase effector T cell and NK cell stimulation. The IL2 variant hAlb-hIL2_A4s8 described herein stimulates T cells already at lower concentrations compared to wild-type IL2. reg The ability to activate cells is significantly reduced, and effector immune cells, preferably CD8 + T cell and NK cell-like IL2Rβγ + In vivo, treatment with nanoparticle mRNA encoding wild-type IL2 or a selected IL2 variant (hAlb-hIL2_A4s8) with mutations affecting both IL2Rα and IL2Rβ binding, targeted to the liver of mice for systemic availability, potently inhibited tumor growth, with hIL2_A4s8 being the most effective. hAlb-hIL2 primarily targeted antigen-specific T cells and T reg hAlb-hIL2_A4s8 activated NK cells and increased T reg Antigen-specific and antigen-nonspecific CD8 without cell expansion + Increases T cell numbers. Treatment with hAlb-hIL2_A4s8 increases T regVaccine-induced CD8 while avoiding cell stimulation and expansion + It potently synergizes with RNA vaccination by expanding T cell responses and inhibiting pre-existing antigen-specific CD8 + Enhancement of the antitumor effects of PD-L1 immune checkpoint blockade by specifically expanding T cells. [Means for solving the problem]

[0008] In a first aspect, provided herein is a polypeptide comprising a mutein of human interleukin-2 (IL2) or a functional variant of human IL2, wherein the human IL2 or functional variant thereof has substitutions at at least positions 80 (leucine), 81 (arginine), 85 (leucine), and 92 (isoleucine), relative to wild-type human IL2 and numbered according to wild-type human IL2, which substitutions enhance affinity for the βγ IL2 receptor complex (IL2Rβγ), and wherein the human IL2 or functional variant thereof does not have a substitution at position 86 (isoleucine), relative to wild-type human IL2 and numbered according to wild-type human IL2.

[0009] In one embodiment, compared to wild-type human IL2 and numbered according to wild-type human IL2, position 80 (leucine) is substituted with phenylalanine, position 81 (arginine) is substituted with glutamic acid, position 85 (leucine) is substituted with valine, and position 92 (isoleucine) is substituted with phenylalanine.

[0010] In one embodiment, human IL2 or a functional variant thereof is further substituted at position 74 (glutamine) compared to and numbered according to wild-type human IL2. In one embodiment, position 74 (glutamine) compared to and numbered according to wild-type human IL2 is substituted with histidine.

[0011] In a second aspect, there is provided herein a polypeptide comprising a mutein of human IL2 or a functional variant of human IL2, wherein the human IL2 or functional variant thereof is substituted at least at position 80 (leucine) by phenylalanine, at position 81 (arginine) by glutamic acid, at position 85 (leucine) by valine, and at position 92 (isoleucine) by phenylalanine compared to wild-type human IL2 and numbered according to wild-type human IL2.

[0012] In one embodiment, the human IL2 or functional variant thereof is further substituted by a histidine at position 74 (glutamine) compared to and numbered according to wild-type human IL2.

[0013] In a third aspect, there is provided herein a polypeptide comprising a mutein of human IL2 or a functional variant of human IL2, wherein the human IL2 or functional variant thereof has at least one substitution at position 74 (glutamine) by histidine, at position 80 (leucine) by phenylalanine, at position 81 (arginine) by glutamic acid, at position 85 (leucine) by valine, and at position 92 (isoleucine) by phenylalanine, compared to wild-type human IL2 and numbered according to wild-type human IL2.

[0014] In one embodiment of the second and third aspects, the substitution enhances affinity for IL2Rβγ.

[0015] In one embodiment, the substituted IL2 or functional variant thereof (IL2 mutein) has an amino acid sequence identical to wild-type IL2 at other unsubstituted residues. In one embodiment, the IL2 mutein has an amino acid modification, such as an amino acid substitution, at one or more sites or other residues in wild-type human IL2. In one embodiment, such amino acid substitution results in a relatively reduced affinity for IL2Rαβγ compared to wild-type IL2 (also referred to herein as a "mutα" mutation). In one embodiment, such amino acid substitution is in an amino acid residue that contacts IL2Rα.

[0016] Thus, in one embodiment, the human IL2 or functional variant thereof further comprises one or more amino acid substitutions that reduce affinity for the alpha subunit of the αβγ IL2 receptor complex (IL2Rαβγ).

[0017] In one embodiment, the one or more amino acid substitutions that reduce affinity for the alpha subunit of IL2Rαβγ reduce affinity for IL2Rαβγ to a greater extent than affinity for IL2Rβγ.

[0018] In one embodiment, the one or more amino acid substitutions that reduce affinity for the alpha subunit of IL2Rαβγ comprise a substitution of human IL2 or a functional variant thereof at at least one of positions 35 (lysine), 43 (lysine), 61 (glutamic acid), and 62 (glutamic acid), compared to 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 with 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 with an acidic amino acid residue.

[0019] In different embodiments, the one or more amino acid substitutions that reduce affinity for the alpha subunit of IL2Rαβγ comprise substitutions of human IL2 or a functional variant thereof at least at the following positions, compared to and numbered according to wild-type human IL2: -35th place, -43rd place, -61st place, -62nd place, -35th and 43rd place, -35th and 61st place, -35th and 62nd place, -43rd and 61st place, -43rd and 62nd place, -61st and 62nd place, -35th, 43rd and 61st places, -35th, 43rd and 62nd places, -35th, 61st and 62nd places, -43rd, 61st and 62nd places, or -35th, 43rd, 61st and 62nd places.

[0020] In one embodiment, position 35 is substituted with glutamic acid. In one embodiment, position 43 is substituted with glutamic acid. In one embodiment, position 61 is substituted with lysine. In one embodiment, position 62 is substituted with lysine.

[0021] In one embodiment, there is a substitution at position 35. In one embodiment, there is a substitution at position 35 with glutamic acid.

[0022] In one embodiment, there is a substitution at position 43. In one embodiment, there is a substitution at position 43 with glutamic acid.

[0023] In one embodiment, there is a substitution at position 61. In one embodiment, there is a substitution at position 61 with lysine.

[0024] In one embodiment, there is a substitution at position 62. In one embodiment, there is a substitution at position 62 with lysine.

[0025] In one embodiment, there are substitutions at positions 43 and 61. In one embodiment, there is a substitution at position 43 with glutamic acid and at position 61 with lysine.

[0026] In one embodiment, there are substitutions at positions 35, 43, and 61. In one embodiment, there is a substitution at position 35 with glutamic acid, a substitution at position 43 with glutamic acid, and a substitution at position 61 with lysine.

[0027] In one embodiment, there are substitutions at positions 61 and 62. In one embodiment, there is a substitution at position 61 with a lysine and a substitution at position 62 with a lysine.

[0028] In one embodiment, the one or more amino acid substitutions that reduce affinity for the alpha subunit of IL2Rαβγ comprise substitutions in human IL2 or a functional variant thereof at positions 43 (lysine) and 61 (glutamic acid) compared to and numbered according to wild-type human IL2. In one embodiment, position 43 (lysine) is substituted with glutamic acid and position 61 (glutamic acid) is substituted with lysine.

[0029] In a fourth aspect, there is provided herein a polypeptide comprising a mutein of human IL2 or a functional variant of human IL2, wherein the human IL2 or functional variant thereof has at least one substitution at position 43 (lysine) by glutamic acid, at position 61 (glutamic acid) by lysine, at position 74 (glutamine) by histidine, at position 80 (leucine) by phenylalanine, at position 81 (arginine) by glutamic acid, at position 85 (leucine) by valine, and at position 92 (isoleucine) by phenylalanine, compared to wild-type human IL2 and numbered according to wild-type human IL2.

[0030] In one embodiment of the second to fourth aspects, the human IL2 or functional variant thereof is not substituted at position 86 (isoleucine) compared to and numbered according to wild-type human IL2.

[0031] In one embodiment, the human IL2 has an amino acid sequence according to SEQ ID NO:1.

[0032] In one embodiment, the mutein of human IL2 or a functional variant thereof has a reduced ability to stimulate regulatory T cells compared to wild-type human IL2.

[0033] In one embodiment, the mutein of human IL2 or a functional variant thereof has an increased ability to stimulate effector T cells compared to wild-type human IL2.

[0034] The IL2 muteins described herein can be linked to a pharmacokinetic-modifying group and therefore can be an "extended pharmacokinetic (PK) IL2."

[0035] In one embodiment, the polypeptide described herein is an extended pharmacokinetic (PK) polypeptide. In one embodiment, the extended PK polypeptide comprises a fusion protein. In one embodiment, the fusion protein comprises a portion of a mutein of human IL2 or a functional variant thereof and a portion heterologous to human IL2 or a functional variant thereof. In one embodiment, the fusion protein comprises a portion of a mutein of human IL2 or a functional variant thereof and a portion selected from the group consisting of serum albumin, an immunoglobulin fragment, transferrin, Fn3, and variants 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.

[0036] In one aspect, provided herein is a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 11, 13, and 22, such as SEQ ID NO: 2, SEQ ID NO: 11, SEQ ID NO: 13, or SEQ ID NO: 22 in the Sequence Listing.

[0037] The above polypeptides are also referred to herein as "IL2 variant polypeptides" or simply "IL2 variants."

[0038] In one aspect, provided herein is a polynucleotide encoding a polypeptide described herein. In one embodiment, the polynucleotide is RNA.

[0039] In one aspect, provided herein is a host cell comprising a polynucleotide described herein.

[0040] In one aspect, provided herein is a polypeptide described herein, a polynucleotide described herein, or a host cell described herein for pharmaceutical uses. In one embodiment, the pharmaceutical use comprises therapeutic or prophylactic treatment of a disease or disorder.

[0041] In one aspect, provided herein is a polypeptide described herein, a polynucleotide described herein, or a host cell described herein for use in a method for treating or preventing cancer in a subject.

[0042] In one aspect, provided herein is a pharmaceutical composition comprising a polypeptide described herein, a polynucleotide described herein, or a host cell described herein.

[0043] In one aspect, provided herein is a method of treating a subject comprising administering to the subject a polypeptide described herein, a polynucleotide described herein, a host cell described herein, or a pharmaceutical composition described herein.

[0044] In one aspect, provided herein is a method for inducing an immune response in a subject, comprising administering to the subject a polypeptide described herein, a polynucleotide described herein, a host cell described herein, or a pharmaceutical composition described herein.

[0045] In one embodiment, the subject is further treated with one or more immunotherapies, e.g., vaccination or adoptive transfer of T cells, e.g., a T cell vaccine or adoptive transfer of (naive or T cell receptor transgenic or chimeric antigen receptor transgenic) T cells or NK cells.

[0046] The efficacy of vaccines in which the antigen is delivered by itself or as a polynucleotide, particularly as RNA encoding the antigen (e.g., RNA encoding a peptide or protein used in vaccination, also referred to herein as a "peptide or protein containing an epitope for inducing an immune response against the antigen," "vaccine antigen," or simply "antigen") can be increased by co-administering an IL2 variant polypeptide described herein, delivered by itself or as a polynucleotide, particularly an RNA encoding the IL2 variant polypeptide. Vaccines are particularly effective when the RNA encoding the IL2 variant polypeptide is targeted to the liver for systemic availability. Hepatocytes can be efficiently transfected and produce large amounts of protein. The RNA encoding the antigen is preferably targeted to secondary lymphoid organs. Furthermore, vaccines are particularly effective when an immune checkpoint inhibitor, such as an anti-PD-L1 antibody, is also administered.

[0047] In one embodiment, the method described herein further comprises administering to the subject a peptide or protein comprising an epitope for inducing an antigen-specific immune response in the subject, or a polynucleotide encoding said peptide or protein. In one embodiment, the polynucleotide encoding the peptide or protein is RNA.

[0048] In one embodiment, the method described herein is a method for treating or preventing cancer in a subject, and optionally, the antigen is a tumor-associated antigen.

[0049] In one aspect, provided herein is a method for treating or preventing cancer in a subject, comprising administering to the subject a polypeptide described herein, a polynucleotide described herein, a host cell described herein, or a pharmaceutical composition described herein.

[0050] In one embodiment, the method further comprises administering to the subject a peptide or protein comprising an epitope for inducing an immune response specific to the tumor-associated antigen in the subject, or a polynucleotide encoding said peptide or protein. In one embodiment, the polynucleotide encoding the peptide or protein is RNA.

[0051] In one embodiment, the methods described herein comprise administering to a subject: a. RNA encoding an IL2 variant polypeptide described herein; and b. RNA encoding a peptide or protein containing an epitope for inducing an antigen-specific immune response in a subject.

[0052] In one embodiment, the cancer is selected from the group consisting of melanoma, leukemia, lymphoma, lung cancer, breast cancer, prostate cancer, ovarian cancer, colon cancer, mesothelioma, renal cell carcinoma, and brain cancer.

[0053] In one embodiment, the methods described herein further comprise administering to the subject an immune checkpoint inhibitor. In one embodiment, the immune checkpoint inhibitor targets the interaction between (i) PD-1 and PD-L1, or (ii) 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.

[0054] 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 against an antigen in a subject, and optionally an immune checkpoint inhibitor are administered simultaneously or sequentially.

[0055] In one aspect, provided herein is a pharmaceutical formulation comprising a polypeptide described herein, a polynucleotide described herein, a host cell described herein, or a pharmaceutical composition described herein.

[0056] In one embodiment, the pharmaceutical formulation further comprises a peptide or protein comprising an epitope for inducing an antigen-specific immune response in a subject, or a polynucleotide encoding said peptide or protein. In one embodiment, the polynucleotide encoding the peptide or protein is RNA.

[0057] In one embodiment, the pharmaceutical preparation comprises the polypeptide, polynucleotide, host cell, or pharmaceutical composition and the epitope-containing peptide or protein or the polynucleotide encoding said peptide or protein, each in separate containers.

[0058] In one embodiment, the pharmaceutical formulation comprises: a. RNA encoding an IL2 variant polypeptide described herein; and b. RNA encoding a peptide or protein containing an epitope for inducing an antigen-specific immune response in a subject.

[0059] In one embodiment of the pharmaceutical preparation, RNA is present in a form selected from liquid form, solid form, or a combination thereof.In one embodiment, the solid form is frozen form or dehydrated form.In one embodiment, the dehydrated form is freeze-dried form or spray-dried form.

[0060] In one embodiment, the pharmaceutical formulation further comprises an immune checkpoint inhibitor. In one embodiment, the immune checkpoint inhibitor targets the interaction between (i) PD-1 and PD-L1, or (ii) 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.

[0061] In one embodiment, the pharmaceutical preparation is a kit. In one embodiment, the pharmaceutical preparation comprises each of components a and b in separate containers.

[0062] In one embodiment, the pharmaceutical formulation is a pharmaceutical composition, which in one embodiment further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0063] In one embodiment, the pharmaceutical formulation further comprises instructions for using the pharmaceutical formulation to treat or prevent cancer, and optionally, the antigen is a tumor-associated antigen.

[0064] In one aspect, provided herein is a pharmaceutical formulation as described herein for medical use. In one embodiment, the medical use includes the therapeutic or prophylactic treatment of a disease or disorder.

[0065] In one aspect, provided herein is a pharmaceutical formulation as described herein for use in a method for treating or preventing cancer in a subject, optionally wherein the antigen is a tumor-associated antigen.

[0066] In one embodiment, the cancer is selected from the group consisting of melanoma, leukemia, lymphoma, lung cancer, breast cancer, prostate cancer, ovarian cancer, colon cancer, mesothelioma, renal cell carcinoma, and brain cancer.

[0067] In a further aspect, the 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 the methods described herein. [Brief explanation of the drawings]

[0068] [Figure 1] Functional activity of hAlb-hIL2_A4 variants in different lymphocyte subsets of human PBMCs, as measured by IL2-mediated phosphorylation of STAT5. Dose-response curves for STAT5 phosphorylation (pSTAT5) in CD4+CD25+ Treg cells (A), CD8+ cytotoxic T cells (B), and CD56+ NK cells (C). Human PBMCs were incubated with serial dilutions of hAlb-hIL2_A4 variant-containing supernatants, and STAT5 phosphorylation was subsequently analyzed in various lymphocyte subsets by flow cytometry. Supernatant from HEK293T / 17 cells lipofected with mRNA encoding hAlb alone was used as a negative control. Data shown are single values ​​from one representative experiment, to which a four-parameter logarithmic fit was applied to calculate EC50 values. [Figure 2] Functional activity of hAlb-hIL2_A4 variants in different lymphocyte subsets of mouse splenocytes, as measured by IL2-mediated phosphorylation of STAT5. Dose-response curves for STAT5 phosphorylation (pSTAT5) in CD4+CD25+ Treg cells (A), CD8+ cytotoxic T cells (B), and NK cells (C). Balb / c splenocytes were incubated with serial dilutions of hAlb-hIL2_A4 variant-containing supernatants, and STAT5 phosphorylation was subsequently analyzed in various lymphocyte subsets by flow cytometry. Supernatant from HEK293T / 17 cells lipofected with mRNA encoding hAlb alone was used as a negative control. Data shown are single values ​​from one representative experiment, to which a four-parameter logarithmic fit was applied to calculate EC50 values. [Figure 3]Relative biological activity of hAlb-hIL2_A4 variants in intermediate-affinity IL2 receptor (IL2Rβγ)- and high-affinity IL2 receptor (IL2Rαβγ)-dependent cell cultures. The proliferative responses of the human cell line TF-1_hIL2Rβγ (A), which expresses the intermediate-affinity IL2 receptor (IL2Rβγ), and the human cell line TF-1_hIL2Rαβγ (B), which expresses the high-affinity IL2 receptor (IL2Rαβγ), are shown. Cell cultures were incubated with serial dilutions of hAlb-hIL2_A4 variant-containing supernatants for 3 days, and proliferation was measured by quantifying viable cells via ATP content using the CellTiter-Glo® 2.0 assay. Data shown are the mean ± SD of n = 2 technical replicates, to which a four-parameter logarithmic fit was applied to calculate EC50 values. RLU = relative luminescence units. [Figure 4] Binding of human IL2Rα (CD25) and human IL2Rβ (CD122) by hAlb-hIL2_A4s8 compared to hAlb-hIL2_A4 and hAlb-hIL2. 100 ng of plate-bound recombinant human CD25-Fc (A) or human CD122-Fc (B) was incubated with a 1:2 dilution of hAlb-hIL2 variant-containing supernatant, and bound protein was detected with an HRP-conjugated anti-human serum albumin antibody. Supernatant from HEK293T / 17 cells lipofected with mRNA encoding hAlb alone was used as a negative control. Data shown are the mean ± SD of n=2 technical replicates. [Figure 5]Comparison of hAlb-hIL2 and hAlb-hIL2_A4s8 for functional activity in different immune cell subsets of human PBMCs, as measured by IL2-mediated phosphorylation of STAT5. Dose-response curves for STAT5 phosphorylation (pSTAT5) in CD4+CD25+ Treg cells (A), CD8+ cytotoxic T cells (B), and CD56+ NK cells (C). Human PBMCs were incubated with serial dilutions of hAlb-hIL2 variant-containing supernatants, and STAT5 phosphorylation was subsequently analyzed in various lymphocyte subsets by flow cytometry. Data shown are the mean ± SD of n=2 technical replicates, to which a four-parameter logarithmic fit was applied to calculate EC50 values. [Figure 6] Functional activity of hAlb-hIL2_A4s8 compared to hAlb-hIL2 in different T cell subsets of mouse PBMCs, as measured by IL2-mediated phosphorylation of STAT5. Dose-response curves for STAT5 phosphorylation (pSTAT5) in CD4+CD25+ Treg cells (A) and CD8+ cytotoxic T cells (B). PBMCs isolated from whole blood of Balb / c mice were incubated with serial dilutions of hAlb-hIL2 variant-containing supernatants, and STAT5 phosphorylation was subsequently analyzed in various T cell subsets by flow cytometry. Data shown are single values ​​from one representative experiment, to which a four-parameter logarithmic fit was applied to calculate EC50 values. [Figure 7]Decreased tumor growth resulted in improved survival when treated with hAlb-hIL2_A4s8 compared with hAlb-hIL2 in combination with a therapeutic RNA vaccine in the murine colon cancer model CT26. BALB / c mice (n=11 per group) were inoculated subcutaneously (sc) with 5x105 CT26 tumor cells and intravenously (iv) with 20µg of RNA-LPX encoding the CD8+ T cell antigen gp70 four times (days 10, 17, 24, and 31) once a week. RNA encoding hAlb-hIL2_A4s8 or hAlb-hIL2 (3µg each), formulated as LNPs, was administered intravenously simultaneously with the RNA vaccine. A control group received an RNA vaccine containing hAlb (no cytokine encoding) formulated as LNPs. (A) Tumor growth in individual mice and (B) survival rate of mice treated with hAlb-hIL2 or hAlb-hIL2_A4s8 in combination with gp70 vaccine. Dotted lines indicate the day of treatment. Ratios in (A) represent the number of tumor-free mice relative to the total number of mice per group. [Figure 8] Expansion of tumor antigen-specific CD8+ T cells and NK cells upon treatment with hAlb-hIL2_A4s8 compared to hAlb-hIL2 in combination with a therapeutic RNA vaccine in the murine colon cancer model CT26. Absolute numbers of (A) gp70-specific T cells and (B) NK cells per μL of blood measured by flow cytometry 7 days (days 17, 24, and 31) after the first three treatments from BALB / c mice as described in Example 6 and Figure 7. Dotted lines indicate treatment days, and dotted numbers indicate the number of treatments. Mean ± SEM. Statistical significance was determined using a two-way ANOVA followed by Dunnett's multiple comparison test. All analyses were two-tailed and performed using GraphPad Prism 8. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 9A]Expansion of tumor antigen-specific CD8+ T cells relative to Treg cells upon treatment with hAlb-hIL2_A4s8 compared to hAlb-hIL2 in combination with a therapeutic RNA vaccine in the murine colon cancer model CT26. (A) Absolute numbers of antigen-nonspecific CD8+ T cells, CD4+ T cells, and Treg cells per μL of blood measured by flow cytometry 7 days after the first treatment (day 17) from BALB / c mice as described in Example 6, Figures 7 and 8. (B) Ratio of antigen-specific CD8+ T cells (left) or antigen-nonspecific CD8+ T cells (right) relative to Treg cells. Points represent individual mice, and lines represent group means. Statistical significance was determined using one-way analysis of variance followed by Dunnett's multiple comparison test. All analyses were two-tailed and performed using GraphPad Prism 8. ns P>0.05, *P<0.05, **P<0.01, ****P<0.0001. [Figure 9B] Expansion of tumor antigen-specific CD8+ T cells relative to Treg cells upon treatment with hAlb-hIL2_A4s8 compared to hAlb-hIL2 in combination with a therapeutic RNA vaccine in the murine colon cancer model CT26. (A) Absolute numbers of antigen-nonspecific CD8+ T cells, CD4+ T cells, and Treg cells per μL of blood measured by flow cytometry 7 days after the first treatment (day 17) from BALB / c mice as described in Example 6, Figures 7 and 8. (B) Ratio of antigen-specific CD8+ T cells (left) or antigen-nonspecific CD8+ T cells (right) relative to Treg cells. Points represent individual mice, and lines represent group means. Statistical significance was determined using one-way analysis of variance followed by Dunnett's multiple comparison test. All analyses were two-tailed and performed using GraphPad Prism 8. ns P>0.05, *P<0.05, **P<0.01, ****P<0.0001. [Figure 10]Decreased tumor growth resulted in improved survival after treatment with hAlb-hIL2_A4s8 alone in the murine colon cancer model CT26. BALB / c mice (n=11 per group) were inoculated subcutaneously (sc) with 5x10 CT26 tumor cells and treated intravenously (iv) four times (days 10, 17, 24, and 31) weekly with RNA encoding hAlb-hIL2_A4s8 formulated as LNPs, with or without concurrent iv vaccination with 20 μg of gp70 RNA-LPX. Control groups received an irrelevant RNA vaccine (non-antigen-encoding vaccine) containing hAlb (no cytokine-encoding) formulated as LNPs, or gp70 vaccine alone. (A) Tumor growth and (B) survival rates of individual mice treated with hAlb-hIL2_A4s8, with or without gp70 vaccine. Dotted lines indicate treatment days. The ratios in (A) represent the number of tumor-free mice relative to the total number of mice per group. [Figure 11AB] Expansion of tumor antigen-specific CD8+ T cells and NK cells without affecting Treg cells upon treatment with hAlb-hIL2_A4s8 in the murine colon cancer model CT26. Absolute numbers of (A) gp70-specific T cells, (B) NK cells, and (C) Treg cells per μL of blood measured by flow cytometry 7 days (days 17, 24, and 31) after the first three treatments from BALB / c mice as described in Example 7 and Figure 10. Dotted lines indicate treatment days, and dotted numbers indicate the number of treatments. Mean ± SEM. Statistical significance was determined using a two-way ANOVA followed by Dunnett's multiple comparison test. All analyses were two-tailed and performed using GraphPad Prism 8. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 11C]Expansion of tumor antigen-specific CD8+ T cells and NK cells without affecting Treg cells upon treatment with hAlb-hIL2_A4s8 in the murine colon cancer model CT26. Absolute numbers of (A) gp70-specific T cells, (B) NK cells, and (C) Treg cells per μL of blood measured by flow cytometry 7 days (days 17, 24, and 31) after the first three treatments from BALB / c mice as described in Example 7 and Figure 10. Dotted lines indicate treatment days, and dotted numbers indicate the number of treatments. Mean ± SEM. Statistical significance was determined using a two-way ANOVA followed by Dunnett's multiple comparison test. All analyses were two-tailed and performed using GraphPad Prism 8. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 12A] Expansion of tumor antigen-specific CD8+ T cells relative to Treg cells upon treatment with hAlb-hIL2_A4s8 in the murine colon cancer model CT26. (A) Absolute numbers of antigen-specific CD8+ T cells, total CD8+ T cells, and CD4+ T cells per μL of blood measured by flow cytometry 7 days after the first treatment (day 17) from BALB / c mice as described in Example 6, Figures 10 and 11. (B) Ratio of antigen-specific CD8+ T cells (left) or total CD8+ T cells (right) relative to Treg cells. (C) Fold change in CD8+ T cell subsets in the treatment group relative to the corresponding median value of the CD8+ T cell subset in the control group. Points represent individual mice, and lines represent group means. Mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Dunnett's multiple comparison test. All analyses were two-tailed and performed using GraphPad Prism 8. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 12B]Expansion of tumor antigen-specific CD8+ T cells relative to Treg cells upon treatment with hAlb-hIL2_A4s8 in the murine colon cancer model CT26. (A) Absolute numbers of antigen-specific CD8+ T cells, total CD8+ T cells, and CD4+ T cells per μL of blood measured by flow cytometry 7 days after the first treatment (day 17) from BALB / c mice as described in Example 6, Figures 10 and 11. (B) Ratio of antigen-specific CD8+ T cells (left) or total CD8+ T cells (right) relative to Treg cells. (C) Fold change in CD8+ T cell subsets in the treatment group relative to the corresponding median value of the CD8+ T cell subset in the control group. Points represent individual mice, and lines represent group means. Mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Dunnett's multiple comparison test. All analyses were two-tailed and performed using GraphPad Prism 8. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 12C] Expansion of tumor antigen-specific CD8+ T cells relative to Treg cells upon treatment with hAlb-hIL2_A4s8 in the murine colon cancer model CT26. (A) Absolute numbers of antigen-specific CD8+ T cells, total CD8+ T cells, and CD4+ T cells per μL of blood measured by flow cytometry 7 days after the first treatment (day 17) from BALB / c mice as described in Example 6, Figures 10 and 11. (B) Ratio of antigen-specific CD8+ T cells (left) or total CD8+ T cells (right) relative to Treg cells. (C) Fold change in CD8+ T cell subsets in the treatment group relative to the corresponding median value of the CD8+ T cell subset in the control group. Points represent individual mice, and lines represent group means. Mean ± SEM. Statistical significance was determined using one-way ANOVA followed by Dunnett's multiple comparison test. All analyses were two-tailed and performed using GraphPad Prism 8. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 13]Decreased tumor growth resulted in improved survival after treatment with hAlb-hIL2_A4s8 compared with hAlb-hIL2 in combination with a therapeutic RNA vaccine in the B16 mouse melanoma model. C57BL / 6 mice (n=15 per group) were inoculated subcutaneously (sc) with 3x10 B16-F10 melanoma cells and treated intravenously (iv) once a week for five days (days 8, 15, 22, 29, and 36) with RNA encoding hAlb-hIL2 or hAlb-hIL2_A4s8 formulated as LNPs, with or without co-vaccination with 20µg of TRP1 RNA-LPX. Control groups received a non-antigen-encoding RNA vaccine (irrelevant vaccine) containing hAlb (not encoding a cytokine) formulated as LNPs, or the TRP1 vaccine plus hAlb. (A) Tumor growth in individual mice and (B) survival rate of mice treated with hAlb-hIL2 or hAlb-hIL2_A4s8 with or without TRP1 vaccine. Dotted lines indicate the treatment days. Ratios in (A) represent the number of tumor-free mice relative to the total number of mice per group. [Figure 14AB] Expansion of tumor antigen-specific CD8+ T cells and NK cells without affecting Treg cells upon treatment with hAlb-hIL2_A4s8 compared with hAlb-hIL2 combined with a therapeutic RNA vaccine in the murine melanoma model B16. Absolute numbers of (A) TRP1-specific T cells, (B) NK cells, and (C) Treg cells per μL of blood measured by flow cytometry 7 days (days 15, 22, and 29) after the first three treatments from C57BL / 6 mice as described in Example 8 and Figure 13. Dotted lines indicate treatment days, and dotted numbers indicate the number of treatments. Mean ± SEM. Statistical significance was determined using a two-way ANOVA followed by Dunnett's multiple comparison test. All analyses were two-tailed and performed using GraphPad Prism 8. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 14C]Expansion of tumor antigen-specific CD8+ T cells and NK cells without affecting Treg cells upon treatment with hAlb-hIL2_A4s8 compared with hAlb-hIL2 combined with a therapeutic RNA vaccine in the murine melanoma model B16. Absolute numbers of (A) TRP1-specific T cells, (B) NK cells, and (C) Treg cells per μL of blood measured by flow cytometry 7 days (days 15, 22, and 29) after the first three treatments from C57BL / 6 mice as described in Example 8 and Figure 13. Dotted lines indicate treatment days, and dotted numbers indicate the number of treatments. Mean ± SEM. Statistical significance was determined using a two-way ANOVA followed by Dunnett's multiple comparison test. All analyses were two-tailed and performed using GraphPad Prism 8. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 15A] Expansion of tumor antigen-specific CD8+ T cells relative to Treg cells upon treatment with hAlb-hIL2_A4s8 and hAlb-hIL2 in combination with a therapeutic RNA vaccine in the murine melanoma model B16. (A) Absolute numbers of antigen-specific CD8+ T cells, total CD8+ T cells, and CD4+ T cells per μL of blood measured by flow cytometry 7 days after the first treatment (day 15) from C57BL / 6 mice described in Example 8, Figures 13 and 14. (B) Ratios of total CD8+ T cells (top left) and antigen-specific CD8+ T cells (top right, bottom left) to Treg cells. (C) Fold changes in CD8+ T cell subsets in treatment groups relative to the corresponding median values ​​of CD8+ T cell subsets in the control group. Points represent individual mice, and lines represent group means. Mean ± SEM. Statistical significance was determined using one-way analysis of variance followed by Dunnett's multiple comparison test. All analyses were two-sided and performed using GraphPad Prism 8. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 15B]Expansion of tumor antigen-specific CD8+ T cells relative to Treg cells upon treatment with hAlb-hIL2_A4s8 and hAlb-hIL2 in combination with a therapeutic RNA vaccine in the murine melanoma model B16. (A) Absolute numbers of antigen-specific CD8+ T cells, total CD8+ T cells, and CD4+ T cells per μL of blood measured by flow cytometry 7 days after the first treatment (day 15) from C57BL / 6 mice described in Example 8, Figures 13 and 14. (B) Ratios of total CD8+ T cells (top left) and antigen-specific CD8+ T cells (top right, bottom left) to Treg cells. (C) Fold changes in CD8+ T cell subsets in treatment groups relative to the corresponding median values ​​of CD8+ T cell subsets in the control group. Points represent individual mice, and lines represent group means. Mean ± SEM. Statistical significance was determined using one-way analysis of variance followed by Dunnett's multiple comparison test. All analyses were two-sided and performed using GraphPad Prism 8. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 15C] Expansion of tumor antigen-specific CD8+ T cells relative to Treg cells upon treatment with hAlb-hIL2_A4s8 and hAlb-hIL2 in combination with a therapeutic RNA vaccine in the murine melanoma model B16. (A) Absolute numbers of antigen-specific CD8+ T cells, total CD8+ T cells, and CD4+ T cells per μL of blood measured by flow cytometry 7 days after the first treatment (day 15) from C57BL / 6 mice described in Example 8, Figures 13 and 14. (B) Ratios of total CD8+ T cells (top left) and antigen-specific CD8+ T cells (top right, bottom left) to Treg cells. (C) Fold changes in CD8+ T cell subsets in treatment groups relative to the corresponding median values ​​of CD8+ T cell subsets in the control group. Points represent individual mice, and lines represent group means. Mean ± SEM. Statistical significance was determined using one-way analysis of variance followed by Dunnett's multiple comparison test. All analyses were two-sided and performed using GraphPad Prism 8. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 16]Reduced tumor growth resulted in improved survival following treatment with hAlb-hIL2_A4s8 in combination with PD-L1 blockade in the MC38 murine colon cancer model. C57BL / 6 mice (n=14 per group) were inoculated subcutaneously (sc) with 7.5 x 105 MC38 colon cancer cells and treated intravenously four times weekly (days 17, 24, 32, and 39) with RNA encoding hAlb-hIL2_A4s8 formulated as LNPs, with or without concurrent intraperitoneal (ip) treatment with anti-PD-L1 antibody (200 μg for the first injection, 100 μg for all subsequent injections). Control groups received hAlb (no cytokine encoding) formulated as LNPs with an isotype control antibody or hAlb with anti-PD-L1. Blood lymphocyte subsets and Adpgk-specific T cell responses were determined by flow cytometry 7 days after the second treatment (day 31). (A) Tumor growth in individual mice and (B) survival rates of mice treated with hIL2_A4s8 with or without anti-PD-L1 antibody. Dotted lines indicate treatment days. Ratios in (A) represent the number of tumor-free mice relative to the total number of mice per group. [Figure 17A] Expansion of tumor antigen-specific CD8+ T cells relative to Treg cells upon treatment with hAlb-hIL2_A4s8 in combination with PD-L1 blockade in the murine colon cancer model MC38. (A) Absolute numbers of total CD8+ T cells, antigen-specific CD8+ T cells, total CD4+ T cells, and Treg cells per μL of blood measured by flow cytometry 7 days after the second treatment (day 31) from C57BL / 6 mice described in Example 9, Figure 16. (B) Ratios of total CD8+ T cells (left) and antigen-specific CD8+ T cells (right) to Treg cells. Points represent individual mice, and lines represent group means. Statistical significance was determined using one-way analysis of variance followed by Dunnett's multiple comparison test. All analyses were two-tailed and performed using GraphPad Prism 8. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 17B]Expansion of tumor antigen-specific CD8+ T cells relative to Treg cells upon treatment with hAlb-hIL2_A4s8 in combination with PD-L1 blockade in the murine colon cancer model MC38. (A) Absolute numbers of total CD8+ T cells, antigen-specific CD8+ T cells, total CD4+ T cells, and Treg cells per μL of blood measured by flow cytometry 7 days after the second treatment (day 31) from C57BL / 6 mice described in Example 9, Figure 16. (B) Ratios of total CD8+ T cells (left) and antigen-specific CD8+ T cells (right) to Treg cells. Points represent individual mice, and lines represent group means. Statistical significance was determined using one-way analysis of variance followed by Dunnett's multiple comparison test. All analyses were two-tailed and performed using GraphPad Prism 8. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 18ABCD]Modulation of tumor and blood lymphocyte counts in the MC38 mouse tumor model upon treatment with hAlb-hIL2_A4s8 in combination with PD-L1 checkpoint blockade. C57BL / 6 mice (n = 7–8 per group) were inoculated subcutaneously (sc) with 7.5 × 105 MC38 colon cancer cells and, 19 days later, were treated intravenously with RNA formulated in LNPs encoding hAlb-hIL2_A4s8 and simultaneously with an anti-PD-L1 antibody i.p. treatment. Control groups received one of two treatments or no treatment at all (control for hAlb-hIL2_A4s8: hAlb RNA formulated as LNPs ("hAlb"); control for anti-PD-L1 antibody: isotype antibody ("iso"). Tumors and blood from mice were harvested on day 24 and analyzed by flow cytometry. (A–H) Analysis of tumor-infiltrating lymphocyte subsets. (A) Number of CD8+ T cells, (B) NK cells, and (C) CD4+ T cells. (D) Percentage of CD25+FoxP3+ Treg cells among CD4+ T cells. (E) Ratio of CD8+ T cells to Treg cells. (F) Number of Adpgk, (G) Rpl18, and (H) N4bp2I2 neoantigen-specific CD8+ T cells. (I-M) Analysis of lymphocyte subsets in blood. Number of (I) CD8+ and (J) CD4+ T cells, and number of (K) Adpgk, (L) Rpl18, and (M) N4bp2I2 neoantigen-specific CD8+ T cells. Statistical analysis was performed using the Kruskal-Wallis test (graphs on a log scale) or one-way analysis of variance (graphs on a linear scale) followed by Dunn's or Dunnett's multiple comparison test, respectively. All analyses were two-tailed and performed using GraphPad Prism 8. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 18EFGH]Modulation of tumor and blood lymphocyte counts in the MC38 mouse tumor model upon treatment with hAlb-hIL2_A4s8 in combination with PD-L1 checkpoint blockade. C57BL / 6 mice (n = 7–8 per group) were inoculated subcutaneously (sc) with 7.5 × 105 MC38 colon cancer cells and, 19 days later, were treated intravenously with RNA formulated in LNPs encoding hAlb-hIL2_A4s8 and simultaneously with an anti-PD-L1 antibody i.p. treatment. Control groups received one of two treatments or no treatment at all (control for hAlb-hIL2_A4s8: hAlb RNA formulated as LNPs ("hAlb"); control for anti-PD-L1 antibody: isotype antibody ("iso"). Tumors and blood from mice were harvested on day 24 and analyzed by flow cytometry. (A–H) Analysis of tumor-infiltrating lymphocyte subsets. (A) Number of CD8+ T cells, (B) NK cells, and (C) CD4+ T cells. (D) Percentage of CD25+FoxP3+ Treg cells among CD4+ T cells. (E) Ratio of CD8+ T cells to Treg cells. (F) Number of Adpgk, (G) Rpl18, and (H) N4bp2I2 neoantigen-specific CD8+ T cells. (I-M) Analysis of lymphocyte subsets in blood. Number of (I) CD8+ and (J) CD4+ T cells, and number of (K) Adpgk, (L) Rpl18, and (M) N4bp2I2 neoantigen-specific CD8+ T cells. Statistical analysis was performed using the Kruskal-Wallis test (graphs on a log scale) or one-way analysis of variance (graphs on a linear scale) followed by Dunn's or Dunnett's multiple comparison test, respectively. All analyses were two-tailed and performed using GraphPad Prism 8. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 18IJKL]Modulation of tumor and blood lymphocyte counts in the MC38 mouse tumor model upon treatment with hAlb-hIL2_A4s8 in combination with PD-L1 checkpoint blockade. C57BL / 6 mice (n = 7–8 per group) were inoculated subcutaneously (sc) with 7.5 × 105 MC38 colon cancer cells and, 19 days later, were treated intravenously with RNA formulated in LNPs encoding hAlb-hIL2_A4s8 and simultaneously with an anti-PD-L1 antibody i.p. treatment. Control groups received one of two treatments or no treatment at all (control for hAlb-hIL2_A4s8: hAlb RNA formulated as LNPs ("hAlb"); control for anti-PD-L1 antibody: isotype antibody ("iso"). Tumors and blood from mice were harvested on day 24 and analyzed by flow cytometry. (A–H) Analysis of tumor-infiltrating lymphocyte subsets. (A) Number of CD8+ T cells, (B) NK cells, and (C) CD4+ T cells. (D) Percentage of CD25+FoxP3+ Treg cells among CD4+ T cells. (E) Ratio of CD8+ T cells to Treg cells. (F) Number of Adpgk, (G) Rpl18, and (H) N4bp2I2 neoantigen-specific CD8+ T cells. (I-M) Analysis of lymphocyte subsets in blood. Number of (I) CD8+ and (J) CD4+ T cells, and number of (K) Adpgk, (L) Rpl18, and (M) N4bp2I2 neoantigen-specific CD8+ T cells. Statistical analysis was performed using the Kruskal-Wallis test (graphs on a log scale) or one-way analysis of variance (graphs on a linear scale) followed by Dunn's or Dunnett's multiple comparison test, respectively. All analyses were two-tailed and performed using GraphPad Prism 8. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. [Figure 18M]Modulation of tumor and blood lymphocyte counts in the MC38 mouse tumor model upon treatment with hAlb-hIL2_A4s8 in combination with PD-L1 checkpoint blockade. C57BL / 6 mice (n = 7–8 per group) were inoculated subcutaneously (sc) with 7.5 × 105 MC38 colon cancer cells and, 19 days later, were treated intravenously with RNA formulated in LNPs encoding hAlb-hIL2_A4s8 and simultaneously with an anti-PD-L1 antibody i.p. treatment. Control groups received one of two treatments or no treatment at all (control for hAlb-hIL2_A4s8: hAlb RNA formulated as LNPs ("hAlb"); control for anti-PD-L1 antibody: isotype antibody ("iso"). Tumors and blood from mice were harvested on day 24 and analyzed by flow cytometry. (A–H) Analysis of tumor-infiltrating lymphocyte subsets. (A) Number of CD8+ T cells, (B) NK cells, and (C) CD4+ T cells. (D) Percentage of CD25+FoxP3+ Treg cells among CD4+ T cells. (E) Ratio of CD8+ T cells to Treg cells. (F) Number of Adpgk, (G) Rpl18, and (H) N4bp2I2 neoantigen-specific CD8+ T cells. (I-M) Analysis of lymphocyte subsets in blood. Number of (I) CD8+ and (J) CD4+ T cells, and number of (K) Adpgk, (L) Rpl18, and (M) N4bp2I2 neoantigen-specific CD8+ T cells. Statistical analysis was performed using the Kruskal-Wallis test (graphs on a log scale) or one-way analysis of variance (graphs on a linear scale) followed by Dunn's or Dunnett's multiple comparison test, respectively. All analyses were two-tailed and performed using GraphPad Prism 8. ns P>0.05, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DETAILED DESCRIPTION OF THE INVENTION

[0069] Although the present disclosure will be described in detail below, it should be understood that the disclosure is not limited to the specific methodology, protocols and reagents described herein, which may vary.It should also be understood that the terms used herein are only intended to describe specific embodiments and are not intended to limit the scope of the present disclosure, which is limited only by the scope of the appended claims.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0070] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0071] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as described 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).

[0072] The elements of the present disclosure are described below. Although these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to create further embodiments. The various described examples and embodiments should not be construed as limiting the disclosure to only the embodiments explicitly described. This description should be understood to disclose and encompass embodiments combining the explicitly described embodiment with any number of the disclosed elements. Furthermore, any permutation and combination of all described elements should be considered disclosed by this description unless the context dictates otherwise.

[0073] The term "about" means approximately or near, and in the context of numerical values ​​or ranges described herein, means, in one embodiment, ±20%, ±10%, ±5%, or ±3% of the recited or claimed numerical value or range.

[0074] As used in the context of describing the present disclosure (particularly in the context of the claims), the terms "a" and "an" and "the" and similar references should be construed to encompass both the singular and the plural unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better explain the disclosure and does not impose limitations on the claims. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0075] Unless otherwise specified, the term "comprises" is used in the context of this document to indicate that additional members may optionally be present in addition to the members of the list introduced by "comprises." However, it is contemplated as a specific embodiment of the present disclosure that the term "comprises" encompasses the possibility that additional members are not present, i.e., for the purposes of this embodiment, "comprises" should be understood to have the meaning of "consisting of."

[0076] Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such disclosure.

[0077] The following provides definitions that apply to all aspects of this disclosure. The following terms have the following meanings unless otherwise indicated. Terms not defined have their art-wide accepted meanings.

[0078] definition As used herein, "reduce," "diminish," or "inhibit" refers to an overall decrease or ability to cause an overall decrease in levels, e.g., binding levels, preferably by 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more.

[0079] Terms such as "increase" or "enhance" preferably relate to an increase or enhancement of at least about 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.

[0080] According to the present disclosure, the term "peptide" includes oligopeptides and polypeptides and refers to a substance comprising about 2 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 joined together by peptide bonds. The term "protein" or "polypeptide" refers to large peptides, particularly peptides having at least about 50 amino acids, although the terms "peptide," "protein," and "polypeptide" are generally used synonymously herein.

[0081] A "therapeutic protein" when provided to a subject in a therapeutically effective amount has a positive or beneficial effect on the subject's condition or pathology. In one embodiment, a therapeutic protein has curative or palliative properties and can be administered to improve, alleviate, relieve, reverse, delay the onset, or reduce the severity of one or more symptoms of a disease or disorder. A therapeutic protein has prophylactic properties and can be used to delay the onset of a disease or reduce the severity of such a disease or pathology. The term "therapeutic protein" includes whole proteins or peptides and can also refer to therapeutically active fragments thereof. It can also include therapeutically active variants of proteins. Examples of therapeutically active proteins include, but are not limited to, cytokines and antigens for vaccination.

[0082] With respect to an amino acid sequence (peptide or protein), a "fragment" refers to a portion of the amino acid sequence, i.e., a sequence that is truncated at the N-terminus and / or C-terminus. A C-terminally truncated fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. An N-terminally truncated fragment (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 an initiation codon that serves to initiate translation. A fragment of an amino acid sequence can, for example, contain at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues from the amino acid sequence. A fragment of an amino acid sequence preferably contains at least 6, particularly 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.

[0083] As used herein, "variant" or "variant protein" or "variant polypeptide" refers to a protein that differs from a wild-type protein by at least one amino acid modification. The parent polypeptide can be a naturally occurring or wild-type (WT) polypeptide, or can be a modified version of a wild-type polypeptide. Preferably, the variant polypeptide has at least one amino acid modification compared to the parent polypeptide, e.g., 1 to about 20 amino acid modifications compared to the parent, preferably 1 to about 10 or 1 to about 5 amino acid modifications.

[0084] 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. A parent polypeptide can be a wild-type polypeptide, or a variant or engineered version of a wild-type polypeptide.

[0085] As used herein, "wild-type" or "WT" or "native" refers to an amino acid sequence found in nature, including allelic variations. A wild-type protein or polypeptide has an amino acid sequence that has not been intentionally modified.

[0086] For purposes of this disclosure, a "variant" of an amino acid sequence (peptide, protein, or polypeptide) includes 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, conformational variants, isoform variants, and species homologs, particularly those naturally expressed by cells. The term "variant" particularly includes fragments of an amino acid sequence.

[0087] Amino acid insertion variants include the insertion of one or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted at a specific site in the amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible. Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, for example, 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, for example, the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion may occur at any position in the protein. Amino acid deletion variants containing deletions at the N- and / or C-termini of a protein are also referred to as N- and / or C-terminal truncation variants. Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Modifications at positions in the amino acid sequence that are not conserved between homologous proteins or peptides and / or substitutions of amino acids with other amino acids with similar properties are preferred. Preferably, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes include substitutions of members of a family of amino acids whose side chains are related. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. In one embodiment, conservative amino acid substitutions include substitutions within the following groups: Glycine, Alanine; valine, isoleucine, leucine; Aspartic acid, glutamic acid; Asparagine, glutamine; Serine, threonine; lysine, arginine; and Phenylalanine, tyrosine.

[0088] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of said given amino acid sequence is at least about 60%, 65%, 70%, 75%, 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 over 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 entire 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 consecutive amino acids. In a preferred embodiment, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using optimal sequence alignment, for example, using Align, with standard settings, preferably EMBOSS::Needle, matrix:Blosum62, gap open 10.0, gap extension 0.5.

[0089] "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 amino acids that are identical between those sequences.

[0090] The term "percent identity" is intended to indicate the percentage of identical amino acid residues between the two sequences being compared, obtained after optimal alignment, and this percentage is purely statistical, with the differences between the two sequences being distributed randomly and over their entire length. Sequence comparison between two amino acid sequences is conventionally carried out by comparing these sequences after optimal alignment, said comparison being carried out segment by segment or "comparison window" to identify and compare local regions of sequence similarity. In addition to being created manually, optimal alignment of sequences for comparison can be achieved 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 using these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0091] The percent identity is calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the number of positions being compared, and multiplying the result by 100 to obtain the percent identity between the two sequences.

[0092] Homologous amino acid sequences, according to the present disclosure, exhibit an identity of at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably at least 95%, at least 98, or at least 99% of the amino acid residues.

[0093] The amino acid sequence variants described herein can be readily prepared by those skilled in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to prepare peptides or proteins with substitutions, additions, insertions, or deletions is described in detail, for example, in Sambrook et al. (1989). Furthermore, the peptides and amino acid variants described herein can be readily prepared using known peptide synthesis techniques, for example, by solid-phase synthesis and similar methods.

[0094] 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 refers to any fragment or variant that exhibits one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., is functionally equivalent. For cytokines such as IL2, 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 by binding to the receptor(s) to which the amino acid sequence from which the fragment or variant is derived binds. The term "functional fragment" or "functional variant" as used herein particularly refers to a variant molecule or sequence that contains an amino acid sequence that has been altered by one or more amino acids compared to the amino acid sequence of a parent molecule or sequence, yet still performs one or more functions of the parent molecule or sequence, e.g., can bind to or contribute to binding to a target molecule. In one embodiment, alterations to the amino acid sequence of the parent molecule or sequence do not significantly affect or change the binding properties of the molecule or sequence. In different embodiments, binding of the functional fragment or functional variant may be reduced but still significant, for example, binding of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence, however, in other embodiments, binding of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0095] An amino acid sequence (peptide, protein, or polypeptide) "derived from" a specified amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the initial amino acid sequence. Preferably, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to the particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence or a fragment thereof. For example, it will be understood by those skilled in the art that antigens and cytokines (e.g., IL2) suitable for use herein can be modified to differ in sequence from the naturally occurring or native sequence from which they are derived while retaining the desired activity of the native sequence.

[0096] As used herein, "instructional material" or "instructions" includes publications, records, drawings, or any other medium of expression that can be used to communicate the usefulness of the compositions and methods of the present invention. The instructional material of the kits of the present invention may, for example, be affixed to a container containing the composition of the present invention or may be shipped together with a container containing the composition. Alternatively, the instructional material may be shipped separately from the container, with the intention that the instructional material and the composition be used in conjunction with each other by the recipient.

[0097] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or it can exist in a non-native environment, such as a host cell.

[0098] In the context of the present invention, the term "recombinant" means "produced through genetic engineering." Preferably, a "recombinant entity," such as a recombinant cell, in the context of the present invention does not occur in nature.

[0099] As used herein, the term "naturally occurring" refers to the fact that an entity can be found in nature. For example, a peptide or nucleic acid that is present in living organisms (including viruses), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is naturally occurring.

[0100] The term "genetic modification" or simply "modification" includes the transfection of cells with nucleic acids. The term "transfection" refers to the introduction of nucleic acids, particularly RNA, into cells. For purposes of the present invention, the term "transfection" also includes the introduction of nucleic acids into cells or the uptake of nucleic acids by such cells, which may be present in a subject, e.g., a patient. Thus, according to the present invention, cells for transfection with nucleic acids described herein can be present in vitro or in vivo; for example, the cells can form part of a patient's organ, tissue, and / or organism. According to the present invention, transfection can be transient or stable. In some transfection applications, transient expression of the transfected genetic material is sufficient. RNA can be transfected into cells to transiently express its encoded protein. Nucleic acids introduced during transfection are usually not integrated into the nuclear genome, resulting in the exogenous nucleic acid being diluted or degraded by mitosis. Cells that allow episomal amplification of nucleic acids significantly reduce the dilution rate. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, stable transfection must occur.Such stable transfection can be achieved by using a virus-based system or a transposon-based system for transfection.Generally, cells genetically modified to express receptor polypeptides, such as IL2R or IL2R variants, and / or antigen receptors, such as TCRs or CARs, are stably transfected with nucleic acids encoding receptor polypeptides and / or nucleic acids encoding antigen receptors, while nucleic acids encoding ligand polypeptides, such as IL2 variants, and / or nucleic acids encoding antigens are generally transiently transfected into cells.

[0101] As used herein, the term "polynucleotide" or "nucleic acid" is intended to include DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA. According to the present invention, polynucleotides are preferably isolated.

[0102] The nucleic acid may be contained in a vector. As used herein, the term "vector" includes any vector known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retrovirus, adenovirus, or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC). Such vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and generally contain a desired coding sequence and appropriate DNA sequences required for expression of the operably linked coding sequence in a specific host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are generally used to manipulate and amplify specific desired DNA fragments and may lack functional sequences required for expression of the desired DNA fragment.

[0103] In one embodiment of all aspects of the invention, a nucleic acid, such as a nucleic acid encoding an IL2 variant, a nucleic acid encoding an IL2R or an IL2R variant, a nucleic acid encoding an antigen receptor, or a nucleic acid encoding a vaccine antigen, is expressed in a cell, particularly in the cells of a subject to be treated, to provide the IL2 variant, IL2R or an IL2R variant, antigen receptor, or vaccine antigen. In one embodiment of all aspects of the invention, the nucleic acid is transiently expressed in the subject's cells. Thus, in one embodiment, the nucleic acid is not integrated into the genome of the cell. In one embodiment of all aspects of the invention, the nucleic acid is RNA, preferably in vitro transcribed RNA.

[0104] The nucleic acids described herein can be recombinant and / or isolated molecules.

[0105] In this disclosure, the term "RNA" refers to a nucleic acid molecule containing ribonucleotide residues. In preferred embodiments, RNA contains all or most ribonucleotide residues. As used herein, "ribonucleotide" 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 such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may refer to the addition of non-nucleotide material to internal RNA nucleotides or to the end(s) of the RNA. It is also contemplated herein that the nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. In this disclosure, these modified RNAs are considered analogs of naturally occurring RNA.

[0106] In some embodiments of the present disclosure, the RNA is messenger RNA (mRNA), which is related to an RNA transcript encoding a peptide or protein. As established 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, the RNA is produced by in vitro transcription or chemical synthesis. In one embodiment, the mRNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid comprising deoxyribonucleotides.

[0107] In one embodiment, the RNA is in vitro transcribed RNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly a cDNA, and introducing it into a suitable vector for in vitro transcription. The cDNA can be obtained by reverse transcription of RNA.

[0108] In one embodiment, the RNA may have modified ribonucleotides. Examples of modified ribonucleotides include, but are not limited to, 5-methylcytidine, pseudouridine, and / or 1-methylpseudouridine.

[0109] In some embodiments, the RNA of the present disclosure includes 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 with a 5' cap analog. The term "5' cap" refers to the structure found at the 5' end of an mRNA molecule and generally consists of a guanosine nucleotide linked to the mRNA by a 5'-5' triphosphate bond. In one embodiment, the guanosine is methylated at position 7. Providing an RNA with a 5' cap or 5' cap analog can be achieved by in vitro transcription, in which the 5' cap is co-transcriptionally expressed on the RNA strand, or can be added to the RNA post-transcriptionally using a capping enzyme.

[0110] In some embodiments, an RNA according to the present disclosure includes 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 a corresponding region in an RNA molecule, such as an mRNA molecule. The untranslated region (UTR) can be located 5' (upstream) of the open reading frame (5'-UTR) and / or 3' (downstream) of the open reading frame (3'-UTR). If present, the 5'-UTR is located at the 5' end upstream of the start codon of the protein-coding region. If present, the 5'-UTR is downstream of the 5' cap (if present), e.g., directly adjacent to the 5' cap. If present, the 3'-UTR is located at the 3' end downstream of the stop codon of the protein-coding region, although the term "3'-UTR" preferably does not include a poly(A) tail. Thus, the 3'-UTR is upstream of the poly(A) sequence (if present), e.g., directly adjacent to the poly(A) sequence.

[0111] In some embodiments, an RNA according to the present disclosure comprises a 3'-poly(A) sequence. As used herein, the term "poly(A) sequence" or "poly(A tail)" refers to a continuous or intermittent sequence of adenylic acid residues typically located at the 3' end of an RNA molecule. Poly(A) sequences are known to those skilled in the art and may follow the 3' UTR of an RNA described herein. The poly(A) sequence may be of any length. In some embodiments, the poly(A) sequence comprises or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 nucleotides, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides, particularly about 100 nucleotides.

[0112] In some embodiments, the poly(A) sequence consists solely of A nucleotides. In some embodiments, the poly(A) sequence consists essentially of A nucleotides but is interrupted by random sequences of four nucleotides (A, C, G, and U), as disclosed in International Publication No. 2016 / 005324 A1, incorporated herein by reference. Such random sequences can be 5-50, 10-30, or 10-20 nucleotides in length. Poly(A) cassettes present in the coding strand of DNA that consist essentially of dA nucleotides but are interrupted by random sequences in which the four nucleotides (dA, dC, dG, dT) are evenly distributed and have a length of, for example, 5-50 nucleotides, have been shown to, at the DNA level, consistently grow plasmid DNA in Escherichia coli (E. coli), while, at the RNA level, still be associated with beneficial properties for supporting RNA stability and translation efficiency.

[0113] In some embodiments, no nucleotides other than A nucleotides flank the poly(A) sequence at its 3' end, i.e., the poly(A) sequence is not masked or followed by a nucleotide other than A at its 3' end.

[0114] In the context of the present disclosure, the term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA, which can then be translated into peptides or proteins.

[0115] "Encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties that result therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0116] As used herein, "endogenous" refers to any substance that is produced from or within an organism, cell, tissue, or system.

[0117] As used herein, the term "exogenous" refers to any substance introduced from or produced outside an organism, cell, tissue, or system.

[0118] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence.

[0119] As used herein, the terms "linked," "fused," or "fusion" are used interchangeably and refer to the joining of two or more elements or components or domains.

[0120] Cytokines are a category of small proteins (approximately 5–20 kDa) that are important in cell signaling. Their release affects the behavior of surrounding cells. Cytokines, as immunomodulators, participate in autocrine, paracrine, and endocrine signaling. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, but generally do not include hormones or growth factors (despite some overlap in terminology). Cytokines are produced by a wide range of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes, and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. A given cytokine can be produced by multiple cell types. Cytokines act through receptors and are particularly important in the immune system; they regulate the balance between humoral and cellular immune responses and regulate the maturation, growth, and responsiveness of specific cell populations. Some cytokines enhance or inhibit the actions of other cytokines in complex ways.

[0121] Interleukin-2 (IL2) is a cytokine that induces proliferation of antigen-activated T cells and stimulates natural killer (NK) cells. IL2's biological activity is mediated through the multisubunit IL2 receptor complex (IL2R), which contains three membrane-spanning polypeptide subunits: p55 (IL2Rα, alpha subunit, also known as CD25 in humans), p75 (IL2Rβ, beta subunit, also known as CD122 in humans), and p64 ​​(IL2Rγ, gamma subunit, also known as CD132 in humans). T cell responses to IL2 depend on various 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” Receptor Activation by Antigens, Cytokines, Hormones, and Growth Factors 766:263-271, 1995). The IL2:IL2R complex is internalized upon ligand binding, and various components undergo differential sorting. 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).

[0122] In eukaryotic cells, human IL2 is synthesized as a 153-amino acid precursor polypeptide, from which 20 amino acids are removed to generate the mature, secreted form. Recombinant human IL2 has been produced in E. coli, insect cells, and mammalian COS cells.

[0123] The results of systemic administration of IL2 in cancer patients are far from ideal. While 15–20% of patients objectively respond to high-dose IL2, the majority do not, and many experience severe and life-threatening side effects, such as nausea, confusion, hypotension, and septic shock. The severe toxicity associated with high-dose IL2 treatment is primarily due to the activation of natural killer (NK) cells. Attempts to lower serum concentrations by reducing the dose and adjusting the dosing regimen have resulted in less toxicity, but such treatments have also been less effective.

[0124] According to the present disclosure, in certain embodiments, the IL2 variant polypeptides described herein comprise a pharmacokinetic-modifying group. In one embodiment, the IL2 variant moieties or muteins described herein are conjugated to a pharmacokinetic-modifying group. The resulting molecule, hereinafter referred to as "extended pharmacokinetic (PK) IL2," has an extended circulating half-life compared to free IL2. The extended circulating half-life of extended PK IL2 allows serum IL2 concentrations to be maintained within the therapeutic range in vivo, potentially leading to enhanced activation of many types of immune cells, including T cells. Due to its favorable pharmacokinetic profile, extended PK IL2 can be administered less frequently and for a longer period of time compared to unmodified IL2.

[0125] As used herein, "half-life" refers to the time required for the serum or plasma concentration of a compound, such as a peptide or protein, to decrease by 50% in vivo, for example, due to degradation and / or clearance or sequestration by natural mechanisms. Extended PK interleukins (ILs) suitable for use herein are stabilized in vivo, and their half-life is increased, for example, by fusion to serum albumin (e.g., HSA or MSA), to resist degradation and / or clearance or sequestration. Half-life can be determined by any method known per se, such as by pharmacokinetic analysis. Suitable techniques will be apparent to those skilled in the art and may, for example, generally include the steps of administering an appropriate dose of an amino acid sequence or compound to a subject; collecting blood or other samples from the subject at regular intervals; determining the level or concentration of the amino acid sequence or compound in the blood samples; and calculating, from a plot of the data thus obtained, the time until the level or concentration of the amino acid sequence or compound decreases by 50% compared to the initial level at the time of administration. Further details are provided in standard handbooks such as, for example, Kenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al., Pharmacokinetic Analysis: A Practical Approach (1996). See also Gibaldi, M. et al., Pharmacokinetics, 2nd Rev. Edition, Marcel Dekker (1982).

[0126] As used herein, "human IL2" or "wild-type human IL2" refers to IL2, whether native or recombinant, having the normally occurring 133 amino acid sequence of native human IL2 (excluding the signal peptide consisting of the additional 20 N-terminal amino acids), with or without the additional N-terminal methionine that is necessarily included when the protein is expressed as an intracellular fraction in E. coli, as described in Fujita, et. al., PNAS USA, 80, 7437-7441 (1983). In one embodiment, human IL2 comprises the amino acid sequence of SEQ ID NO: 1. In one embodiment, a 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: 1. In one embodiment, the functional variant of human IL2 binds to the IL2 receptor.

[0127] In certain embodiments described herein, the IL2 variant portion or mutein 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 more detail below, the polypeptide that increases the circulating half-life can be serum albumin, such as human or mouse serum albumin.

[0128] As used herein, "IL2 mutein" refers to a variant of IL2 (including functional variants thereof), particularly a polypeptide in which specific substitutions have been made into the IL2 protein.

[0129] In one embodiment, substitutions are made into the human IL2 protein to enhance IL2Rβγ ("mutβγ") binding, particularly CD122 binding. For example, an IL2 mutein can be characterized by amino acid substitutions in the native IL2 polypeptide chain that result in a relatively increased affinity for IL2Rβγ, e.g., compared to wild-type IL2, such that IL2-mediated stimulation no longer requires engagement of IL2Rα. Such mutants are potent IL2 signaling agonists. Particularly preferred embodiments include a leucine (Leu) residue at position 80, an arginine (Arg) residue at position 81, a leucine (Leu) residue at position 85, and an isoleucine (Ile) residue at position 92, numbered relative to and according to wild-type human IL2.

[0130] In one embodiment, additional substitutions ("mutα") are made in the human IL2 protein that affect IL2Rαβγ binding, particularly CD25 binding. For example, an IL2 mutein can also be characterized by amino acid substitutions in the native IL2 polypeptide chain that result in a relatively reduced affinity for IL2Rαβγ, particularly its α subunit, when compared to, for example, wild-type IL2 (i.e., the IL2 mutein includes a "mutα" mutation in addition to a "mutβγ" mutation). These mutations can be present in amino acid residues that contact IL2Rα. Particularly preferred embodiments include lysine (Lys) residues at positions 35, 43, glutamic acid (Glu) residues at positions 61, and 62, or any combination thereof, numbered relative to and according to wild-type human IL2.

[0131] An IL2 mutein may have an amino acid sequence identical to wild-type IL2 at otherwise unsubstituted residues (i.e., an IL2 mutein includes a "mutβγ" and optionally a "mutα" mutation, e.g., a mutation in which the sequence of SEQ ID NO:2 or 11 differs from the sequence of SEQ ID NO:1). However, an IL2 mutein may also be characterized by amino acid insertions, deletions, substitutions, and modifications at one or more sites or at other residues in the native IL2 polypeptide chain. In accordance with the present invention, such insertions, deletions, substitutions, and modifications may result in an IL2 mutein with enhanced affinity for IL2Rβγ, optionally with reduced affinity for IL2Rαβγ.

[0132] The substituted amino acid residue(s) may, but need not necessarily, be a conservative substitution.

[0133] "Numbered according to wild-type IL2" means that the selected amino acid is identified by reference to the position at which that amino acid normally occurs in the mature sequence of wild-type IL2. When insertions or deletions are made in an IL2 mutein, one skilled in the art will understand that the amino acid normally occurring at a particular position may be shifted in position in the mutein. However, the position of the shifted amino acid can be readily determined by inspection and correlation of the adjacent amino acids with those adjacent to that amino acid in wild-type IL2.

[0134] The IL2 variant polypeptides and polynucleotides encoding them described herein can be produced by any suitable method known in the art. Such methods include introducing appropriate nucleotide changes into a nucleic acid encoding IL2 or by in vitro synthesis of IL2 polynucleotides or proteins. For example, DNA sequences encoding the IL2 variant polypeptides described herein can be constructed and expressed in an appropriately transformed host or any other suitable expression system. This method produces the IL2 variant polypeptides described herein and / or RNA encoding them. However, the IL2 variant polypeptides and polynucleotides encoding them described herein can also be produced by chemical synthesis, although this is less preferred.

[0135] The IL2 variant polypeptides described herein may bind to IL2Rβγ with an affinity that is higher than that with which wild-type IL2 binds to IL2Rβγ. In one embodiment, the IL2 variant polypeptides described herein may bind to IL2Rαβγ with an affinity that is lower than that with which wild-type IL2 binds to IL2Rαβγ.

[0136] 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 higher than the affinity with which wild-type IL2 binds to IL2Rβγ. Furthermore, 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 with which wild-type IL2 binds to IL2Rαβγ.

[0137] The IL2 variant polypeptides described herein may have a reduced ability to stimulate regulatory T cells compared to wild-type IL2, particularly when compared to their ability to stimulate effector T cells and / or NK cells.

[0138] 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 compared to wild-type IL2.

[0139] 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.

[0140] In one embodiment, the IL2 variant polypeptides described herein have one or more, preferably all, of the following properties: 1) Agonistic activity at IL2Rβγ: This property can be directly assessed in an in vitro proliferation assay using IL2-dependent cell lines.

[0141] 2) Loss of ability to stimulate in vitro and / or in vivo populations of regulatory T cells compared to wild-type IL2. This property can be assessed, for example, by examining the ability of the mutein to induce expansion of regulatory T cells compared to the ability of wild-type IL2.

[0142] 3) Increased therapeutic efficacy relative to native IL2 in animal models. This property can be assessed, 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 assessed through the enhancement of cellular and / or humoral responses to the vaccine of interest.

[0143] Many immune cells transiently upregulate IL2Rαβγ upon activation to enhance their sensitivity to IL2 in initiating immune responses, including priming CD8 T cells. Because some IL2Rαβγ binding by IL2 may be necessary, the present invention contemplates the use of mixtures of IL2Rβγ-selective IL2 variant polypeptides described herein in combination with IL2 (including functional variants thereof) that do not exhibit selective affinity for IL2Rβγ, such as wild-type IL2. In certain embodiments, the molar ratio of IL2Rβγ-selective IL2 variant polypeptides described herein to IL2 that does not exhibit selective 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.

[0144] The IL2 variant polypeptides described herein can be prepared as fusion or chimeric polypeptides comprising an IL2 variant moiety and a heterologous polypeptide (i.e., a polypeptide that is not IL2 or a variant thereof). The IL2 variant may be fused to an extended PK group that increases circulating half-life. Non-limiting examples of extended PK groups are described below. It should be understood that other PK groups that increase the circulating half-life of a cytokine or its variants are also applicable to the present disclosure. In certain embodiments, the extended PK group is a serum albumin domain (e.g., mouse serum albumin, human serum albumin).

[0145] As used herein, the term "PK" is an acronym for "pharmacokinetics" and encompasses the properties of a compound, including, by way of example, absorption, distribution, metabolism, and excretion by a subject. As used herein, a "prolonged PK group" refers to a protein, peptide, or moiety that, when fused to or administered together with a biologically active molecule, increases the circulating half-life of the biologically active molecule. Examples of prolonged PK groups include serum albumin (e.g., HSA), immunoglobulin Fc or Fc fragments and variants thereof, transferrin and variants thereof, and human serum albumin (HSA) binders (disclosed in U.S. Patent Application Publication Nos. 2005 / 0287153 and 2007 / 0003549). Other exemplary prolonged PK groups are disclosed in Kontermann, Expert Opin Biol Ther, 2016 Jul;16(7):903-15, the entire contents of which are incorporated herein by reference. As used herein, "extended PK IL" refers to an interleukin (IL) moiety (including an IL variant moiety) combined with an extended PK group. In one embodiment, the extended PK IL is a fusion protein in which the IL moiety is linked or fused to the extended PK group. An exemplary fusion protein is an HSA / IL2 fusion in which the IL2 moiety is fused to HSA.

[0146] In some embodiments, the serum half-life of the extended PK IL is increased compared to the IL alone (i.e., an IL not fused to an extended PK group). In some embodiments, the serum half-life of the extended PK IL is at least 20, 40, 60, 80, 100, 120, 150, 180, 200, 400, 600, 800, or 1000% longer than the serum half-life of the IL alone. In some embodiments, the serum half-life of the extended PK IL is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 10-fold, 12-fold, 13-fold, 15-fold, 17-fold, 20-fold, 22-fold, 25-fold, 27-fold, 30-fold, 35-fold, 40-fold, or 50-fold longer than the serum half-life of the IL alone. In certain embodiments, the serum half-life of the extended PK 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.

[0147] In some embodiments, the extended PK group comprises serum albumin, or a fragment thereof, or a variant of serum albumin or a fragment thereof (all of which are included in the term "albumin" for purposes of this disclosure). The polypeptides described herein can be fused to albumin (or a fragment or variant thereof) to form an albumin fusion protein. Such albumin fusion proteins are described in U.S. Patent Application Publication No. 20070048282.

[0148] As used herein, "albumin fusion protein" refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) with at least one molecule of a protein, such as a Therapeutic protein, particularly IL2 (or a variant thereof). Albumin fusion proteins can be produced by translation of a nucleic acid in which a polynucleotide encoding a Therapeutic protein is joined in-frame with a polynucleotide encoding albumin. Once part of the albumin fusion protein, the Therapeutic protein and albumin can be referred to as a "portion," "region," or "moiety," respectively, of the albumin fusion protein (e.g., a "Therapeutic protein portion" or an "albumin protein portion"). In a highly preferred embodiment, the albumin fusion protein comprises at least one molecule of a Therapeutic protein (including, but not limited to, the mature form of a Therapeutic protein) and at least one molecule of albumin (including, but not limited to, the mature form of albumin). In one embodiment, the albumin fusion protein is processed by host cells, such as hepatocytes, in the target organ of the administered RNA and secreted into the circulation. Processing of the nascent albumin fusion protein 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, carbohydrate addition and processing (e.g., N-linked and O-linked glycosylation), specific proteolytic cleavage, and / or assembly into a multimeric protein. The albumin fusion protein is preferably encoded by RNA in an unprocessed form, particularly with a signal peptide at its N-terminus, and, after secretion by the cell, preferably exists in a processed form, particularly with the signal peptide cleaved. In the most preferred embodiment, the term "processed form of the albumin fusion protein" refers to the albumin fusion protein product that has undergone N-terminal signal peptide cleavage, also referred to herein as the "mature albumin fusion protein."

[0149] In preferred embodiments, albumin fusion proteins containing a therapeutic protein have higher plasma stability compared to the plasma stability of the same therapeutic protein when not fused to albumin. Plasma stability typically refers to the period from when a therapeutic protein is administered in vivo and transported into the bloodstream, until the therapeutic protein is degraded and removed from the bloodstream to organs such as the kidneys or liver, and finally, when the therapeutic protein is removed from the body. Plasma stability is calculated in terms of the half-life of the therapeutic protein in the bloodstream. The half-life of a therapeutic protein in the bloodstream can be easily determined by common assays known in the art.

[0150] As used herein, "albumin" collectively refers to an albumin protein or amino acid sequence, or an albumin fragment or variant, having one or more functional activities (e.g., biological activities) of albumin. In particular, "albumin" refers to human albumin or a fragment or variant thereof, particularly the mature form of human albumin, or albumin or a fragment thereof from another vertebrate, or a variant of these molecules. Albumin can be derived from any vertebrate, particularly any mammal, such as human, mouse, cow, sheep, or pig. Non-mammalian albumins include, but are not limited to, hen and salmon. The albumin portion of the albumin fusion protein can be derived from a different animal than the therapeutic protein portion.

[0151] In one embodiment, the albumin is human serum albumin (HSA), or a fragment or variant thereof, such as those disclosed in U.S. Pat. No. 5,876,969, WO 2011 / 124718, WO 2013 / 075066, and WO 2011 / 0514789.

[0152] 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.

[0153] As used herein, a fragment of albumin sufficient to prolong the therapeutic activity or plasma stability of a Therapeutic protein refers to a fragment of albumin of sufficient length or structure 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 extended or expanded compared to its plasma stability in the unfused state.

[0154] 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 can stabilize or extend therapeutic activity or plasma stability. Such fragments may be 10 or more amino acids in length, or may comprise approximately 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 spanning the first two immunoglobulin-like domains may be used. In a preferred embodiment, the HSA fragment is the mature form of HSA.

[0155] Generally speaking, an albumin fragment or variant is at least 100 amino acids in length, preferably at least 150 amino acids in length.

[0156] 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, particularly any mammal. In one embodiment, the albumin comprises the amino acid sequence of SEQ ID NO: 23 or 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: 23.

[0157] Preferably, the albumin fusion protein comprises albumin as the N-terminal moiety and a Therapeutic protein as the C-terminal moiety. Alternatively, albumin fusion proteins comprising albumin as the C-terminal moiety and a Therapeutic protein as the N-terminal moiety may also be used. In other embodiments, the albumin fusion protein has Therapeutic proteins fused to both the N- and C-termini of albumin. In a preferred embodiment, the Therapeutic proteins fused at the N- and C-termini are the same Therapeutic protein. In another preferred embodiment, the Therapeutic proteins fused at the N- and C-termini are different Therapeutic proteins. In one embodiment, the different Therapeutic proteins may be useful for treating or preventing the same or related diseases, disorders, or conditions.

[0158] In one embodiment, the therapeutic protein(s) are linked to albumin via a peptide linker(s). A linker peptide between the fusion moieties can provide greater physical separation between the moieties, thus maximizing the accessibility of the therapeutic protein moiety to bind to, for example, its cognate receptor. The linker peptide can be composed of amino acids so that it is flexible or more rigid. The linker sequence can be cleavable by protease or chemically.

[0159] As used herein, the term "Fc region" refers to the portion of a native immunoglobulin formed by the Fc domains (or Fc portions) of each of the two heavy chains of the immunoglobulin. As used herein, the term "Fc domain" refers to a portion or fragment of a single immunoglobulin (Ig) heavy chain in which the Fc domain does not include an Fv domain. In certain embodiments, the Fc domain begins at the hinge region immediately upstream of the papain cleavage site and ends at the C-terminus of the antibody. Thus, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments, an Fc domain comprises at least one of a hinge (e.g., upper, middle, and / or lower hinge region), a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragment thereof. In certain embodiments, an Fc domain comprises a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In certain embodiments, an Fc domain comprises a hinge domain (or a portion thereof) fused to a CH3 domain (or a portion thereof). In some embodiments, the Fc domain comprises a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof). In some embodiments, the Fc domain consists of a CH3 domain or a portion thereof. In some embodiments, the Fc domain consists of a hinge domain (or a portion thereof) and a CH3 domain (or a portion thereof). In some embodiments, the Fc domain consists of a CH2 domain (or a portion thereof) and a CH3 domain. In some embodiments, the Fc domain consists of a hinge domain (or a portion thereof) and a CH2 domain (or a portion thereof). In some embodiments, the Fc domain lacks at least a portion of the CH2 domain (e.g., all or a portion of the CH2 domain). An Fc domain, as used herein, generally refers to a polypeptide comprising all or a portion of the Fc domain of an immunoglobulin heavy chain. This includes, but is not limited to, polypeptides comprising the entire CH1, hinge, CH2, and / or CH3 domains, as well as fragments of such peptides, e.g., comprising only the 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 encompasses natural Fc and Fc variant molecules. As described herein, those skilled in the art will understand that any Fc domain can be modified such that its amino acid sequence differs from that of a naturally occurring Fc domain of an immunoglobulin molecule. In certain embodiments, the Fc domain has reduced effector function (e.g., FcγR binding).

[0160] The Fc domain of the polypeptide described herein can be derived from different immunoglobulin molecules.For example, the Fc domain of the polypeptide can 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 can 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 can include a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule.

[0161] In some embodiments, the extended PK 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 in the term "Fc domain" for purposes of this disclosure). The Fc domain does not include the variable region that binds to the antigen. Fc domains suitable for use in this disclosure may be obtained from several different sources. In some embodiments, the Fc domain is derived from a human immunoglobulin. In some embodiments, the Fc domain is derived from a human IgG1 constant region. However, it is understood that the Fc domain may be derived from an immunoglobulin of another mammalian species, including, for example, a rodent species (e.g., mouse, rat, rabbit, guinea pig) or a non-human primate species (e.g., chimpanzee, macaque).

[0162] Furthermore, the Fc domain (or a fragment or variant thereof) can be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4.

[0163] Various Fc domain gene sequences (e.g., mouse and human constant region gene sequences) are available in the form of publicly accessible deposits. Constant region domains can be selected, including Fc domain sequences that lack specific effector functions and / or have specific modifications that reduce immunogenicity. Many sequences of antibodies and antibody-encoding genes have been published, and appropriate Fc domain sequences (e.g., hinge, CH2, and / or CH3 sequences, or fragments or variants thereof) can be derived from these sequences using techniques widely recognized in the art.

[0164] In some embodiments, the extended PK group is a serum albumin-binding protein, such as those described in U.S. Patent Application Nos. 2005 / 0287153, 2007 / 0003549, 2007 / 0178082, 2007 / 0269422, 2010 / 0113339, WO 2009 / 083804, and WO 2009 / 133208, the entire contents of which are incorporated herein by reference. In some embodiments, the extended PK group is transferrin, as disclosed in U.S. Patent Nos. 7,176,278 and 8,158,579, the entire contents of which are incorporated herein by reference. In some embodiments, the extended PK group is a serum immunoglobulin-binding protein, as disclosed in U.S. Patent Application No. 2007 / 0178082, the entire contents of which are incorporated herein by reference. In some embodiments, the extended PK group is a fibronectin (Fn)-based scaffold domain protein that binds to serum albumin, as disclosed in U.S. Patent Application No. 2012 / 0094909, the entire contents of which are incorporated herein by reference. Methods for producing fibronectin-based scaffold domain proteins are also disclosed in U.S. Patent Application No. 2012 / 0094909. A non-limiting example of an Fn3-based extended PK group is Fn3 (HSA), i.e., an Fn3 protein that binds to human serum albumin.

[0165] In certain embodiments, an extended PK IL suitable for use according to the present disclosure may use one or more peptide linkers. As used herein, the term "peptide linker" refers to a peptide or polypeptide sequence that links two or more domains (e.g., an extended PK portion and an IL portion, such as IL2) in the linear amino acid sequence of a polypeptide chain. For example, a peptide linker may be used to link the IL2 portion to the HSA domain.

[0166] Linkers suitable for fusing an extended PK group to, for example, IL2 are well known in the art. Exemplary linkers include a glycine-serine polypeptide linker, a glycine-proline polypeptide linker, and a proline-alanine polypeptide linker. In one embodiment, the linker is a glycine-serine polypeptide linker, i.e., a peptide consisting of glycine and serine residues.

[0167] In addition to, or instead of, the heterologous polypeptides described above, the IL2 variant polypeptides described herein can include a sequence encoding a "marker" or "reporter." 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).

[0168] The IL2 variant polypeptides described herein can act on immune effector cells that may be endogenously present in a subject or that may be present after administration of immune effector cells. Thus, immune effector cells can be provided to a subject by administration of immune effector cells, e.g., naive or antigen receptor-transgenic immune effector cells. Particularly preferred "immune effector cells" are cells that respond to IL2 naturally or after transfection with a nucleic acid encoding one or more IL2R polypeptides. Such responsiveness includes activation, differentiation, proliferation, survival, and / or display of one or more immune effector functions. Cells include, in particular, cytotoxic cells, such as cells with lytic capacity, particularly lymphoid cells, and preferably T cells, particularly effector T cells, such as cytotoxic lymphocytes, preferably selected from cytotoxic T cells, natural killer (NK) cells, and lymphokine-activated killer (LAK) cells. Upon activation, these cells can cause destruction of target cells. For example, cytotoxic T cells cause destruction of target cells by one or both of the following means: First, upon activation, T cells release cytotoxins such as perforin, granzymes, and granulysin. Perforin and granulysin create pores in target cells, and granzymes enter the cells, triggering the cytoplasmic caspase cascade that induces apoptosis (programmed cell death) of the cells. Second, apoptosis can be induced through Fas-Fas ligand interaction between T cells and target cells. Cells used in connection with the present invention are preferably autologous cells, but xenogeneic or allogeneic cells can also be used.

[0169] In the context of the present invention, the term "effector function" includes any function mediated by a component of the immune system that results in the inhibition of tumor growth and / or tumorigenesis, including the killing of diseased cells, e.g., tumor cells, or the suppression of tumor dissemination and metastasis. Preferably, the effector function in the context of the present invention is a T cell-mediated effector function. Such a function is mediated by helper T cells (CD4 +T cells), cytokine release and / or CD8 + It involves the activation of lymphocytes (CTLs) and / or B cells, and in the case of CTLs, the elimination of cells, i.e., cells characterized by expression of the antigen, e.g., via apoptosis or perforin-mediated cytolysis, the production of cytokines such as IFN-γ and TNF-α, and the specific cytolytic killing of target cells expressing the antigen.

[0170] The term "immune effector cells" or "immunoreactive cells" in the context of the present invention relates to cells that exert effector functions during an immune response. In one embodiment, "immune effector cells" are capable of binding to antigens, such as antigens presented in association with MHC on cells or expressed on the surface of cells, and mediating an immune response. For example, immune effector cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in the context of the present invention, "immune effector cells" are T cells, preferably CD4 + and / or CD8 + According to the present invention, the term "immune effector cells" also includes cells that can mature into immune cells (such as T cells, especially T helper cells, or cytolytic T cells) upon appropriate stimulation. Immune effector cells are CD34 + They include hematopoietic stem cells, immature and mature T cells, and immature and mature B cells. The differentiation of T cell precursors into cytolytic T cells resembles the clonal selection of the immune system upon exposure to antigen.

[0171] Preferably, "immune effector cells" recognize antigens with some degree of specificity, especially when presented in the context of MHC or present on the surface of diseased cells such as cancer cells. Preferably, said recognition enables the cells that recognize the antigen to be responsive or reactive. The cells are called helper T cells (CD4 + T cells), such responsiveness or reactivity may be mediated by the release of cytokines and / or CD8 +The activation of lymphocytes (CTLs) and / or B cells may be involved. When the cells are CTLs, such responsiveness or reactivity may include the elimination of cells, i.e., cells characterized by antigen expression, via, for example, apoptosis or perforin-mediated cytolysis. According to the present invention, CTL responsiveness may include sustained calcium flux, cell division, production of cytokines such as IFN-γ and TNF-α, upregulation of activation markers such as CD44 and CD69, and specific cytolytic killing of target cells expressing the antigen. CTL responsiveness may also be determined using an artificial reporter that accurately indicates CTL responsiveness. Such CTLs that recognize and are responsive or reactive to an antigen are also referred to herein as "antigen-responsive CTLs."

[0172] In one embodiment, the immune effector cell is an antigen receptor (e.g., a chimeric antigen receptor (CAR) or a T cell receptor (TCR)) expressing immune effector cell. In one embodiment, the immune effector cell is a CAR-expressing immune effector cell. In one embodiment, the immune effector cell is a TCR-expressing immune effector cell. In one embodiment, the immune effector cell is an antigen receptor (e.g., a chimeric antigen receptor (CAR) or a T cell receptor (TCR)) transgenic immune effector cell.

[0173] Immune effector cells used in accordance with the present invention may express an endogenous antigen receptor, such as a T cell receptor or a B cell receptor, or may lack expression of an endogenous antigen receptor.

[0174] "Lymphoid cells" are cells or precursors of such cells that can generate immune responses, such as cellular immune responses, optionally after appropriate modification, for example, after introduction of antigen receptors such as TCRs or CARs, and include lymphocytes, preferably T lymphocytes, lymphoblasts, and plasma cells. Lymphoid cells can be immune effector cells as described herein. Preferred lymphoid cells are T cells that can be modified to express antigen receptors on their cell surface. In one embodiment, lymphoid cells lack endogenous expression of T cell receptors.

[0175] The terms "T cell" and "T lymphocyte" are used interchangeably herein and refer to T helper cells (CD4 + Cytotoxic T cells (CTL, CD8 + The term "antigen-specific T cells" or similar terms refers to T cells that recognize the antigen targeted by the T cell and preferably exert T cell effector functions. A T cell is considered specific for an antigen if it kills a target cell expressing the antigen. The specificity of a T cell can be assessed using any of a variety of standard techniques, for example, in a chromium release assay or proliferation assay. Alternatively, the synthesis of lymphokines (such as IFN-γ) can be measured.

[0176] T cells belong to a group of white blood cells known as lymphocytes and play a central role in cell-mediated immunity. They can be distinguished from other types of lymphocytes, such as B cells and natural killer cells, by the presence of a special receptor on their cell surface called the T cell receptor (TCR). The thymus is the primary organ responsible for the maturation of T cells. Several different subsets of T cells have been discovered, each with distinct functions.

[0177] T helper cells assist other white blood cells in immunological processes, including, among other functions, the maturation of B cells into plasma cells and the activation of cytotoxic T cells and macrophages. These cells express the CD4 glycoprotein on their surface and are therefore CD4 + Also known as T cells, helper T cells are activated when presented with peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or support the active immune response.

[0178] Cytotoxic T cells destroy virus-infected and tumor cells and are also involved in transplant rejection. These cells express the CD8 glycoprotein on their surface and are therefore CD8 + Also known as T cells, these cells recognize their targets by binding to antigens associated with MHC class I, which are present on the surface of almost every cell in the body.

[0179] As used herein, "effector T cells" or "T eff The term "regulatory T cells" refers to non-regulatory T cells, including T helper cells and cytotoxic T cells. Additionally, "effector T cells" or "T eff The term "effector T cells" includes activated and non-activated T cells, and therefore includes cells that have encountered antigen and / or costimulatory molecules, as well as cells that have not encountered antigen and / or costimulatory molecules. Thus, the term "effector T cells" also includes naive T cells. T cells express CD25 upon activation. According to the present invention, effector T cells express CD25 + T cells or CD25 - T cells, preferably CD25 - T cells.

[0180] “Regulatory T cell”, “T reg "Treg" or "Treg" cells are a subpopulation of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. Tregs are immunosuppressive and generally suppress or downregulate the induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FoxP3, and CD25.

[0181] As used herein, the term "naive T cells" refers to mature T cells that, unlike activated or memory T cells, have not encountered their cognate antigen in the periphery. Naive T cells are generally 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.

[0182] As used herein, the term "memory T cells" refers to a subgroup or subpopulation of T cells that have previously encountered and responded to their cognate antigen. Upon a second encounter with the antigen, memory T cells can be regenerated to mount a faster and stronger immune response than when the immune system first responded to the antigen. Memory T cells are CD4 + or CD8 + and typically expresses CD45RO.

[0183] All T cells have a T cell receptor (TCR) that exists as a complex of several proteins. In the majority of T cells, the actual T cell receptor is produced by independent T cell receptor alpha and beta (TCRα and TCRβ) genes and is composed of two separate peptide chains called the α- and β-TCR chains. A much less common group of T cells (2% of all T cells), γδ T cells (gamma delta T cells), have a separate T cell receptor (TCR) on their surface that is composed of one γ chain and one δ chain.

[0184] All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells derived from hematopoietic stem cells reside in the thymus and expand by cell division to generate a large population of immature thymocytes. The earliest thymocytes express neither CD4 nor CD8 and are therefore double-negative (CD4 - CD8 - ) cells. As development progresses, they become double-positive thymocytes (CD4 + CD8 + ) and eventually become single positive (CD4 + CD8 - or CD4 - CD8 + ) mature into thymocytes, which are then released from the thymus into peripheral tissues.

[0185] T cells can generally be prepared in vitro or ex vivo using standard procedures.For example, T cells can be isolated from the bone marrow, peripheral blood, or bone marrow or peripheral blood fraction of mammals such as patients using commercially available cell separation systems.Alternatively, T cells can be derived from related or unrelated humans, non-human animals, cell lines, or cultures.The sample containing T cells can be, for example, peripheral blood mononuclear cells (PBMCs).

[0186] As used herein, the term "NK cells" or "natural killer cells" refers to a subset of peripheral blood lymphocytes defined by expression of CD56 or CD16 and the absence of a T cell receptor. As provided herein, NK cells can also be differentiated from stem or progenitor cells.

[0187] Immune effector cells may naturally or after modification (e.g., ex vivo / in vitro or in vivo in the subject being treated) express an IL2R or an IL2R variant. Additionally, immune effector cells may naturally or after modification (e.g., ex vivo / in vitro or in vivo in the subject being treated) express an antigen receptor or its processing product, such as a T cell receptor (TCR) or chimeric antigen receptor (CAR)-bound antigen, particularly when present on or presented by a target cell.

[0188] Adoptive cell transfer therapy using CAR-engineered T cells expressing chimeric antigen receptors is a promising anticancer treatment because CAR-modified T cells can be engineered to target virtually any tumor antigen. For example, a patient's T cells can be genetically engineered (modified) to express a CAR that specifically targets an antigen on the patient's tumor cells, and then infused back into the patient.

[0189] The term "CAR" (or "chimeric antigen receptor") is synonymous with the terms "chimeric T cell receptor" and "artificial T cell receptor" and relates to an artificial receptor comprising a single molecule or complex of molecules that can recognize, i.e., bind to, a target structure (e.g., an antigen) on a target cell, such as a cancer cell (e.g., by binding of an antigen-binding domain to an antigen expressed on the surface of the target cell), and confer specificity to an immune effector cell, such as a T cell, that expresses the CAR on its cell surface. Preferably, recognition of the target structure by a CAR results in activation of the immune effector cell that expresses the CAR. A CAR can comprise one or more protein units comprising one or more domains described herein. The term "CAR" does not include T cell receptors.

[0190] CARs generally contain a target-specific binding element, also referred to as an antigen-binding portion or antigen-binding domain, which is part of the extracellular domain of the CAR. The antigen-binding domain recognizes a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Specifically, CARs target antigens, such as tumor antigens, on diseased cells, such as tumor cells.

[0191] In one embodiment, the binding domain of the CAR specifically binds to an antigen. In one embodiment, the antigen to which the binding domain in the CAR binds is expressed on cancer cells (tumor antigen). In one embodiment, the antigen is expressed on the surface of cancer cells. In one embodiment, the binding domain binds to the extracellular domain of the antigen or an epitope of the extracellular domain. In one embodiment, the binding domain binds to a natural epitope of the antigen present on the surface of living cells.

[0192] In one embodiment, the antigen-binding domain comprises a heavy chain variable region (VH) of an immunoglobulin having specificity for an antigen and a light chain variable region (VL) of an immunoglobulin having specificity for an antigen. In one embodiment, the immunoglobulin is an antibody. In one embodiment, the heavy chain variable region (VH) and the corresponding light chain variable region (VL) are linked by a peptide linker. Preferably, the antigen-binding portion in the CAR is an scFv.

[0193] CAR is designed to include a transmembrane domain fused to the extracellular domain of CAR.In one embodiment, the transmembrane domain is not naturally associated with one of the domains in CAR.In one embodiment, the transmembrane domain is naturally associated with one of the domains in CAR.In one embodiment, the transmembrane domain is modified by amino acid substitution to prevent such domain from binding to the transmembrane domain of the same or different surface membrane protein, thereby minimizing interaction with other members of the receptor complex.The transmembrane domain can be derived from either natural or synthetic sources.If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane regions particularly useful in the present invention may be derived from (i.e., comprise at least one or more of) the α, β, or ζ chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domain may be synthetic, in which case it comprises primarily hydrophobic residues such as leucine and valine. Preferably, triplets of phenylalanine, tryptophan, and valine are found at each end of the synthetic transmembrane domain.

[0194] In some cases, the CAR of the present invention comprises a hinge domain that forms the link between the transmembrane domain and the extracellular domain.

[0195] The cytoplasmic domain or other intracellular signaling domain of a CAR is involved in activating at least one of the normal effector functions of the immune cell in which the CAR is placed. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. While the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, as long as it transmits the effector function signal. Thus, the term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0196] It is known that signals generated solely through the TCR are insufficient for full activation of T cells, and that secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and those that act antigen-independently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).

[0197] In one embodiment, the CAR comprises a primary cytoplasmic signaling sequence derived from CD3ζ. Additionally, the cytoplasmic domain of the CAR may comprise a CD3ζ signaling domain combined with a costimulatory signaling region.

[0198] The identity of the costimulatory domain is limited only by its ability to enhance cell proliferation and survival upon binding of the targeting moiety by the CAR. Suitable costimulatory domains include CD28, CD137 (4-1BB), a member of the tumor necrosis factor receptor (TNFR) superfamily, CD134 (OX40), a member of the TNFR superfamily of receptors, and CD278 (ICOS), a costimulatory molecule of the CD28 superfamily expressed on activated T cells. Those skilled in the art will understand that sequence variants of these described costimulatory domains can be used without adversely affecting the present invention if they have the same or similar activity as the domain they are modeled after. Such variants have at least about 80% sequence identity with the amino acid sequence of the domain from which they are derived. In some embodiments of the present invention, the CAR construct comprises two costimulatory domains. Specific combinations include all possible variations of the four described domains, with specific examples including CD28+CD137(4-1BB) and CD28+CD134(OX40).

[0199] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other in random or specific order. Optionally, a short oligopeptide or polypeptide linker, preferably 2-10 amino acids in length, can form the linkage. A glycine-serine doublet provides a particularly suitable linker.

[0200] In one embodiment, the CAR comprises a signal peptide that targets the nascent protein to the endoplasmic reticulum. In one embodiment, the signal peptide precedes the antigen-binding domain. In one embodiment, the signal peptide is derived from an immunoglobulin, such as IgG.

[0201] The term "antibody" includes immunoglobulins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with antigens. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Antibodies bind, preferably specifically, to antigens. Antibodies can be intact immunoglobulins derived from natural or recombinant sources, or can be immunoreactive portions or fragments of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies in the present invention can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab')2, as well as single-chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0202] Antibodies expressed by B cells are sometimes called BCRs (B cell receptors) or antigen receptors. The five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody present in bodily secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses and is important for defense against bacteria and viruses. IgD is an immunoglobulin whose antibody function is unknown but which can function as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by triggering the release of mediators from mast cells and basophils upon exposure to allergens.

[0203] Antibodies may be derived from a variety of species, including but not limited to mouse, rat, rabbit, guinea pig, and human.

[0204] Antibodies described herein include IgA, such as 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 kappa or IgG1 lambda 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).

[0205] The term "antibody fragment" refers to a portion of an intact antibody, typically comprising the antigen-determining variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0206] As used herein, "antibody heavy chain" refers to the larger of the two polypeptide chains present in antibody molecules in their native conformation.

[0207] As used herein, "antibody light chain" refers to the smaller of the two polypeptide chains present in antibody molecules in their native conformation; kappa light chain and lambda light chain refer to the two major antibody light chain isotypes.

[0208] According to the present disclosure, a CAR recognizes an antigen, such as on the surface of an antigen-presenting cell or a diseased cell, such as a cancer cell, such that when present on a T cell, the T cell is stimulated and / or expanded or exerts an effector function, as described above.

[0209] Various methods can be used to introduce an IL2 receptor polypeptide and / or an antigen receptor, such as a CAR construct, into cells, such as T cells, to produce cells genetically modified to express the IL2 receptor polypeptide and / or antigen receptor. Such methods include non-viral DNA transfection, non-viral RNA transfection (e.g., mRNA transfection), transposon-based systems, and viral-based systems. Non-viral DNA transfection has a lower risk of insertional mutagenesis. Transposon-based systems can integrate transgenes more efficiently than plasmids that do not contain integration elements. Viral-based systems include the use of gamma-retroviruses and lentiviral vectors. Gamma-retroviruses are relatively easy to produce, efficiently and persistently transduce T cells, and have been preliminarily proven to be safe in terms of integration into primary human T cells. Lentiviral vectors also efficiently and persistently transduce T cells, but are more expensive to produce. They are also potentially safer than retroviral-based systems. In one embodiment of all aspects of the present invention, cells are transfected with a nucleic acid encoding an IL2 receptor polypeptide and / or a nucleic acid encoding an antigen receptor either ex vivo or in vivo. In one embodiment, a combination of ex vivo and in vivo transfection may be used. After ex vivo modification, the cells may be administered to the subject to be treated.

[0210] In one embodiment of all aspects of the present invention, the cells described herein can be autologous, allogeneic, or syngeneic to the subject being treated. In one embodiment, the present disclosure contemplates the removal of cells from the patient and the subsequent re-delivery of the cells (e.g., modified and / or expanded ex vivo) to the patient. In one embodiment, the present disclosure does not contemplate the removal of cells from the patient. In the latter case, all steps of genetic modification of the cells are performed in vivo.

[0211] The term "autologous" is used to refer to something derived from the same subject. For example, "autologous transplantation" refers to the transplantation of tissue or organs derived from the same subject. Such procedures are advantageous because they overcome immunological barriers that would otherwise result in rejection.

[0212] The term "allogeneic" is used to describe something that is derived from different individuals of the same species. Two or more individuals are said to be allogeneic to one another if the genes at one or more loci are not identical.

[0213] The term "syngeneic" is used to describe individuals or tissues that have the same genotype, i.e., derived from identical twins or the same inbred strain of animals, or tissues thereof.

[0214] The term "xenogeneic" is used to describe something that is made up of multiple dissimilar elements. As an example, transferring bone marrow from one individual to another constitutes a xenogeneic transplant. A xenogeneic gene is a gene that originates from a source other than the subject.

[0215] The present disclosure also provides for providing, e.g., administering, an antigenic molecule, such as a peptide or protein antigen, to contact immune effector cells, particularly immune effector cells expressing an antigen receptor, e.g., immune effector cells genetically engineered to express an antigen receptor ex vivo or in a subject to be treated, with a cognate antigenic molecule, wherein the antigenic molecule or a processing product thereof, e.g., a fragment thereof, binds to an antigen receptor, such as a TCR or CAR, carried by the immune effector cells. In one embodiment, the cognate antigenic molecule is selected from the group consisting of an antigen or fragment thereof expressed by a target cell targeted by the immune effector cells, or a variant of said antigen or fragment. In one embodiment, the immune effector cells are contacted with the cognate antigenic molecule under conditions such that expansion and / or activation of the immune effector cells occurs. In one embodiment, the step of contacting the immune effector cells with the cognate antigenic molecule is performed in vivo or ex vivo.

[0216] Peptide and protein antigens suitable for use according to the present disclosure typically include peptides or proteins that contain epitopes for inducing an immune response. The peptides or proteins or epitopes can be derived from target antigens, i.e., antigens against which an immune response should be elicited. For example, the peptide or protein antigen or the epitope contained within the peptide or protein antigen can be the target antigen or a fragment or variant of the target antigen.

[0217] In one embodiment, the method described herein comprises administering an antigen molecule or a nucleic acid encoding the same to a subject. In one embodiment, the nucleic acid encoding the antigen molecule is expressed in the subject's cells to provide the antigen molecule. In one embodiment, the expression of the antigen molecule is on the cell surface. In one embodiment, the antigen molecule is presented in the context of MHC. In one embodiment, the nucleic acid encoding the antigen molecule is transiently expressed in the subject's cells. In one embodiment, the nucleic acid encoding the antigen molecule is RNA. In one embodiment, the antigen molecule or the nucleic acid encoding the same is administered systemically. In one embodiment, systemic administration of the nucleic acid encoding the antigen molecule results in expression of the nucleic acid encoding the antigen molecule in the spleen. In one embodiment, systemic administration of the nucleic acid encoding the antigen molecule results in expression of the nucleic acid encoding the antigen molecule in antigen-presenting cells, preferably professional antigen-presenting cells. In one embodiment, the antigen-presenting cells are selected from the group consisting of dendritic cells, macrophages, and B cells. In one embodiment, systemic administration of the nucleic acid encoding the antigen molecule results in no or essentially no expression of the nucleic acid encoding the antigen molecule in the lung and / or liver. In one embodiment, systemic administration of the nucleic acid encoding the antigen molecule results in expression of the nucleic acid encoding the antigen molecule in the spleen that is at least five times higher than the expression level in the lung.

[0218] The peptide and protein antigens provided to a subject according to the present invention (by administering the peptide and protein antigens or nucleic acids, particularly RNA, encoding said peptide and protein antigens), i.e., vaccine antigens, preferably result in the stimulation, priming, and / or expansion of immune effector cells in the subject receiving the peptide or protein antigen or nucleic acid. The stimulated, primed, and / or expanded immune effector cells are preferably directed against a target antigen, particularly a target antigen expressed by diseased cells, tissues, and / or organs, i.e., a disease-associated antigen. Thus, 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 the disease-associated antigen. In the context of the present disclosure, the term "antigen fragment" or "antigen variant" refers to an agent that results in the stimulation, priming, and / or expansion of immune effector cells, and the stimulated, primed, and / or expanded immune effector cells target the antigen, i.e., the disease-associated antigen, particularly when presented by diseased cells, tissues, and / or organs. Thus, a vaccine antigen may correspond to or comprise a disease-associated antigen, a fragment of a disease-associated antigen, or an antigen homologous to a disease-associated antigen or its fragment. When a vaccine antigen 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 of the disease-associated antigen or a sequence homologous to an epitope of the disease-associated antigen. Thus, according to the present disclosure, a vaccine antigen 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 capable of stimulating, priming, and / or expanding immune effector cells bearing an antigen receptor that binds to the antigen or cells expressing the antigen. Vaccine antigens (like disease-associated antigens) preferably provide a relevant epitope for binding by an antigen-binding domain present on immune effector cells.In one embodiment, vaccine antigens (as well as disease-associated antigens) are expressed on the surface of cells, such as antigen-presenting cells, to provide relevant epitopes for binding by immune effector cells. Vaccine antigens can be recombinant antigens.

[0219] In one embodiment of all aspects of the invention, a nucleic acid encoding a vaccine antigen is expressed in cells of a subject to provide the antigen or its processing product for binding by an antigen receptor expressed by immune effector cells, said binding resulting in stimulation, priming and / or expansion of the immune effector cells.

[0220] The term "immunologically equivalent" means that an immunologically equivalent molecule, such as an immunologically equivalent amino acid sequence, exhibits the same or essentially the same immunological properties and / or exerts the same or essentially the same immunological effect, e.g., with respect to the type of immunological effect. In the context of the present disclosure, the term "immunologically equivalent" is preferably used with respect to the immunological effect or properties of an antigen or antigen variant used for immunization. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if, when exposed to a subject's immune system, such as T cells that bind to the reference amino acid sequence or cells that express the reference amino acid sequence, it induces an immune response with specificity that reacts with the reference amino acid sequence. Thus, a molecule that is immunologically equivalent to an antigen exhibits the same or essentially the same properties and / or exerts the same or essentially the same effect as the antigen targeted by the T cells with respect to stimulating, priming, and / or expanding T cells.

[0221] As used herein, "activation" or "stimulation" refers to the state of immune effector cells, such as T cells, that are stimulated sufficiently to induce detectable cell proliferation. Activation can also involve the initiation of signal transduction pathways, the induction of cytokine production, and detectable effector function. The term "activated immune effector cells" refers, among other things, to immune effector cells undergoing cell division.

[0222] The term "priming" refers to the process by which an immune effector cell, such as a T cell, first contacts its specific antigen, leading to differentiation into an effector cell, such as an effector T cell.

[0223] The term "clonal expansion" or "expansion" refers to the process of increasing a specific entity. In the context of the present disclosure, the term is preferably used in the context of an immunological response in which lymphocytes are stimulated by an antigen, proliferate, and the specific lymphocytes that recognize said antigen are amplified. Preferably, clonal expansion results in differentiation of lymphocytes.

[0224] The term "antigen" refers to an agent containing an epitope capable of generating an immune response. The term "antigen" includes, inter alia, proteins and peptides. In one embodiment, an antigen is presented or present on the surface of a cell of the immune system, such as an antigen-presenting cell, such as a dendritic cell or macrophage. In one embodiment, an antigen or its processing product, such as a T cell epitope, is bound by an antigen receptor. Thus, the antigen or its processing product can specifically react with immune effector cells, such as T lymphocytes (T cells). In one embodiment, the antigen is a disease-related antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, and the epitope is derived from such an antigen.

[0225] 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 containing an epitope that stimulates the host's immune system to generate a cellular antigen-specific immune response and / or a humoral antibody response against the disease. Thus, a disease-associated antigen or its epitope can be used for therapeutic purposes. A disease-associated antigen can be associated with infection by a microorganism, typically a microbial antigen, or can be associated with cancer, typically a tumor.

[0226] The term "tumor antigen" refers to components of cancer cells that can originate from the cytoplasm, cell surface, and cell nucleus. In particular, this term refers to antigens produced intracellularly or as surface antigens on tumor cells. Tumor antigens are typically selectively expressed by cancer cells (e.g., expressed at higher levels in cancer cells than in non-cancerous cells), and in some cases, are expressed only by cancer cells. 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, claudin family cell surface proteins 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 / MelanA, MC1R, myosin / m, MUC These include 1, MUM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 minor 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.

[0227] The term "viral antigen" refers to any viral component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. A viral antigen can be a viral ribonucleoprotein or an envelope protein.

[0228] The term "bacterial antigen" refers to any bacterial component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. Bacterial antigens can be derived from the bacterial cell wall or cytoplasmic membrane.

[0229] The terms "expressed on the cell surface" or "associated with the cell surface" mean that a molecule, such as a receptor or antigen, is located in association with the plasma membrane of a cell, with at least a portion of the molecule facing the extracellular space of the cell and accessible from the outside of the cell, for example, by an antibody located on the outside of the cell. In this context, a portion is preferably at least 4, preferably at least 8, preferably at least 12, more preferably at least 20 amino acids. The association can be direct or indirect. For example, the association can be via one or more transmembrane domains, one or more lipid anchors, or by interaction with any other protein, lipid, saccharide, or other structure that can be found on the outer leaflet of the plasma membrane of the cell. For example, a molecule associated with the surface of a cell can be a transmembrane protein having an extracellular portion, or a protein that associates with the surface of a cell by interacting with another protein that is a transmembrane protein.

[0230] "Cell surface" or "surface of a cell" is used according to its ordinary meaning in the art and thus includes the outside of a cell that is accessible to binding by proteins and other molecules.

[0231] The term "extracellular portion" or "exodomain" in the context of the present invention refers to a part of a molecule, such as a protein, that faces the extracellular space of a cell and is preferably accessible from outside the cell, for example by binding to a molecule, such as an antibody, that is located on the outside of said cell. Preferably, the term refers to one or more extracellular loops or domains or fragments thereof.

[0232] The term "epitope" refers to a portion or fragment of a molecule, such as an antigen, that is recognized by the immune system. For example, an epitope can be recognized by T cells, B cells, or an antibody. An epitope of an antigen can include a continuous or discontinuous portion of the antigen and can be about 5 to about 100, e.g., about 5 to about 50, more preferably about 8 to about 30, and most preferably about 10 to about 25 amino acids in length. For example, an 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 length. In one embodiment, an epitope is about 10 to about 25 amino acids in length. The term "epitope" includes T cell epitopes.

[0233] The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in the context of an MHC molecule. The terms "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes present in all vertebrates, including MHC class I and MHC class II molecules. MHC proteins or molecules are important in signaling between lymphocytes and antigen-presenting or diseased cells during the immune response. MHC proteins or molecules bind to peptide epitopes and present them for recognition by T cell receptors on T cells. Proteins encoded by MHC are expressed on the surface of cells and display both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. For class I MHC / peptide complexes, the binding peptide is typically about 8 to about 10 amino acids long, although longer or shorter peptides can also be effective. For class II MHC / peptide complexes, the binding peptide is typically about 10 to about 25 amino acids long, particularly about 13 to about 18 amino acids long, although longer and shorter peptides can also be effective.

[0234] In one embodiment, the target antigen is a tumor antigen, and the epitope-containing peptide or protein, or a fragment thereof (e.g., epitope), is derived from the tumor antigen. The tumor antigen may be a "standard" antigen generally known to be expressed in various cancers. The tumor antigen may also be a "neoantigen" that is specific to an individual's tumor and has not previously been recognized by the immune system. The neoantigen or neoepitope may result from one or more cancer-specific mutations in the genome of a cancer cell that result in amino acid changes. When the tumor antigen is a neoantigen, the epitope-containing peptide or protein preferably comprises an epitope or fragment of the neoantigen that contains one or more amino acid changes.

[0235] Cancer mutations vary from individual to individual. Therefore, cancer mutations encoding novel epitopes (neoepitopes) are attractive targets for the development of vaccine compositions and immunotherapies. The effectiveness of tumor immunotherapy depends on the selection of cancer-specific antigens and epitopes that can induce a strong immune response in the host. RNA can be used to deliver patient-specific tumor epitopes to patients. Dendritic cells (DCs) present in the spleen are particularly interesting antigen-presenting cells for RNA expression of immunogenic epitopes or antigens, such as tumor epitopes. The use of multiple epitopes has been shown to enhance therapeutic efficacy in tumor vaccine compositions. Rapid sequencing of tumor mutagenesis can provide multiple epitopes for personalized vaccines that can be encoded by the RNA described herein, for example, as a single polypeptide in which the epitopes are optionally separated by linkers. In certain embodiments of the present disclosure, the RNA encodes at least 1 epitope, at least 2 epitopes, at least 3 epitopes, at least 4 epitopes, at least 5 epitopes, at least 6 epitopes, at least 7 epitopes, at least 8 epitopes, at least 9 epitopes, or at least 10 epitopes. Exemplary embodiments include RNA encoding at least 5 epitopes (called "pentatopes") and RNA encoding at least 10 epitopes (called "decatopes").

[0236] According to various aspects of the present invention, the objective is preferably to provide an immune response against cancer cells expressing tumor antigens and to treat cancer diseases in which cells expressing tumor antigens are involved. Preferably, the present invention involves the administration of immune effector cells, such as T cells, that target cancer cells expressing tumor antigens and / or vaccine antigens recognized by such cells.

[0237] Peptide and protein antigens can be 2 to 100 amino acids in length, including, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. In some embodiments, peptides can be greater than 50 amino acids. In some embodiments, peptides can be greater than 100 amino acids.

[0238] The peptide or protein antigen can be any peptide or protein that is capable of inducing or increasing the ability of the immune system to generate antibody and T cell responses against the peptide or protein.

[0239] In one embodiment, the vaccine antigen is recognized by immune effector cells. Preferably, when recognized by immune effector cells, the vaccine antigen can induce stimulation, priming, and / or expansion of immune effector cells bearing antigen receptors that recognize the vaccine antigen in the presence of appropriate costimulatory signals. In the context of this embodiment of the present invention, the vaccine antigen is preferably presented or present on the surface of a cell, preferably on the surface of an antigen-presenting cell. Recognition of a disease-associated antigen on the surface of a disease cell by immune effector cells can result in an immune response against the antigen (or cells expressing the antigen).

[0240] 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 an epitope-containing peptide or protein).

[0241] As used herein, "immune checkpoint" refers to costimulatory and inhibitory signals that regulate the magnitude and quality of T cell receptor recognition of antigens. In some embodiments, the immune checkpoint is an inhibitory signal. In some embodiments, the inhibitory signal is the interaction between PD-1 and PD-L1. In some embodiments, the inhibitory signal is the interaction between CTLA-4 and CD80 or CD86, which displaces CD28 binding. In some embodiments, the inhibitory signal is the interaction between LAG3 and an MHC class II molecule. In some embodiments, the inhibitory signal is the interaction between TIM3 and galectin-9.

[0242] As used herein, "immune checkpoint inhibitor" refers to a molecule that completely or partially reduces, inhibits, prevents, or modulates one or more checkpoint proteins. In some embodiments, the immune checkpoint inhibitor prevents inhibitory signals associated with an immune checkpoint. In some embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that interferes with inhibitory signaling associated with an immune checkpoint. In some embodiments, the immune checkpoint inhibitor is a small molecule that interferes with inhibitory signaling. In some embodiments, the immune checkpoint inhibitor is an antibody, fragment thereof, or antibody mimetic that prevents interactions between checkpoint blocker proteins, e.g., an antibody or fragment thereof that prevents the interaction between PD-1 and PD-L1. In some embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that prevents the interaction between CTLA-4 and CD80 or CD86. In some embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that prevents the interaction between LAG3 and its ligand, or TIM-3 and its ligand. The checkpoint inhibitor can also be in the form of a soluble form of the molecule (or variant thereof) itself, e.g., a soluble PD-L1 or a PD-L1 fusion.

[0243] The "programmed death 1 (PD-1)" receptor refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is primarily expressed on pre-activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. As used herein, the term "PD-1" includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, as well as analogs that share at least one epitope with hPD-1.

[0244] "Programmed death-ligand 1 (PD-L1)" is one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that downregulates T cell activation and cytokine secretion upon binding to PD-1. As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, and analogs that share at least one epitope with hPD-L1.

[0245] "Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4)" is a T-cell surface molecule and a member of the immunoglobulin superfamily. This protein downregulates the immune system by binding to CD80 and CD86. As used herein, the term "CTLA-4" includes human CTLA-4 (hCTLA-4), variants, isoforms, and species homologs of hCTLA-4, as well as analogs that share at least one epitope with hCTLA-4.

[0246] "Lymphocyte activation gene 3 (LAG3)" is an inhibitory receptor involved in the inhibition of lymphocyte activity by binding to MHC class II molecules. This receptor enhances the function of Treg cells and inhibits the function of CD8+ effector T cells. As used herein, the term "LAG3" includes human LAG3 (hLAG3), variants, isoforms, and species homologs of hLAG3, as well as analogs that share at least one common epitope.

[0247] "T-cell membrane protein 3 (TIM3)" is an inhibitory receptor involved in inhibiting lymphocyte activity by inhibiting TH1 cell responses. Its ligand is galectin 9, which is upregulated in various types of cancer. As used herein, the term "TIM3" includes human TIM3 (hTIM3), variants, isoforms, and species homologs of hTIM3, as well as analogs that share at least one common epitope.

[0248] "B7 family" refers to inhibitory ligands with undefined receptors. The B7 family includes B7-H3 and B7-H4, both of which are upregulated in tumor cells and tumor-infiltrating cells.

[0249] In certain embodiments, immune checkpoint inhibitors suitable for use in the methods disclosed herein are antagonists of inhibitory signals, such as antibodies targeting 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.

[0250] In some embodiments, the immune checkpoint inhibitor is an antibody or antigen-binding portion thereof that disrupts or inhibits signaling from an inhibitory immunoregulator. In some embodiments, the immune checkpoint inhibitor is a small molecule that disrupts or inhibits signaling from an inhibitory immunoregulator.

[0251] In some embodiments, the inhibitory immunomodulator is a component of the PD-1 / PD-L1 signaling pathway. Accordingly, some embodiments of the present disclosure provide for administering to a subject an antibody, or antigen-binding portion thereof, that interferes with the interaction between the PD-1 receptor and its ligand, PD-L1. Antibodies that bind to PD-1 and interfere with the interaction between PD-1 and its ligand, PD-L1, are known in the art. In some embodiments, the antibody, or antigen-binding portion thereof, specifically binds to PD-1. In some embodiments, the antibody, or antigen-binding portion thereof, specifically binds to PD-L1 and inhibits its interaction with PD-1, thereby increasing immune activity.

[0252] In some embodiments, the inhibitory immunomodulator is a component of the CTLA4 signaling pathway. Accordingly, some embodiments of the present disclosure provide for administering to a subject an antibody or antigen-binding portion thereof that targets CTLA4 and interferes with its interaction with CD80 and CD86.

[0253] 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 interferes with its interaction with MHC class II molecules.

[0254] In some embodiments, the inhibitory immunomodulator is a component of the B7 family signaling pathway. In some embodiments, the B7 family members are B7-H3 and B7-H4. Accordingly, some embodiments of the present disclosure provide for administering to a subject an antibody or antigen-binding portion thereof that targets B7-H3 or B7-H4. While the B7 family does not have a defined receptor, 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.

[0255] 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 interferes with its interaction with galectin-9.

[0256] It will be understood by those skilled in the art that other immune checkpoint targets can also be targeted by antagonists or antibodies, provided that targeting results in stimulation of an immune response, such as an anti-tumor immune response as reflected, for example, in increased T cell proliferation, enhanced T cell activation, and / or increased cytokine production (e.g., IFN-γ, IL2).

[0257] According to the present invention, it is particularly preferred that the peptides, proteins or polypeptides described herein, in particular the IL2 variant polypeptides and / or vaccine 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.

[0258] In one embodiment, the RNA is formulated in a delivery vehicle. In one embodiment, the delivery vehicle comprises a particle. In one embodiment, the delivery vehicle comprises at least one lipid. In one embodiment, the at least one lipid comprises at least one cationic lipid. In one embodiment, the lipid complexes with and / or encapsulates the RNA. In one embodiment, the lipid is included in a vesicle that encapsulates the RNA. In one embodiment, the RNA is formulated in a liposome.

[0259] According to the present disclosure, after administering the RNA described herein, at least a portion of the RNA is delivered to target cells.In one embodiment, at least a portion of the RNA is delivered to the cytosol of target cells.In one embodiment, the RNA is translated by target cells to produce the encoded peptide or protein.

[0260] Some embodiments of the present disclosure involve targeted delivery of the RNA disclosed herein (RNA encoding an IL2 variant polypeptide and / or RNA encoding a peptide or protein comprising an epitope).

[0261] In one embodiment, the present disclosure includes targeting the lymphatic system, particularly secondary lymphatic organs, more particularly the spleen.When the administered RNA is an RNA encoding a peptide or protein containing an epitope, it is particularly preferred to target the lymphatic system, particularly secondary lymphatic organs, more particularly the spleen.

[0262] 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.

[0263] The "lymphatic system" is part of the circulatory system and an important part of the immune system, including the network of lymphatic vessels that transport lymph. The lymphatic system consists of lymphoid organs, the conducting network of lymphatic vessels, and circulating lymph. Primary or central lymphoid organs generate lymphocytes from immature precursor cells. The thymus and bone marrow constitute the primary lymphoid organs. Secondary or peripheral lymphoid organs, including the lymph nodes and spleen, maintain mature naive lymphocytes and initiate adaptive immune responses.

[0264] RNA can be delivered to the spleen via so-called lipoplex formulations, in which the RNA is bound to liposomes containing cationic lipids and, optionally, additional lipids or helper lipids, to form injectable nanoparticle formulations. Liposomes can be obtained by injecting a solution of lipids in ethanol into water or a suitable aqueous phase. RNA lipoplex particles can be prepared by mixing liposomes with RNA. Spleen-targeted RNA lipoplex particles are described in International Publication No. 2013 / 143683, which is incorporated herein by reference. It has been discovered that RNA lipoplex particles with a net negative charge can be used to selectively target spleen tissue or spleen cells, such as antigen-presenting cells, particularly dendritic cells. Thus, RNA accumulation and / or RNA expression occurs in the spleen after administration of the RNA lipoplex particles. Thus, the RNA lipoplex particles of the present disclosure can be used to express RNA in the spleen. In one embodiment, no or essentially no RNA accumulation and / or RNA expression occurs in the lung and / or liver after administration of the RNA lipoplex particles. In one embodiment, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression occurs in antigen-presenting cells, such as professional antigen-presenting cells in the spleen.Therefore, the RNA lipoplex 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.

[0265] In the context of the present disclosure, the term "RNA lipoplex particle" refers to a particle comprising a lipid, particularly a cationic lipid, and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA result in the complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes can generally be synthesized using a cationic lipid, such as DOTMA, and an additional lipid, such as DOPE. In one embodiment, the RNA lipoplex particle is a nanoparticle.

[0266] As used herein, "cationic lipid" refers to a lipid with a net positive charge. Cationic lipids bind negatively charged RNA to lipid matrices through electrostatic interactions. Generally, cationic lipids have a lipophilic moiety such as a sterol, acyl, or diacyl chain, and the lipid head group typically carries a positive charge. Examples of cationic lipids include 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); 1,2-dioleoyl-3-dimethylammonium propane (DODAP); 1,2-diacyloxy-3-dimethylammonium propane; 1,2-dialkyloxy-3-dimethylammonium propane; dioctadecyldimethylammonium chloride (DODAC), 2,3-di(tetradecoxy)propane. Cationic lipids include, but are not limited to, pyr-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA).Preferably, DOTMA, DOTAP, DODAC, and DOSPA are cationic lipids.In a specific embodiment, the cationic lipid is DOTMA and / or DOTAP.

[0267] Additional lipids may be incorporated to adjust the overall ratio of positive to negative charges and the physical stability of the RNA lipoplex particles. In certain embodiments, the additional lipid is a neutral lipid. As used herein, "neutral lipid" refers to a lipid with a zero net charge. Examples of neutral lipids include, but are not limited to, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, and cerebroside. In specific embodiments, the additional lipid is DOPE, cholesterol, and / or DOPC.

[0268] In certain embodiments, the RNA lipoplex particles comprise both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

[0269] In some embodiments, the molar ratio of the at least one cationic lipid to the 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 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 the at least one cationic lipid to the at least one additional lipid is about 2:1.

[0270] The RNA lipoplex particles described herein, in one embodiment, have an average diameter in the range of about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 250 nm to about 700 nm, about 400 nm to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm. In specific embodiments, the RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm. In one embodiment, the RNA lipoplex particles have an average diameter in the range of about 250 nm to about 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter ranging from about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.

[0271] The charge of the RNA lipoplex particles of the present disclosure is the sum of the charge present in at least one cationic lipid and the charge present in RNA.The charge ratio is the ratio of the positive charge present in at least one cationic lipid to the negative charge present in RNA.The charge ratio of the positive charge present in at least one cationic lipid to the negative charge present in RNA is calculated by the following formula: charge ratio = [(cationic lipid concentration (mol)) * (total number of positive charges in cationic lipid)] / [(RNA concentration (mol)) * (total number of negative charges in RNA)].

[0272] The spleen-targeted RNA lipoplex particles described herein at physiological pH preferably have a net negative charge, such as a positive to negative charge ratio of about 1.9:2 to about 1:2. In specific embodiments, the positive to negative charge ratio in the RNA lipoplex 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.

[0273] RNA delivery systems have inherent liver selectivity. This is related to lipid nanoparticles, including lipid-based particles, cationic and neutral nanoparticles, particularly liposomes, nanomicelles, and lipid nanoparticles containing lipophilic ligands in bioconjugates. Liver accumulation is caused by the discontinuous nature of the hepatic vasculature or lipid metabolism (liposomes and lipid or cholesterol conjugates).

[0274] In one embodiment of the targeted delivery of IL2 variant polypeptides described herein, the target organ is the liver and the target tissue is hepatic tissue. Delivery to such a target tissue is preferred, particularly when the presence of the IL2 variant polypeptide in this organ or tissue is desired, and / or when it is desired to express large amounts of the IL2 variant polypeptide, and / or when the systemic presence of the IL2 variant polypeptide, particularly in significant amounts, is desired or required.

[0275] In one embodiment, the RNA encoding the IL2 variant polypeptide is administered in a formulation for targeting to the liver. Such formulations are described herein above.

[0276] For in vivo delivery of RNA to the liver, drug delivery systems can be used to transport RNA to the liver by preventing its degradation. For example, polyplex nanomicelles, consisting of a poly(ethylene glycol) (PEG)-coated surface and an mRNA-containing core, are useful systems because the nanomicelles provide exceptional in vivo stability of RNA under physiological conditions. Furthermore, the stealth properties provided by the polyplex nanomicelle surface, composed of a dense PEG pallisade, effectively evade the host immune defenses.

[0277] The peptides, proteins, polypeptides, RNA, RNA particles, immune effector cells and additional agents, e.g., immune checkpoint inhibitors, described herein may be administered in pharmaceutical compositions or medicaments for therapeutic or prophylactic treatment, and may be administered in the form of any suitable pharmaceutical composition, which may include a pharmaceutically acceptable carrier, and may optionally include one or more adjuvants, stabilizers, etc. In one embodiment, the pharmaceutical composition is for therapeutic or prophylactic treatment, e.g., for use in the treatment or prevention of a disease in which an antigen is involved, such as a cancer disease, as described herein.

[0278] The term "pharmaceutical composition" relates to a formulation comprising a therapeutically effective agent, preferably together with a pharmaceutically acceptable carrier, diluent and / or excipient. The pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease or disorder by administering the pharmaceutical composition to a subject. A pharmaceutical composition is also known in the art as a pharmaceutical formulation. In the context of the present disclosure, a pharmaceutical composition comprises a peptide, protein, polypeptide, RNA, RNA particle, immune effector cell, and / or additional agent as described herein.

[0279] The pharmaceutical compositions of the present disclosure may contain or be administered with one or more adjuvants. The term "adjuvant" refers to a compound that prolongs, enhances, or accelerates an immune response. Adjuvants include a heterogeneous group of compounds, such as oil emulsions (e.g., Freund's adjuvant), inorganic compounds (e.g., alum), bacterial products (e.g., Bordetella pertussis toxin), or immune-stimulating complexes. Examples of adjuvants include, but are not limited to, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, and chemokines. Cytokines may be IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IFNα, IFNγ, GM-CSF, or LT-α. Further known adjuvants are aluminum hydroxide, Freund's adjuvant, or oils such as Montanide® ISA 51. Other suitable adjuvants for use in the present disclosure include lipopeptides such as Pam3Cys.

[0280] Pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and a "pharmaceutically acceptable formulation."

[0281] The term "pharmaceutically acceptable" refers to the non-toxicity of a substance that does not interact with the action of the active ingredients of the pharmaceutical composition.

[0282] The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount that, alone or together with further doses, achieves a desired response or a desired effect. In the case of the treatment of a specific disease, the desired response preferably relates to the inhibition of the course of the disease. This includes slowing the progression of the disease, particularly halting or reversing the progression of the disease. The desired response in the treatment of a disease can also be delaying or preventing the onset of the disease or condition. The effective amount of the compositions described herein depends on the condition being treated, the severity of the disease, individual patient parameters including age, physiological condition, size, and weight, the duration of treatment, the type of concomitant treatment (if any), the specific route of administration, and similar factors. Thus, the dosage of the compositions described herein can depend on such various parameters. If the patient's response is inadequate with the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) can be used.

[0283] The pharmaceutical compositions of the present disclosure may include salts, buffering agents, preservatives, and optionally other therapeutic agents. In one embodiment, the pharmaceutical compositions of the present disclosure include one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0284] Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0285] The term "excipient" as used herein refers to a substance that may be present in the pharmaceutical compositions of the present disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

[0286] The term "diluent" refers to an agent that is diluted and / or diluted. Furthermore, the term "diluent" includes any one or more of a fluid, liquid or solid suspension and / or mixture medium. Examples of suitable diluents include ethanol, glycerol, and water.

[0287] The term "carrier" refers to a component, which may be natural, synthetic, organic, or inorganic, with which an active ingredient is combined to facilitate, enhance, or enable administration of a pharmaceutical composition. As used herein, a carrier may be one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a subject. 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, in particular, biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers. In one embodiment, the pharmaceutical composition of the present disclosure comprises isotonic saline.

[0288] 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).

[0289] Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0290] In one embodiment, the pharmaceutical compositions described herein may be administered intravenously, intraarterially, subcutaneously, intradermally, or intramuscularly. In certain embodiments, the pharmaceutical compositions are formulated for local or systemic administration. Systemic administration may include enteral administration, including absorption via the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to administration by any means other than via 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 by intravenous administration. In one embodiment of all aspects of the invention, RNA encoding an IL2 variant polypeptide described herein, and optionally RNA encoding an antigen, is administered systemically.

[0291] As used herein, the term "co-administration" refers to the process of administering different compounds or compositions (e.g., RNA encoding an IL2 variant polypeptide, RNA encoding an epitope-containing peptide or protein, and optionally, an immune checkpoint inhibitor) to the same patient. The RNA encoding an IL2 variant polypeptide and the RNA encoding the epitope-containing peptide or protein can be administered simultaneously, essentially simultaneously, or sequentially. When administration is sequential, the RNA encoding the IL2 variant polypeptide can be administered before or after the administration of the RNA encoding the epitope-containing peptide or protein. When administration is simultaneous, the RNA encoding the IL2 variant polypeptide and the RNA encoding the epitope-containing peptide or protein do not need to be administered in the same composition. The RNA encoding the IL2 variant polypeptide and the RNA encoding the epitope-containing peptide or protein may be administered more than once, and the number of administrations of each component may be the same or different. Furthermore, the RNA encoding the IL2 variant polypeptide and the RNA encoding the epitope-containing peptide or protein do not need to be administered at the same site.

[0292] The IL2 variant polypeptides, polynucleotides encoding IL2 variant polypeptides, host cells comprising polynucleotides encoding IL2 variant polypeptides, pharmaceutical compositions, and therapeutic methods described herein may be used in the therapeutic or prophylactic treatment of various diseases, particularly cancer, autoimmune diseases, and infectious diseases, for which providing IL2, particularly the IL2 variant polypeptides described herein, to a subject provides a therapeutic or prophylactic effect; in cancer vaccines and conventional vaccine therapies for immune stimulation in elderly or immunocompromised individuals, as well as in HIV or human SCID patients; or in other therapeutic applications requiring general stimulation of the immune system in any suitable animal, preferably a mammal, and most preferably a human. IL2 has many actions. Some of these are the stimulation of T cells, particularly memory T cells, naive T cells, and / or effector T cells, and / or NK cells. The IL2 variant polypeptides described herein are active against cell types that express only intermediate-affinity IL2 receptors, such as memory T cells, naive T cells, and / or effector T cells, but not against high-affinity IL2 receptors, such as regulatory T cells. Thus, the use of IL2 variant polypeptides, polynucleotides encoding IL2 variant polypeptides, host cells comprising polynucleotides encoding IL2 variant polypeptides, pharmaceutical compositions and treatment methods described herein in the treatment of diseases for which IL2, due to its T cell activity, is expected to provide effective therapy is contemplated.

[0293] Alternatively, or in addition to methods of direct administration to a patient, in some embodiments, IL2 variant polypeptides can be used in ex vivo methods. For example, cells (e.g., peripheral blood lymphocytes or purified lymphocyte populations isolated from a patient and placed or maintained in culture) can be cultured in vitro in a culture medium, and the contacting step can be carried out by adding an IL2 variant polypeptide and / or a polynucleotide encoding it to the culture medium. The culturing step can include a further step of stimulating the cells or treating them with other agents, for example, to stimulate proliferation or to expand the population of cells reactive to an antigen of interest (e.g., a cancer antigen or a viral antigen). The cells are then administered to the patient after treatment.

[0294] The term "disease" refers to an abnormal condition affecting an individual's body. Disease is often interpreted as a medical condition associated with specific symptoms and signs. Diseases can be caused by external factors, such as infection, or by internal dysfunction, such as autoimmune disease. In humans, "disease" is often used more broadly to refer to conditions that cause pain, impairment, distress, social problems, or death in the affected individual, or that cause similar problems in those who come into contact with the individual. In this broader sense, disease sometimes includes impairment, incapacity, disability, syndrome, infection, isolated symptoms, deviant behavior, and atypical changes in structure and function, although in other contexts and for other purposes, these may be considered distinct categories. Because suffering from and living with many illnesses can alter one's outlook on life and personality, illnesses typically affect individuals not only physically but also emotionally.

[0295] In the present context, the terms "treatment", "treating" or "therapeutic intervention" relate to the management and care of a subject with the aim of combating a condition, such as a disease or disorder. This term is intended to include the full range of treatments for a given condition from which a subject is suffering, such as the administration of therapeutically effective compounds to alleviate symptoms or complications, slow the progression of a disease, disorder or condition, relieve or reduce symptoms and complications, and / or cure or eliminate a disease, disorder or condition, as well as to prevent a condition, where prevention is to be understood as the management and care of an individual with the aim of combating a disease, condition or disorder and includes the administration of active compounds to prevent the onset of symptoms or complications.

[0296] The term "therapeutic treatment" relates to any treatment that improves the health status and / or prolongs (increases) the lifespan of an individual. The treatment may eliminate the disease in an individual, halt or delay the onset of the disease in an individual, inhibit or delay 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 who currently has or has previously had the disease.

[0297] The term "prophylactic treatment" or "preventative treatment" relates to any treatment intended to prevent a disease from occurring in an individual. The terms "prophylactic treatment" or "preventative treatment" are used interchangeably herein.

[0298] The terms "individual" and "subject" are used interchangeably herein. They refer to a human or another mammal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate) that may or may not have a disease or disorder (e.g., cancer), but that may or may not have the disease or disorder. In many embodiments, the individual is a human. Unless otherwise specified, the terms "individual" and "subject" do not denote a particular age and thus encompass adults, elderly people, children, and newborns. In embodiments of the present disclosure, an "individual" or "subject" is a "patient."

[0299] The term "patient" refers to an individual or subject for treatment, particularly an afflicted individual or subject.

[0300] In one embodiment of the present disclosure, the objective is to provide an immune response against diseased cells that express an antigen, such as cancer cells that express a tumor antigen, to treat a disease, such as a cancer disease, involving cells that express an antigen, such as a tumor antigen.

[0301] The pharmaceutical composition comprising RNA encoding the peptide or protein comprising epitope can be administered to a subject to induce the immune response against the antigen comprising said epitope in the subject, which can be therapeutic or partially or completely protective.Those skilled in the art will understand that one of the principles of immunotherapy and vaccination is based on the fact that immunizing a subject with the immunologically related antigen or epitope for the disease to be treated generates an immune protective response against the disease.Therefore, the pharmaceutical composition described herein can be applied to induce or enhance immune response.Therefore, the pharmaceutical composition described herein is useful in the preventive and / or therapeutic treatment of the disease that antigen or epitope is involved in.

[0302] As used herein, "immune response" refers to an integrated body response against an antigen or a cell expressing the antigen, and refers to a cellular immune response and / or a humoral immune response. Cellular immune responses include, but are not limited to, cellular responses directed against cells expressing an antigen and characterized by antigen presentation by class I or class II MHC molecules. Cellular responses involve T lymphocytes, which can be classified as helper T cells (also called CD4+ T cells), which play a central role by regulating the immune response, or killer cells (also called cytotoxic T cells, CD8+ T cells, or CTLs), which induce apoptosis in infected or cancer cells. In one embodiment, administering a pharmaceutical composition of the present disclosure includes 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 with a class I MHC molecule.

[0303] "Cell-mediated immunity," "cellular immunity," "cellular immune response," or similar terms are intended to include cellular responses directed against cells characterized by the expression of antigens, particularly those characterized by the presentation of antigens by class I or class II MHC. The cellular response involves cells called T cells or T lymphocytes that act as either "helpers" or "killers." Helper T cells (CD4 + T cells (also called T cells) play a central role by regulating the immune response and are responsible for the production of killer cells (cytotoxic T cells, cytolytic T cells, CD8 + T cells or CTLs) kill diseased cells, such as cancer cells, and prevent the production of further diseased cells.

[0304] The present disclosure contemplates an immune response that can be protective, defensive, preventative, and / or therapeutic. As used herein, "inducing an immune response (or inducing)" can indicate that an immune response to a particular antigen was not present before induction, or that there was a basal level of immune response to a particular antigen before induction, which was enhanced after induction. Thus, "inducing an immune response (or inducing)" includes "enhancing an immune response (or enhancing)."

[0305] 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.

[0306] The term "immunization" or "vaccination" refers to the process of administering an antigen to an individual with the intent of inducing an immune response, for example, for therapeutic or prophylactic reasons.

[0307] The peptides, proteins, polypeptides, RNA, RNA particles and additional agents, such as immune checkpoint inhibitors, described herein can be used for the therapeutic or preventive treatment of diseases in which providing a subject with a peptide or protein containing an epitope for inducing an immune response against an antigen in the subject results in a therapeutic or preventive effect.For example, providing an antigen or epitope derived from a virus can be useful for treating viral diseases caused by the virus.Providing a tumor antigen or epitope can be useful for treating cancer diseases in which cancer cells express the tumor antigen.

[0308] In one embodiment, the present disclosure contemplates an embodiment in which an RNA formulation, such as the RNA lipoplex particles described herein, is administered to target spleen tissue. The RNA encodes, for example, a peptide or protein containing an epitope as described herein. The RNA is taken up by antigen-presenting cells in the spleen, such as dendritic cells, to express the peptide or protein. Following optional processing and presentation by the antigen-presenting cells, an immune response against the epitope can be generated, resulting in preventive and / or therapeutic treatment of a disease involving the epitope or an antigen containing the epitope. In one embodiment, the immune response induced by the RNA described herein includes presentation of an antigen, such as an epitope, or a fragment thereof by antigen-presenting cells, such as dendritic cells and / or macrophages, and activation of cytotoxic T cells through this presentation. For example, the peptide or protein encoded by the RNA, or its processing product, can be presented by major histocompatibility complex (MHC) proteins expressed on antigen-presenting cells. The MHC-peptide complex can then be recognized by immune cells, such as T cells or B cells, resulting in their activation.

[0309] Thus, the present disclosure relates to the RNA described herein for use in the prophylactic and / or therapeutic treatment of antigen-associated diseases, preferably cancer diseases.

[0310] The term "macrophage" refers to a subgroup of phagocytes generated by differentiation of monocytes. Activated by inflammation, immune cytokines, or microbial products, macrophages nonspecifically engulf foreign pathogens within the macrophage and kill them through hydrolytic and oxidative attack, resulting in their degradation. Peptides from degraded proteins are displayed on the macrophage cell surface where they can be recognized by T cells and interact directly with antibodies on the surface of B cells, leading to T and B cell activation and further stimulation of the immune response. Macrophages belong to a class of antigen-presenting cells. In one embodiment, the macrophages are splenic macrophages.

[0311] The term "dendritic cell" (DC) refers to another subtype of phagocyte belonging to the class of antigen-presenting cells. In one embodiment, dendritic cells are derived from hematopoietic bone marrow progenitors. These progenitor cells initially transform into immature dendritic cells. These immature cells are characterized by high phagocytosis and low T cell activation capacity. Immature dendritic cells constantly sample their environment for pathogens, such as viruses and bacteria. Upon contact with presentable antigens, they are activated to become mature dendritic cells and begin migrating to the spleen or lymph nodes. Immature dendritic cells phagocytose pathogens, degrade their proteins into small fragments, and upon maturation, present these fragments on their cell surface using MHC molecules. At the same time, they upregulate cell surface receptors that function as coreceptors in T cell activation, such as CD80, CD86, and CD40, greatly enhancing their ability to activate T cells. They also upregulate CCR7, a chemotactic receptor that directs dendritic cells to migrate through the bloodstream to the spleen or through the lymphatic system to lymph nodes. Here, they function as antigen-presenting cells, activating helper T cells, killer T cells, and B cells by presenting antigens together with non-antigen-specific costimulatory signals. Thus, dendritic cells can actively induce T cell- or B cell-related immune responses. In one embodiment, the dendritic cells are splenic dendritic cells.

[0312] The term "antigen-presenting cell" (APC) refers to any of a variety of cells that can display, acquire, and / or present at least one antigen or antigen fragment on (or at) their cell surface. Antigen-presenting cells can be distinguished into professional and non-professional antigen-presenting cells.

[0313] The term "professional antigen-presenting cells" refers to antigen-presenting cells that constitutively express major histocompatibility complex class II (MHC class II) molecules, which are necessary for interaction with naive T cells. When T cells interact with the MHC class II molecule complex on the membrane of the antigen-presenting cell, the antigen-presenting cell produces costimulatory molecules that induce T cell activation. Professional antigen-presenting cells include dendritic cells and macrophages.

[0314] The term "non-professional antigen-presenting cells" refers to antigen-presenting cells that do not constitutively express MHC class II molecules but do so upon stimulation with certain cytokines, such as interferon gamma. Exemplary non-professional antigen-presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells, or vascular endothelial cells.

[0315] "Antigen processing" refers to the breakdown of an antigen into processing products that are fragments of the antigen (e.g., breakdown of a protein into peptides), and the association (e.g., by binding) of one or more of these fragments with an MHC molecule for presentation to specific T cells by a cell, such as an antigen-presenting cell.

[0316] The term "antigen-associated disease" or "epitope-associated disease" refers to any disease in which an antigen or epitope is associated, e.g., a disease characterized by the presence of an antigen or epitope. The antigen- or epitope-associated disease may be an infectious disease, or a cancer disease or simply cancer. As noted 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. In one embodiment, the antigen-associated disease is a disease involving cells that preferably express the antigen on their cell surface.

[0317] The term "infectious disease" refers to any disease (e.g., the common cold) that can be transmitted from individual to individual or from organism to organism and is 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 immunodeficiency syndrome (AIDS), diphtheria, hepatitis B, hepatitis C, cholera, severe acute respiratory syndrome (SARS), avian influenza, and influenza.

[0318] The term "cancer disease" or "cancer" refers to or describes a physiological condition in an individual that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specifically, examples of such cancer include bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, cancer of the genital and reproductive organs, Hodgkin's disease, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, neoplasms of the central nervous system (CNS), neuroectodermal carcinoma, spinal axis tumor, glioma, meningioma, and pituitary adenoma. The term "cancer" according to the present disclosure also includes cancer metastasis.

[0319] Combination strategies in cancer treatment can be desirable due to the resulting synergistic effects, which can be significantly more potent than the effects of monotherapy approaches. In one embodiment, the pharmaceutical composition is administered with an immunotherapeutic agent. As used herein, "immunotherapeutic agent" refers to any agent that can be involved in activating a specific immune response and / or immune effector function(s). Examples of immunotherapeutic agents include vaccines, such as T cell vaccines, immune effector cells, or combinations thereof. The present disclosure also contemplates the use of antibodies as immunotherapeutic agents. Without wishing to be bound by theory, antibodies can achieve therapeutic effects against cancer cells through various mechanisms, including inducing apoptosis, blocking components of signaling pathways, or inhibiting tumor cell proliferation. In certain embodiments, the antibody is a monoclonal antibody. Monoclonal antibodies can induce cell death via antibody-dependent cell-mediated cytotoxicity (ADCC) or can bind to complement proteins, resulting in direct cytotoxicity, known as complement-dependent cytotoxicity (CDC). Non-limiting examples of anti-cancer antibodies and potential antibody targets (in parentheses) that may be used in combination with the present disclosure include abagovomab (CA-125), abciximab (CD41), adecatumumab (EpCAM), afutuzumab (CD20), alacizumab pegol (VEGFR2), altumomab pentetate (CEA), amatuximab (MORAb-009), anatumomab mafenatox (TAG-72), apolizumab (HLA-DR), arcitumomab (CEA), atezolizumab (PD-L1), bavituximab (phosphatidylserine), bectumomab (CD22), belimumab (BAFF), bevacizumab (VEGF-A), bivatuzumab mertansine (CD44 v6), blinatumomab (CD 19), brentuximab vedotin (CD30TNFRSF8), cantuzumab mertansine (mucin CanAg), cantuzumab mertansine (MUC1), capromab pendetide (prostate cancer cells), carlumab (CNT0888), catumaxomab (EpCAM, CD3), cetuximab (EGFR), sitatuzumab bogatox (EpCAM), cixutumumab (IGF-1 receptor), claudiximab (claudin), clivatuzumab tetraxetan (MUC1), conatumumab (TRAIL-R2), dacetuzumab (CD40), dalotuzumab (insulin -like growth factor I receptor), denosumab (RANKL), detumomab (B lymphoma cells), drozitumab (DR5), ecloneximab (GD3 ganglioside), edrecolomab (EpCAM), elotuzumab (SLAMF7), enavatuzumab (PDL192), ensituximab (NPC-1C), epratuzumab (CD22), ertumaxomab (HER2 / neu, CD3), etaracizumab (integrin ανβ3), farletuzumab (folate receptor 1), FBTA05 (CD20), ficlatuzumab (SCH900105), figitumumab (IGF-1 receptor), framvotumab (glycoprotein 75), fresolimumab (TGF-β), galiximab (CD80), ganitumab (IGF-I), gemtuzumab ozogamicin (CD33), gevokizumab (ILΙβ), girentuximab (carbonic anhydrase 9 (CA-IX)), glembatumumab vedotin ( GPNMB), ibritumomab tiuxetan (CD20), icrucumab (VEGFR-1), igovoma (CA-125), indatuximab ravtansine (SDC1), intetumumab (CD51), inotuzumab ozogamicin (CD22), ipilimumab (CD152), iratumumab (CD30), labetuzumab (CEA), lexatumumab (TRAIL-R2), ribivirumab (hepatitis B surface antigen), lintuzumab (CD33), lorvotuzumab mertansine (CD56), lucatumumab (CD40), rumiliximab (CD23), mapatumumab (TRAIL-R1), matuzumab (EGFR), mepolizumab (IL5), milatuzumab (CD74), mitsumomab (GD3 ganglionic Osido), mogamulizumab (CCR4), moxetumomab pasudotox (CD22), nacolomab butafenatox (C242 antigen), naptumomab estafenatox (5T4), namatumab (RON), necitumumab (EGFR), nimotuzumab (EGFR), nivolumab (IgG4), ofatumumab (CD20), olaratumab (PDGF-Ra), onartuzumab (human scatter factor receptor kinase), oportuzumab monatox (EpCAM), oregovomab (CA-125), oxelumab (OX-40), panitumumab (EGFR), patritumab (HER3), pemtumomab (MUC1), pertuzumab (HER2 / neu), pintumomab (adenocarcinoma antigen), pritumumab (vimentin), racotumomab (N-glycolylneuraminic acid), radletumab (fibronectin extra domain B), rafivirumab (rabies virus glycoprotein), ramucirumab (VEGFR2), rilotumumab (HGF), rituximab (CD20), lobatumumab (IGF-1 receptor) body), samaryzumab (CD200), sibrotuzumab (FAP), siltuximab (IL6), tabalumab (BAFF), tacatuzumab tetraxetan (alpha-fetoprotein), taplitumomab paptox (CD19), tenatumomab (tenascin-C), teprotumumab (CD221), ticilimumab (CTLA-4), tigatuzumab (TRAIL-R2), TNX-650 (IL13), tositumomab (CD20), trastuzumab (HER2 / neu), TRBS07 (GD2), tremelimumab (CTLA-4), tucotuzumab celmoleukin (EpCAM), ublituximab (MS4A1), urelumab (4-1 BB), boroximab (integrin α5β1), votumumab (tumor antigen CTAA 16.88), zalutumumab (EGFR), and zanolimumab (CD4).

[0320] Citation of documents and works referenced herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements regarding the contents of these documents are based on information available to applicant and do not constitute an admission as to the accuracy of the contents of these documents.

[0321] The following description is presented to enable those skilled in the art to make and use various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications to the examples described herein will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Accordingly, the various embodiments are not intended to be limited to the examples described and shown herein, but are to be accorded the scope consistent with the appended claims. [Example]

[0322] Example 1: Construct design and mRNA production To design a variant of human IL2 (hIL2) that combines reduced binding and activation to hIL2Rα (CD25) with increased binding and activation to hIL2Rβ (CD122), various mutations were introduced into the hIL2Rβ binding site of hIL2 mutein A4, which has been described to have reduced CD25 binding. In addition to the amino acid substitutions K43E and E61K, which are hallmark mutations of hIL2 mutein A4, the following additional mutations were introduced into the mature domain of hIL2: -hIL2_A4s:L80F, R81D, L85V, I86V, I92F -hIL2_A4s1:L80F, R81E, L85V, I86V, I92F -hIL2_A4s2:L80F, R81E, L85V, I86V, I92W -hIL2_A4s3:L80F, R81E, I92F -hIL2_A4s4:L80F, R81D, I92F -hIL2_A4s6:Q74H, L80F, R81E, I92F -hIL2_A4s7:Q74H, L80F, R81E, I92W -hIL2_A4s8:Q74H, L80F, R81E, L85V, I92F.

[0323] The cytokine-encoding mRNAs for in vitro transcription were based on the pST1-T7-AGA-dEarI-hAg-MCS-FI-A30LA70 plasmid backbone and derivative DNA constructs. These plasmid constructs contained the 5' UTR (untranslated region, a derivative of the 5'-UTR of Homo sapiens hemoglobin subunit α1 (hAg)), the 3' FI element (F is a 136-nucleotide 3'-UTR fragment of the amino-terminal enhancer of split mRNA, and I is a 142-nucleotide fragment of mitochondrially encoded 12S RNA, both identified in Homo sapiens; WO 2017 / 060314), and a 100-nucleotide poly(A) tail followed by a 70-nucleotide linker. The cytokine and serum albumin (hAlb) coding sequences were derived from Homo sapiens, and no changes were introduced into the resulting amino acid sequences, except for the intended mutations in the hIL2 variants described above (hIL2:NP_000577.2; NCBI Protein Resource; https: / / www.ncbi.nlm.nih.gov / protein / ). For the cytokine constructs, the hIL2 variants were added to the C-terminus of hAlb, and the encoded proteins contained an N-terminal signal peptide (SP) that was the native signal peptide of the respective proteins. For the fusion proteins, only the SP in the N-terminal portion was maintained, and for the further portions, only the mature portion (protein without the SP) was encoded. A stop codon was introduced only for the most C-terminal portion. The different protein portions of the cytokine and hAlb fusion constructs were separated by a 30-nucleotide-long linker sequence encoding glycine and serine residues.

[0324] mRNA was generated by in vitro transcription as described by Kreiter et al. (Kreiter, S. et al. Cancer Immunol. Immunother. 56, 1577-87 (2007)), substituting the normal nucleoside uridine with 1-methylpseudouridine. The resulting mRNA contained a cap structure and was depleted of double-stranded (dsRNA) molecules. Purified mRNA was eluted with HO 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 in subsequent experiments is shown in Table 1. [Table 1] TIFF2026027331000002.tif208153TIFF2026027331000003.tif204153TIFF2026027331000004.tif203153TIFF2026027331000005.tif205153

[0325] Example 2: Functional activity of novel hAlb-hIL2_A4 variants with mutations in the hIL2Rβ (CD122) binding region compared to parental hAlb-hIL2_A4 on different cell subsets of human PBMCs and mouse splenocytes as measured by IL2-mediated phosphorylation of STAT5. To evaluate the functional activity of novel hAlb-hIL2_A4 variants that were additionally mutated in the hIL2Rβ (CD122) binding region compared with hAlb-hIL2_A4 mutated only in the hIL2Rα (CD25) binding region, we analyzed the different CD25 binding regions of bulk human PBMCs (Figure 1) or mouse splenocytes (Figure 2). + and CD25 - T cell subsets and NK cells were stimulated with cytokine-containing supernatants and assayed for STAT5 phosphorylation. To generate cytokine-containing supernatants, 1.2 × 10 6HEK293T / 17 cells were seeded in 3 mL DMEM (Life Technologies GmbH, Cat. No. 31966-021) + 10% fetal bovine serum (FBS, Biochrom GmbH, Cat. No. S0115) in 6-well plates and incubated overnight at 37 °C, 5% CO. The next day, 3 μg of cytokine-encoding mRNA was formulated under sterile, RNase-free conditions using 400 ng of mRNA per μL of Lipofectamine MessengerMax (Thermo Fisher Scientific, Cat. No. LMRNA015) and transfected into 10 cm wells. 2The IgG was applied to HEK293T / 17 cells at approximately 80% confluence per culture dish. After 20 hours of expression, the supernatant was collected under sterile conditions and stored at -20°C until further use. PBMCs were obtained from the buffy coat of healthy donors by Ficoll-Paque (1.073 g / mL, VWR International, catalog no. 17-1440-03) density gradient separation. PBMCs were washed twice with D-PBS (Life Technologies GmbH, catalog no. 14190250) and collected by centrifugation at 300 × g for 8 minutes at room temperature. To isolate mouse splenocytes, one Balb / c mouse (Balb / c JRj, Janvier Labs) was euthanized by cervical dislocation, and the spleen was removed from the abdominal cavity. Spleens were mechanically dissociated using a 70 μm cell strainer, and the resulting cell suspension was washed once with D-PBS before cell separation by Ficoll-Paque (1.084 g / mL, VWR International, catalog no. 17-5446-02) density gradient centrifugation. Isolated splenocytes were washed twice with D-PBS and collected by centrifugation at 300 × g for 8 minutes at room temperature. For STAT5 phosphorylation assays, PBMCs were resuspended in Iscove's modified Dulbecco's medium (IMDM; Life Technologies GmbH, catalog no. 12440-053) supplemented with 5% plasma-derived human serum (PHS; One Lambda Inc., catalog no. A25761), and splenocytes were resuspended in RPMI 1640 (Life Technologies GmbH, catalog no. 61870010) supplemented with 10% FBS. Both were incubated at 37°C and 5% CO2 for 1 hour. Next, 125,000 PBMCs or splenocytes were seeded per well of a 96-well V-bottom plate (Greiner Bio-One GmbH, catalog no. 651101) in the respective medium. In parallel, six 4-fold serial dilutions of cytokine-containing supernatants were generated in IMDM supplemented with 5% PHS or RPMI 1640 supplemented with 10% FBS. The seeded cells were mixed 1:1 with cytokine-containing supernatants (see medium volume of seeded cells) and stimulated for 10 min at 37°C and 5% CO2.Next, 1:1000 of the fixable viability dye eFluor™ 780 was added, and the cells were stimulated for an additional 5 minutes at 37°C and 5% CO2. Formaldehyde (Carl Roth GmbH+Co.KG, Cat. No. P087.4) was added to 2% to fix the cells and incubated on ice for 10 minutes. Fixed cells were washed with ice-cold D-PBS and permeabilized with 100% ice-cold methanol on ice for 30 minutes. Permeabilized PBMCs were washed twice with D-PBS supplemented with 2% FBS and 2 mM EDTA (Sigma-Aldrich, Cat. No. 03690-100ML) and subsequently incubated with 1:5 Alexa Fluor® 488 anti-Stat5 (pY694) (Becton Dickinson GmbH, Cat. No. 612598), 1:25 PerCP-Cy™ 5.5 mouse anti-human CD25 (Becton Dickinson GmbH, Cat. No. 560503), 1:50 BV421 mouse anti-human CD4 (Becton Dickinson GmbH, Cat. No. 565997), 1:25 BV510 mouse anti-human CD8 (Becton Dickinson GmbH, Cat. No. 563256), and 1:12.5 APC mouse anti-human CD56 (Becton Dickinson GmbH, Cat. No. 555518) in 2% FBS and 2 mM EDTA. Staining was carried out in D-PBS containing EDTA for 30 minutes at 2 to 8°C in the dark.Permeabilized splenocytes were washed twice with D-PBS supplemented with 2% FBS and 2 mM EDTA (Sigma-Aldrich, Cat. No. 03690-100ML) and subsequently incubated in D-PBS supplemented with 2% FBS and 2 mM EDTA for 30 min at 2–8°C protected from light with 1:5 Alexa Fluor® 488 anti-Stat5 (pY694) (Becton Dickinson GmbH, Cat. No. 612598), 1:25 PerCP-Cy™ 5.5 rat anti-mouse CD25 (Becton Dickinson GmbH, Cat. No. 551071), 1:50 BV786 rat anti-mouse CD4 (Becton Dickinson GmbH, Cat. No. 563727), 1:25 BV605 rat anti-mouse CD8 (Becton Dickinson GmbH, Cat. No. 563727), and 1:25 BV605 rat anti-mouse CD8 (Becton Dickinson GmbH, Cat. No. 563727). The PBMCs and splenocytes were stained with 1:25 BV421 rat anti-mouse CD49b (Becton Dickinson GmbH, Cat. No. 563152) and 1:25 BV421 rat anti-mouse CD49b (Becton Dickinson GmbH, Cat. No. 563063). Stained PBMCs and splenocytes were washed twice and finally resuspended in D-PBS supplemented with 2% FBS and 2 mM EDTA. Flow cytometry analysis was performed on a BD FACSCanto™ II or FACSCelesta flow cytometer (Becton Dickinson GmbH), and acquired data were analyzed using FlowJo software version 10. Dose-response curves and EC2 were analyzed. 50 Values ​​were calculated with GraphPad Prism version 6.04 (GraphPad Software, Inc.).

[0326] In all PBMC subsets analyzed, all novel hAlb-hIL2_A4 variants designed for increased CD122 binding, except for hAlb-hIL2_A4s7, showed superior potency to hAlb-hIL2_A4, regardless of CD25 expression. Specifically, the biological activity of hAlb-hIL2_A4s, hAlb-hIL2_A4s1, and hAlb-hIL2_A4s8 was significantly greater than that of hAlb-hIL2_A4(T reg EC 50 1.247% supernatant, CD8 + T cells and ECs 50>20% supernatant) compared with at least one log phase (CD4 + CD25 + T reg , E.C. 50 Approximately 0.1% of the supernatant) or up to 2 logarithmic steps (CD8 + T cells, EC 50 hAlb-hIL2_A4s, hAlb-hIL2_A4s1, and hAlb-hIL2_A4s8 significantly increased CD4 + CD25 + T reg It functions equally well on CD8 cells. + T cells and CD56 + They were similarly effective in NK cells. In addition to hAlb-hIL2_A4s7, hAlb-hIL2_A4s2 showed the least increase in biological activity, while the other variants, hAlb-hIL2_A4s3, hAlb-hIL2_A4s4, and hAlb-hIL2_A4s6, displayed intermediate phenotypes (Figure 1). In mouse splenocytes, the performance of the various hAlb-hIL2_A4 variants was comparable to each other, but mouse splenocytes showed an approximately 30-fold decrease in overall sensitivity to hAlb-hIL2_A4 variant-induced STAT5 phosphorylation (Figure 2). [Table 2]

[0327] Example 3: Relative biological activity of novel hAlb-hIL2_A4 variants with mutations in the hIL2Rβ (CD122) binding region on IL-2-dependent reporter cell lines expressing the intermediate-affinity IL2 receptor (IL2Rβγ) and the high-affinity IL2 receptor (IL2Rαβγ). To analyze whether the hIL2Rα chain (CD25) specifically influences the biological activity of the novel hAlb-hIL2_A4 variants, we determined the proliferative responses of TF-1_hIL2Rβγ and TF-1_hIL2Rαβγ cells derived from the human erythroleukemia cell line TF-1 (ATCC CRL-2003), which naturally expresses the IL2R common γ chain (Figure 3). The cell lines were generated by transduction with a retroviral vector (Gene ID: 3560) encoding the sequence of the human IL2Rβ chain and, optionally, a second retroviral vector (Gene ID: 3559) encoding the sequence of the human hIL2Rα chain similar to that described by Farner, N. Lett. et al., Blood 86, 4568-4578 (1995). Briefly, TF-1_IL2Rβγ and TF-1_IL2Rαβγ cells were harvested from continuous culture, washed twice with D-PBS, and resuspended in RPMI 1640 supplemented with 10% FBS and 1 mM sodium pyruvate (Life Technologies GmbH, catalog no. 11360070). A total of 5,000 cells / well were seeded into white 96-well flat-bottom plates (Fisher Scientific GmbH, catalog no. 10072151) and incubated with eight 4-fold serial dilutions of hAlb-hIL2_A4 variant-containing supernatants (generated as described in Example 2). After 3 days of culture, proliferation was measured by quantifying viable cells via ATP content using the CellTiter-Glo® 2.0 assay (Promega, catalog no. G9242). Luminescence was recorded with a Tecan Infinite® F200 PRO reader (Tecan Deutschland GmbH), and dose-response curves were plotted with GraphPad Prism version 6.04 (GraphPad Software, Inc.) to obtain EC 50 values ​​were calculated.

[0328] In TF-1_hIL2Rβγ cells expressing CD25-independent IL2Rβγ, hAlb-hIL2_A4s, hAlb-hIL2_A4s1, and hAlb-hIL2_A4s8 performed comparably, with dose-response curves that were nearly superimposable (Figure 3A), as indicated by calculated EC values ​​ranging from 0.712% supernatant for hAlb-hIL2_A4s to 0.755% supernatant for hAlb-hIL2_A4s1 and 0.807% supernatant for hAlb-hIL2_A4s8. 50 This is also reflected in the EC values ​​(Table 3). Variants hAlb-hIL2_A4s2 and hAlb-hIL2_A4s7 showed a more than 10-fold shift toward decreased biological activity, as expected from the results of Example 2. The remaining variants, hAlb-hIL2_A4s3, hAlb-hIL2_A4s4, and hAlb-hIL2_A4s6, showed a EC 50 The EC values ​​ranged from 1.270% to 2.557% supernatant, indicating an intermediate phenotype (Figure 3A, Table 3). In contrast, in TF-1_hIL2Rαβγ cells expressing CD25-dependent IL2Rαβγ, the dose-response curves of all hAlb-hIL2 variants were slightly shifted toward increased biological activity, and the differences between individual variants were less pronounced (Figure 3B). hAlb-hIL2_A4s1 and hAlb-hIL2_A4s8 exhibited EC 50 The best performing variants were still identified with EC values ​​of 0.216% and 0.236% supernatant, respectively, while all other variants except hAlb-hIL2_A4s7 (EC50 2.689% supernatant) showed similar EC values ​​ranging from 0.314% to 0.566% supernatant. 50 The values ​​are shown (Fig. 3B, Table 3). [Table 3]

[0329] Among the hAlb-hIL2_A4s variants tested in Examples 2 and 3, hAlb-hIL2_A4s8 was selected for further characterization due to the overall encouraging results shown in Figures 1-3.

[0330] Example 4 Binding of hAlb-hIL2, hAlb-hIL2_A4, and hAlb-hIL2_A4s8 to recombinant hIL2Rα (CD25) and hIL2Rβ (CD122) The binding abilities of mRNA-encoded wild-type hAlb-hIL2, hAlb-hIL2_A4 (designed for reduced CD25 binding), and hAlb-hIL2_A4s8 (designed for reduced CD25 binding and increased CD122 binding) to hIL2Rα (CD25) and hIL2Rβ (CD122) were analyzed by ELISA (Figure 4). Here, 1 μg / mL of recombinant human CD25 (C-Fc, Novoprotein catalog number CJ78) or human CD122 (C-Fc, Novoprotein catalog number CJ82) was coated overnight onto a high-protein-binding 96-well plate (Nunc MaxiSorp™, Thermo Fisher Scientific, catalog number 439454) in 100 μL D-PBS. hAlb-hIL2 variant-containing supernatants produced as described in Example 2 were applied to coated CD25 or CD122, and bound proteins were detected with an HRP-conjugated anti-human serum albumin antibody (Abcam, catalog no. ab8941). General ELISA reagents and procedures were applied according to the protocol of DuoSet ELISA Ancillary Reagent Kit 2 (R&D Systems, catalog no. DY008).

[0331] Binding to hIL2Rα (CD25) was detected only with wild-type hAlb-hIL2, whereas two variants, hAlb-hIL2_A4 and hAlb-hIL2_A4s8, carrying K43E and E61K mutations for reduced hIL2Rα binding, showed no binding (Figure 4A). In contrast, hIL2Rβ (CD122) was bound only by hAlb-hIL2_A4s8 with mutations (Q74H, L80F, R81E, L85V, I92F) for increased hIL2Rβ binding, but not by hAlb-hIL2 or hAlb-hIL2_A4 (Figure 4B). Together, both results clearly confirm the functionality of the mutations introduced into hAlb-hIL2_A4s8 to reduce binding to hIL2Rα and increase binding to hIL2Rβ.

[0332] Example 5: Functional activity and "regulatory T cell bias" of hAlb-hIL2_A4s8 compared to hAlb-hIL2 on different cell subsets of human and mouse PBMCs as measured by IL2-mediated phosphorylation of STAT5. The functional activity of hAlb-hIL2_A4s8 compared to wild-type hAlb-hIL2 was measured using CD4 + CD25 + T reg cells, CD25 negative CD8 + T cells and CD56 + NK cells (Fig. 5) and CD4 in mouse PBMCs + CD25 + T reg cells and CD25-negative CD8 + The IL2-mediated phosphorylation of STAT5 induced by hAlb-hIL2 variant-containing supernatants was analyzed in human PBMCs as described in Example 2. The resulting dose-response curves were analyzed using GraphPad Prism version 6.04 (GraphPad Software, Inc.) to calculate EC 50 The values ​​were fitted using a four-parameter logarithmic fit to calculate EC 50 To estimate the value, CD8 + T cells and CD56+ The dose-response curve of hAlb-hIL2 on NK cells was interpolated for supernatant concentrations above 12.5% ​​(Table 4). + T cells or CD56 + EC determined for hAlb-hIL2 or hAlb-hIL2_A4s8 on NK cells 50 value and CD4 + CD25 + T reg Cell-determined EC 50 The ratio of these values ​​was calculated to determine the "regulatory T cell bias" of hAlb-hIL2 and hAlb-hIL2_A4s8 (Table 5). To isolate mouse PBMCs, two to three Balb / c mice (Balb / c JRj, Janvier Labs) were euthanized by cervical dislocation and terminally bled. Whole blood was collected into Li-heparin tubes (Microvette®, VWR International, catalog number SARS20.1309). 2.5 mL of blood was mixed 1:1 with D-PBS, and PBMCs were isolated by Ficoll-Paque (1.084, catalog number 17-5446-02) density gradient centrifugation. The isolated PBMCs were washed twice with D-PBS and collected by centrifugation at 300 × g for 8 minutes at room temperature. IL2-mediated phosphorylation of STAT5 in mouse PBMCs treated with hAlb-hIL2 variant-containing supernatants was analyzed as described for mouse splenocytes in Example 2.

[0333] Consistent with the ELISA data presented in Example 4, hAlb-hIL2 binds to human CD4 + CD25 + T reg showed excellent efficacy in CD25-negative CD8 + T cells and CD56 + hAlb-hIL2_A4s8 showed very limited activity on NK cells (Fig. 5B-C). In contrast, hAlb-hIL2_A4s8 showed significantly higher activity on CD4 + CD25 + T reg Although it showed 10-fold lower biological activity in CD25-negative immune cells (CD8 + T cells and CD56+ NK cells (Figure 5B-C) exceeded hAlb-hIL2. + CD25 + T reg ECs of CD25-negative cells and subsets of CD25-negative cells 50 Comparison of values ​​was used to calculate the "regulatory T cell bias" for both hAlb-hIL2 and hAlb-hIL2_A4s8 (Tables 4 and 5). hAlb-hIL2 was associated with a CD8 + Cytotoxic T cells and CD56 + Compared to the biological effects of NK cells, CD4 + CD25 + T reg shows a calculated bias of over 400-fold against hAlb-hIL2_A4s8. In contrast, the regulatory T cell bias is significantly reduced to only 1.3-3.1-fold for hAlb-hIL2_A4s8, depending on the respective comparator immune cell subset. Consequently, hAlb-hIL2_A4s8, engineered with point mutations (K43E, E61K) that decrease hIL2Rα binding while increasing hIL2Rβ binding (Q74H, L80F, R81E, L85V, I92F), already targets effector immune cells (e.g., CD8 + T cells) and CD4 + CD25 + This confirmed that hAlb-hIL2 is a hIL2Rβ-biased variant of hAlb-hIL2, which exhibits a significantly reduced ability to activate Treg cells. In mouse PBMCs, the performance of hAlb-hIL2_A4s8 and hAlb-hIL2 was comparable. hAlb-hIL2 inhibited CD4 + CD25 + T reg hAlb-hIL2_A4s8 was significantly superior to mouse CD8 + It was superior to hAlb-hIL2 in T cells (Fig. 6A-B). [Table 4] [Table 5]

[0334] Example 6: The IL2 variant hAlb-hIL2_A4s8 with combined mutations in the hILRα and hILRβ binding regions is superior to hAlb-hIL2 in enhancing anti-tumor immunity in combination with a therapeutic RNA vaccine in the murine colon cancer model CT26. We then characterized the potency of the selected IL2 variant, hAlb-hIL2_A4s8, to improve the therapeutic antitumor efficacy of RNA vaccines in vivo. BALB / c mice (n=11 per group) were inoculated with 5 × 10 5 CT26 tumor cells were inoculated subcutaneously (sc) and CD8 + Mice were vaccinated intravenously (iv) four times (days 10, 17, 24, and 31) with 20 μg of RNA-LPX encoding the T cell antigen gp70 (SPSYAYHQF). gp70 is a tumor antigen found in the colon cancer cell line CT26. The antitumor effect of gp70-targeted vaccines increases with the number of gp70-specific T cells induced (Kranz, LM et al. Nature 534, 396-401 (2016) and unpublished data). Concurrently with the RNA vaccine, RNA encoding hAlb-hIL2_A4s8 or hAlb-hIL2 (3 μg each) formulated as lipid nanoparticles (LNPs) was administered intravenously. A control group received the RNA vaccine plus hAlb RNA (not encoding a cytokine) formulated as LNPs. Blood lymphocyte subsets and gp70-specific T cell responses were determined by flow cytometry (BD FACSCelesta) (staining as described in Kranz, LM et al. Nature 534, 396-401 (2016)) 7 days after the first three treatments (days 17, 24, and 31). Antitumor efficacy was determined as tumor growth inhibition in the test group compared with the control group, and overall survival during the observation period up to day 104 after tumor inoculation.

[0335] Combined treatment with the vaccine and hAlb-hIL2 and hAlb-hIL2_A4s8 resulted in a significant reduction in tumor growth and an extension of survival compared with the control group (Figure 7A, B). In the hAlb-hIL2-treated group, 7 of 11 (64%) animals showed a complete response. The most robust effect was observed in the hAlb-hIL2_A4s8-treated group, which resulted in a complete response in 11 of 11 (100%) animals. In comparison, all mice in the control group had to be sacrificed by day 39.

[0336] Tumor antigen-specific T cells and NK cells play important roles in tumor control, but T reg gp70 tumor antigen-specific CD8 cells are known to inhibit anti-tumor immunity. + Increased T cell numbers were observed in the groups treated with hAlb-hIL2 and hAlb-hIL2_A4s8 across all measurement days (Figure 8A). Consistent with the IL2R binding profile, only hAlb-hIL2_A4s8 resulted in a strong proliferation of NK cells 7 days after the first treatment (day 17). Once activated, NK cells quickly disappeared from the blood, explaining the significant decrease in NK cell numbers on day 24 (Figure 8B). Both hAlb-hIL2 and hAlb-hIL2_A4s8 stimulated the proliferation of CD8 T cells, which are not specific for gp70 (antigen non-specific). + hAlb-hIL2_A4s8 induced the most potent cell expansion, resulting in a significant increase in CD4 T cells (Figure 9A). + T cells were not increased by treatment with hAlb-hIL2 or hAlb-hIL2_A4s8. Only hAlb-hIL2 increased T reg hAlb-hIL2_A4s8 led to the expansion of effector T cells and T reg The ratio of hAlb-hIL2 and hAlb-hIL2_A4s8 cells significantly influences the efficacy of T cell-based immunotherapy. reg Furthermore, treatment with hAlb-hIL2_A4s8 improved the ratio of gp70-specific T cells to non-gp70-specific CD8 + T cells and T regincreased the proportion of cells (Fig. 9B).

[0337] In summary, in combination with antigen-specific vaccines, both hAlb-hIL2 and the variant hAlb-hIL2_A4s8 conferred tumor control, with hAlb-hIL2_A4s8 being the most effective. hAlb-hIL2 primarily stimulated antigen-specific T cells and T reg hAlb-hIL2_A4s8 activated NK cells and increased T reg Antigen-specific and antigen-nonspecific CD8 without cell expansion + Increases T cell numbers.

[0338] Example 7: IL2 variant hAlb-hIL2_A4s8 improves anti-tumor immunity as monotherapy in the murine colon cancer model CT26. Furthermore, we investigated whether hAlb-hIL2_A4s8 was effective as a monotherapy. BALB / c mice (n = 11 per group) were injected with 5 × 10 5 Mice were inoculated subcutaneously (sc) with CT26 tumor cells and treated intravenously (iv) four times (days 10, 17, 24, and 31) weekly with RNA encoding hAlb-hIL2_A4s8 formulated as LNPs, with or without concurrent iv vaccination with 20 μg of gp70 RNA-LPX, as described in Example 6. Control groups received hAlb RNA (not encoding a cytokine) formulated as LNPs and an RNA vaccine (irrelevant vaccine) encoding no antigen, or gp70 vaccine alone. Blood lymphocyte subsets and gp70-specific T cell responses were determined by flow cytometry 7 days (days 17, 24, and 31) after the first three treatments, as described in Example 6. Antitumor efficacy was determined as tumor growth inhibition in the test group compared with the control group, and overall survival during the observation period up to day 100 after tumor inoculation.

[0339] Treatment with hAlb-hIL2_A4s8 resulted in a significant reduction in tumor growth and prolonged survival compared with controls vaccinated with either an irrelevant vaccine or the hAlb or gp70 vaccine alone (Figure 10A, B). In the group treated with hAlb and the irrelevant vaccine, 1 of 11 animals (9%) rejected the tumor and survived until day 100. In the group treated with the gp70 vaccine, 2 of 11 animals (18%) survived during this period. In the group receiving hAlb-hIL2_A4s8 as monotherapy, tumor growth inhibition and survival improved in 5 of 11 mice (45%). Combining hAlb-hIL2_A4s8 and gp70 vaccination further increased the rate of complete responses, with 10 of 11 animals (91%) rejecting tumors and surviving.

[0340] Similar to previous studies (Example 6), hAlb-hIL2_A4s8 treatment in combination with gp70 vaccine significantly increased antigen-specific CD8 + hAlb-hIL2_A4s8 alone resulted in a dramatic increase in the number of T cells and NK cells (Fig. 11A, B). + There was a significant, but modest, increase in the number of T cells (Fig. 11A, Fig. 12A). hAlb-hIL2_A4s8 alone also expanded NK cells comparable to the combination. Again, NK cells were rapidly lost from the blood after the initial expansion. reg As previously shown, hAlb-hIL2_A4s8 treatment did not expand the cells, and they further decreased with continued treatment (Fig. 11C). Similar to the combination with gp70 vaccine, hAlb-hIL2_A4s8 suppressed CD4 + without changing the number of T cells + Increased the number of T cells (Fig. 12A). reg Stabilizing the number of antigen-specific and total CD8 cells + Expanding T cells increases T reg Antigen-specific CD8 cells + The proportion of T cells, and T reg CD8 on cells +hAlb-hIL2_A4s8 monotherapy resulted in a highly significant increase in the proportion of CD8 T cells regardless of antigen specificity (Fig. 12B). + T cells, but the combination with gp70 vaccine increased non-gp70-specific CD8 + gp70-specific CD8 T cells + The gp70 vaccination alone did not induce any measurable changes in the cell subsets analyzed, demonstrating a synergistic effect in selectively expanding T cells (Fig. 12C).

[0341] In summary, hAlb-hIL2_A4s8 treatment significantly improved T reg without a measurable increase in CD8 + It has been shown to lead to a strong increase in T and NK cells, thereby conferring antitumor effects as monotherapy or in combination with tumor antigen-specific T cell vaccines.

[0342] Example 8: The IL2 variant hAlb-hIL2_A4s8 is superior to hAlb-hIL2 in enhancing anti-tumor immunity in combination with a therapeutic RNA vaccine in the murine melanoma model B16. To confirm the efficacy of hAlb-hIL2_A4s8 in a low-immunogenic mouse tumor model, C57BL / 6 mice (n=15 per group) were treated with 3 × 10 5Mice were inoculated subcutaneously (sc) with B16-F10 melanoma cells and treated intravenously (iv) with RNA encoding hAlb-hIL2_A4s8 or hAlb-hIL2 formulated as LNPs, with or without simultaneous iv vaccination with 20 μg of TRP1 RNA-LPX, for five weekly doses (days 8, 15, 22, 29, and 36). TRP1 is a murine melanosomal antigen, tyrosine-linked protein 1, constitutively expressed in B16-F10 melanoma cells and normal melanocytes. The antitumor efficacy of this TRP1 vaccine was demonstrated by Kranz et al. (Kranz, LM et al. Nature 534, 396-401 (2016)). Control groups received hAlb RNA (no cytokine encoding) formulated as LNPs plus an RNA vaccine (irrelevant vaccine) encoding no antigen, or the TRP1 vaccine plus hAlb. Blood lymphocyte subsets and TRP1-specific T cell responses were determined 7 days after the first three treatments (days 15, 22, and 29) by flow cytometry as described in Example 6. Antitumor efficacy was determined as tumor growth inhibition in the test group compared with the control group, and overall survival during the observation period up to day 75 after tumor inoculation.

[0343] Compared with the control group treated with hAlb and an unrelated vaccine, TRP1 vaccination alone showed little therapeutic effect and showed a modest trend toward reduced tumor growth and increased median survival (Figure 13A, B). Treatment with both hAlb-hIL2 and Alb-hIL2_A4s8 led to significant tumor growth reduction, with both treatments resulting in complete tumor rejection in 5 of 15 (33%) animals. While TRP1 vaccination did not improve the survival rate of hAlb-hIL2-treated animals, hAlb-hIL2_A4s8 treatment benefited from simultaneous TRP1 vaccination, resulting in further tumor growth reduction and significantly enhanced survival compared to the individual treatments. Among animals treated with the combination of hAlb-hIL2_A4s8 and TRP1 vaccines, 6 of 15 (40%) rejected tumors, and 50% survived the observation period.

[0344] Treatment with hAlb-hIL2 or hAlb-hIL2-A4s8 alone did not significantly increase TRP1-specific CD8 + Although TRP1 vaccination did not induce T cells, it did induce weak TRP1-specific CD8 + As observed in the colon cancer model (Examples 6 and 7), the combination of hAlb-hIL2_A4s8 and TRP1 vaccine resulted in TRP1-specific CD8 T cell responses over time (Figure 14A). + Strongly increased T cell numbers. TRP1-specific CD8 + Despite the high initial number of T cells, the combination of hAlb-hIL2 and TRP1 vaccine did not increase TRP1-specific CD8 + It took longer for T cells to reach similar levels over time as hAlb-hIL2_A4s8. Again, hAlb-hIL2_A4s8 expanded NK cells after the first treatment, which then declined to baseline levels (Figure 14B). Either alone or in combination with the TRP1 vaccine, hAlb-hIL2_A4s8 significantly increased T reg hAlb-hIL2 did not expand T cells after the first treatment. reg hAlb-hIL2 increased the number of T cells (Fig. 14C). reg The initial expansion of cells is more T reg Based on a sensitive and susceptible mouse model, the TRP1-specific CD8 + This may explain the delayed T cell expansion. Confirming previous experiments, both hAlb-hIL2 and hAlb-hIL2_A4s8, alone or in combination, stimulated CD8 + hAlb-hIL2_A4s8 significantly increased the total number of T cells, while only hAlb-hIL2 significantly increased the total number of CD4 + As previously observed, these CD8 + Expansion of T cell subsets was observed in response to treatment with hAlb-hIL2_A4s8 alone or in combination with the TRP1 vaccine. reg Selective total CD8 cells + Treatment with either hAlb-hIL2 alone, hAlb-hIL2_A4s8 alone, or TRP1 vaccine alone resulted in a Treg Antigen-specific CD8 cells + The combination had no or little effect on the proportion of T cells, but did not significantly affect this proportion of antigen-specific CD8 + The ratio of hAlb-hIL2 to hAlb-hIL2_A4s8 was further increased with sequential treatment (Fig. 15B). Similar to the observations in the colon cancer model (Example 7, Fig. 12C), hAlb-hIL2_A4s8 monotherapy significantly increased the CD8 T cell ratio regardless of antigen specificity. + T cells, but the combination with the TRP1 vaccine increased TRP1-nonspecific CD8 + TRP1-specific CD8 over T cells + The combination of hAlb-hIL2 and TRP1 vaccines demonstrated a synergistic effect in selectively expanding T cells (Fig. 15C).

[0345] In summary, hAlb-hIL2_A4s8 is effective as monotherapy even in low-immunogenic tumor models and, in contrast to hAlb-hIL2, inhibits T reg It synergizes with T cell vaccination by expanding vaccine-induced T cell responses while avoiding cell stimulation and expansion.

[0346] Example 9: IL2 variant hAlb-hIL2_A4s8 enhances anti-tumor immunity alone and in combination with PD-L1 blockade in the murine colon cancer model MC38. Because multiple studies have shown that IL2 or modified IL2 variants synergize with PD-1 / PD-L1 blocking antibodies (Moynihan, KD et al. Nat. Med. 22(12):1402-10(2016)), Castro, DT et al. SITC Abstract #P557(2018)), we anticipated that the efficacy of hAlb-hIL2_A4s8 could be further enhanced by combining it with immune checkpoint blockade. C57BL / 6 mice (n=14 per group) were administered 7.5 × 10 5MC38 colon cancer cells were inoculated subcutaneously (sc) and treated intravenously (iv) with RNA encoding hAlb-hIL2_A4s8 formulated as LNPs four times weekly (days 17, 24, 32, and 39) with or without concurrent intraperitoneal (ip) treatment with anti-PD-L1 antibody (200 μg for the first injection, 100 μg for all subsequent injections). Control groups received hAlb RNA (no cytokine encoding) formulated as LNPs with an isotype control antibody, and hAlb with anti-PD-L1. Blood lymphocyte subsets and Adpgk-specific T cell responses were determined 7 days after the second treatment (day 31) by flow cytometry as described in Example 6. Adpgk is a neoantigen specifically expressed by MC38 tumor cells but not by normal cells (Yadav, M et al., Nature 515(7528):572-6(2014)). Antitumor efficacy was determined as tumor growth inhibition in the test group compared with the control group, and overall survival during the observation period up to 74 days after tumor inoculation.

[0347] Compared with the control group treated with hAlb and an isotype control antibody, treatment with hAlb-hIL2_A4s8 and anti-PD-L1 alone showed a trend toward decreased tumor growth and increased survival (Figure 16A, B). The combination of hAlb-hIL2_A4s8 and anti-PD-L1 strongly synergized and improved the anti-tumor effect of anti-PD-L1. This combination led to tumor rejection in 9 of 14 animals (64%). None of the tumors in the control group were rejected.

[0348] Treatment with hAlb-hIL2_A4s8 alone inhibited CD8 + The number of T cells increased, which was significantly higher than the control group when combined with anti-PD-L1 antibody treatment (Figure 17A). Similarly, antigen-specific CD8 T cells specific for the tumor-specific neoantigen Adpgk were also increased. + T cells were expanded above background levels by hAlb-hIL2_A4s8 alone and were significantly enhanced when combined with anti-PD-L1 antibodies. + It does not affect the number of T cells,reg Cell numbers similarly remained unaffected by treatment with hAlb-hIL2_A4s8 (Figure 17A). reg Total CD8 on cells + T cells and antigen-specific CD8 + Proportions of T cells benefited from treatment with hAlb-hIL2_A4s8, with anti-PD-L1 antibodies tending to be most beneficial (Figure 17B). Anti-PD-L1 alone had no effect on any of the cell subsets or proportions analyzed.

[0349] In summary, hAlb-hIL2_A4s8 is a CD8 + T cells, especially pre-existing antigen-specific CD8 + Expanding T cells improved the antitumor effect of PD-L1 immune checkpoint blockade.

[0350] Example 10: Treatment with hAlb-hIL2-A4s8 monotherapy or in combination with PD-L1 checkpoint inhibition or therapeutic RNA vaccination modulates lymphocyte numbers in tumors of the murine tumor model MC38. In the previous examples, the effects of hAlb-hIL2_A4s8 alone or in combination with RNA vaccination or PD-L1 checkpoint inhibition on tumor growth, survival, and blood lymphocyte counts were determined. Therapeutic activity was assessed by CD8 + and CD4 + This was associated with an increase in T lymphocytes, NK cells, and tumor antigen-specific T cells. reg No significant increase in T cells was observed, and no significant increase in T cells or T reg However, it has not been shown whether the increase in effector lymphocytes in the blood, which are thought to promote tumor cell killing, also corresponds to an increase in tumors.

[0351] In this study, modulation of the tumor microenvironment was investigated during hAlb-hIL2_A4s8 treatment in the highly immunogenic colon cancer model MC38. MC38 tumors contain CD8 markers derived from point mutations in the IL-1 / IL-2 gene. +These include several mutated neoantigens recognized by T cells (Yadav, M. et al. Nature 515, 572-576 (2014); Capietto, A. et al. The Journal of Experimental Medicine 217, e20190179 (2020)). As described in Example 9, treatment with hAlb-hIL2_A4s8 synergized with PD-L1 checkpoint inhibition and increased the induction of T cell responses against tumor-specific neoantigens in the blood (Figure 17). Subsequent experiments in MC38 tumor-bearing mice were performed to analyze the infiltration of tumor neoantigen-specific T cells into tumors.

[0352] C57BL / 6 mice (n = 7–8 per group) received 7.5 × 10 5Mice were inoculated subcutaneously (sc) with MC38 colon cancer cells and, 19 days later, were treated intravenously with 3 μg of RNA formulated in LNPs encoding hAlb-hIL2_A4s8, followed by simultaneous i.p. treatment with 200 μg of an anti-PD-L1 antibody. Control groups received one of two treatments or no treatment at all (control for hAlb-hIL2_A4s8: hAlb RNA formulated as LNPs ("hAlb"); control for anti-PD-L1: isotype antibody ("iso"). Tumors and blood from mice were collected on day 24 and analyzed by flow cytometry (BD FACSCelesta). Blood was processed and stained as previously described (Kranz, LM et al. Nature 534, 396-401 (2016)). Tumors were digested using a mouse Tumor Dissociation Kit (Miltenyi Biotec) and a GentleMACS™ Dissociator (Miltenyi Biotec, catalog number 130-093-235). After lysing red blood cells with standard ACK buffer (8.25 g NH₄CL, 1 g KHCO₃, 0.2 ml EDTA, 1 L distilled H₂O), cells were stained for flow cytometry analysis as previously described (Kranz, LM et al. Nature 534, 396-401 (2016)). Analysis was performed using FlowJo software version 10 and GraphPad Prism version 8.

[0353] As shown in Figure 18A and B, intratumoral CD8 + T cells and NK cells were significantly increased in all hAlb-hIL2_A4s8-treated groups. Combination with anti-PD-L1 antibody (αPD-L1) further increased T cell numbers, but PD-L1 blockade alone had no effect. In this model, intratumoral CD4 + No significant increase in T cells was detected (Fig. 18C). As observed in TC-1 tumors, CD4 + Intratumoral T cells reg The fraction did not change with any treatment, and the T reg CD8 on cells +This resulted in an overall increase in the proportion of tumor-specific CD8 T cells that recognized neoantigens derived from mutated Adpgk, Rpl18, or N4pb2I2 (Figures 18D and E). + T cells were significantly increased in both hAlb-hIL2_A4s8-treated groups (Fig. 18F-H). Similarly, hAlb-hIL2_A4s8 treatment significantly increased blood CD8 + and CD4 + This resulted in an increase in the number of T cells and Adpgk, Rpl18, and N4pb2I2 neoantigen-specific T cells (Figures 18I-M).

[0354] In summary, hAlb-hIL2_A4s8 treatment alone or in combination with PD-L1 checkpoint inhibition and / or RNA vaccination was consistent with an increase in (tumor antigen-specific) effector T cells and NK cells in the tumor and blood.

Claims

1. 1. A polypeptide comprising a mutein of human interleukin-2 (IL2) or a functional variant of human IL2, wherein the human IL2 or functional variant thereof has substitutions at at least positions 80 (leucine), 81 (arginine), 85 (leucine), and 92 (isoleucine), numbered as compared to and according to wild-type human IL2, such substitutions having enhanced affinity for the βγ IL2 receptor complex (IL2Rβγ), and wherein the human IL2 or functional variant thereof does not have a substitution at position 86 (isoleucine), numbered as compared to and according to wild-type human IL2.

2. 2. The polypeptide of claim 1, wherein, compared to wild-type human IL2 and numbered according to wild-type human IL2, position 80 (leucine) is substituted with phenylalanine, position 81 (arginine) is substituted with glutamic acid, position 85 (leucine) is substituted with valine, and position 92 (isoleucine) is substituted with phenylalanine.

3. 3. The polypeptide of claim 1 or 2, wherein the human IL2 or functional variant thereof further comprises a substitution at position 74 (glutamine) compared to wild-type human IL2 and numbered according to wild-type human IL2.

4. 4. The polypeptide of claim 3, wherein position 74 (glutamine) as compared to and numbered according to wild-type human IL2 is substituted with histidine.

5. A polypeptide comprising a mutein of human IL2 or a functional variant of human IL2, wherein said human IL2 or functional variant thereof is substituted by at least phenylalanine at position 80 (leucine), by glutamic acid at position 81 (arginine), by valine at position 85 (leucine), and by phenylalanine at position 92 (isoleucine) compared to wild-type human IL2 and numbered according to wild-type human IL2.

6. 6. The polypeptide of claim 5, wherein the human IL2 or functional variant thereof is further substituted by histidine at position 74 (glutamine) numbered as compared to wild-type human IL2 and according to wild-type human IL2.

7. A polypeptide comprising a mutein of human IL2 or a functional variant of human IL2, wherein said human IL2 or functional variant thereof is substituted with at least histidine at position 74 (glutamine), phenylalanine at position 80 (leucine), glutamic acid at position 81 (arginine), valine at position 85 (leucine), and phenylalanine at position 92 (isoleucine) compared to wild-type human IL2 and numbered according to wild-type human IL2.

8. The polypeptide of any one of claims 5 to 7, wherein the substitution enhances affinity for IL2Rβγ.

9. 9. The polypeptide of claim 1, wherein the human IL2 or functional variant thereof further comprises one or more amino acid substitutions that reduce affinity for the alpha subunit of the αβγ IL2 receptor complex (IL2Rαβγ).

10. The polypeptide of claim 9, wherein the one or more amino acid substitutions that reduce affinity for the alpha subunit of IL2Rαβγ reduce affinity for IL2Rαβγ to a greater extent than affinity for IL2Rβγ.

11. 11. The polypeptide of claim 9 or 10, wherein the one or more amino acid substitutions that reduce affinity for the alpha subunit of IL2Rαβγ include substitutions of the human IL2 or a functional variant thereof at positions 43 (lysine) and 61 (glutamic acid) compared to and numbered according to wild-type human IL2.

12. 12. The polypeptide of claim 11, wherein position 43 (lysine) is substituted with glutamic acid and position 61 (glutamic acid) is substituted with lysine.

13. 1. A polypeptide comprising a mutein of human IL2 or a functional variant of human IL2, wherein said human IL2 or functional variant thereof is substituted at least at position 43 (lysine) by glutamic acid, at position 61 (glutamic acid) by lysine, at position 74 (glutamine) by histidine, at position 80 (leucine) by phenylalanine, at position 81 (arginine) by glutamic acid, at position 85 (leucine) by valine, and at position 92 (isoleucine) by phenylalanine, compared to wild-type human IL2 and numbered according to wild-type human IL2.

14. 14. The polypeptide of any one of claims 5 to 13, wherein said human IL2 or functional variant thereof is not substituted at position 86 (isoleucine) compared to and numbered according to wild-type human IL2.

15. The polypeptide according to any one of claims 1 to 14, wherein said human IL2 has an amino acid sequence according to SEQ ID NO:

1.

16. The polypeptide according to any one of claims 1 to 15, wherein the mutein of human IL2 or a functional variant thereof has a reduced ability to stimulate regulatory T cells compared to wild-type human IL2.

17. The polypeptide according to any one of claims 1 to 16, wherein the mutein of human IL2 or a functional variant thereof has an increased ability to stimulate effector T cells compared to wild-type human IL2.

18. 18. The polypeptide of any one of claims 1 to 17, which is an extended pharmacokinetic (PK) polypeptide.

19. 20. The polypeptide of claim 18, wherein the extended PK polypeptide comprises a fusion protein.

20. 20. The polypeptide of claim 19, wherein the fusion protein comprises a portion that is a mutein of human IL2 or a functional variant thereof and a portion that is heterologous to human IL2 or a functional variant thereof.

21. 21. The polypeptide of claim 19 or 20, wherein the fusion protein comprises a portion of a mutein of human IL2 or a functional variant thereof, and a portion selected from the group consisting of serum albumin, an immunoglobulin fragment, transferrin, Fn3, and variants thereof.

22. 22. The polypeptide of claim 21, wherein the serum albumin comprises mouse serum albumin or human serum albumin.

23. 22. The polypeptide of claim 21, wherein the immunoglobulin fragment comprises an immunoglobulin Fc domain.

24. A polynucleotide encoding the polypeptide of any one of claims 1 to 23.

25. 25. The polynucleotide of claim 24, which is RNA.

26. A host cell comprising the polynucleotide of claim 24 or 25.

27. A polypeptide according to any one of claims 1 to 23, a polynucleotide according to claim 24 or 25, or a host cell according to claim 26, for pharmaceutical use.

28. 28. The polypeptide, polynucleotide or host cell of claim 27, wherein the pharmaceutical use comprises the therapeutic or prophylactic treatment of a disease or disorder.

29. 29. A polypeptide, polynucleotide or host cell according to claim 27 or 28 for use in a method for treating or preventing cancer in a subject.

30. A pharmaceutical composition comprising a polypeptide according to any one of claims 1 to 23, a polynucleotide according to claim 24 or 25, or a host cell according to claim 26.

31. A method of treating a subject, comprising administering to the subject a polypeptide according to any one of claims 1 to 23, a polynucleotide according to claim 24 or 25, a host cell according to claim 26, or a pharmaceutical composition according to claim 30.

32. A method for inducing an immune response in a subject, comprising administering to the subject a polypeptide according to any one of claims 1 to 23, a polynucleotide according to claim 24 or 25, a host cell according to claim 26, or a pharmaceutical composition according to claim 30.

33. 33. The method of claim 31 or 32, further comprising administering to the subject a peptide or protein comprising an epitope for inducing an antigen-specific immune response in the subject, or a polynucleotide encoding said peptide or protein.

34. 34. The method of claim 33, wherein the polynucleotide encoding the peptide or protein is RNA.

35. 35. The method of any one of claims 31 to 34, for treating or preventing cancer in a subject, optionally wherein the antigen is a tumor-associated antigen.

36. A method for treating or preventing cancer in a subject, comprising administering to the subject a polypeptide according to any one of claims 1 to 23, a polynucleotide according to claim 24 or 25, a host cell according to claim 26, or a pharmaceutical composition according to claim 30.

37. 37. The method of claim 36, further comprising administering to the subject a peptide or protein comprising an epitope for inducing an immune response specific to a tumor-associated antigen in the subject, or a polynucleotide encoding said peptide or protein.

38. 38. The method of claim 37, wherein the polynucleotide encoding the peptide or protein is RNA.

39. A pharmaceutical preparation comprising a polypeptide according to any one of claims 1 to 23, a polynucleotide according to claim 24 or 25, a host cell according to claim 26, or a pharmaceutical composition according to claim 30.

40. 40. The pharmaceutical formulation of claim 39, further comprising a peptide or protein comprising an epitope for inducing an antigen-specific immune response in a subject, or a polynucleotide encoding said peptide or protein.

41. 41. The pharmaceutical formulation of claim 40, wherein the polynucleotide encoding the peptide or protein is RNA.

42. 42. The pharmaceutical formulation of claim 40 or 41, wherein the polypeptide, polynucleotide, host cell, or pharmaceutical composition and the peptide or protein containing the epitope or the polynucleotide encoding the peptide or protein are each contained in separate containers.

43. 43. The pharmaceutical preparation of any one of claims 39 to 42, further comprising instructions for using said pharmaceutical preparation for treating or preventing cancer, and optionally wherein said antigen is a tumor-associated antigen.

44. A pharmaceutical formulation according to any one of claims 39 to 43 for pharmaceutical use.

45. 45. The pharmaceutical formulation of claim 44, wherein the pharmaceutical use comprises the therapeutic or prophylactic treatment of a disease or disorder.

46. 46. ​​The pharmaceutical preparation of any one of claims 39 to 45 for use in a method for treating or preventing cancer in a subject, optionally wherein the antigen is a tumor-associated antigen.

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