Treatment involving interleukin-2 (IL2) and interferon (IFN)

Abstract

To provide methods and agents for enhancing the effect of immune effector cells, in particular immune effector cells that respond to interleukin-2 (IL2), for example effector T cells such as CD8+ T cells.SOLUTION: A method for inducing an immune response in a subject, comprises administering to the subject: a. a polypeptide comprising IL2 or a functional variant thereof, or a polynucleotide encoding a polypeptide comprising IL2 or a functional variant thereof; and b. a polypeptide comprising type I interferon or a functional variant thereof, or a polynucleotide encoding a polypeptide comprising type I interferon or a functional variant thereof.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to methods and agents for enhancing the effectiveness of immune effector cells, particularly immune effector cells that respond to interleukin 2 (IL2), e.g., effector T cells such as CD8+ T cells. These methods and agents are particularly useful for treating diseases characterized by diseased cells that express an antigen to which immune effector cells are directed. Specifically, the disclosure relates to methods comprising administering to a subject a polypeptide comprising IL2 or a functional variant thereof (commonly referred to herein as "IL2") or a polynucleotide encoding a polypeptide comprising IL2 or a functional variant thereof, and a polypeptide comprising type I interferon (IFN) or a functional variant thereof (commonly referred to herein as "interferon" or "IFN") or a polynucleotide encoding a polypeptide comprising type I interferon or a functional variant thereof. IL2 provided to a subject acts on immune effector cells, such as effector T cells, resulting in enhanced immune effector cell action, for example by promoting immune effector cell expansion, while interferon prevents or reduces IL2-mediated expansion of regulatory T cells (Tregs), which antagonize the action of immune effector cells. The method of the present disclosure may further include administering a vaccine antigen or a polynucleotide encoding the same to a subject (optionally after expression of the polynucleotide by an appropriate target cell) to provide the antigen for stimulation, priming, and / or expansion of immune effector cells. In one embodiment, the immune effector cells bear an antigen receptor, such as a T cell receptor (TCR) or chimeric antigen receptor (CAR), with binding specificity for the antigen or its processing product. In one embodiment, the immune effector cells are genetically modified to express the antigen receptor. Alternatively or additionally, the immune effector cells are genetically modified to express an IL2 receptor (IL2R). Such genetic modification can be performed ex vivo or in vitro, after which the immune effector cells are administered to a subject in need of treatment, and / or can be performed in vivo in a subject in need of treatment. The immune effector cells can be derived from the subject in need of treatment or can be endogenous to the subject in need of treatment.Antigen receptors of immune effector cells can target disease-associated antigens. In a particularly preferred embodiment, the polynucleotide encoding IL2, the polynucleotide encoding IFN, and / or the polynucleotide encoding a vaccine antigen administered in accordance with the present disclosure is RNA. [Background technology]

[0002] The immune system plays an important role not only in pathogen-related diseases but also in cancer, autoimmunity, and allergies. T cells and NK cells are important mediators of antitumor immune responses. CD8+ T cells and NK cells can directly lyse tumor cells. Meanwhile, CD4+ T cells can mediate the influx of various immune subsets, including CD8+ T cells and NK cells, into tumors. CD4+ T cells can allow dendritic cells (DCs) to prime antitumor CD8+ T cell responses and can directly act on tumor cells by upregulating MHC and inhibiting proliferation via IFNγ. CD8+ and CD4+ tumor-specific T cell responses can be induced by vaccination or adoptive transfer of T cells. In the context of mRNA-based vaccine platforms, mRNA can be delivered to antigen-presenting cells located in secondary lymphoid organs by liposomal formulations (RNA-lipoplexes, RNA-LPX) without the need for additional adjuvants for immune stimulation (Kreiter, S. et al. Nature 520, 692-696 (2015); Kranz, L. M. et al. Nature 534, 396-401 (2016)).

[0003] One possible way to further improve the clinical effectiveness of T cells is to support and regulate them through cytokines that affect cell survival and function. For example, interleukin 2 (IL2) is a potent immunostimulator and activates various cells of the immune system. IL2 is known to support the differentiation, proliferation, survival and effector function of T cells and NK cells (Blattman, J. N. Nat. Med. 9, 540-7 (2003)), and has been used for decades in the treatment of late-stage malignant melanoma (Maas, RA, Dullens, HF & Den Otter, W. Cancer Immunother. 36, 141-8 (1993)).

[0004] However, there are several difficulties associated with cytokine administration. Recombinant cytokines have a very short plasma half-life, which necessitates frequent injection of large amounts of cytokine. In the case of IL2, this can result in severe side effects such as vascular leak syndrome (VLS) (Rosenberg, SN Engl. J. Med. 316, 889-97 (1987)). Furthermore, cytokine administration can cause undesirable effects on immune cells. For example, the high-affinity IL2 receptor (IL2Rαβγ), consisting of CD25 (IL2Rα), CD122 (IL2Rβ), and CD132 (IL2Rγ), mediates the expression of regulatory T cells (Treg) and activated CD4 + and CD8 +While expressed on T cells, the intermediate-affinity receptor (IL2Rβγ) lacking CD25 is predominant on naive and memory T cells and NK cells. IL2 is known for its ability to stimulate Tregs more potently than effector T cells (Todd, J. PLoS Med. 13, e1002139 (2016)). Tregs can suppress the function of antitumor effector T cells and NK cells, which correlates with decreased survival in cancer patients (Nishikawa, H. & Sakaguchi. Curr. Opin 27, 1-7 (2014)). Attempts to modify IL2 in a way that loses its selectivity for CD25-expressing cells, thereby increasing its relative ability to stimulate naive and memory T cells and NK cells, have been shown to improve its antitumor potential (Arenas-Ramirez, N. et al. Sci. Transl. Med. 8, 1-13 (2016)). [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Kreiter,S.et al.Nature 520,692-696(2015) [Non-patent document 2] Kranz,LMet al.Nature 534,396-401(2016) [Non-patent document 3] Blattman, J.NNat.Med.9,540-7(2003) [Non-patent document 4] Maas, RA, Dullens, HF & Den Otter, W. Cancer Immunother. 36, 141-8 (1993) [Non-patent document 5] Rosenberg, SNEngl. J. Med. 316, 889-97 (1987) [Non-patent document 6] Todd,J.PLoS Med.13,e1002139(2016) [Non-Patent Document 7] Nishikawa, H. & Sakaguchi. Curr. Opin27, 1-7 (2014) [Non-patent document 8] Arenas-Ramirez,N.et al.Sci.Transl.Med.8,1-13(2016) Summary of the Invention [Problem to be solved by the invention]

[0006] Clearly, novel strategies are needed to enhance the efficacy of immunotherapies, particularly vaccines, such as cancer vaccines, and / or cell-based immunotherapies, such as cell-based cancer immunotherapies involving the adoptive transfer of T cells (naive or T cell receptor transgenic or chimeric antigen receptor transgenic) and NK cells.

[0007] To address the limitations that arise with cytokine therapy, novel strategies are provided herein to enhance the efficacy of immunotherapy, including IL2 treatment.

[0008] The present disclosure provides means and methods for preventing IL2-mediated Treg expansion while maintaining the beneficial effects of IL2 treatment, such as the expansion of antigen-specific T cell responses.

[0009] We demonstrate that mRNA encoding IL2 fused to serum albumin (Alb-IL2) for long-term systemic availability significantly activated CD8+ T cells and potently expanded vaccine-induced antigen-specific T cells in vivo in mice. At the same time, Alb-IL2 led to massive proliferation of Tregs and strongly restricted the subsequent expansion of antigen-specific T cells. Coadministration of IFNα-encoding mRNA with Alb-IL2 prevented Treg expansion and enabled a sustained boost of vaccine-induced antigen-specific T cells. In vitro studies using isolated human T cells confirmed that IFNα prevented IL2-mediated activation of Tregs without significantly affecting CD8+ T cell activation. [Means for solving the problem]

[0010] The present invention generally encompasses immunotherapy treatment of a subject, including administration of a polypeptide comprising IL2 or a functional variant thereof, or a polynucleotide encoding a polypeptide comprising IL2 or a functional variant thereof, to enhance the efficacy of immunotherapy, and administration of a polypeptide comprising type I interferon or a functional variant thereof, or a polynucleotide encoding a polypeptide comprising type I interferon or a functional variant thereof, to reduce or prevent IL2-mediated undesirable effects, particularly Treg expansion. Immunotherapy may include vaccine therapy and / or cell-based cancer immunotherapy, such as TIL or T cell-based therapy, e.g., TCR or CAR transgenic T cell-based therapy using autologous cells. Generally, immune effector cells stimulated using the treatments described herein may target antigen-expressing cells, such as diseased cells, particularly cancer cells expressing tumor antigens. Target cells may express the antigen on their cell surface or display a processed product of the antigen. In one embodiment, the antigen is a tumor-associated antigen and the disease is cancer. Such treatment provides selective eradication of antigen-expressing cells, thereby minimizing adverse effects on normal cells that do not express the antigen. Immune effector cells (optionally genetically modified to express an antigen receptor) stimulated by IL2 administration, preferably having an endogenous IL2 receptor (IL2R) (or optionally genetically modified to express IL2R), target the antigen or its processing product, and thus the target cell population or target tissue expressing the antigen. Such immune effector cells can be administered to a subject in need of treatment or can be endogenous to the subject in need of treatment. In one embodiment, the immune effector cells bear an IL2 receptor (IL2R). In one embodiment, the immune effector cells are genetically modified to express IL2R. In one embodiment, the immune effector cells bear an antigen receptor, such as a T cell receptor (TCR) or chimeric antigen receptor (CAR), that has binding specificity for the target antigen or its processing product. In one embodiment, the immune effector cells are genetically modified to express an antigen receptor.Such genetic modification to express IL2R and / or antigen receptors can be performed ex vivo or in vitro, followed by administration of immune effector cells to a subject in need of treatment, or can be performed in vivo in a subject in need of treatment, or can be performed by a combination of ex vivo or in vitro and in vivo modification. In one embodiment, a vaccine antigen or a polynucleotide encoding it is administered to provide an antigen for stimulation, priming, and / or expansion of immune effector cells that target the target antigen or its processing product (optionally after expression of the polynucleotide by appropriate target cells). In one embodiment, the immune response induced in accordance with the present disclosure is an immune response against a target cell population or target tissue expressing the antigen to which the immune effector cells are directed. In one embodiment, the immune response induced in accordance with the present disclosure is a T cell-mediated immune response. In one embodiment, the immune response is an anti-tumor immune response, and the target cell population or target tissue is a tumor cell or tumor tissue.

[0011] The methods and medicaments described herein are particularly effective when IL2 is conjugated to a pharmacokinetic-modifying group (hereinafter referred to as "extended pharmacokinetic (PK) IL2"). The methods and medicaments described herein are particularly effective when the polynucleotide encoding IL2, such as extended PK IL2, is RNA and / or the polynucleotide encoding type I interferon is RNA. In one embodiment, the RNA is targeted to the liver for systemic availability. Hepatocytes can be efficiently transfected and produce large amounts of protein. RNA encoding a vaccine antigen is preferably targeted to secondary lymphoid organs.

[0012] In one aspect, there is provided a method for inducing an immune response in a subject, comprising: a. a polypeptide comprising IL2 or a functional variant thereof or a polynucleotide encoding a polypeptide comprising IL2 or a functional variant thereof; and Polypeptide containing type I interferon or a functional variant thereof or a polynucleotide encoding a polypeptide containing type I interferon or a functional variant thereof Provided herein are methods comprising administering to a subject

[0013] In one embodiment, the method comprises: c. A peptide or protein containing an epitope for inducing an immune response to an antigen in a subject, or a polynucleotide encoding said peptide or protein and administering to the subject.

[0014] In one embodiment, the polynucleotide encoding the polypeptide comprising IL2 or a functional variant thereof is RNA, the polynucleotide encoding the polypeptide comprising a type I interferon or a functional variant thereof is RNA, and optionally the polynucleotide encoding said peptide or protein is RNA.

[0015] In one aspect, there is provided a method for inducing an immune response in a subject, comprising: a. RNA encoding a polypeptide comprising IL2 or a functional variant thereof; and RNA encoding a polypeptide containing type I interferon or a functional variant thereof Provided herein are methods comprising administering to a subject

[0016] In one embodiment, the method comprises: c. RNA encoding a peptide or protein containing an epitope for inducing an immune response to the antigen in a subject. and administering to the subject.

[0017] In one embodiment, the immune response is a T cell-mediated immune response.

[0018] In one embodiment, the subject has a disease, disorder or condition associated with expression or up-regulation of the antigen.

[0019] In one aspect, there is provided a method for treating a subject having a disease, disorder, or condition associated with expression or up-regulation of an antigen, comprising: a. a polypeptide comprising IL2 or a functional variant thereof or a polynucleotide encoding a polypeptide comprising IL2 or a functional variant thereof; b A polypeptide comprising a type I interferon or a functional variant thereof or a polynucleotide encoding a polypeptide comprising a type I interferon or a functional variant thereof; and c. A peptide or protein containing an epitope for inducing an immune response to an antigen in a subject, or a polynucleotide encoding said peptide or protein Provided herein are methods comprising administering to a subject

[0020] In one embodiment, the polynucleotide encoding the polypeptide comprising IL2 or a functional variant thereof is RNA, the polynucleotide encoding the polypeptide comprising a type I interferon or a functional variant thereof is RNA, and the polynucleotide encoding said peptide or protein is RNA.

[0021] In one aspect, there is provided a method for treating a subject having a disease, disorder, or condition associated with expression or up-regulation of an antigen, comprising: a. RNA encoding a polypeptide comprising IL2 or a functional variant thereof; RNA encoding a polypeptide comprising type I interferon or a functional variant thereof; and c. RNA encoding a peptide or protein containing an epitope for inducing an immune response to the antigen in a subject. Provided herein are methods comprising administering to a subject

[0022] In one embodiment, the disease, disorder, or condition is cancer and the antigen is a tumor-associated antigen.

[0023] In one embodiment, the polypeptide comprising IL2 or a functional variant thereof is extended pharmacokinetic (PK) IL2. In one embodiment, the extended PK IL2 comprises a fusion protein. In one embodiment, the fusion protein comprises a portion of IL2 or a functional variant thereof and a moiety 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.

[0024] In one embodiment, the method of any aspect is a method for treating or preventing cancer in a subject, and optionally the antigen is a tumor-associated antigen.

[0025] In one embodiment, administration of a polypeptide comprising IL2 or a functional variant thereof or a polynucleotide, particularly RNA, encoding a polypeptide comprising IL2 or a functional variant thereof, and optionally a peptide or protein comprising an epitope for inducing an immune response against an antigen in a subject, or a polynucleotide, particularly RNA encoding said peptide or protein, induces antigen-specific T cells.

[0026] In one embodiment, administration of a polypeptide comprising a type I interferon or a functional variant thereof or a polynucleotide, particularly RNA, encoding a polypeptide comprising a type I interferon or a functional variant thereof reduces the number of regulatory T cells.

[0027] In one embodiment, administration of a polypeptide comprising a type I interferon or a functional variant thereof or a polynucleotide, particularly RNA, encoding a polypeptide comprising a type I interferon or a functional variant thereof reduces IL2-mediated expansion of regulatory T cells.

[0028] In one embodiment, administration of a polypeptide comprising a type I interferon or a functional variant thereof or a polynucleotide, particularly RNA, encoding a polypeptide comprising a type I interferon or a functional variant thereof increases the ratio of antigen-specific T cells to regulatory T cells.

[0029] In one embodiment of all aspects, the type I interferon is interferon alpha.

[0030] In one aspect, a. a polypeptide comprising IL2 or a functional variant thereof or a polynucleotide encoding a polypeptide comprising IL2 or a functional variant thereof; and Polypeptide containing type I interferon or a functional variant thereof or a polynucleotide encoding a polypeptide containing type I interferon or a functional variant thereof Provided herein is a pharmaceutical formulation comprising:

[0031] In one embodiment, the pharmaceutical formulation comprises: c. A peptide or protein containing an epitope for inducing an immune response to an antigen in a subject, or a polynucleotide encoding said peptide or protein Further includes:

[0032] In one embodiment, the polynucleotide encoding the polypeptide comprising IL2 or a functional variant thereof is RNA, the polynucleotide encoding the polypeptide comprising a type I interferon or a functional variant thereof is RNA, and optionally the polynucleotide encoding said peptide or protein is RNA.

[0033] In one embodiment, the pharmaceutical preparation is a kit.

[0034] In one embodiment, the pharmaceutical formulation comprises a polypeptide comprising IL2 or a functional variant thereof or a polynucleotide encoding a polypeptide comprising IL2 or a functional variant thereof, a polypeptide comprising a type I interferon or a functional variant thereof or a polynucleotide encoding a polypeptide comprising a type I interferon or a functional variant thereof, and optionally said peptide or protein or a polynucleotide encoding said peptide or protein, in separate containers.

[0035] 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.

[0036] In one embodiment, the pharmaceutical formulation is a pharmaceutical composition.

[0037] In one embodiment, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0038] In one aspect, a. RNA encoding a polypeptide comprising IL2 or a functional variant thereof; and RNA encoding a polypeptide containing type I interferon or a functional variant thereof Provided herein is a pharmaceutical formulation comprising:

[0039] In one embodiment, the pharmaceutical formulation comprises: c. RNA encoding a peptide or protein containing an epitope for inducing an immune response to the antigen in a subject. Further includes:

[0040] In one embodiment, the pharmaceutical preparation is a kit.

[0041] In one embodiment, the pharmaceutical formulation comprises, in separate containers, RNA encoding a polypeptide comprising IL2 or a functional variant thereof, RNA encoding a polypeptide comprising a type I interferon or a functional variant thereof, and optionally RNA encoding a peptide or protein.

[0042] 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.

[0043] In one embodiment, the pharmaceutical formulation is a pharmaceutical composition.

[0044] In one embodiment, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

[0045] In one embodiment of the pharmaceutical preparation, the immune response is a T cell-mediated immune response.

[0046] In one embodiment, the polypeptide comprising IL2 or a functional variant thereof is extended pharmacokinetic (PK) IL2. In one embodiment, the extended PK IL2 comprises a fusion protein. In one embodiment, the fusion protein comprises a portion of IL2 or a functional variant thereof and a moiety 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.

[0047] In one aspect, provided herein is a pharmaceutical formulation as described herein for pharmaceutical use.

[0048] In one embodiment, the medical use includes the therapeutic or prophylactic treatment of a disease or disorder.

[0049] 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.

[0050] In one embodiment of all aspects, the type I interferon is interferon alpha. [Brief explanation of the drawings]

[0051] [Figure 1] Combined treatment with mAlb-mIL2 and T cell vaccination results in a transient boost of ovalbumin-specific CD8+ T cells. A, Experimental overview. B–D, Number of ovalbumin (OVA)-specific CD8+ T cells in peripheral blood on days 7 (B), 14 (C), and 21 (D). E, Frequency of OVA-specific T cells over time. F, Frequency of CD4+CD25+FoxP3+ Tregs among CD4+ T cells over time. Statistical analysis was performed using unpaired two-tailed Student's t-test (B–D) or two-way ANOVA with Sidak's multiple comparison test (E, F). ns; P > 0.05, *; P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001, ***; P ≤ 0.0001. Mean ± standard error is shown. [Figure 2] Combined treatment with mAlb-mIL2 and T cell vaccination results in a transient boost of gp70-specific CD8+ T cells. A, Experimental overview. B–D, Number of gp70-specific CD8+ T cells in peripheral blood on days 7 (B), 14 (C), and 21 (D). E, Frequency of gp70-specific T cells over time. F, Frequency of CD4+CD25+FoxP3+ Tregs among CD4+ T cells over time. Statistical analysis was performed using an unpaired two-tailed Student's t-test (B–D) or two-way ANOVA with Sidak's multiple comparison test (E, F). ns; P > 0.05, *; P ≤ 0.05, **; P ≤ 0.01, ***; P ≤ 0.001, ***; P ≤ 0.0001. Mean ± standard error is shown. [Figure 3]IFNα limits IL2-mediated Treg expansion and leads to robust priming of OVA-specific T cells in vivo. A, Experimental overview. B, D, Frequency of OVA-specific CD8+ T cells in peripheral blood on days 7 (B) and 14 (D). C, Frequency of CD4+CD25+FoxP3+ Tregs among CD4+ T cells on day 7. Statistical analysis was performed by one-way ANOVA followed by Sidak's multiple comparison test. ns; P > 0.05, *: P ≤ 0.05, **: P ≤ 0.01, ***; P ≤ 0.001, ***; P ≤ 0.0001. Mean ± standard error is shown. [Figure 4] IFNα limits IL2-mediated Treg expansion and leads to robust priming of gp70-specific T cells in vivo. A, Experimental overview. B, D, Number of gp70-specific CD8+ T cells in peripheral blood on days 7 (B) and 21 (D). C, Frequency of CD4+CD25+FoxP3+ Tregs among CD4+ T cells on day 7. Statistical analysis was performed by one-way ANOVA followed by Sidak's multiple comparison test. ns; P > 0.05, *: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001, ***: P ≤ 0.0001. Mean ± standard error is shown. [Figure 5] IFNα limits IL2-mediated Treg but not CD8+ T cell expansion in vitro. CellTrace FarRed-labeled isolated Tregs (CD4+CD25+) were cocultured at a 1:1 ratio with autologous CFSE-labeled PBMCs in the presence of a suboptimal concentration of anti-CD3 antibody (clone UCHT1) and treated with 5% hAlb-hIL2-containing supernatant. Cocultures were incubated with 10,000 U / mL hIFNα2b, 625 U / mL hIFNα2b, or maintained without hIFNα2b. CD4+CD25+ Treg and CD8+ T cell proliferation was measured by flow cytometry after 4 days of incubation. Data from two different PBMC donors (A, B) are shown as the mean value of the proliferation index calculated using FlowJo v10.5 software. Error bars indicate intra-experimental variation (two replicates). [Figure 6ABC]Combination therapy with IFNα and IL2 produces synergistic antitumor effects in mice. A, Treatment regimen and analysis schedule. B, Median tumor size per group. For statistical analysis, all groups were compared with the mAlb control, and two-way ANOVA followed by Dunnett's test was performed. C, Tumor growth curves of single mice. Vertical dotted lines indicate treatment. D, Mouse survival. A log-rank test was performed to statistically compare survival rates between the IL2 and IFNα combination group and the IL2 monotherapy group. E, CD8+ T cell fraction among CD45+ cells on days 29 (left) and 35 (right). Means (lines) and individual values ​​(symbols) are shown. The horizontal dotted line indicates the mean of the mAlb group. To identify significant differences, one-way ANOVA followed by Tukey's test was performed. ns; P > 0.05, *: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001, ***: P ≤ 0.0001. [Figure 6DE] Combination therapy with IFNα and IL2 produces synergistic antitumor effects in mice. A, Treatment regimen and analysis schedule. B, Median tumor size per group. For statistical analysis, all groups were compared with the mAlb control, and two-way ANOVA followed by Dunnett's test was performed. C, Tumor growth curves of single mice. Vertical dotted lines indicate treatment. D, Mouse survival. A log-rank test was performed to statistically compare survival rates between the IL2 and IFNα combination group and the IL2 monotherapy group. E, CD8+ T cell fraction among CD45+ cells on days 29 (left) and 35 (right). Means (lines) and individual values ​​(symbols) are shown. The horizontal dotted line indicates the mean of the mAlb group. To identify significant differences, one-way ANOVA followed by Tukey's test was performed. ns; P > 0.05, *: P ≤ 0.05, **: P ≤ 0.01, ***: P ≤ 0.001, ***: P ≤ 0.0001. DETAILED DESCRIPTION OF THE INVENTION

[0052] 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.

[0053] 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).

[0054] 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).

[0055] 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.

[0056] The term "about" means approximately or approximately, 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.

[0057] As used in the context of describing this disclosure (particularly in the context of the claims), the terms "a," "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 separate value is incorporated herein as if it were individually listed 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.

[0058] 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."

[0059] 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 incorporated herein by reference in its entirety. No citation herein should be construed as an admission that the present disclosure was not entitled to antedate such disclosure.

[0060] 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.

[0061] definition As used herein, terms such as "reduce," "diminish," "inhibit," or "impair" relate 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.

[0062] Terms such as "increase," "enhance," or "exceed" 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.

[0063] According to the present disclosure, the term "peptide" 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 linked together by peptide bonds. The terms "protein" or "polypeptide" refer to large peptides, particularly peptides having at least about 150 amino acids, although the terms "peptide," "protein," and "polypeptide" are generally used synonymously herein.

[0064] 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 pathological condition. 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.

[0065] A "fragment" of an amino acid sequence (peptide or protein) refers to a portion of the amino acid sequence, i.e., a sequence representing an amino acid sequence 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 contains, for example, 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.

[0066] 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 polypeptide, preferably 1 to about 10 or 1 to about 5 amino acid modifications.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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 replacement 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.

[0071] 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%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably given 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.

[0072] "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.

[0073] The term "percent identity" is intended to indicate the percentage of amino acid residues that are identical between the two sequences being compared, obtained after optimal alignment, and this percentage is purely statistical, with the differences between the two sequences being randomly distributed 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 that use 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.).

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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 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 is altered by one or more amino acids compared to the amino acid sequence of the parent molecule or sequence, but that is still capable of performing one or more functions of the parent molecule or sequence, such as binding to or contributing to binding to a target molecule. In one embodiment, alterations in the amino acid sequence of the parent molecule or sequence do not significantly affect or alter 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, the 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.

[0078] 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 may 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.

[0079] 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.

[0080] "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.

[0081] The term "recombinant" in the context of the present invention means "produced through genetic engineering." Preferably, a "recombinant subject," such as a recombinant cell in the context of the present invention, does not occur in nature.

[0082] As used herein, the term "naturally occurring" refers to the fact that an object can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is naturally occurring.

[0083] The term "genetic 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, e.g., the cells can form part of an organ, tissue, and / or organism of a patient. According to the present invention, transfection can be transient or stable. For some applications of transfection, it is sufficient for the transfected genetic material to be expressed only transiently. Because nucleic acids introduced during transfection are not usually integrated into the nuclear genome, the foreign nucleic acid is diluted or degraded by mitosis. Cells that allow episomal amplification of nucleic acids significantly reduce the dilution rate. If the transfected nucleic acid is desired to actually remain 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 a receptor polypeptide, such as an antigen receptor or IL2 receptor, are stably transfected with a nucleic acid encoding the receptor, whereas nucleic acids encoding cytokines, such as IL2 or type I interferon, and / or antigens are generally transiently transfected into the cells. RNA can be transfected into cells to transiently express the encoded protein.

[0084] immune effector cells As used herein, immune effector cells, particularly IL2-responsive immune effector cells (IL2R-bearing immune effector cells), can be administered to a subject in need of treatment or can be endogenously present in the subject in need of treatment. Administering IL2 or a polynucleotide encoding IL2 to a subject allows for stimulation of immune effector cells. The methods and medicaments described herein are particularly useful for treating diseases characterized by disease cells expressing an antigen against which immune effector cells are directed. In one embodiment, the immune effector cells bear an antigen receptor, such as a T cell receptor (TCR) or chimeric antigen receptor (CAR), with binding specificity for the antigen or its processing product. In one embodiment, the immune effector cells are present in the subject to be treated and are genetically modified in vivo in the subject to express the antigen receptor. In one embodiment, immune effector cells from either the subject to be treated or a different subject are administered to the subject to be treated. The administered immune effector cells can be genetically modified ex vivo before administration or can be genetically modified in vivo in the subject to express the antigen receptor after administration. In one embodiment, antigen receptor is endogenous to immune effector cells.In one embodiment, immune effector cells are genetically modified ex vivo or in vivo to express IL2R.Therefore, such genetic modification with IL2R can be carried out in vitro (optionally together with genetic modification with antigen receptor), and then immune effector cells can be administered to the subject in need of treatment, or can be carried out in vivo in the subject in need of treatment (optionally together with genetic modification with antigen receptor).

[0085] Thus, immune effector cells stimulated by IL2 include any cells that are responsive to IL2, either naturally or after transfection with one or more IL2R polypeptides. Such responsiveness includes the activation, differentiation, proliferation, survival, and / or adaptation of one or more immune effector functions. Cells include, in particular, lytic cells, particularly lymphoid cells, preferably T cells, particularly cytotoxic lymphocytes, preferably selected from cytotoxic T cells, natural killer (NK) cells, and lymphokine-activated killer (LAK) cells. Upon activation, these cytotoxic lymphocytes each cause the destruction of target cells. For example, cytotoxic T cells cause the 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 the target cell, and granzymes enter the cell and trigger a cytoplasmic caspase cascade that induces apoptosis (programmed cell death). Second, apoptosis can be induced through Fas-Fas ligand interaction between T cells and target cells. The cells used in connection with the present invention are preferably autologous cells, but xenogeneic or allogeneic cells can also be used. In one embodiment, the immune effector cells stimulated by IL2 are endogenous to the subject being treated.

[0086] The term "effector function" in the context of the present invention 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.

[0087] 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, particularly 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.

[0088] Preferably, "immune effector cells" recognize antigens with a 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 included. When the cells are CTLs, such responsiveness or reactivity may include the elimination of the 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."

[0089] 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.

[0090] 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.

[0091] "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.

[0092] The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper cells (CD4+ T cells) and cytotoxic T cells (CTL, CD8+ T cells), including cytolytic T cells. The term "antigen-specific T cell" or similar terms refers to a T cell that recognizes the antigen targeted by the T cell and preferably exerts the effector function of the T cell. A T cell is considered to be 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.

[0093] 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.

[0094] 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 also known as CD4+ T cells. Helper T cells become 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, which regulate or support active immune responses.

[0095] Cytotoxic T cells destroy virus-infected and tumor cells and are also involved in transplant rejection. These cells are also known as CD8+ T cells because they express the CD8 glycoprotein on their surface. 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.

[0096] "Regulatory T cells" or "Tregs" 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.

[0097] As used herein, the term "naive T cells" refers to mature T cells that, unlike activated or memory T cells, have never 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.

[0098] As used herein, the term "memory T cells" refers to a subgroup or subpopulation of T cells that previously encountered and responded to their cognate antigen. Upon a second encounter with the antigen, memory T cells can regenerate to mount a faster and more potent immune response than when the immune system first responded to the antigen. Memory T cells are CD4 + or CD8 + and typically express CD45RO.

[0099] All T cells possess a T cell receptor (TCR), which exists as a complex of several proteins. The TCR of a T cell binds to major histocompatibility complex (MHC) molecules and can interact with immunogenic peptides (epitopes) displayed on the surface of target cells. Specific binding of the TCR triggers a signaling cascade within the T cell, leading to proliferation and differentiation into mature effector T cells. 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 consists of two distinct peptide chains, termed the α-TCR chain and the β-TCR chain. A much less common group of T cells (2% of all T cells), γδ T cells (gamma delta T cells), possess a distinct T cell receptor (TCR) on their surface, consisting of one γ chain and one δ chain.

[0100] 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 classified as double-negative (CD4-CD8-) cells. As development progresses, they become double-positive thymocytes (CD4+CD8+) and ultimately mature into single-positive (CD4+CD8- or CD4-CD8+) thymocytes, which are then released from the thymus into peripheral tissues.

[0101] 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).

[0102] 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.

[0103] nucleic acid 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.

[0104] 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.

[0105] In one embodiment of all aspects of the invention, a nucleic acid, such as a nucleic acid encoding a cytokine (e.g., IL2 or IFN), a nucleic acid encoding an IL2R, a nucleic acid encoding an antigen receptor, or a nucleic acid encoding a vaccine antigen, is expressed in cells of a subject to be treated to provide the cytokine, IL2R, 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.

[0106] The nucleic acids described herein can be recombinant and / or isolated molecules.

[0107] 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 either or both ends 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.

[0108] In certain 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.

[0109] 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.

[0110] In one embodiment, the RNA described herein can have modified nucleosides. In some embodiments, the RNA includes a modified nucleoside in place of at least one (e.g., all) uridines.

[0111] The term "uracil" as used herein refers to one of the nucleic acid bases that can occur in RNA nucleic acids. The structure of uracil is: [ka] is.

[0112] As used herein, the term "uridine" refers to one of the nucleosides that can occur in RNA. The structure of uridine is: [ka] is.

[0113] UTP (uridine 5'-triphosphate) has the following structure: [ka] It has.

[0114] Pseudo-UTP (pseudouridine 5'-triphosphate) has the following structure: [ka] It has.

[0115] "Pseudouridine" is an example of a modified nucleoside that is an isomer of uridine in which uracil is attached to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.

[0116] Another exemplary modified nucleoside is N1-methylpseudouridine (m1Ψ), which has the structure: [ka] It has.

[0117] N1-methyl pseudo-UTP has the following structure: [ka] It has.

[0118] Another exemplary modified nucleoside is 5-methyluridine (m5U), which has the structure: [ka] It has.

[0119] In some embodiments, one or more uridines in the RNA described herein are replaced with a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.

[0120] In some embodiments, the RNA comprises a modified nucleoside in place of at least one uridine. In some embodiments, the RNA comprises a modified nucleoside in place of each uridine.

[0121] In some embodiments, the modified nucleosides are independently selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methyluridine (m5U). In some embodiments, the modified nucleoside comprises pseudouridine (ψ). In some embodiments, the modified nucleoside comprises N1-methylpseudouridine (m1ψ). In some embodiments, the modified nucleoside comprises 5-methyluridine (m5U). In some embodiments, the RNA may comprise two or more modified nucleosides, wherein the modified nucleosides are independently selected from pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methyluridine (m5U). In some embodiments, the modified nucleosides comprise pseudouridine (ψ) and N1-methylpseudouridine (m1ψ). In some embodiments, modified nucleosides include pseudouridine (ψ) and 5-methyluridine (m5U). In some embodiments, modified nucleosides include N1-methylpseudouridine (m1ψ) and 5-methyluridine (m5U). In some embodiments, modified nucleosides include pseudouridine (ψ), N1-methylpseudouridine (m1ψ), and 5-methyluridine (m5U).

[0122] In some embodiments, the modified nucleoside that replaces one or more uridines in the RNA is 3-methyluridine (m 3 U), 5-methoxyuridine (mo 5 U), 5-azauridine, 6-azauridine, 2-thio-5-azauridine, 2-thiouridine (s 2 U), 4-thiouridine (s 4 U), 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine (ho 5U), 5-aminoallyl uridine, 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-hydroxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyluridine (cm 5 U), 1-carboxymethylpseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thiouridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 1-ethylpseudouridine, 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-selenouridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyluridine, 1-propynylpseudouridine, 5-taurinomethyluridine (τm 5 U), 1-taurinomethylpseudouridine, 5-taurinomethyl-2-thiouridine (τm5s2U), 1-taurinomethyl-4-thiopseudouridine, 5-methyl-2-thiouridine (m 5 s 2 U), 1-methyl-4-thiopseudouridine (m 1 s 4 ψ), 4-thio-1-methylpseudouridine, 3-methylpseudouridine (m 3ψ), 2-thio-1-methylpseudouridine, 1-methyl-1-deazapseudouridine, 2-thio-1-methyl-1-deazapseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyldihydrouridine (m 5 D), 2-thiodihydrouridine, 2-thiodihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxypseudouridine, 4-methoxy-2-thiopseudouridine, N1-methylpseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 Ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine (inm 5 s 2 U), α-thiouridine, 2'-O-methyluridine (Um), 5,2'-O-dimethyluridine (m 5 Um), 2'-O-methylpseudouridine (Ψm), 2-thio-2'-O-methyluridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyluridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm 5 Um), 3,2'-O-dimethyluridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyluridine (inm 5 Um), 1-thiouridine, deoxythymidine, 2'-F-aruridine, 2'-F-uridine, 2'-OH-aruridine, 5-(2-carbomethoxyvinyl)uridine, 5-[3-(1-E-propenylamino)uridine, or any other modified uridine known in the art.

[0123] 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 attached to the RNA post-transcriptionally using a capping enzyme.

[0124] In some embodiments, the building block cap for RNA is m2 7,3'-O Gppp(m1 2'-O ) ApG (sometimes m2 7,3'O G(5')ppp(5')m 2'-O ApG), which has the following structure: [ka] It has.

[0125] The following are RNA and m2 7,3`O G(5')ppp(5')m 2'-O An exemplary Cap1 RNA containing ApG. [ka]

[0126] Below is another exemplary Cap1 RNA (no cap analog): [ka]

[0127] In some embodiments, the RNA has, in one embodiment, the structure: [ka] Cap analogues with anti-reverse cap (ARCA cap (m2 7,3`O G(5')ppp(5')G)) is modified with a "cap 0" structure.

[0128] The following are RNA and m2 7,3`O An exemplary cap 0 RNA containing G(5')ppp(5')G. [ka]

[0129] In some embodiments, the "cap 0" structure has the structure: [ka] Cap analogue β-S-ARCA (m2 7,2`O G(5')ppSp(5')G).

[0130] The following is β-S-ARCA(m2 7,2`O G(5')ppSp(5')G) and RNA. [ka]

[0131] In some embodiments, an RNA according to the present disclosure comprises a 5'-UTR and / or a 3'-UTR. The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or 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) sequence. Thus, the 3'-UTR is upstream of the poly(A) sequence (if present), e.g., directly adjacent to the poly(A) sequence.

[0132] 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 can follow the 3'UTR of an RNA described herein. The poly(A) sequence can be of any length. In some aspects, 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 110 nucleotides. In some aspects, the poly(A) sequence consists exclusively 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. WO 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. 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.

[0133] 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.

[0134] "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 when 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.

[0135] As used herein, "endogenous" refers to any substance that is produced from or within an organism, cell, tissue, or system.

[0136] As used herein, the term "exogenous" refers to any substance that is introduced into or produced outside of an organism, cell, tissue, or system.

[0137] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence.

[0138] 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.

[0139] cytokines 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.

[0140] IL2 and IL2R 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," In Receptor Activation by Antigens, Cytokines, Hormones, and Growth Factors 766:263-271, 1995). The IL2:IL2R complex is internalized upon ligand binding, and different 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).

[0141] 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 IL2. Recombinant human IL2 has been produced in E. coli, insect cells, and mammalian COS cells.

[0142] 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, 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 activity of natural killer (NK) cells. Attempts to lower serum concentrations by reducing the dose and adjusting the dosing regimen have been made, resulting in less toxicity, but such treatments have also been less effective.

[0143] According to the present disclosure, IL2 (optionally as part of extended PK IL2) can be naturally occurring IL2 or a fragment or variant thereof. IL2 can be human IL2 and can be derived from any vertebrate, particularly any mammal. As used herein, "human IL2" or "wild-type human IL2," whether natural or recombinant, has 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).

[0144] In one embodiment, the IL2 comprises the amino acid sequence of SEQ ID NO: 1 or 2. In one embodiment, a functional variant of 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 or 2. In one embodiment, a functional variant of IL2 binds to the IL2 receptor or a subunit of the IL2 receptor, e.g., the α subunit and / or the β / γ subunit. In general, for purposes of this disclosure, the term "IL2," as used herein, unless contradicted by context, includes any polypeptide comprising a naturally occurring IL2 portion or a functional variant thereof.

[0145] According to the present disclosure, in certain embodiments, IL2 is 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 in vivo serum IL2 concentrations to be maintained within the therapeutic range, 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.

[0146] Thus, in certain embodiments described herein, the IL2 portion is fused to a heterologous polypeptide (i.e., a polypeptide that is not IL2, and preferably is not a variant of IL2), and is therefore an extended PK IL2. In certain embodiments, the IL2 portion of the extended PK IL2 is human IL2. In other embodiments, the IL2 portion of the extended PK IL2 is a fragment or variant of human 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.

[0147] According to the present disclosure, the IL2 receptor (IL2R) may be a naturally occurring IL2R or a fragment or variant thereof. The IL2R may be a human IL2R and may be derived from any vertebrate, particularly any mammal. When IL2 as used herein includes an IL2 variant, the IL2R may be a variant receptor that binds to the IL2 variant.

[0148] interferon Interferons (IFNs) are a group of signaling proteins produced and released by host cells in response to the presence of several pathogens, such as viruses, bacteria, and parasites, as well as tumor cells. In a typical scenario, a virus-infected cell releases interferons to enhance the antiviral defenses of nearby cells.

[0149] Based on the type of receptor through which the interferons signal, interferons are typically divided into three classes: type I interferons, type II interferons, and type III interferons.

[0150] All type I interferons bind to a specific cell surface receptor complex known as the IFN-α / β receptor (IFNAR), which consists of the IFNAR1 and IFNAR2 chains.

[0151] The type I interferons present in humans are IFNα, IFNβ, IFNε, IFNκ, and IFNω. Generally, type I interferons are produced when the body recognizes an invading virus. They are produced by fibroblasts and monocytes. Once released, type I interferons bind to specific receptors on target cells, resulting in the expression of proteins that prevent the virus from producing and replicating its RNA and DNA.

[0152] IFNα proteins are primarily produced by plasmacytoid dendritic cells (pDCs). They are primarily involved in innate immunity against viral infections. The genes responsible for their synthesis belong to 13 subtypes, designated IFNA1, IFNA2, IFNA4, IFNA5, IFNA6, IFNA7, IFNA8, IFNA10, IFNA13, IFNA14, IFNA16, IFNA17, and IFNA21. These genes are found together in a cluster on chromosome 9.

[0153] IFNβ proteins are produced in large amounts by fibroblasts. They have antiviral activity and are primarily involved in the innate immune response. Two types of IFNβ have been described: IFNβ1 and IFNβ3. Both native and recombinant forms of IFNβ1 possess antiviral, antibacterial, and anticancer properties.

[0154] Type II interferons (IFNγ in humans), also known as immune interferons, are activated by IL12. Additionally, type II interferons are released by cytotoxic T cells and T helper cells.

[0155] Type III interferons signal through a receptor complex consisting of IL10R2 (also known as CRF2-4) and IFNLR1 (also known as CRF2-12). Although more recently discovered than type I and type II IFNs, recent information has demonstrated the importance of type III IFNs in several types of viral or fungal infections.

[0156] In general, type I and type II interferons are responsible for regulating and activating the immune response.

[0157] According to the present disclosure, the type I interferon is preferably IFNα or IFNβ, more preferably IFNα.

[0158] According to the present disclosure, the IFNα can be naturally occurring IFNα or a fragment or variant thereof. The IFNα can be human IFNα and can be derived from any vertebrate, particularly any mammal. In one embodiment, the IFNα comprises the amino acid sequence of SEQ ID NO: 3. In one embodiment, a functional variant of IFNα 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: 3. In one embodiment, the functional variant of IFNα binds to the IFN-α / β receptor. In general, for purposes of the present disclosure, the term "IFNα" as used herein includes any polypeptide comprising a naturally occurring IFNα portion or a functional variant thereof, unless contradicted by context.

[0159] extended PK group The IL2 polypeptides described herein can be prepared as fusion or chimeric polypeptides comprising an IL2 moiety and a heterologous polypeptide (i.e., a polypeptide that is not IL2 or a variant thereof). IL2 can be fused to an extended PK group, which increases its 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).

[0160] 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, an "extended 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 extended 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 extended 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, an "extended PK cytokine" refers to a cytokine moiety combined with an extended PK group. In one embodiment, the extended PK cytokine is a fusion protein in which the cytokine portion is linked or fused to an extended PK group. As used herein, "extended PK IL" refers to an interleukin (IL) portion (including an IL variant portion) combined with an extended PK group. In one embodiment, the extended PK IL is a fusion protein in which the IL portion is linked or fused to an extended PK group. An exemplary fusion protein is an HSA / IL2 fusion in which the IL2 portion is fused to HSA.

[0161] In certain 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 certain 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 certain 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.

[0162] 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 cytokines, such as 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), which resists 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).

[0163] In certain 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 may 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.

[0164] 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 "portions," "regions," or "moieties," 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, specifically bearing a signal peptide at its N-terminus, and, following secretion by the cell, preferably exists in a processed form, specifically in which the signal peptide has been cleaved. In the most preferred embodiment, the term "processed form of the albumin fusion protein" refers to an albumin fusion protein product that has undergone N-terminal signal peptide cleavage, also referred to herein as a "mature albumin fusion protein."

[0165] 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.

[0166] 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, bovine, ovine, or porcine. 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.

[0167] In certain embodiments, 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.

[0168] 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.

[0169] 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.

[0170] 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 specific 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.

[0171] Generally speaking, an albumin fragment or variant is at least 100 amino acids in length, preferably at least 150 amino acids in length.

[0172] According to the present disclosure, the albumin can be a naturally occurring albumin or a fragment or variant thereof. The albumin can be human albumin and can be derived from any vertebrate, particularly any mammal. In one embodiment, the albumin comprises the amino acid sequence of SEQ ID NO: 4 or 5 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: 4 or 5.

[0173] 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 a therapeutic protein fused to both the N-terminus and the C-terminus of albumin. In a preferred embodiment, the therapeutic proteins fused at the N-terminus and the C-terminus are the same therapeutic protein. In another preferred embodiment, the therapeutic proteins fused at the N-terminus and the C-terminus are different therapeutic proteins. In one embodiment, both different therapeutic proteins are cytokines.

[0174] 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.

[0175] 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 native 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 certain embodiments, an Fc domain comprises a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof). In certain embodiments, an Fc domain consists of a CH3 domain or a portion thereof. In certain embodiments, an Fc domain consists of a hinge domain (or a portion thereof) and a CH3 domain (or a portion thereof). In certain embodiments, an Fc domain consists of a CH2 domain (or a portion thereof) and a CH3 domain. In certain embodiments, an Fc domain consists of a hinge domain (or a portion thereof) and a CH2 domain (or a portion thereof). In certain embodiments, an 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. Fc domains encompass 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).

[0176] 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.

[0177] In certain 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 encompassed by the term "Fc domain" for purposes of this disclosure). The Fc domain does not comprise the variable region that binds to the antigen. Fc domains suitable for use in the present disclosure can be obtained from several different sources. In certain embodiments, the Fc domain is derived from a human immunoglobulin. In certain embodiments, the Fc domain is derived from a human IgG1 constant region. However, it is understood that the Fc domain may also be derived from the 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).

[0178] 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.

[0179] 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.

[0180] In certain 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, which are incorporated by reference in their entireties. In certain embodiments, the extended PK group is transferrin, as disclosed in U.S. Patent Nos. 7,176,278 and 8,158,579, which are incorporated by reference in their entireties. In certain embodiments, the extended PK group is a serum immunoglobulin binding protein, such as those disclosed in US Patent Application No. 2007 / 0178082, US Patent Application No. 2014 / 0220017, and US Patent Application No. 2017 / 0145062, the entire contents of which are incorporated herein by reference. In certain embodiments, the extended PK group is a fibronectin (Fn)-based scaffold domain protein that binds to serum albumin, such as those disclosed in US Patent Application No. 2012 / 0094909, the entire contents of which are incorporated herein by reference. A method for producing a fibronectin-based scaffold domain protein is also disclosed in US 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.

[0181] 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.

[0182] 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 certain embodiments, the linker is a glycine-serine polypeptide linker, i.e., a peptide consisting of glycine and serine residues.

[0183] 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).

[0184] antigen receptor The immune effector cells described herein can express an antigen receptor, such as a T cell receptor (TCR) or a chimeric antigen receptor (CAR)-bound antigen, or a processed product thereof, particularly when present on or presented by a target cell. The cells can naturally express the antigen receptor or can be modified to express the antigen receptor (e.g., ex vivo / in vitro or in vivo in the subject to be treated). Additionally, the immune effector cells can express an IL2R or can be modified to express an IL2R (e.g., ex vivo / in vitro or in vivo in the subject to be treated). In one embodiment, the modification to express the IL2R and the modification to express the antigen receptor are performed ex vivo / in vitro, either simultaneously or at different times. The modified cells can then be administered to a patient. In one embodiment, the modification to express the IL2R is performed ex vivo / in vitro, and the modification to express the antigen receptor is performed in vivo after the cells are administered to the patient. In one embodiment, the modification to express the antigen receptor is performed ex vivo / in vitro, and after the cells are administered to a patient, the modification to express the IL2R is performed in vivo. In one embodiment, the modification to express the IL2R and the modification to express the antigen receptor are performed in vivo at the same time or at different times. The cells may be endogenous cells of the patient or may have been administered to the patient.

[0185] Chimeric Antigen Receptor 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 (genetically modified) to express a CAR that specifically targets an antigen on the patient's tumor cells, and then infused back into the patient.

[0186] According to the present invention, 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.

[0187] 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, the CARs of the present invention target antigens, such as tumor antigens, on diseased cells, such as tumor cells.

[0188] In one embodiment, the binding domain in the CAR specifically binds to an antigen. In one embodiment, the antigen to which the binding domain in the CAR binds is expressed in 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.

[0189] In one embodiment of the present invention, the antigen-binding domain comprises a variable region (VH) of an immunoglobulin heavy chain having specificity for an antigen and a variable region (VL) of an immunoglobulin light chain 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, part of the antigen-binding portion in the CAR is an scFv.

[0190] 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.When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane domains 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 contains 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.

[0191] In some cases, the CAR of the present invention comprises a hinge domain that forms a link between the transmembrane domain and the extracellular domain.

[0192] The cytoplasmic domain of a CAR, or alternatively, the intracellular signaling domain, is responsible for 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.

[0193] 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).

[0194] 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.

[0195] 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).

[0196] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other in random or specified 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.

[0197] 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.

[0198] The term "antibody" includes immunoglobulins comprising at least two heavy (H) chains and two light (L) chains inter-connected 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).

[0199] 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 in 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.

[0200] The term "antibody fragment" refers to a portion of an intact antibody, typically comprising the antigen-determining variable region of the intact antibody.

[0201] 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.

[0202] As used herein, "antibody heavy chain" refers to the larger of the two polypeptide chains present in antibody molecules in their native conformations.

[0203] 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.

[0204] 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.

[0205] Genetic modification of immune effector cells Various methods can be used to introduce antigen receptors, such as IL2 receptors and / or CAR constructs, into cells, such as T cells, to produce cells genetically modified to express the IL2 receptor and / or antigen receptor. These 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 generate and efficiently and persistently transduce T cells, and have been preliminarily proven to be safe in terms of integration in 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.

[0206] In one embodiment of all aspects of the invention, T cells or T cell precursors are transfected either ex vivo or in vivo with a nucleic acid encoding an IL2 receptor and / or a nucleic acid encoding an antigen receptor. In one embodiment, a combination of ex vivo and in vivo transfection may be used. In one embodiment of all aspects of the invention, the T cells or T cell precursors are derived from a subject to be treated. In one embodiment of all aspects of the invention, the T cells or T cell precursors are derived from a subject different from the subject to be treated.

[0207] CAR T cells can be generated in vivo, thus almost instantly, using nanoparticles targeted to T cells. For example, poly(β-amino ester)-based nanoparticles can be coupled to anti-CD3e F(ab) fragments to bind to CD3 on T cells. Upon binding to T cells, these nanoparticles are endocytosed. Their contents, such as plasmid DNA encoding the antitumor antigen CAR, contain peptides containing microtubule-associated sequences (MTAS) and nuclear localization signals (NLS), and thus can be targeted to the T cell nucleus. The inclusion of a separate plasmid encoding a transposon and hyperactive transposase flanking the CAR gene expression cassette can enable efficient integration of the CAR vector into chromosomes. Such a system, enabling in vivo production of CAR T cells after nanoparticle injection, is described in Smith et al. (2017) Nat. Nanotechnol. 12:813-820.

[0208] Another possibility is to use CRISPR / Cas9 technology to deliberately place a CAR coding sequence at a specific genetic locus, for example, knocking out an existing T cell receptor (TCR) while knocking in the CAR and placing it under the dynamic regulatory control of an endogenous promoter that would otherwise silence TCR expression; see, e.g., Eyquem et al. (2017) Nature 543:113-117.

[0209] In one embodiment of all aspects of the invention, cells genetically modified to express one or more IL2 receptor polypeptides and / or antigen receptors are stably or transiently transfected with nucleic acids encoding the IL2 receptor polypeptides and / or antigen receptors. In one embodiment, the cells are stably transfected with some nucleic acids and transiently transfected with other nucleic acids. Thus, the nucleic acids encoding the IL2 receptor polypeptides and / or antigen receptors may or may not be integrated into the genome of the cells.

[0210] In one embodiment of all aspects of the invention, the cells genetically modified to express an antigen receptor are inactivated with respect to expression of endogenous T cell receptors and / or endogenous HLA.

[0211] 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 subsequent re-delivery of the cells 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, if any, can be performed in vivo.

[0212] 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.

[0213] 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.

[0214] 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.

[0215] 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.

[0216] antigen In one embodiment, the method described herein further comprises contacting 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 the subject being treated, with a cognate antigen molecule, wherein the antigen molecule or a processing product thereof, e.g., a fragment thereof, binds to the antigen receptor, such as a TCR or CAR, carried by the immune effector cells. Such cognate antigen molecules are also referred to herein as "vaccine antigens" or "peptides or proteins containing an epitope for inducing an immune response against an antigen." In one embodiment, the cognate antigen 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 antigen molecule under conditions that result in the expansion and / or activation of the immune effector cells. In one embodiment, the step of contacting the immune effector cells with the cognate antigen molecule is performed in vivo or ex vivo.

[0217] In one embodiment, the method described herein comprises administering a cognate antigen molecule or a nucleic acid encoding the same to a subject. In one embodiment, the nucleic acid encoding the cognate antigen molecule is expressed in the subject's cells to provide the cognate antigen molecule. In one embodiment, the cognate antigen molecule is expressed on the cell surface. In one embodiment, the nucleic acid encoding the cognate antigen molecule is transiently expressed in the subject's cells. In one embodiment, the nucleic acid encoding the cognate antigen molecule is RNA. In one embodiment, the cognate antigen molecule or a nucleic acid encoding the same is administered systemically. In one embodiment, systemic administration of the nucleic acid encoding the cognate antigen molecule results in expression of the nucleic acid encoding the cognate antigen molecule in the spleen. In one embodiment, systemic administration of the nucleic acid encoding the cognate antigen molecule results in expression of the nucleic acid encoding the cognate 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 cognate antigen molecule results in no or essentially no expression of the nucleic acid encoding the cognate antigen molecule in the lung and / or liver. In one embodiment, following systemic administration of nucleic acid encoding a cognate antigen molecule, expression of nucleic acid encoding a cognate antigen molecule in the spleen is at least 5-fold higher than expression 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 the peptide and protein antigens), i.e., vaccine antigens, preferably result in the stimulation, priming, and / or expansion of immune effector cells in the subject to which the peptide or protein antigen or nucleic acid is administered. 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, where 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 targeted by an antigen receptor on an immune effector cell 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. Preferably, a vaccine antigen (similar to a disease-associated antigen) provides a relevant epitope for binding by an antigen-binding domain present on immune effector cells.In one embodiment, the vaccine antigen (similar to the disease-associated antigen) is expressed on the surface of a cell, such as an antigen-presenting cell, to provide relevant epitopes for binding by immune effector cells. The vaccine antigen may be a recombinant antigen.

[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 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" particularly includes 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. An antigen or its processing product, such as a T cell epitope, is bound by an antigen receptor in one embodiment. 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 a 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 the outside of said 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; they 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 vaccine antigen 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 vaccine antigen preferably comprises an epitope or fragment of the neoantigen containing 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 (termed "pentatopes") and RNA encoding at least 10 epitopes (termed "decatopes").

[0236] According to various aspects of the 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. In one embodiment, the invention involves the administration of antigen receptor-engineered immune effector cells, such as T cells, that target cancer cells expressing tumor antigens.

[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 some embodiments, peptides can be greater than 50 amino acids. In some embodiments, peptides can be greater than 100 amino acids.

[0238] According to the present invention, vaccine antigens must be recognizable by immune effector cells. Preferably, when recognized by immune effector cells, the antigen can induce stimulation, priming, and / or expansion of immune effector cells bearing antigen receptors that recognize the antigen in the presence of appropriate costimulatory signals. In the context of embodiments of the present invention, the antigen is preferably present on the surface of a cell, preferably on the surface of an antigen-presenting cell. Recognition of the antigen on the surface of diseased cells can result in an immune response against the antigen (or cells expressing the antigen).

[0239] In one embodiment of all aspects of the present invention, the antigen is expressed on a diseased cell, such as a cancer cell. In one embodiment, the antigen is expressed on the surface of a diseased cell, such as a cancer cell. In one embodiment, the antigen receptor is a CAR that binds to the extracellular domain of the antigen or an epitope of the extracellular domain. In one embodiment, the CAR binds to a native epitope of the antigen present on the surface of a living cell. In one embodiment, binding of a CAR, when expressed by and / or present on a T cell, to an antigen present on a cell, such as an antigen-presenting cell, results in stimulation, priming, and / or expansion of the T cell. In one embodiment, binding of a CAR, when expressed by and / or present on a T cell, to an antigen present on a diseased cell, such as a cancer cell, results in cytolysis and / or apoptosis of the diseased cell, and the T cell preferably releases cytotoxic factors, such as perforin and granzymes.

[0240] Immune checkpoint inhibitors In certain embodiments, immune checkpoint inhibitors are used in combination with other therapeutic agents described herein.

[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 certain embodiments, the immune checkpoint is an inhibitory signal. In certain embodiments, the inhibitory signal is the interaction between PD-1 and PD-L1. In certain embodiments, the inhibitory signal is the interaction between CTLA-4 and CD80 or CD86, which displaces CD28 binding. In certain embodiments, the inhibitory signal is the interaction between LAG3 and an MHC class II molecule. In certain 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 certain embodiments, an immune checkpoint inhibitor prevents inhibitory signals associated with an immune checkpoint. In certain embodiments, an immune checkpoint inhibitor is an antibody or fragment thereof that interferes with inhibitory signaling associated with an immune checkpoint. In certain embodiments, an immune checkpoint inhibitor is a small molecule that interferes with inhibitory signaling. In certain embodiments, an immune checkpoint inhibitor is an antibody, fragment thereof, or antibody mimetic that interferes with the interaction between checkpoint blocker proteins, e.g., an antibody or fragment thereof that interferes with the interaction between PD-1 and PD-L1. In certain embodiments, an immune checkpoint inhibitor is an antibody or fragment thereof that interferes with the interaction between CTLA-4 and CD80 or CD86. In certain embodiments, an immune checkpoint inhibitor is an antibody or fragment thereof that interferes with the interaction between LAG3 and its ligand, or TIM-3 and its ligand. A checkpoint inhibitor may also be in the form of a soluble form of the molecule (or a 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 certain embodiments, the immune checkpoint inhibitor is an antibody or antigen-binding portion thereof that disrupts or inhibits signaling from an inhibitory immunoregulator. In certain embodiments, the immune checkpoint inhibitor is a small molecule that disrupts or inhibits signaling from an inhibitory immunoregulator.

[0251] In certain embodiments, the inhibitory immunomodulator is a component of the PD-1 / PD-L1 signaling pathway. Accordingly, certain 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 certain embodiments, the antibody, or antigen-binding portion thereof, specifically binds to PD-1. In certain 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 certain embodiments, the inhibitory immunomodulator is a component of the CTLA4 signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administering to a subject an antibody or antigen-binding portion thereof that targets CTLA4 and 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 certain embodiments, the inhibitory immunomodulator is a component of the B7 family signaling pathway. In certain embodiments, the B7 family members are B7-H3 and B7-H4. Therefore, certain embodiments of the present disclosure provide for administering to a subject an antibody or antigen-binding portion thereof that targets B7-H3 or B7-H4. Although 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] RNA targeting According to the present invention, it is particularly preferred that the peptides, proteins or polypeptides described herein, in particular IL2 polypeptides, IFN 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 forms a complex 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 include targeted delivery of an RNA disclosed herein (eg, an RNA encoding an IL2 polypeptide, an RNA encoding an IFN polypeptide, and / or an RNA encoding a vaccine antigen).

[0261] In one embodiment, the present disclosure includes targeting the lymphatic system, particularly secondary lymphoid organs, more particularly the spleen.When the administered RNA is an RNA encoding a vaccine antigen, it is particularly preferred to target the lymphatic system, particularly secondary lymphoid 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 using so-called lipoplex formulations, in which 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 recognized 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, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression occurs in the spleen. Thus, the RNA lipoplex particles of the present disclosure can be used to express RNA in the spleen. In one embodiment, after administration of the RNA lipoplex particles, no or essentially no RNA accumulation and / or RNA expression occurs in the lungs and / or liver. 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. Thus, 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 certain embodiments, cationic lipids are DOTMA and / or DOTAP.

[0267] Additional lipids may be incorporated to adjust the overall ratio of positive and 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 certain 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 certain 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] In one embodiment, the RNA lipoplex particles described herein 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 certain 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] At physiological pH, the spleen-targeted RNA lipoplex particles described herein preferably have a net negative charge, such as a positive to negative charge ratio of about 1.9:2 to about 1:2. In certain 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] Cytokines such as extended PK cytokines, particularly as described herein, extended PK interleukins can be provided to subjects by administering to subjects the RNA encoding cytokines in the formulation for selective delivery of RNA to liver or liver tissue.The delivery of RNA to such target organ or tissue is preferred, particularly when it is desired to express a large amount of cytokine, and / or when it is desired or required that cytokines are present systemically, particularly in significant amounts.

[0274] 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 complexes).

[0275] 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 excellent 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.

[0276] Pharmaceutical Composition The agents described herein may be administered in a pharmaceutical composition or medicament, or may be administered in the form of any suitable pharmaceutical composition.

[0277] In one embodiment of all aspects of the invention, the components described herein, such as a nucleic acid encoding a cytokine (IL2 or IFN) or a nucleic acid encoding an antigen, together or separately from each other, may be administered in a 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 the antigen is involved, such as a cancer disease as described herein.

[0278] The term "pharmaceutical composition" relates to a formulation containing a therapeutically active agent, preferably together with a pharmaceutically acceptable carrier, diluent and / or excipient. Said pharmaceutical composition is useful for treating, preventing or lessening the severity of a disease or disorder by administering said pharmaceutical composition to a subject. Pharmaceutical compositions are also known in the art as pharmaceutical formulations.

[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 "pharmacologically 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 a substantially 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 digestive tract, or parenteral administration. As used herein, "parenteral administration" refers to administration by any means other than via the digestive tract, such as by intravenous injection. In a preferred embodiment, the pharmaceutical composition is formulated for systemic administration. In another preferred embodiment, the systemic administration is by intravenous administration. In one embodiment of all aspects of the present invention, the RNA encoding the cytokine and optionally the RNA encoding the antigen described herein is administered systemically.

[0291] As used herein, the term "co-administration" refers to administering different compounds or compositions (e.g., RNA encoding an IL2 polypeptide, RNA encoding an IFN polypeptide, and optionally RNA encoding a vaccine antigen) to the same patient. The different compounds or compositions can be administered simultaneously, essentially simultaneously, or sequentially. In one embodiment, the IL2 polypeptide or nucleic acid encoding the IL2 polypeptide is administered first, followed by the IFN polypeptide or nucleic acid encoding the IFN polypeptide.

[0292] treatment The agents, compositions and methods described herein can be used to treat subjects with diseases, such as diseases characterized by the presence of diseased cells that express antigens.Particularly preferred diseases are cancer diseases.For example, when the antigen is derived from a virus, the agents, compositions and methods can be useful for treating viral diseases caused by the virus.When the antigen is a tumor antigen, the agents, compositions and methods can be useful for treating cancer diseases in which cancer cells express the tumor antigen.

[0293] 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 factors from external sources, such as infection, or by internal malfunctions, such as autoimmune diseases. 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 damage, disability, disorder, 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.

[0294] In the present context, the terms "treatment", "treating" or "therapeutic intervention" relate to the management and care of a subject with the purpose 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 afflicted, such as the administration of therapeutically effective compounds to alleviate the symptoms or complications, slow the progression of the disease, disorder or condition, relieve or relieve the symptoms and complications, and / or cure or eliminate the disease, disorder or condition, as well as to prevent the condition, where prevention is to be understood as the management and care of an individual with the purpose of combating the disease, condition or disorder and includes the administration of active compounds to prevent the onset of symptoms or complications.

[0295] The term "therapeutic treatment" relates to any treatment that improves the health status and / or prolongs (increases) the lifespan of an individual. Said 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.

[0296] 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.

[0297] 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."

[0298] The term "patient" refers to an individual or subject for treatment, particularly an afflicted individual or subject.

[0299] 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.

[0300] As used herein, "immune response" refers to the body's orchestrated response to an antigen or a cell expressing an antigen, and refers to a cellular immune response and / or a humoral immune response.

[0301] "Cell-mediated immunity," "cellular immunity," "cellular immune response," or similar terms are intended to include a cellular response to 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 known as killer cells (cytotoxic T cells, cytolytic T cells, CD8 + T cells (also called CTLs) kill diseased cells, such as cancer cells, and prevent the production of further diseased cells.

[0302] 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)."

[0303] 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.

[0304] 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.

[0305] The term "macrophage" refers to a subgroup of phagocytes produced by differentiation of monocytes. Activated by inflammation, immune cytokines, or microbial products, macrophages nonspecifically engulf and kill foreign pathogens within the macrophage through hydrolytic and oxidative attack, resulting in the degradation of the pathogen. 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 the activation of T and B cells and further stimulation of the immune response. Macrophages belong to a class of antigen-presenting cells. In one embodiment, the macrophages are splenic macrophages.

[0306] 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 progenitors are first converted into immature dendritic cells (DCs). These immature cells are characterized by high phagocytic activity and low T cell activation capacity. Immature dendritic cells constantly sample the 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 act 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 act 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.

[0307] 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 antigenic fragment on (or at) their cell surface. Antigen-presenting cells can be distinguished into professional and non-professional antigen-presenting cells.

[0308] 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.

[0309] 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.

[0310] "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.

[0311] The term "antigen-associated disease" refers to any disease related to an antigen, e.g., a disease characterized by the presence of an antigen. The antigen-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. In one embodiment, the antigen-associated disease is a disease involving cells that express the antigen, preferably on their cell surface.

[0312] The term "infectious disease" refers to any disease (e.g., the common cold) that can be transmitted from individual to individual or 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.

[0313] 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.

[0314] 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). The present disclosure contemplates the use of antibodies as immunotherapeutic agents. Without wishing to be bound by theory, antibodies can achieve therapeutic effects on 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), bivacizumab mertansine (CD44 v6), blinatumomab (CD19), 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), samalizumab (CD200), sinostat Brotuzumab (FAP), siltuximab (IL6), tabalumab (BAFF), tacatuzumab tetraxetan (α-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 (4-1BB), boroximab (integrin α5β1), votumumab (tumor antigen CTAA 16.88), zalutumumab (EGFR), and zanolimumab (CD4).

[0315] Citation of documents and tests 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 any admission as to the accuracy of the contents of these documents.

[0316] 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]

[0317] Example 1: Construct design and mRNA production In vitro transcription of cytokine-encoding mRNA was based on the pST1-T7-AGA-dEarI-hAg-MCS-FI-A30LA70 plasmid backbone and derivative DNA constructs. These plasmid constructs contained a 5' UTR (untranslated region, a derivative of the 5'-UTR of human hemoglobin subunit α1 (hAg)), a 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 humans; WO 2017 / 060314), and a 100-nucleotide poly(A) tail with a linker after 70 nucleotides. The cytokine and serum albumin (Alb) coding sequences were derived from Mus musculus (abbreviated as m, i.e., mAlb, mIL2, or mIFNα) or human (abbreviated as h, i.e., hAlb, hIL2, or hIFNα), and no changes were introduced into the resulting amino acid sequences. Alb was introduced at the N-terminus of the mature IL2 sequence (the IL2 signal peptide was not encoded). A stop codon was introduced only at the most C-terminal end. The different protein moieties in the cytokine and hAlb fusion constructs were separated by a 30-nucleotide linker sequence encoding glycine and serine residues. mRNA was produced by in vitro transcription as described by Kreiter et al. (Kreiter, S. et al. Cancer Immunol. Immunother. 56, 1577-87 (2007)). For all cytokine / albumin-encoding RNAs, the normal nucleoside uridine was replaced with 1-methylpseudouridine. The resulting mRNA was equipped with a Cap1 structure and depleted of double-stranded (dsRNA) molecules. The purified cytokine / albumin mRNA was eluted in 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] TIFF2025131724000016.tif212153TIFF2025131724000017.tif85153

[0318] Example 2: Effect of RNA-encoded IL2 on vaccine-induced T cell responses in vivo Female C57BL / 6 mice (9 weeks old) (n = 8 mice per group) were purchased from Envigo and transfected with chicken ovalbumin (OVA)-derived H2-Kb-restricted CD8 +Mice were vaccinated intravenously (iv) with 10 μg of mRNA lipoplex (RNA-L) encoding the T cell epitope SIINFEKL (Kranz, LM et al. Nature 534, 396-401 (2016)). Three days after each vaccination, 1 μg of mRNA encoding a mAb fusion protein formulated with TransIT (Mirrus) was administered intravenously. Mice were administered either mAb or mIL2 fused to mAb (mAb-mIL2). On days 7, 14, and 21, mouse blood was collected and analyzed for antigen-specific T cell responses and regulatory T cells (Tregs) by flow cytometry. 50 μl of blood was stained with fluorochrome-conjugated antibodies or MHC tetramers for 30 minutes at 2-8°C. Antigen-specific T cells were detected by co-staining with CD45-specific antibodies (30-F11, BD) and CD8-specific antibodies (clone 5H10, BD) and MHC tetramer conjugated to the SIINFEKL peptide (MBL International). Tregs were identified using antibodies specific for CD45 (30-F11, BD), CD4 (clone RM4-5, Biolegend), CD25 (clone PC61, BD), and FoxP3 (clone FJK-16s, eBioscience) using the FoxP3 staining kit from eBioscience according to the manufacturer's instructions. Blood was lysed using lysis solution (BD FACS™). To determine absolute cell counts, cells were transferred to Trucount® tubes (BD). Flow cytometry data were acquired using an LSRFortessa flow cytometer (BD) and analyzed with FlowJo X software (Tree Star). Results were displayed and statistically analyzed using GraphPad Prism 7. Figure 1A shows an overview of the treatment and analysis schedule. The combination of RNA-L vaccination and treatment with mAlb-mIL2 resulted in a significant increase in OVA-specific T cells on days 7 and 14 compared with the control group that received RNA-L vaccination and mAlb alone (Figures 1B and 1C). However, analysis on day 21 revealed a significant decrease in antigen-specific T cell responses in mAlb-mIL2-treated animals, with frequencies significantly lower than even those of mAlb controls (Figures 1D and 1E).We hypothesized that this decrease in antigen-specific T cells was caused by mAlb-mIL2-induced Tregs. Indeed, mAlb-mIL2-treated mice showed significantly higher Treg frequencies at each time point measured, peaking on day 14 (Fig. 1F). Presumably, at early time points, the stimulatory ability of mAlb-mIL2 exceeds the inhibitory ability of Tregs. At later time points, the increased number of Tregs provides a stronger inhibitory signal to activated CD8+ T cells, reducing the stimulatory ability of mAlb-mIL2.

[0319] To confirm these results in a second model of a different background, BALB / c mice (n = 7-8 per group, female, 9 weeks old, purchased from Janvier Labs) were treated with 20 μg of gp70 RNA-L vaccination encoding the H2-Kd-restricted CD8 T cell antigen SPSYAYHQF (Kranz, LM et al. Nature 534, 396-401 (2016)) combined with 1 μg of mAlb-mIL2 or mAlb, as described in Figure 2A. Again, treatment with mAlb-mIL2 resulted in a significant but transient increase in gp70-specific T cells only on days 7 and 14 (Figures 2B, 2C). By day 21, antigen-specific T cell numbers had returned to the level of mAlb controls (Figures 2D, 2E). As previously shown, Treg frequencies were significantly elevated in all measurements (Figure 2F).

[0320] Example 3: IFNα limits IL2-mediated Treg expansion and leads to robust priming of antigen-specific T cells in vivo It has been demonstrated that IFNα administration can reduce Treg frequency and impair Treg function in mice (Gangaplara, A. et al. PLoS Pathog. 14, 1-27 (2018)) and humans (Tatsugami, K., Eto, M. & Naito, S.J. Interferon Cytokine Res. 30, 43-48 (2010)). However, IFNα also plays a positive role in Treg development (Metidji, A. et al. J. Immunol. 194, 4265-4276 (2015)). We hypothesized that IFNα may enhance the effect of IL2 on vaccine-induced T cell priming by antagonizing immediate IL2-mediated Treg activation and expansion, thereby preventing subsequent T cell proliferation inhibition by Tregs. Therefore, C57BL / 6 mice (9 weeks old, n = 5 mice per group, purchased from Envigo) were vaccinated intravenously with 10 μg of OVA RNA-L in 100 μl on days 0, 7, and 14, as described in Example 2. Three days later, mice were stimulated with 1 μg of mRNA encoding mAlb or mAlb-mIL2 in 100 μl formulated with TransIT (Mirrus). Concurrently, one mAlb-mIL2-treated group received 2 μg of mRNA encoding IFNα in 100 μl formulated with TransIT (Mirrus). As shown in Figure 3A, a second treatment with 10 μg of OVA RNA-L and cytokine-encoding RNA was administered on day 7. On days 7 and 14, mouse blood was collected and analyzed for antigen-specific T cell responses and Tregs by flow cytometry, as described in Example 2. As expected, on day 7, vaccine-induced OVA-specific T cells and Tregs were more frequent when mice were treated with mAlb-mIL2 (Fig. 3B, 3C). Coadministration of IFNα did not limit or increase the number of antigen-specific T cells (Fig. 3B), but did reduce the number of Tregs to baseline levels in mAlb controls (Fig. 3C).As a result, analysis on day 14 showed that OVA-specific T cells were significantly elevated in the group administered IFNα together with mAlb-mIL2, whereas treatment with mAlb-mIL2 alone had no advantage compared to the control group (Figure 3D).

[0321] Confirmation of these results was performed in the gp70 model described in Example 2 and Figure 2. Experiments were performed similarly to those described above. BALB / c mice (n = 5 per group) were vaccinated with 20 μg of gp70 RNA-L in 100 μl on days 0, 7, and 14. Cytokine RNA (1 μg mAlb, 1 μg mAlb-mIL2, or 1 μg mAlb-mIL2 + 2 μg IFNα) was injected in 100 μl on days 3, 7, and 14, and mouse blood was examined on days 7 and 21 (Figure 4A). Again, IFNα was able to normalize the mAlb-mIL2-mediated increase in Treg frequency without limiting the expansion of antigen-specific T cells (Figures 4B and 4C), resulting in an increase in antigen-specific T cells on day 21 (Figure 4D).

[0322] Example 4: IFNα limits IL2-mediated Treg expansion without impairing CD8+ T cell expansion in vitro To confirm the beneficial effects of IFNα observed in vivo, we evaluated the effects of hIFNα on IL2-stimulated CD4+CD25+ Tregs and CD8+ T cells in vitro. Tregs and autologous bulk PBMCs were cocultured at a 1:1 ratio in the presence of a suboptimal concentration of anti-CD3 antibody (clone UCHT1) and 5% hAlb-hIL2-containing supernatant, and were either further treated with IFNα or maintained without IFNα. Briefly, human PBMCs were obtained from the buffy coat of healthy donors by Ficoll-Paque (VWR International, catalog no. 17-1440-03) density gradient separation, and CD4+CD25+ Tregs were isolated from freshly prepared PBMCs (CD4 + CD25 +Regulatory T Cell Isolation Kit Human, Miltenyi Biotec, Catalog No. 130-091-301). Bulk PBMCs were labeled using 1.6 μM carboxyfluorescein succinimidyl ester (CFSE; Thermo Fisher, Catalog No. C34554), and Tregs were labeled using 1 μM CellTrace FarRed dye (Thermo Fisher, Catalog No. C34564). 30,000 CFSE-labeled PBMCs and 30,000 FarRed-labeled Tregs were cocultured 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) per well in 96-well round-bottom plates (Costar, catalog no. 734-1797) and incubated with a suboptimal concentration of anti-CD3 antibody (UCHT1; R&D Systems, catalog no. MAB100; final concentration 0.09 μg / mL). PBMC:Treg cocultures were treated with hAlb-hIL2-containing supernatant (final concentration 5%) and supplemented with either no recombinant IFNα2b, 625 U / mL, or 10,000 U / mL recombinant IFNα2b (pbl assay science, catalog no. 11105-1). Co-cultures were stimulated for 4 days at 37°C and 5% CO2. Cells were harvested and analyzed by flow cytometry, excluding dead cells with eFluor 780 staining (eBioscience, catalog no. 65-0865-18). CD8+ T cells were identified by staining with anti-human CD8 PE-Cy7 antibody (TONBO Biosciences, catalog no. 60-0088) diluted 1:100 in FACS buffer (DPBS + 2% FBS + 2mM EDTA). Flow cytometry analysis was performed on a BD FACS Canto II flow cytometer (Becton Dickinson) using CFSE dilutions (CD8+ T cells) and FarRed dilutions (Tregs) as proliferation readouts.The acquired proliferation data were analyzed using FlowJo 10.5 software (TreeStar, Inc.), and the exported proliferation index values ​​were plotted in GraphPad Prism 6 (GraphPad Software, Inc.).

[0323] Both CD8+ T cells and CD4+CD25+ Tregs responded with robust proliferation to combined anti-CD3 and hAlb-hIL2 treatment. Addition of recombinant IFNα reduced Treg proliferation by approximately 50-55%, whereas CD8+ T cell proliferation was reduced by only approximately 10-20%. The selective inhibitory effect of Treg proliferation was observed for both PBMC donors tested, but not for CD8+ T cells (Figures 5A and 5B), and was largely independent of the IFNα concentration used.

[0324] Example 5: Combination therapy of IFNα and IL2 produces synergistic antitumor effects in mice Next, we evaluated whether the addition of IFNα to IL2 therapy would improve the antitumor effect. Female BALB / c mice (9 weeks old, n = 12 mice per group) were purchased from Janvier Labs SAS and inoculated with 5 × 10 5 CT26 colon cancer cells were injected subcutaneously (sc). 15 days after tumor cell inoculation, 10–11 mice per group were stratified based on tumor size. Mice were treated five times with 1 μg of mAlb-mIL2-encoding RNA and 2 μg of IFNα-encoding mRNA, separately formulated in 100 μL of TransIT (Mirrus). Control groups received either IFNα, IL2, or irrelevant RNA (1 μg of mAlb-encoding RNA) formulated in TransIT. Tumor size was measured three times weekly with calipers and calculated using the formula (a 2 The calculation was made using (a, width; b, length) / 2(a × b). Patients were excluded if they showed signs of health problems or if the tumor volume exceeded 1500 mm. 3 On days 29 and 35, blood was collected from the mice and CD8 + T cells were analyzed by flow cytometry as described in Example 2. Figure 6A shows the experimental outline.

[0325] Of note, because CT26 tumors are immunogenic in themselves and tumor-specific T cells can be primed without vaccination, especially under IL2 treatment, we did not add a vaccine in this experiment. Furthermore, in murine tumors, early intervention, especially in these tumors, determines treatment outcomes. Thus, IL2-mediated boosting of vaccine-induced T cells leads to potent tumor control, and subsequent inhibition of T cells by IL2-stimulated Tregs is not significant. In contrast, without vaccination, the boosting effect of IL2 on tumor-specific T cells is less pronounced, and therefore the inhibitory effect of Tregs on tumor-specific T cells is more significant.

[0326] Significant therapeutic activity was detected in the group treated with the combination of IFNα and IL2, whereas IL2 or IFNα monotherapy had no effect on tumor growth (Fig. 6B). In total, 4 of 11 mice (36%) rejected their tumors under combination therapy, whereas only 2 (18%) or none of the mice were tumor-free after monotherapy with IFNα or IL2, respectively (Fig. 6C). IL2 and IFNα treatment conferred a significant survival benefit compared with IL2 monotherapy (Fig. 6D). Importantly, the therapeutic activity of IL2 and IFNα combination therapy was significantly enhanced by the increase in blood CD8 + As in Figures 2E and 3D, IL2 monotherapy resulted in a consistent and significant increase in CD8 T cells at day 29. + It mediated only a transient increase in T cells, which normalized by day 35 (Fig. 6E).

Claims

1. 1. A method for inducing an immune response in a subject, comprising: a. a polypeptide comprising IL2 or a functional variant thereof or a polynucleotide encoding a polypeptide comprising IL2 or a functional variant thereof; and b. A polypeptide comprising a type I interferon or a functional variant thereof, or a polynucleotide encoding a polypeptide comprising a type I interferon or a functional variant thereof to said subject.

2. c. A peptide or protein containing an epitope for inducing an immune response against the antigen in the subject, or a polynucleotide encoding said peptide or protein.

10. The method of claim 1, further comprising administering to the subject

3. 3. The method of claim 1 or 2, wherein the polynucleotide encoding a polypeptide comprising IL2 or a functional variant thereof is RNA, the polynucleotide encoding a polypeptide comprising a type I interferon or a functional variant thereof is RNA, and optionally the polynucleotide encoding the peptide or protein is RNA.

4. 1. A method for inducing an immune response in a subject, comprising: a. RNA encoding a polypeptide comprising IL2 or a functional variant thereof; and b. RNA encoding a polypeptide comprising a type I interferon or a functional variant thereof to said subject.

5. c. RNA encoding a peptide or protein containing an epitope for inducing an immune response to the antigen in the subject.

5. The method of claim 4, further comprising administering to the subject

6. The method of any one of claims 1 to 5, wherein the immune response is a T cell-mediated immune response.

7. The method of any one of claims 1 to 6, wherein the subject has a disease, disorder or condition associated with expression or elevated expression of an antigen.

8. 1. A method for treating a subject having a disease, disorder, or condition associated with expression or up-regulation of an antigen, comprising: a. a polypeptide comprising IL2 or a functional variant thereof or a polynucleotide encoding a polypeptide comprising IL2 or a functional variant thereof; b. a polypeptide comprising a type I interferon or a functional variant thereof or a polynucleotide encoding a polypeptide comprising a type I interferon or a functional variant thereof; and c. A peptide or protein comprising an epitope for inducing an immune response against the antigen in the subject, or a polynucleotide encoding the peptide or protein to said subject.

9. 9. The method of claim 8, wherein the polynucleotide encoding a polypeptide comprising IL2 or a functional variant thereof is RNA, the polynucleotide encoding a polypeptide comprising a type I interferon or a functional variant thereof is RNA, and the polynucleotide encoding the peptide or protein is RNA.

10. 1. A method for treating a subject having a disease, disorder, or condition associated with expression or up-regulation of an antigen, comprising: a. RNA encoding a polypeptide comprising IL2 or a functional variant thereof; b. RNA encoding a polypeptide comprising a type I interferon or a functional variant thereof; and c. RNA encoding a peptide or protein containing an epitope for inducing an immune response against the antigen in the subject. to said subject.

11. The method of any one of claims 7 to 10, wherein the disease, disorder or condition is cancer and the antigen is a tumor-associated antigen.

12. The method according to any one of claims 1 to 11, wherein the polypeptide comprising IL2 or a functional variant thereof is extended pharmacokinetic (PK) IL2.

13. The method of claim 12 , wherein the extended PK IL2 comprises a fusion protein.

14. 14. The method of claim 13, wherein the fusion protein comprises a portion of 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.

15. 15. The method of claim 14, wherein the serum albumin comprises mouse serum albumin or human serum albumin.

16. 15. The method of claim 14, wherein the immunoglobulin fragment comprises an immunoglobulin Fc domain.

17. 17. A method for treating or preventing cancer in a subject according to any one of claims 1 to 16, wherein optionally the antigen is a tumor-associated antigen.

18. 18. The method according to any one of claims 1 to 17, wherein administration of a polypeptide comprising IL2 or a functional variant thereof or a polynucleotide, in particular RNA, encoding a polypeptide comprising IL2 or a functional variant thereof, and optionally a peptide or protein comprising an epitope for inducing an immune response against an antigen in said subject, or a polynucleotide, in particular RNA encoding said peptide or protein, induces antigen-specific T cells.

19. 19. The method according to any one of claims 1 to 18, wherein the administration of a polypeptide comprising a type I interferon or a functional variant thereof or a polynucleotide, in particular RNA, encoding a type I interferon or a polypeptide comprising a functional variant thereof reduces the number of regulatory T cells.

20. 20. The method according to any one of claims 1 to 19, wherein the administration of a polypeptide comprising a type I interferon or a functional variant thereof or a polynucleotide, in particular RNA, encoding a polypeptide comprising a type I interferon or a functional variant thereof reduces IL2-mediated expansion of regulatory T cells.

21. 21. The method according to any one of claims 1 to 20, wherein the administration of a polypeptide comprising a type I interferon or a functional variant thereof or a polynucleotide, in particular RNA, encoding a polypeptide comprising a type I interferon or a functional variant thereof increases the ratio of antigen-specific T cells to regulatory T cells.

22. The method according to any one of claims 1 to 21, wherein the type I interferon is interferon alpha.

23. a. a polypeptide comprising IL2 or a functional variant thereof or a polynucleotide encoding a polypeptide comprising IL2 or a functional variant thereof; and b. A polypeptide comprising a type I interferon or a functional variant thereof, or a polynucleotide encoding a polypeptide comprising a type I interferon or a functional variant thereof 10. A pharmaceutical formulation comprising:

24. c. A peptide or protein containing an epitope for inducing an immune response to an antigen in a subject, or a polynucleotide encoding said peptide or protein.

24. The pharmaceutical formulation of claim 23, further comprising:

25. 25. The pharmaceutical formulation of claim 23 or 24, wherein the polynucleotide encoding a polypeptide comprising IL2 or a functional variant thereof is RNA, the polynucleotide encoding a polypeptide comprising a type I interferon or a functional variant thereof is RNA, and optionally the polynucleotide encoding the peptide or protein is RNA.

26. The pharmaceutical preparation according to any one of claims 23 to 25, which is a kit.

27. 27. The pharmaceutical formulation of claim 26, wherein the polypeptide comprising IL2 or a functional variant thereof or a polynucleotide encoding a polypeptide comprising IL2 or a functional variant thereof, the polypeptide comprising a type I interferon or a functional variant thereof or a polynucleotide encoding a polypeptide comprising a type I interferon or a functional variant thereof, and optionally the peptide or protein or the polynucleotide encoding the peptide or protein are contained in separate containers.

28. 28. The pharmaceutical preparation of claim 26 or 27, further comprising instructions for using said pharmaceutical preparation for treating or preventing cancer, and optionally wherein said antigen is a tumor-associated antigen.

29. The pharmaceutical preparation according to any one of claims 23 to 25, which is a pharmaceutical composition.

30. 30. The pharmaceutical formulation of claim 29, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

31. a. RNA encoding a polypeptide comprising IL2 or a functional variant thereof; and b. RNA encoding a polypeptide comprising a type I interferon or a functional variant thereof 10. A pharmaceutical formulation comprising:

32. c. RNA encoding a peptide or protein containing an epitope for inducing an immune response to an antigen in a subject.

32. The pharmaceutical formulation of claim 31, further comprising:

33. 33. The pharmaceutical preparation of claim 31 or 32, which is a kit.

34. 34. The pharmaceutical formulation of claim 33, wherein the RNA encoding a polypeptide comprising IL2 or a functional variant thereof, the RNA encoding a polypeptide comprising a type I interferon or a functional variant thereof, and optionally the RNA encoding a peptide or protein are contained in separate containers.

35. 35. The pharmaceutical preparation of claim 33 or 34, further comprising instructions for using said pharmaceutical preparation for treating or preventing cancer, and optionally wherein said antigen is a tumor-associated antigen.

36. 33. The pharmaceutical preparation of claim 31 or 32, which is a pharmaceutical composition.

37. 37. The pharmaceutical formulation of claim 36, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents and / or excipients.

38. The pharmaceutical formulation of any one of claims 24 to 30 and 32 to 37, wherein the immune response is a T cell-mediated immune response.

39. The pharmaceutical formulation of any one of claims 23 to 38, wherein the polypeptide comprising IL2 or a functional variant thereof is extended pharmacokinetic (PK) IL2.

40. 40. The pharmaceutical formulation of claim 39, wherein the extended PK IL2 comprises a fusion protein.

41. 41. The pharmaceutical formulation of claim 40, wherein the fusion protein comprises a portion of 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.

42. 42. The pharmaceutical formulation of claim 41, wherein the serum albumin comprises mouse serum albumin or human serum albumin.

43. 42. The pharmaceutical preparation of claim 41, wherein the immunoglobulin fragment comprises an immunoglobulin Fc domain.

44. A pharmaceutical formulation according to any one of claims 23 to 43 for pharmaceutical use.

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

46. 46. ​​The pharmaceutical preparation of any one of claims 23 to 45, wherein the antigen is optionally a tumor-associated antigen, for use in a method for treating or preventing cancer in a subject.

47. The pharmaceutical formulation according to any one of claims 23 to 46, wherein the type I interferon is interferon alpha.

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