Treatment involving car-engineered t cells and cytokines

Abstract

To provide methods and agents for enhancing the effect of T cells engineered to express chimeric antigen receptors (CARs), wherein these methods and agents are, in particular, useful for the treatment of diseases characterized by diseased cells expressing an antigen which the CAR is directed to.SOLUTION: The present disclosure relates to methods comprising: providing to a subject T cells genetically modified to express a chimeric antigen receptor (CAR); and administering to the subject IL2 or a polynucleotide encoding IL2. The methods may comprise administering a further cytokine or a polynucleotide encoding a further cytokine, wherein the further cytokine may be IL7 or IL21.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to methods and agents for enhancing the effect of T cells engineered to express a chimeric antigen receptor (CAR). These methods and agents are particularly useful for the treatment of diseases characterized by diseased cells that express an antigen to which the CAR is directed. Specifically, the present disclosure relates to methods comprising providing to a subject T cells genetically modified to express a chimeric antigen receptor (CAR), and administering to the subject an interleukin-2 (IL-2) or a polynucleotide encoding IL-2. The methods of the present disclosure may comprise administering an IL-2 or a polynucleotide encoding IL-2, and a further cytokine or a polynucleotide encoding a further cytokine, which further cytokine may be IL-7 or IL-21. T cells genetically modified to express a CAR can be provided to a subject by administering T cells genetically modified to express a CAR, or by generating in a subject T cells genetically modified to express a CAR. The methods of the present disclosure may further comprise administering to the subject an antigen or a variant thereof, or a polynucleotide encoding an antigen or a variant thereof, wherein the T cells genetically modified to express a CAR target the antigen. In a particularly preferred embodiment, the polynucleotide administered according to the present disclosure is RNA.

Background Art

[0002] The immune system plays an important role in cancer, autoimmunity, allergy, and pathogen-related diseases. T cells play a central role in human and animal cell-mediated immunity. Recognition and binding of a specific antigen by T cells is mediated by the T cell receptor (TCR) expressed on the surface of T cells. The TCR of T cells can bind to major histocompatibility complex (MHC) molecules and interact with immunogenic peptides (epitopes) presented on the surface of target cells. Specific binding of the TCR initiates a signal cascade within the T cell that leads to proliferation and differentiation into mature effector T cells.

[0003] The diversity of TCRs is obtained by gene rearrangement of various discontinuous segments of genes encoding various structural regions of TCRs. TCRs are composed of one α chain and one β chain, or one γ chain and one δ chain. The TCR α / β chains are composed of a highly polymorphic variable region at the N-terminus involved in antigen recognition and a constant region that is invariant. At the gene level, these chains are divided into several regions, namely the variable (V) region, the diversity (D) region (only for the β and δ chains), the joining (J) region, and the constant (C) region. During T cell differentiation, a specific T cell receptor gene is created by rearranging one V region, one D region (only for the β and δ chains), one J region, and one C region gene. The diversity of TCRs is further amplified by inaccurate V-(D)-J rearrangement in which random nucleotides are introduced and / or deleted at the recombination sites. Since the rearrangement of TCR gene loci occurs within the genome during T cell maturation, each mature T cell expresses only one specific α / β TCR or γ / δ TCR. TCRs are part of a complex signaling mechanism that includes the heterodimeric complex of TCR α and β chains, the co-receptors CD4 or CD8, and the CD3 signaling module. The CD3 chains transmit activation signals intracellularly, while the TCR α / β heterodimer is only involved in antigen recognition.

[0004] Adoptive cell transfer (ACT)-based immunotherapy can be broadly defined as a form of passive immunity by previously sensitized T cells that are expanded ex vivo from low precursor cell frequencies to clinically relevant cell numbers and then transferred into non-immune recipients or autologous hosts. The cell types that have been used in ACT experiments are lymphokine-activated killer (LAK) cells (Mule, J. J. et al. (1984) Science 225, 1487-1489; Rosenberg, S. A. et al. (1985) N. Engl. J. Med. 313, 1485-1492), tumor-infiltrating lymphocytes (TIL) (Rosenberg, S. A. et al. (1994) J. Natl. Cancer Inst. 86, 1159-1166), donor lymphocytes after hematopoietic stem cell transplantation (HSCT), and tumor-specific T cell lines or clones (Dudley, M. E. et al. (2001) J. Immunother. 24, 363-373; Yee, C. et al. (2002) Proc. Natl. Acad. Sci. U.S.A 99, 16168-16173). Adoptive T cell transfer has been shown to have therapeutic activity against human virus infections such as CMV. CMV infection and reactivation of endogenous latent viruses are controlled by the immune system in healthy individuals but result in significant morbidity and mortality in immunocompromised individuals such as transplant recipients or AIDS patients. Riddell and co-workers demonstrated viral immune reconstitution by adoptive T cell therapy in immunosuppressed patients after transfer of CD8+ CMV-specific T cell clones derived from HLA-matched CMV-seropositive transplant donors (Riddell, S. R. (1992) Science 257, 238-241). As an alternative approach, CMV- or EBV-specific T cell populations derived from polyclonal donors were transferred into transplant recipients to increase the persistence of the transferred T cells (Rooney, C. M. et al. (1998) Blood 92, 1549-1555; Peggs, K. S. et al. (2003) Lancet 362, 1375-1377).For adoptive immunotherapy of melanoma, Rosenberg and co-workers established an ACT approach based on the continuous infusion of autologous tumor-infiltrating lymphocytes (TILs) isolated from resected tumors and expanded in vitro, in combination with non-myeloablative lymphocyte-depleting chemotherapy and high-dose IL-2. In a recently published clinical trial, an objective response rate of approximately 50% was obtained in treated patients with metastatic melanoma (Dudley, M.E. et al. (2005) J. Clin. Oncol. 23:2346-2357).

[0005] An alternative approach is the adoptive transfer of autologous T cells reprogrammed to express tumor-reactive immunoreceptors of defined specificity during short-term ex vivo culture, followed by reinfusion into the patient (Kershaw M.H. et al. (2013) Nature Reviews Cancer 13(8):525-41). This strategy enables the application of ACT to a variety of common malignancies, even when tumor-reactive T cells are not present in the patient. Since the antigen specificity of T cells depends entirely on the heterodimeric complex of the TCR α and β chains, the transfer of cloned TCR genes into T cells offers the potential to redirect them to the antigen of interest. Thus, TCR gene therapy provides an attractive strategy for developing antigen-specific immunotherapy using autologous lymphocytes as a treatment option. The main advantages of TCR gene transfer are the ability to produce therapeutic amounts of antigen-specific T cells within a few days and the ability to introduce specificities that do not exist in the patient's endogenous TCR repertoire.

[0006] Several groups have demonstrated that TCR gene transfer is an attractive strategy for redirecting the antigen specificity of primary T cells (Morgan, R. A. et al. (2003) J. Immunol. 171, 3287-3295; Cooper, L. J. et al. (2000) J. Virol. 74, 8207-8212; Fujio, K. et al. (2000) J. Immunol. 165, 528-532; Kessels, H. W. et al. (2001) Nat. Immunol. 2, 957-961; Dembic, Z. et al. (1986) Nature 320, 232-238). The feasibility of TCR gene therapy in humans was first demonstrated in a clinical trial for the treatment of melanoma by Rosenberg and his group. Adoptive transfer of autologous lymphocytes transduced with a retrovirus encoding a melanoma / melanocyte antigen-specific TCR resulted in tumor regression in up to 30% of treated melanoma patients (Morgan, R. A. et al. (2006) Science 314, 126-129; Johnson, L. A. et al. (2009) Blood 114, 535-546). On the other hand, clinical trials of TCR gene therapy have been extended to cancers other than melanoma, targeting a number of different tumor antigens (Park, T. S. et al., (2011) Trends Biotechnol. 29, 550-557).

[0007] The use of genetic engineering approaches to insert antigen-targeting receptors of defined specificities into T cells has greatly expanded the potential of ACT. Chimeric antigen receptors (CARs) are a type of antigen-targeting receptor consisting of an extracellular antigen-binding portion, most commonly a single-chain variable fragment (scFv) from a monoclonal antibody, fused to an intracellular T cell signaling domain. CARs recognize cell surface antigens directly, independent of MHC-mediated presentation, and enable the use of a single receptor construct specific for a given antigen in all patients. The first CARs fused the antigen recognition domain to the CD3ζ activation chain of the T cell receptor (TCR) complex. Subsequent CAR iterations included secondary co-stimulatory signals in tandem with CD3ζ, including intracellular domains from various TNF receptor family molecules such as CD28 or 4-1BB (CD137) and OX40 (CD134). Furthermore, third-generation receptors include two co-stimulatory signals, most commonly from CD28 and 4-1BB, in addition to CD3ζ. Second- and third-generation CARs have dramatically improved the antitumor effect and, in some cases, induced complete remission in patients with advanced cancer.

Prior Art Documents

Non-Patent Documents

[0008]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 18

Summary of the Invention

Problems to be Solved by the Invention

[0009] Generally, the number of transferred T cells is considered to correlate with the therapeutic response. However, the number of cells that can be administered to a patient for adoptive T cell transfer is limited, and the generation of a large amount of T cells for adoptive T cell transfer remains a problem. When a patient receives a lymphodepletion preparative regimen prior to injection of either TIL or receptor-engineered T cells, a substantial increase in cell persistence can be achieved. However, transferring a large number of engineered T cells into an empty host also poses a risk of serious adverse events if the target antigen is unexpectedly expressed in normal tissues associated with it. Therefore, it would be desirable to transfer a limited amount of engineered T cells that can proliferate in a patient after it has been proven to be safe.

[0010] The inventors have found that it is possible to expand CAR-T cells in a subject by administering RNA encoding IL2, optionally in combination with RNA encoding additional cytokines such as IL7 or IL21, and providing an antigen for CAR-T cell stimulation using optional RNA vaccination. The method of the present invention enables providing only a small amount of CAR-engineered T cells to a patient and then expanding the T cells in vivo.

Means for Solving the Problems

[0011] The present invention generally encompasses the treatment of diseases by targeting cells that express an antigen on their cell surface, particularly diseased cells that express an antigen on their cell surface such as cancer cells that express a tumor antigen on their cell surface. This method provides for the selective eradication of cells that express an antigen on their own surface, thereby minimizing harmful effects on normal cells that do not express the antigen. T cells genetically modified to express a chimeric antigen receptor (CAR) that targets cells via binding to the antigen are provided to a subject, such as by administration of the T cells. IL2 or a nucleic acid encoding the same is administered. In one embodiment, an additional cytokine such as IL7 or IL21 or a nucleic acid encoding the same is administered. In one embodiment, an antigen or a variant thereof or a nucleic acid encoding the same is administered to provide an antigen for stimulation, priming and / or expansion of the T cells (optionally after expression of the nucleic acid by a suitable target cell). T cells that have been stimulated, primed and / or expanded in a patient are capable of recognizing cells that express an antigen on their cell surface, such as diseased cells, and effecting the eradication of the diseased cells. This approach can be considered to include passive immunization and active immunization. Treatment comprising administration of T cells genetically modified to express a CAR can be considered a form of passive immunization. Treatment comprising administration of an antigen or a variant thereof, thereby stimulating a T cell-mediated immune response against a target cell population or tissue, can be considered a form of active immunization.

[0012] The immune response according to the present disclosure is against a target cell population or target tissue that expresses an antigen in a mammal, and T cells genetically modified to express a chimeric antigen receptor (CAR) target the antigen. The method optionally also includes administration of an antigen or a variant thereof. In one embodiment, the immune response 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 tumor cells or tumor tissue.

[0013] The methods and agents described herein are particularly effective when RNA encoding IL2 conjugated to a pharmacokinetic modifying group (hereinafter referred to as "extended pharmacokinetic (PK) IL2") is administered in combination with RNA encoding an additional cytokine such as IL7 or IL21 optionally conjugated to a pharmacokinetic modifying group (hereinafter referred to as "extended pharmacokinetic (PK) cytokine"). The methods and agents described herein are particularly effective when the RNA encoding extended PK IL2 and / or the RNA encoding extended PK cytokine target the liver for systemic availability. Hepatocytes can be efficiently transfected and can produce large amounts of protein. RNA encoding an antigen preferably targets secondary lymphoid organs.

[0014] In one aspect, the invention is a method for inducing an immune response in a subject, comprising: a. providing to the subject T cells genetically modified to express a chimeric antigen receptor (CAR), and b. administering to the subject IL2 or a polynucleotide encoding IL2. A method is provided that includes the steps above.

[0015] In one embodiment, the method includes administering an IL2 or a polynucleotide encoding IL2, and a polynucleotide encoding an additional cytokine or an additional cytokine. In one embodiment, the additional cytokine is selected from the group consisting of IL7 and IL21. In one embodiment, the method includes administering an IL2 or a polynucleotide encoding IL2, and a polynucleotide encoding IL7 or IL7. In one embodiment, the method includes administering an IL2 or a polynucleotide encoding IL2, and a polynucleotide encoding IL21 or IL21.

[0016] In one embodiment, the polynucleotide encoding IL2 is RNA, and optionally, the polynucleotide encoding an additional cytokine is RNA.

[0017] In one embodiment, T cells genetically modified to express a CAR are provided to a subject by administering T cells genetically modified to express a CAR or by generating T cells genetically modified to express a CAR in the subject.

[0018] In one embodiment, the method further comprises administering to the subject an antigen or a variant thereof, or a polynucleotide encoding the antigen or variant, wherein the T cells genetically modified to express a CAR target the antigen and the immune response is an immune response against a target cell population or target tissue expressing the antigen. In one embodiment, the polynucleotide encoding the antigen or variant is RNA.

[0019] In one aspect, the present invention is a method for inducing an immune response in a subject, comprising: a. providing to the subject T cells genetically modified to express a chimeric antigen receptor (CAR), and b. administering to the subject RNA encoding IL2 A method comprising is provided.

[0020] In one embodiment, the method comprises administering RNA encoding IL2 and RNA encoding an additional cytokine. In one embodiment, the additional cytokine is selected from the group consisting of IL7 and IL21. In one embodiment, the method comprises administering RNA encoding IL2 and RNA encoding IL7. In one embodiment, the method comprises administering RNA encoding IL2 and RNA encoding IL21.

[0021] In one embodiment, T cells genetically modified to express a CAR are provided to a subject by administering T cells genetically modified to express a CAR or by generating T cells genetically modified to express a CAR in the subject.

[0022] In one embodiment, the method further comprises administering RNA encoding an antigen or a variant thereof, wherein the T cells genetically modified to express the CAR target the antigen, and the immune response is an immune response against a target cell population or target tissue expressing the antigen.

[0023] In one embodiment of all aspects, the immune response is a T cell-mediated immune response.

[0024] In one aspect, the present invention is a method for treating a subject having a disease, disorder or condition associated with the expression or upregulation of an antigen, comprising: a. providing to the subject T cells genetically modified to express a chimeric antigen receptor (CAR) that targets the antigen, and b. administering to the subject IL2 or a polynucleotide encoding IL2. A method comprising is provided.

[0025] In one embodiment, the method comprises administering IL2 or a polynucleotide encoding IL2, and a further cytokine or a polynucleotide encoding a further cytokine. In one embodiment, the further cytokine is selected from the group consisting of IL7 and IL21. In one embodiment, the method comprises administering IL2 or a polynucleotide encoding IL2, and IL7 or a polynucleotide encoding IL7. In one embodiment, the method comprises administering IL2 or a polynucleotide encoding IL2, and IL21 or a polynucleotide encoding IL21.

[0026] In one embodiment, the polynucleotide encoding IL2 is RNA, and optionally, the polynucleotide encoding the further cytokine is RNA.

[0027] In one embodiment, T cells genetically modified to express a CAR are provided to a subject by administering T cells genetically modified to express a CAR or by generating T cells genetically modified to express a CAR in the subject.

[0028] In one embodiment, the method further comprises administering to the subject an antigen or a variant thereof, or a polynucleotide encoding the antigen or variant. In one embodiment, the polynucleotide encoding the antigen or variant is RNA.

[0029] In one aspect, the present invention is a method for treating a subject having a disease, disorder or condition associated with the expression or upregulation of an antigen, comprising: a. providing to the subject T cells genetically modified to express a chimeric antigen receptor (CAR) that targets the antigen, and b. administering to the subject RNA encoding IL2 The method provided includes.

[0030] In one embodiment, the method comprises administering RNA encoding IL2 and RNA encoding an additional cytokine. In one embodiment, the additional cytokine is selected from the group consisting of IL7 and IL21. In one embodiment, the method comprises administering RNA encoding IL2 and RNA encoding IL7. In one embodiment, the method comprises administering RNA encoding IL2 and RNA encoding IL21.

[0031] In one embodiment, T cells genetically modified to express a CAR are provided to a subject by administering T cells genetically modified to express a CAR or by generating T cells genetically modified to express a CAR in the subject.

[0032] In one embodiment, the method further comprises administering to the subject RNA encoding the antigen or a variant thereof.

[0033] In one embodiment of all aspects, the disease, disorder or condition is cancer and the antigen is a tumor-associated antigen.

[0034] In one embodiment of all aspects, the IIL2 is extended pharmacokinetic (PK) IL2. In one embodiment, the extended PK IL2 comprises a fusion protein. In one embodiment, the fusion protein comprises an IL2 moiety and a moiety selected from the group consisting of serum albumin, immunoglobulin fragments, transferrin, Fn3, and variants thereof.

[0035] In one embodiment of all aspects, a further cytokine, particularly IL7 or IL21, is an extended pharmacokinetic (PK) cytokine, particularly extended PK IL7 or extended PK IL21. In one embodiment, the extended PK cytokine, particularly extended PK IL7 or extended PK IL21, comprises a fusion protein. In one embodiment, the fusion protein comprises a moiety of the further cytokine, particularly an IL7 moiety or an IL21 moiety, and a moiety selected from the group consisting of serum albumin, immunoglobulin fragments, transferrin, Fn3, and variants thereof.

[0036] 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.

[0037] In one embodiment of all aspects, the method is a method for treating or preventing cancer in a subject and the antigen is a tumor-associated antigen.

[0038] In one aspect, the present invention provides a. T cells genetically modified to express a chimeric antigen receptor (CAR), and b. IL2 or a polynucleotide encoding IL2 in a pharmaceutical formulation.

[0039] In one embodiment, the pharmaceutical preparation comprises IL2 or a polynucleotide encoding IL2, and a further cytokine or a polynucleotide encoding a further cytokine. In one embodiment, the further cytokine is selected from the group consisting of IL7 and IL21. In one embodiment, the pharmaceutical preparation comprises IL2 or a polynucleotide encoding IL2, and IL7 or a polynucleotide encoding IL7. In one embodiment, the pharmaceutical preparation comprises IL2 or a polynucleotide encoding IL2, and IL21 or a polynucleotide encoding IL21.

[0040] In one embodiment, the polynucleotide encoding IL2 is RNA, and optionally, the polynucleotide encoding a further cytokine is also RNA.

[0041] In one embodiment, the pharmaceutical preparation further comprises an antigen or a variant thereof, or a polynucleotide encoding an antigen or a variant, and the T cells genetically modified to express CAR target the antigen. In one embodiment, the polynucleotide encoding the antigen or variant is RNA.

[0042] In one embodiment, the pharmaceutical preparation is a kit.

[0043] In one embodiment, the pharmaceutical preparation comprises, in separate containers, T cells genetically modified to express CAR, IL2 or a polynucleotide encoding IL2, optionally a further cytokine or a polynucleotide encoding a further cytokine, and optionally an antigen or a variant thereof or a polynucleotide encoding an antigen or a variant.

[0044] In one embodiment, the pharmaceutical preparation further comprises instructions for use of the pharmaceutical preparation for treating or preventing cancer, and the antigen is a tumor-associated antigen.

[0045] In one embodiment, the pharmaceutical preparation is a pharmaceutical composition.

[0046] In one embodiment, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0047] In one aspect, the present invention provides a. T cells genetically modified to express a chimeric antigen receptor (CAR), and b. RNA encoding IL2 in a pharmaceutical formulation.

[0048] In one embodiment, the pharmaceutical formulation comprises RNA encoding IL2 and RNA encoding a further cytokine. In one embodiment, the further cytokine is selected from the group consisting of IL7 and IL21. In one embodiment, the pharmaceutical formulation comprises RNA encoding IL2 and RNA encoding IL7. In one embodiment, the pharmaceutical formulation comprises RNA encoding IL2 and RNA encoding IL21.

[0049] In one embodiment, the pharmaceutical formulation further comprises RNA encoding an antigen or a variant thereof, and the T cells genetically modified to express CAR target the antigen.

[0050] In one embodiment, the pharmaceutical formulation is a kit.

[0051] In one embodiment, the pharmaceutical formulation comprises, in separate containers, T cells genetically modified to express CAR, RNA encoding IL2, optionally RNA encoding a further cytokine, and optionally RNA encoding an antigen or a variant thereof.

[0052] In one embodiment, the pharmaceutical formulation further comprises instructions for use of the pharmaceutical formulation for treating or preventing cancer, and the antigen is a tumor-associated antigen.

[0053] In one embodiment, the pharmaceutical formulation is a pharmaceutical composition.

[0054] In one embodiment, the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0055] In one embodiment of all aspects, IIL2 is extended pharmacokinetic (PK) IL2. In one embodiment, the extended PK IL2 comprises a fusion protein. In one embodiment, the fusion protein comprises an IL2 portion and a portion selected from the group consisting of serum albumin, an immunoglobulin fragment, transferrin, Fn3, and variants thereof.

[0056] In one embodiment of all aspects, the additional cytokine is an extended pharmacokinetic (PK) cytokine. In one embodiment, the extended PK cytokine comprises a fusion protein. In one embodiment, the fusion protein comprises a cytokine portion and a portion selected from the group consisting of serum albumin, an immunoglobulin fragment, transferrin, Fn3, and variants thereof.

[0057] In one embodiment, the serum albumin comprises mouse serum albumin or human serum albumin.

[0058] In one embodiment, the immunoglobulin fragment comprises an immunoglobulin Fc domain.

[0059] In one aspect, the present invention provides a pharmaceutical formulation described herein for pharmaceutical use. In one embodiment, the pharmaceutical use includes therapeutic or prophylactic treatment of a disease or disorder.

[0060] In one aspect, the present invention provides a pharmaceutical formulation described herein for use in a method for treating or preventing cancer in a subject, wherein the antigen is a tumor-associated antigen.

[0061] In one aspect, the present invention provides a drug and composition described herein for use in the method described herein.

[0062] In one aspect, the present invention provides a T cell genetically modified to express a chimeric antigen receptor (CAR) targeting an antigen for use in the method described herein.

[0063] In one aspect, the present invention provides IL2 or a polynucleotide encoding IL2 for use in the methods described herein.

[0064] In one aspect, the present invention provides a cytokine other than IL2, such as IL7 or IL21, or a polynucleotide encoding the same, for use in the methods described herein.

[0065] In one aspect, the present invention provides an antigen or a variant thereof, or a nucleic acid encoding the antigen or a variant thereof, for use in the methods described herein.

[0066] In one embodiment of the pharmaceutical formulation, the RNA is present in a form selected from a liquid form, a solid form, or a combination thereof. In one embodiment, the solid form is a frozen form or a dehydrated form. In one embodiment, the dehydrated form is a lyophilized form or a spray-dried form.

[0067] In one embodiment, the cancer described herein is selected from the group consisting of melanoma, leukemia, lymphoma, lung cancer, breast cancer, prostate cancer, ovarian cancer, colon cancer, mesothelioma, renal cell carcinoma, and brain cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0068]

Figure 1AB

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Mode for Carrying Out the Invention

[0069] The present disclosure will be described in detail below, but it should be understood that this disclosure is not limited to the specific methodologies, protocols, and reagents described herein, and these may vary. It should also be understood that the terms used herein are for the purpose of describing only particular embodiments and are not intended to limit the scope of the present disclosure, which is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

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

[0071] The practice of the present disclosure uses conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as described in the literature of the art (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989), unless otherwise indicated.

[0072] In the following, elements of the present disclosure are described. While these elements are recited with specific embodiments, it should be understood that they may be combined in any manner and in any number to create further embodiments. The examples and embodiments described variously should not be construed as limiting the present disclosure to only the explicitly described embodiments. This description is to be understood as disclosing and encompassing embodiments that combine the explicitly described embodiments with any number of the disclosed elements. Further, any rearrangement and combination of all the elements described are to be considered as disclosed by this description, unless otherwise specifically indicated in the context.

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

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

[0075] Unless otherwise specified, the term "comprising" is used in the context of this document to indicate that in addition to the members of the list introduced by "comprising", further members may optionally be present. However, the term "comprising" is contemplated as a specific embodiment of the present disclosure to encompass the possibility that no further members are present; that is, for the purposes of this embodiment, "comprising" should be understood to have the meaning of "consisting of".

[0076] Throughout the text of this specification, several materials are cited. Each material cited in this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.) is hereby incorporated by reference in its entirety into this specification, whether above or below. Nothing in this specification should be construed as an admission that the present disclosure has any right to precedence over such disclosures.

[0077] In the following, definitions applicable to all aspects of the present disclosure are provided. The following terms have the following meanings, unless otherwise indicated. Undefined terms have the meanings generally recognized in their technical fields.

[0078] According to the present disclosure, the term "peptide" includes oligopeptides and polypeptides and refers to a substance comprising from 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, and up to about 50, about 100, or about 150 consecutive amino acids linked to each other by peptide bonds. The term "protein" or "polypeptide" refers to a large peptide, particularly a peptide having at least about 151 amino acids, but the terms "peptide", "protein", and "polypeptide" are generally used interchangeably herein.

[0079] A "therapeutic protein", when provided to a subject in a therapeutically effective amount, has a positive or beneficial effect on the condition or disease state of the subject. In one embodiment, the therapeutic protein has curative or palliative properties and can be administered to improve, alleviate, soothe, reverse, delay the onset of, or reduce the severity of one or more symptoms of a disease or disorder. The therapeutic protein has prophylactic properties and can be used to delay the onset of a disease or to reduce the severity of such a disease or pathological condition. The term "therapeutic protein" includes the entire protein or peptide and can also refer to its therapeutically active fragments. It can also include therapeutically active variants of the protein. Examples of therapeutically active proteins include, but are not limited to, cytokines.

[0080] A "fragment" with respect to 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 shortened at the N-terminus and / or C-terminus. A fragment shortened at the C-terminus (N-terminal fragment) can be obtained, for example, by translation of a truncated open reading frame lacking the 3'-end of the open reading frame. A fragment shortened at the N-terminus (C-terminal fragment) can be obtained, for example, by translation of a truncated open reading frame lacking the 5'-end of the open reading frame as long as the truncated open reading frame contains a start codon that functions to initiate translation. Fragments of an amino acid sequence include, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues from the amino acid sequence. Fragments of an amino acid sequence preferably contain 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.

[0081] For the purposes of the present disclosure, a "variant" of an amino acid sequence (peptide or protein) includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants and / or amino acid substitution variants. The term "variant" specifically includes fragments of amino acid sequences.

[0082] Amino acid insertion mutants contain the insertion of one or more amino acids in a specific amino acid sequence. In the case of amino acid sequence variants having an insertion, one or more amino acid residues are inserted at a specific site in the amino acid sequence, but random insertions with appropriate screening of the resulting products are also possible. Amino acid addition mutants include the amino-terminal and / or carboxy-terminal fusion of one or more amino acids, such as 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion mutants are characterized by the removal of one or more amino acids from the sequence, such as the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion can be at any position in the protein. Amino acid deletion mutants containing deletions at the N-terminal and / or C-terminal of the protein are also called N-terminal and / or C-terminal truncation mutants. Amino acid substitution mutants are characterized in that at least one residue within the sequence is removed and another residue is inserted in its place. Modifications at positions in the amino acid sequence that are not conserved between homologous proteins or peptides, and / or replacing amino acids with other amino acids having similar properties are preferred. Preferably, the amino acid changes in peptide and protein mutants are conservative amino acid changes, that is, substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes include substitutions of one of the families 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), non-polar (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 grouped together as aromatic amino acids.

[0083] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of the given amino acid sequence is at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably given for an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the full length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, preferably contiguous amino acids. In a preferred embodiment, the degree of similarity or identity is given for the full length of the reference amino acid sequence. The alignment for determining sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using optimal sequence alignment, for example using Align, with standard settings, preferably EMBOSS::needle, matrix: Blosum62, gap open 10.0, gap extend 0.5.

[0084] "Sequence similarity" indicates the percentage of amino acids that are either identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between these sequences.

[0085] The term "percent identity" is intended to indicate the percentage of amino acid residues that are identical between two sequences being compared, obtained after optimal alignment, this percentage being purely statistical and the differences between the two sequences being randomly distributed over their entire length. The sequence comparison between two amino acid sequences has conventionally been done by comparing these sequences after they have been optimally aligned, said comparison identifying local regions of sequence similarity and being done segment by segment or for each "comparison window". The optimal alignment of the sequences for comparison can be made by hand, in addition to by the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by the similarity search method of Pearson and Lipman, 1988, Proc. Natl Acad. Sci. USA 85, 2444, or by computer programs using these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA of the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0086] The percent identity is calculated by determining the number of positions that are identical between two sequences being compared, dividing this number by the number of positions being compared, and multiplying the resulting result by 100 to obtain the percent identity between these two sequences.

[0087] According to the present disclosure, homologous amino acid sequences exhibit at least 40%, particularly 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% identity.

[0088] The amino acid sequence variants described herein can be readily prepared by those skilled in the art, for example, by recombinant DNA manipulation. Manipulation of DNA sequences to prepare peptides or proteins having substitutions, additions, insertions or deletions is described in detail, for example, in Sambrook et al. (1989). Further, the peptides and amino acid variants described herein can be readily prepared using known peptide synthesis techniques such as solid phase synthesis and similar methods.

[0089] In one embodiment, a fragment or variant of an amino acid sequence (peptide or protein) is preferably a "functional fragment" or "functional variant". The terms "functional fragment" or "functional variant" of an amino acid sequence refer 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., it is functionally equivalent. With respect to cytokines, one particular function is one or more immunomodulatory activities exhibited by the amino acid sequence from which the fragment or variant is derived and / or by binding of the amino acid sequence from which the fragment or variant is derived to the receptor(s) to which it binds.

[0090] An amino acid sequence (peptide or protein) "derived from" a specified amino acid sequence (peptide or protein) refers to the origin of the first 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 that particular sequence or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence can be a variant or a fragment of that particular sequence. For example, those skilled in the art will understand that antigens and cytokines suitable for use herein (e.g., IL2, IL7 or IL21) can be modified such that they have a sequence different from the naturally occurring or native sequence from which they are derived while retaining the desired activity of the native sequence.

[0091] T cell T cells belong to a group of white blood cells known as lymphocytes and play a central role in cellular immunity. They can be distinguished from other types of lymphocytes, such as B cells and natural killer cells, by the presence of special receptors on their cell surface called T cell receptors (TCRs). The thymus is the main organ involved in the maturation of T cells. Several different subsets of T cells, each with distinct functions, have been discovered.

[0092] Most T cells have a T cell receptor (TCR) that exists as a complex of several proteins. The actual T cell receptor is produced from separate T cell receptor alpha and beta (TCRα and TCRβ) genes and is composed of two distinct peptide chains called the α-TCR chain and the β-TCR chain. Gamma-delta T cells (γδ T cells) are a small subset of T cells that have a different T cell receptor (TCR) on their surface. However, in γδ T cells, the TCR is composed of one gamma chain and one delta chain. This group of T cells is much rarer than alpha-beta T cells (2% of all T cells).

[0093] All T cells are derived from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells derived from hematopoietic stem cells are present in the thymus and expand by cell division to give rise to a large population of immature thymocytes. The earliest thymocytes do not express either CD4 or CD8 and are thus classified as double-negative (CD4-CD8-) cells. As development progresses, they become double-positive thymocytes (CD4+CD8+) and finally mature into single-positive (CD4+CD8- or CD4-CD8+) thymocytes, which are released from the thymus into the peripheral tissues.

[0094] The terms "T cell" and "T lymphocyte" are used interchangeably herein and include helper T cells (CD4+ T cells) and cytotoxic T cells (CTLs, CD8+ T cells) including cytolytic T cells. The term "antigen-specific T cell" or similar terms relate to T cells that recognize an antigen targeted by the T cell, preferably exerting effector functions of the T cell, particularly when presented on the surface of antigen-presenting cells or diseased cells such as cancer cells. When a T cell kills a target cell expressing an antigen, the T cell is considered to be specific for the antigen. The specificity of T cells can be evaluated using any of a variety of standard techniques, for example in a chromium release assay or a proliferation assay. Alternatively, the synthesis of lymphokines (such as interferon γ) can be measured.

[0095] Helper T 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 are also known as CD4+ T cells because they express the CD4 protein on their surface. Helper T cells are activated when presented with a peptide antigen by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they rapidly divide and secrete small proteins called cytokines that regulate or assist the active immune response.

[0096] Cytotoxic T cells destroy virus-infected cells and tumor cells and are also involved in graft 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 all cells of the body.

[0097] T cell-mediated effector functions, in the case of helper T cells (CD4 + T cells), include cytokine release and / or CD8 +Including the activation of lymphocytes (CTL) and / or B cells, in the case of CTL, for example, the removal of cells, i.e., cells characterized by the expression of an antigen, through apoptosis or perforin-mediated cell lysis, the production of cytokines such as IFN-γ and TNF-α, and the specific cytolytic killing of target cells expressing the antigen.

[0098] According to the present invention, the term "T cell" also includes cells that can mature into T cells with appropriate stimulation.

[0099] 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 fractions of bone marrow or peripheral blood 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. A sample containing T cells can be, for example, peripheral blood mononuclear cells (PBMC).

[0100] The T cells used according to the present invention may express an endogenous T cell receptor or may lack the expression of an endogenous T cell receptor.

[0101] CAR Nucleic acids such as RNA encoding CAR can be introduced into T cells or other cells with lytic ability, particularly lymphoid cells.

[0102] According to the present disclosure, when CAR is present on T cells, it recognizes antigens on the surface of antigen-presenting cells or diseased cells such as cancer cells so that the T cells are stimulated, primed, and / or expanded as described above, or exert effector functions.

[0103] According to the present invention, the term "chimeric antigen receptor (CAR)" is synonymous with the terms "chimeric T cell receptor" and "artificial T cell receptor".

[0104] Preferably, the CAR is expressed on the cell surface.

[0105] According to the present invention, the term "CAR" (or "chimeric antigen receptor") refers to an artificial receptor comprising a single molecule or a complex of molecules that recognizes, i.e., binds 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 can confer specificity to immune effector cells such as T cells expressing the CAR on the cell surface. Such cells do not necessarily require antigen processing and presentation for recognition of the target cell, but rather can preferably specifically recognize any antigen present on the target cell. Preferably, recognition of the target structure by the CAR results in activation of the immune effector cell expressing the CAR. The CAR can comprise one or more protein units comprising one or more domains described herein. The term "CAR" does not include a T cell receptor.

[0106] According to the present invention, a CAR generally can comprise several domains. In one embodiment of all aspects of the present invention, the CAR comprises an antigen-binding domain, a transmembrane domain, and a T cell signaling domain.

[0107] The binding domain recognizes and binds to an antigen. In one embodiment, a single-chain variable fragment (scFv) derived from a monoclonal antibody is used as the binding domain. Antigen recognition domains that can be similarly used include, among others, T cell receptor (TCR) alpha and beta single chains. In fact, almost anything that binds to a given target with high affinity can be used as an antigen recognition domain. In one embodiment of all aspects of the present invention, the CAR includes an antigen-binding domain. In one embodiment, the antigen-binding domain is composed of the extracellular domain of the CAR. In one embodiment, the antigen-binding domain includes a single-chain variable fragment (scFv) of an antibody against the antigen. In one embodiment, the antigen-binding domain includes a variable region (VH) of the heavy chain of an immunoglobulin having specificity for the antigen (VH(antigen)) and a variable region (VL) of the light chain of an immunoglobulin having specificity for the antigen (VL(antigen)). In one embodiment, the heavy chain variable region (VH) and the corresponding light chain variable region (VL) are connected by a peptide linker, preferably a peptide linker containing the amino acid sequence (GGGGS)3.

[0108] In one embodiment of all aspects of the present invention, the CAR includes a transmembrane domain. In one embodiment, the transmembrane domain is a hydrophobic alpha helix spanning the membrane. In one embodiment, the transmembrane domain includes the CD28 transmembrane domain or a fragment thereof.

[0109] The activation signaling domain (or T cell signaling domain) functions to activate cytotoxic lymphocytes when the CAR binds to the antigen. The identity of the activation signaling domain is limited only in terms of having the ability to induce the activation of cytotoxic lymphocytes selected upon binding of the antigen by the CAR. Suitable activation signaling domains include the T cell CD3ζ chain and the Fc receptor gamma. Those skilled in the art will understand that sequence variants of these described activation signaling domains can be used without adversely affecting the present invention, provided that the variants have the same or similar activity as the domain they model. Such variants have at least about 80% sequence identity with the amino acid sequence of the domain from which they are derived.

[0110] In one embodiment, the T cell signaling domain is located intracellularly. In one embodiment, the T cell signaling domain optionally includes, in combination with CD28, CD3ζ, preferably the endodomain of CD3ζ.

[0111] A further domain that may be present is a co-stimulatory domain. The co-stimulatory domain serves to enhance the proliferation and survival of cytotoxic lymphocytes when the CAR binds to the target moiety. The identity of the co-stimulatory domain is limited only to the point of having the ability to enhance cell proliferation and survival upon binding of the target moiety by the CAR. Suitable co-stimulatory domains include CD28, CD137 (4-1BB), which is a member of the tumor necrosis factor (TNF) receptor family, CD134 (OX40), which is a member of the receptor of the TNFR superfamily, and CD278 (ICOS), which is a co-stimulatory molecule of the CD28 superfamily expressed on activated T cells. Those skilled in the art will understand that sequence variants of these described co-stimulatory domains can be used without adversely affecting the present invention, and that the variants have the same or similar activity as the domain they model. 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 includes two co-stimulatory domains. Specific combinations include all possible permutations of the four described domains, but specific examples include CD28 + CD137 (4-1BB) and CD28 + CD134 (OX40).

[0112] The CAR of the present invention may include the above domains together in the form of a fusion protein. Such fusion proteins generally include a binding domain, one or more co-stimulatory domains, and an activation signaling domain, linked in the direction from the N-terminus to the C-terminus. However, the CAR of the present invention is not limited to this arrangement, and other arrangements are also acceptable, including a binding domain, an activation signaling domain, and one or more co-stimulatory domains. Since the binding domain must be able to bind freely to the antigen, it will be understood that the arrangement of the binding domain in the fusion protein is generally an arrangement in which surface display of the region is achieved outside the cell. Similarly, since the co-stimulatory domain and the activation signaling domain function to induce the activity and proliferation of cytotoxic lymphocytes, the fusion protein generally presents these two domains inside the cell. The CAR may further include additional elements, such as a signal peptide to ensure proper transport of the fusion protein to the cell surface, a transmembrane domain to ensure that the fusion protein is maintained as an integral membrane protein, and a hinge domain (or spacer region) that confers flexibility to the binding domain and enables strong binding to the antigen.

[0113] In one embodiment of all aspects of the present invention, the CAR includes a signal peptide that directs the nascent protein towards the endoplasmic reticulum. In one embodiment, the signal peptide precedes the antigen-binding domain.

[0114] In one embodiment of all aspects of the present invention, the CAR includes a spacer region that links the antigen-binding domain to the transmembrane domain. In one embodiment, the spacer region allows the antigen-binding domain to be oriented in different directions to facilitate antigen recognition. In one embodiment, the spacer region includes a hinge region derived from IgG1.

[0115] In one embodiment of all aspects of the present invention, the CAR has the structure: NH2-signal peptide - antigen-binding domain - spacer region - transmembrane domain - T cell signaling domain - COOH and includes.

[0116] In one embodiment of all aspects of the present invention, the CAR is preferably specific for the antigen it targets, particularly when present on the surface of cells such as diseased cells or antigen-presenting cells.

[0117] In one embodiment of all aspects of the present invention, the CAR can be expressed by T cells, preferably cytotoxic T cells, and / or can be present on the surface of T cells, preferably cytotoxic T cells. In one embodiment, the T cells are reactive with the antigen targeted by the CAR.

[0118] The cells used in connection with the CAR system of the present invention are preferably T cells, particularly cytotoxic lymphocytes, preferably selected from T cells, particularly cytotoxic T cells, natural killer (NK) cells, and lymphokine-activated killer (LAK) cells. When activated, these cytotoxic lymphocytes each cause the destruction of target cells. For example, cytotoxic T cells cause the destruction of target cells by any or both of the following means. First, when activated, T cells release cytotoxins such as perforin, granzyme, and granulysin. Perforin and granulysin create pores in the target cells, and granzyme enters the cells and causes a cytoplasmic caspase cascade that induces apoptosis (programmed cell death) of the cells. Second, apoptosis can be induced via Fas-Fas ligand interactions between the T cells and the target cells. The cytotoxic lymphocytes are preferably autologous cells, but heterologous or allogeneic cells can be used.

[0119] Adoptive cell transfer therapy using T cells expressing a chimeric antigen receptor is a promising anti-cancer treatment because the CAR-modified T cells can be engineered to target substantially any tumor antigen. For example, a patient's T cells can be genetically engineered (genetically modified) to express a CAR that is specifically directed against an antigen on the patient's tumor cells and then infused back into the patient.

[0120] According to the present invention, a CAR can replace the function of a T cell receptor, and in particular, can confer reactivity such as cytolytic activity on cells such as T cells. However, in contrast to the binding of a T cell receptor to an antigen peptide-MHC complex, a CAR can bind to an antigen, especially when expressed on the cell surface.

[0121] A CAR construct can be introduced into T cells using various methods including non-viral-based DNA transfection, transposon-based systems, and viral-based systems. Non-viral-based DNA transfection has a low risk of insertional mutagenesis. Transposon-based systems can integrate transgenes more efficiently than plasmids without integration elements. Viral-based systems include the use of γ-retroviral and lentiviral vectors. γ-Retroviruses have been previously demonstrated to be safe from the perspective of integration in primary human T cells, produce T cells relatively easily, and transduce them efficiently and persistently. Lentiviral vectors also transduce T cells efficiently and persistently, but have a higher manufacturing cost. They are also potentially safer than retrovirus-based systems.

[0122] In one embodiment of all aspects of the present invention, the method further comprises transfecting a T cell or T cell precursor, either ex vivo or in vivo, with a nucleic acid encoding a CAR to provide a T cell genetically modified to express the CAR.

[0123] CAR T cells can be produced in vivo, and thus almost instantaneously, using nanoparticles that target T cells. For example, poly(β-amino ester)-based nanoparticles can be conjugated to anti-CD3e f(ab) fragments for binding to CD3 on T cells. For this purpose, anti-CD3e f(ab) fragments can be covalently conjugated to polyglutamic acid (PGA). PGA surrounds the particle core containing nucleic acids and excess poly(β-amino ester) (PBAE) polymer and attaches to it by charge interactions. When bound to T cells, these nanoparticles are endocytosed. Their contents, such as plasmid DNA encoding an anti-tumor antigen CAR, can be directed to the T cell nucleus because they contain peptides with microtubule-associated sequences (MTAS) and nuclear localization signals (NLS) covalently linked to the PBAE polymer. Efficient integration of the CAR vector into the chromosome can be enabled by including a transposon adjacent to the CAR gene expression cassette and a separate plasmid encoding a highly active transposase. 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.

[0124] Another possibility is to use the CRISPR / Cas9 method to deliberately place the CAR coding sequence at a specific locus. For example, an existing T cell receptor (TCR) can be knocked out while a CAR is knocked in and placed under the dynamic regulatory control of an endogenous promoter that otherwise suppresses TCR expression; see, for example, Eyquem et al. (2017) Nature 543:113-117.

[0125] In one embodiment of all aspects of the present invention, T cells genetically modified to express a CAR are stably or transiently transfected with a nucleic acid encoding the CAR. Thus, the nucleic acid encoding the CAR is either integrated into the genome of the T cell or not.

[0126] In one embodiment of all aspects of the present invention, the T cells or T cell precursors are derived from the subject to be treated. In one embodiment of all aspects of the present invention, the T cells or T cell precursors are derived from a subject different from the subject to be treated.

[0127] In one embodiment of all aspects of the present invention, the T cells can be autologous, allogeneic or syngeneic to the subject to be treated. The T cells can be genetically modified in vitro to express a chimeric antigen receptor (CAR) that targets an antigen.

[0128] In one embodiment of all aspects of the present invention, T cells genetically modified to express a CAR are inactivated for the expression of the endogenous T cell receptor and / or endogenous HLA.

[0129] The term "autologous" is used to denote 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 as they overcome the immunological barriers that would otherwise result in rejection.

[0130] The term "allogeneic" is used to denote something derived from different individuals of the same species. Two or more individuals are said to be allogeneic to each other if the genes at one or more loci are not identical.

[0131] The term "syngeneic" is used to denote something derived from an individual or tissue having the same genotype, i.e., identical twins or animals of the same inbred strain, or their tissues.

[0132] The term "heterologous" is used to denote something consisting of a plurality of different elements. As an example, transplanting the bone marrow of one individual into a different individual constitutes a heterologous transplantation. A heterologous gene is a gene derived from a source other than the subject.

[0133] RNA As used herein, the terms "polynucleotide" or "nucleic acid" are 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, the polynucleotide is preferably isolated.

[0134] Nucleic acids can be contained in vectors. 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 λ phage, viral vectors such as adenovirus or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BACs), yeast artificial chromosomes (YACs), or P1 artificial chromosomes (PACs). The said vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and generally contain the appropriate DNA sequences necessary for the expression of a desired coding sequence operably linked to 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 a desired DNA fragment and may lack the functional sequences necessary for the expression of the desired DNA fragment.

[0135] In one embodiment of all aspects of the present invention, a nucleic acid encoding a cytokine or encoding an antigen or a variant thereof is expressed in the cells of a subject to be treated to provide the cytokine or antigen or a variant thereof. In one embodiment of all aspects of the present invention, the nucleic acid is transiently expressed in mammalian 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 present invention, the nucleic acid is RNA, preferably in vitro transcribed RNA. In one embodiment of all aspects of the present invention, the expression of the antigen or a variant thereof occurs on the cell surface.

[0136] In one embodiment of all aspects of the present invention, a nucleic acid encoding an antigen or a variant thereof is expressed in mammalian cells to provide an antigen or a variant thereof for binding by T cells genetically modified to express a CAR, and said binding results in the stimulation, priming and / or expansion of T cells genetically modified to express a CAR.

[0137] The term "expression" is used in its broadest sense in accordance with the present invention and includes, for example, the production of RNA and / or peptide or protein by transcription and / or translation. Expression can be transient or stable. According to the present invention, the term "expression" also includes "ectopic expression" or "abnormal expression".

[0138] According to the present invention, the term "encoded by a nucleic acid" means that the nucleic acid can be expressed to produce the protein or peptide it encodes when present in a suitable environment such as within a cell.

[0139] The nucleic acids described herein can be recombinant and / or isolated molecules.

[0140] As used herein, the term "isolated molecule" is intended to refer to a molecule that is substantially free of other molecules such as other cellular materials.

[0141] The term "recombinant" in the context of the present invention means "produced by genetic engineering". Preferably, a "recombinant", such as a recombinant cell, in the context of the present invention does not occur naturally.

[0142] 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 exists in a living organism (including a virus), can be isolated from a natural source, and has not been intentionally modified by humans in the laboratory is naturally occurring.

[0143] The term "transfection" relates to the introduction of nucleic acids, particularly RNA, into cells. For the 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, where the cells may be present in a subject, such as a patient. Thus, according to the present invention, cells for transfection of the nucleic acids described herein can be present in vitro or in vivo, for example 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. In some applications of transfection, it is sufficient for the transfected genetic material to be transiently expressed. Since the nucleic acids introduced during the transfection process are usually not integrated into the nuclear genome, the foreign nucleic acids are diluted or degraded by mitosis. Cells that allow episomal amplification of nucleic acids greatly reduce the dilution rate. Stable transfection must occur if it is desired for the transfected nucleic acids to actually remain in the genome of the cells and their daughter cells. RNA can be transfected into cells to transiently express the encoded protein.

[0144] In one embodiment of all aspects of the present invention, nucleic acids encoding cytokines, or encoding antigens or variants thereof, are formulated into delivery vehicles such as particles. 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 nucleic acid. In one embodiment, the lipid is contained in vesicles that encapsulate the nucleic acid. In one embodiment of all aspects of the present invention, the nucleic acid is formulated into liposomes.

[0145] In the present disclosure, the term "RNA" relates to nucleic acid molecules containing ribonucleotide residues. In preferred embodiments, the RNA comprises all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2'-position of the β-D-ribofuranosyl group. RNA includes, without limitation, isolated RNA such as double-stranded RNA, single-stranded RNA, 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 can refer to the addition of non-nucleotide substances to internal RNA nucleotides or to the ends (one or both) of the RNA. It is also contemplated herein that the nucleotides in the RNA can be non-standard nucleotides such as chemically synthesized nucleotides or deoxynucleotides. In the present disclosure, these modified RNAs are considered analogs of naturally occurring RNA.

[0146] In certain embodiments of the present disclosure, the RNA is messenger RNA (mRNA) associated with an RNA transcript encoding a peptide or protein. As is 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 generated by in vitro transcription or chemical synthesis. In one embodiment, the mRNA is generated by in vitro transcription using a DNA template, where DNA refers to a nucleic acid containing deoxyribonucleotides.

[0147] In one embodiment, the RNA is in vitro transcribed RNA (IVT-RNA) and 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 cDNA, and introducing it into a suitable vector for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

[0148] In one embodiment, the RNA can have modified ribonucleotides. Examples of modified ribonucleotides include, without limitation, 5-methylcytidine, pseudouridine and / or 1-methylpseudouridine.

[0149] In some embodiments, the RNA according to 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 can 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 connected to the mRNA by a 5'-5' triphosphate bond. In one embodiment, this guanosine is methylated at the 7 position. Providing a 5' cap or 5' cap analog to the RNA can be achieved by in vitro transcription where the 5' cap is co-transcriptionally expressed on the RNA strand or can be ligated to the RNA post-transcriptionally using a capping enzyme.

[0150] In some embodiments, the RNA according to the present disclosure includes a 5'-UTR and / or a 3'-UTR. The term "untranslated region" or "UTR" relates to a region within a DNA molecule that is transcribed but not translated into an amino acid sequence, or the corresponding region within an RNA molecule such as an mRNA molecule. The untranslated region (UTR) can be present on the 5' side (upstream) (5'-UTR) and / or the 3' side (downstream) (3'-UTR) of the open reading frame. The 5'-UTR, when present, is located at the 5' end, upstream of the start codon of the protein-coding region. The 5'-UTR is downstream of the 5' cap (when present), for example, immediately adjacent to the 5' cap. The 3'-UTR, when present, is located at the 3' end, downstream of the stop codon of the protein-coding region, but the term "3'-UTR" preferably does not include the poly(A) tail. Thus, the 3'-UTR is upstream of the poly(A) sequence (when present), for example, immediately adjacent to the poly(A) sequence.

[0151] In some embodiments, the RNA according to the present disclosure comprises a 3'-poly(A) sequence. The term "poly(A) sequence" typically relates to a sequence of adenyl (A) residues located at the 3' end of an RNA molecule. According to the present disclosure, in one embodiment, the poly(A) sequence comprises at least about 20, at least about 40, at least about 80, or at least about 100, and up to about 500, up to about 400, up to about 300, up to about 200, or up to about 150 A nucleotides, particularly about 120 A nucleotides.

[0152] In the context of the present disclosure, the term "transcription" relates to the process by which the genetic code in a DNA sequence is transcribed into RNA. Subsequently, the RNA can be translated into a peptide or protein.

[0153] With respect to RNA, the term "expression" or "translation" relates to the process in the ribosomes of a cell by which a strand of mRNA directs the assembly of an amino acid sequence to produce a peptide or protein.

[0154] According to the present disclosure, the term "encoded by the RNA" means that when the RNA is present in a suitable environment, such as within the cells of a target tissue, it can direct the assembly of amino acids to produce the peptide or protein that it encodes during the translation process. In one embodiment, the RNA can interact with the cellular translation machinery that enables the translation of the peptide or protein. The cell can produce the encoded peptide or protein intracellularly (e.g., within the cytoplasm and / or nucleus), secrete the encoded peptide or protein, or produce it on the surface.

[0155] As used herein, the terms "linked", "fused", or "fusion" are used interchangeably. These terms refer to the joining of two or more elements or components or domains.

[0156] As used herein, "half-life" refers to the time required for the serum or plasma concentration of 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), and are resistant to degradation and / or clearance or sequestration. The 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 generally include, for example, the step of appropriately administering a suitable dose of an amino acid sequence or compound to a subject; the step of collecting blood samples or other samples from the subject at regular intervals; the step of determining the level or concentration of the amino acid sequence or compound in the blood sample; and the step of calculating from the data (plots) thus obtained the time until the level or concentration of the amino acid sequence or compound has decreased by 50% compared to the initial level at administration. Further details are provided, for example, in standard handbooks such as 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).

[0157] Cytokine Cytokines are a category of small proteins (about 5 - 20 kDa) that are important for cell signaling. The release of cytokines affects the behavior of the cells around them. Cytokines are involved in autocrine signaling, paracrine signaling, and endocrine signaling as immunomodulators. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, but generally do not include hormones or growth factors (although there is some overlap in terminology). Cytokines are produced by a wide variety of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes, and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. A given cytokine can be produced by multiple types of cells. Cytokines act via receptors and are particularly important in the immune system; cytokines regulate the balance between the humoral and cellular immune responses and control the maturation, growth, and responsiveness of specific cell populations. Some cytokines enhance or inhibit the actions of other cytokines in complex ways.

[0158] IL2 Interleukin 2 (IL2) is a cytokine that induces the proliferation of antigen-activated T cells and stimulates natural killer (NK) cells. The biological activity of IL2 is mediated by the multi-subunit IL2 receptor complex (IL2R), which consists of three polypeptide subunits spanning the cell membrane: p55 (IL2Rα, the α subunit, also known as CD25 in humans), p75 (IL2Rβ, the β subunit, also known as CD122 in humans), and p64 (IL2Rγ, the γ subunit, also known as CD132 in humans). The T cell response to IL2 depends 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 various components undergo different sorting. When administered as an intravenous (i.v.) bolus, IL2 has rapid systemic clearance (an initial clearance phase with a half-life of 12.9 minutes, followed by a more gradual clearance phase with a half-life of 85 minutes) (Konrad et al., Cancer Res. 50:2009-2017, 1990).

[0159] The results of systemic administration of IL2 in cancer patients are far from ideal. 15-20% of patients respond objectively to high-dose IL2, but the majority do not respond, and many experience severe and life-threatening side effects such as nausea, confusion, hypotension, and septic shock. The severe toxicity associated with high-dose IL2 treatment is mainly due to the activation of natural killer (NK) cells. Attempts have been made to reduce the dose and adjust the dosing regimen to lower serum concentrations, which reduces toxicity but such treatments have also been less effective.

[0160] 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 the serum IL2 concentration to be maintained within the therapeutic range in vivo and potentially leads 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.

[0161] According to the present disclosure, IL2 (optionally as part of extended PK IL2) can be a naturally occurring IL2 or a fragment or variant thereof. IL2 can be human IL2 and can be derived from any vertebrate, particularly any mammal. In one embodiment, IL2 comprises the amino acid sequence of SEQ ID NO: 1 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: 1. In one embodiment, IL2 or an IL2 fragment or variant binds to an IL2 receptor or a subunit of an IL2 receptor, such as the α subunit and / or the β / γ subunit.

[0162] In certain embodiments, the IL2 portion of extended PK IL2 is human IL2. In other embodiments, the IL2 portion of extended PK IL2 is a fragment or variant of human IL2.

[0163] In certain embodiments described herein, IL2 is fused to a heterologous polypeptide (i.e., a polypeptide that is not IL2). The heterologous polypeptide can increase the circulating half-life of IL2. As discussed in more detail below, polypeptides that increase the circulating half-life can be serum albumins such as human (e.g., SEQ ID NO: 4) or mouse (e.g., SEQ ID NO: 8, 11) serum albumin.

[0164] IL7 IL7 is a hematopoietic growth factor secreted by stromal cells of the bone marrow and thymus. It is also produced by keratinocytes, dendritic cells, hepatocytes, neurons, and epithelial cells, but not by normal lymphocytes. IL7 is an important cytokine for the development of B cells and T cells. The IL7 cytokine and hepatocyte growth factor form a heterodimer that functions as a pre-pro B cell growth stimulating factor. Knockout studies in mice have suggested that IL7 plays an essential role in the survival of lymphoid cells.

[0165] IL7 binds to the IL7 receptor, which is a heterodimer consisting of the IL7 receptor α and the common γ-chain receptor. The binding results in a cascade of signals that are important for T cell development in the thymus and survival in the periphery. Knockout mice genetically deficient in the IL7 receptor show thymic atrophy, arrest at the double-positive stage of T cell development, and severe lymphopenia. Administration of IL7 to mice results in an increase in thymic emigrant T cells, an increase in B cells and T cells, and an increase in the recovery of T cells after cyclophosphamide administration or bone marrow transplantation.

[0166] According to the present disclosure, in certain embodiments, IL7 is conjugated to a pharmacokinetic modifying group. The resulting molecule, hereinafter referred to as "extended pharmacokinetic (PK) IL7", has an extended circulating half-life compared to free IL7. The extended circulating half-life of extended PK IL7 allows serum IL7 concentrations to be maintained within the therapeutic range in vivo and potentially leads to enhanced survival of many types of immune cells, including T cells. Due to its favorable pharmacokinetic profile, extended PK IL7 can be administered less frequently and for a longer period of time compared to unmodified IL7.

[0167] According to the present disclosure, IL7 (optionally as part of an extended PK IL7) can be a naturally occurring IL7 or a fragment or variant thereof. IL7 can be human IL7 and can be derived from any vertebrate, particularly any mammal. In one embodiment, IL7 has the amino acid sequence of SEQ ID NO: 2, 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: 2. In one embodiment, IL7 or an IL7 fragment or variant binds to the IL7 receptor.

[0168] In certain embodiments, the IL7 portion of the extended PK IL7 is human IL7. In other embodiments, the IL7 portion of the extended PK IL7 is a fragment or variant of human IL7.

[0169] In certain embodiments described herein, IL7 is fused to a heterologous polypeptide (i.e., a polypeptide that is not IL7). The heterologous polypeptide can increase the circulating half-life of IL7. As discussed in more detail below, polypeptides that increase the circulating half-life can be serum albumins such as human (e.g., SEQ ID NO: 4) or mouse (e.g., SEQ ID NOs: 8, 11) serum albumin.

[0170] IL21 Interleukin 21 (IL21) is a cytokine that has a potent regulatory effect on cells of the immune system, including natural killer (NK) cells and cytotoxic T cells. This cytokine induces cell division / proliferation in its target cells. IL21 is expressed in activated human CD4+ T cells but not in most other tissues. Furthermore, IL21 expression is upregulated in the Th2 and Th17 subsets of T helper cells, as well as in T follicular cells. Additionally, IL21 is expressed in NK T cells that regulate the function of these cells. Interleukin 21 is also produced by Hodgkin lymphoma (HL) cancer cells.

[0171] The interleukin-21 receptor (IL21R) is expressed on the surface of T cells, B cells, and NK cells. IL21R has a structure similar to that of other type I cytokine receptors such as IL2 or IL15 and requires dimerization with the common gamma chain (γc) to bind IL21. When bound to IL21, the IL21 receptor acts via the Jak / STAT pathway and utilizes Jak1 and Jak3 as well as STAT3 homodimers to activate its target genes.

[0172] According to the present disclosure, in certain embodiments, IL21 is conjugated to a pharmacokinetic modifying group. The resulting molecule, hereinafter referred to as "extended pharmacokinetic (PK) IL21", has an extended circulating half-life compared to free IL21. The extended circulating half-life of extended PK IL21 enables the serum IL21 concentration to be maintained within the therapeutic range in vivo and potentially leads to enhanced activation of many types of immune cells, including T cells. Due to its favorable pharmacokinetic profile, extended PK IL21 can be administered less frequently and for a longer period compared to unmodified IL21.

[0173] According to the present disclosure, IL21 (optionally as part of extended PK IL21) can be a naturally occurring IL21 or a fragment or variant thereof. IL21 can be human IL21 and can be derived from any vertebrate, particularly any mammal. In one embodiment, IL21 has the amino acid sequence of SEQ ID NO: 3 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: 3. In one embodiment, IL21 or an IL21 fragment or variant binds to the IL21 receptor.

[0174] In certain embodiments, the IL21 portion of extended PK IL21 is human IL21. In other embodiments, the IL21 portion of extended PK IL21 is a fragment or variant of human IL21.

[0175] In certain embodiments described herein, IL21 is fused to a heterologous polypeptide (i.e., a polypeptide that is not IL21). The heterologous polypeptide can increase the circulating half-life of IL21. As discussed in further detail below, polypeptides that increase the circulating half-life can be serum albumins such as human (e.g., SEQ ID NO: 4) or mouse (e.g., SEQ ID NO: 8, 11) serum albumin.

[0176] Extended PK group Cytokines described herein, such as interleukins such as IL2, IL7 or IL21, can be fused to an extended PK group that increases the 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 variant 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).

[0177] As used herein, the term "PK" is an acronym for "pharmacokinetics" and encompasses, by way of example, the properties of a compound including absorption, distribution, metabolism, and excretion by a subject. As used herein, an "extended PK moiety" refers to a protein, peptide, or portion that, when fused to or co-administered with a biologically active molecule, increases the circulating half-life of the biologically active molecule. Examples of extended PK moieties include serum albumin (e.g., HSA), 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 moieties are disclosed in Kontermann et al., Current Opinion in Biotechnology 2011;22:868-876, which is incorporated herein by reference in its entirety. As used herein, an "extended PK cytokine" refers to a cytokine moiety combined with an extended PK moiety. In one embodiment, the extended PK cytokine is a fusion protein in which the cytokine moiety is linked or fused to the extended PK moiety. As used herein, "extended PK IL" refers to an interleukin (IL) moiety combined with an extended PK moiety. In one embodiment, the extended PK IL is a fusion protein in which the IL moiety is linked or fused to the extended PK moiety. Exemplary fusion proteins include an HSA / IL2 fusion in which the IL2 moiety is fused to HSA. Another exemplary fusion protein is an HSA / IL7 fusion in which the IL7 moiety is fused to HSA. Another exemplary fusion protein is an HSA / IL21 fusion in which the IL21 moiety is fused to HSA.

[0178] In certain embodiments, the serum half-life of the extended PK cytokine is increased compared to the cytokine alone (i.e., the cytokine not fused to the extended PK moiety). In certain embodiments, the serum half-life of the extended PK cytokine 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 cytokine alone. In certain embodiments, the serum half-life of the extended PK cytokine 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 cytokine alone. In certain embodiments, the serum half-life of the extended PK cytokine 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.

[0179] In certain embodiments, the extended PK moiety comprises serum albumin, or a fragment thereof, or a variant of serum albumin or a fragment thereof (for the purposes of the present disclosure, all of which are included in the term "albumin"). The polypeptides described herein can be fused to albumin (or a fragment or variant thereof) to form an albumin fusion protein. Such albumin fusion proteins are described in U.S. Patent Application Publication No. 20070048282.

[0180] 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 therapeutic protein, particularly a protein such as IL2, IL7 or IL21 (or a fragment or variant thereof). An albumin fusion protein can be produced by translation of a nucleic acid in which a polynucleotide encoding a therapeutic protein is ligated in-frame with a polynucleotide encoding albumin. When a therapeutic protein and albumin become part of an albumin fusion protein, they can be referred to, respectively, as a "portion", "region" or "part" of the albumin fusion protein (e.g., "therapeutic protein portion" or "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 the 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 of the target organ of the administered RNA, such as hepatocytes, and secreted into the circulation. Processing of the nascent albumin fusion protein that occurs in the secretory pathway of the host cell used for RNA expression can include, but is not limited to, signal peptide cleavage, disulfide bond formation, appropriate folding, carbohydrate addition and processing (e.g., N-linked and O-linked glycosylation, etc.), specific proteolytic cleavage, and / or assembly into multimeric proteins. The albumin fusion protein is preferably encoded by an unprocessed form of RNA that particularly has a signal peptide at its N-terminus and, after secretion by the cell, preferably exists in a processed form in which the signal peptide is cleaved. In the most preferred embodiment, "processed form of albumin fusion protein" refers to an albumin fusion protein product that has undergone N-terminal signal peptide cleavage and is also referred to herein as "mature albumin fusion protein".

[0181] In a preferred embodiment, an albumin fusion protein comprising a therapeutic protein has higher plasma stability compared to 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 carried in the bloodstream until the therapeutic protein is degraded, cleared from the bloodstream to an organ such as the kidney or liver, and ultimately cleared from the body. Plasma stability is calculated with respect to the half-life of the therapeutic protein in the bloodstream. The half-life of the therapeutic protein in the bloodstream can be readily determined by common assays known in the art.

[0182] 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 (such as biological activity) 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 a human, cow, sheep, or pig. Non-mammalian albumins include, but are not limited to, hen and salmon. The albumin portion of an albumin fusion protein can be derived from an animal different from the therapeutic protein portion.

[0183] In one embodiment, the albumin is human serum albumin (HSA), or a fragment or variant thereof, such as those disclosed in U.S. Patent No. 5,876,969, International Publication No. WO 2011 / 124718, International Publication No. WO 2013 / 075066, and International Publication No. WO 2011 / 0514789.

[0184] The terms "human serum albumin (HSA)" and "human albumin (HA)" are used interchangeably herein. The terms "albumin" and "serum albumin" are broader and include human serum albumin (and its fragments and variants) as well as albumin from other species (and its fragments and variants).

[0185] As used herein, an albumin fragment sufficient to extend the therapeutic activity or plasma stability of a therapeutic protein refers to an albumin fragment of a length or structure sufficient to stabilize or extend the therapeutic activity or plasma stability of the therapeutic protein portion of an albumin fusion protein such that the plasma stability of the therapeutic protein portion of the albumin fusion protein is extended or increased compared to its plasma stability in the unfused state.

[0186] The albumin portion of an albumin fusion protein can include the full length of the albumin sequence or one or more fragments thereof that can stabilize or extend therapeutic activity or plasma stability. Such fragments can be at least 10 amino acids in length or can include about 15, 20, 25, 30, 50 or more contiguous amino acids from the albumin sequence or can include a part or all of a particular domain of albumin. For example, one or more fragments of HSA spanning the first two immunoglobulin-like domains can be used. In a preferred embodiment, the HSA fragment is the mature form of HSA.

[0187] Generally speaking, an albumin fragment or variant is at least 100 amino acids in length, preferably at least 150 amino acids in length.

[0188] 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 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.

[0189] Preferably, the albumin fusion protein comprises albumin as the N-terminal portion and a therapeutic protein as the C-terminal portion. Alternatively, an albumin fusion protein that comprises albumin as the C-terminal portion and a therapeutic protein as the N-terminal portion can also be used. In other embodiments, the albumin fusion protein has a therapeutic protein fused to both the N-terminal and C-terminal of albumin. In a preferred embodiment, the therapeutic proteins fused at the N-terminal and C-terminal are the same therapeutic protein. In another preferred embodiment, the therapeutic proteins fused at the N-terminal and C-terminal are different therapeutic proteins. In one embodiment, the different therapeutic proteins can be useful for treating or preventing the same or related diseases, disorders, or conditions. In one embodiment, both of the different therapeutic proteins are cytokines.

[0190] In one embodiment, the therapeutic protein(s) is / are bound to the albumin via a peptide linker(s). The linker peptide between the fusion portions provides a greater physical separation between the portions and can thus maximize the accessibility of the therapeutic protein portion for binding to its cognate receptor, for example. The linker peptide can be composed of amino acids such that it is flexible or more rigid. The linker sequence can be cleavable by proteases or chemically.

[0191] As used herein, the term "Fc region" refers to the portion of a native immunoglobulin formed by the Fc domain (or Fc portion) of each of the two heavy chains of the immunoglobulin. As used herein, the term "Fc domain" refers to a part or fragment of a single immunoglobulin (Ig) heavy chain in which the Fc domain does not contain the 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 includes at least the hinge domain, CH2 domain, and CH3 domain. In certain embodiments, the Fc domain includes at least one of the hinge (e.g., upper, middle and / or lower hinge region) domain, CH2 domain, CH3 domain, CH4 domain, or variants, portions or fragments thereof. In certain embodiments, the Fc domain includes the complete Fc domain (i.e., the hinge domain, CH2 domain, and CH3 domain). In certain embodiments, the Fc domain includes a hinge domain (or a portion thereof) fused to a CH3 domain (or a portion thereof). In certain embodiments, the Fc domain includes a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof). In certain embodiments, the Fc domain consists of a CH3 domain or a portion thereof. In certain embodiments, the Fc domain consists of a hinge domain (or a portion thereof) and a CH3 domain (or a portion thereof). In certain embodiments, the Fc domain consists of a CH2 domain (or a portion thereof) and a CH3 domain. In certain embodiments, the Fc domain consists of a hinge domain (or a portion thereof) and a CH2 domain (or a portion thereof). In certain embodiments, the Fc domain lacks at least a portion of the CH2 domain (e.g., all or a portion of the CH2 domain). The Fc domain herein generally refers to a polypeptide that includes all or a portion of the Fc domain of an immunoglobulin heavy chain. This includes polypeptides that include the entire CH1, hinge, CH2, and / or CH3 domains, as well as fragments of such peptides that include, for example, only the hinge, CH2, and CH3 domains, but is not limited thereto.The Fc domain can be derived from immunoglobulins of any species and / or any subtype, including but not limited to human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies. The Fc domain encompasses native Fc and Fc variant molecules. As described herein, it will be understood by those skilled in the art that any Fc domain can be modified such that its amino acid sequence is different from that of the native Fc domain of a naturally occurring immunoglobulin molecule. In certain embodiments, the Fc domain has reduced effector function (e.g., FcγR binding).

[0192] The Fc domain of the polypeptides described herein can be derived from different immunoglobulin molecules. For example, the Fc domain of a polypeptide can include the CH2 and / or CH3 domains derived from an IgG1 molecule, as well as the hinge region derived from an IgG3 molecule. In another example, the Fc domain can include a chimeric hinge region that is partially derived from an IgG1 molecule and partially derived from an IgG3 molecule. In another example, the Fc domain can include a chimeric hinge that is partially derived from an IgG1 molecule and partially derived from an IgG4 molecule.

[0193] In certain embodiments, the extended PK group includes the Fc domain or a fragment thereof, or a variant of the Fc domain or a fragment thereof (for the purposes of this disclosure, all of which are included in the term "Fc domain"). The Fc domain does not include a variable region that binds to an antigen. Suitable Fc domains 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 the human IgG1 constant region. However, it is understood that the Fc domain can be derived from immunoglobulins of another mammalian species, including, for example, rodent species (e.g., mouse, rat, rabbit, guinea pig) or non-human primate species (e.g., chimpanzee, macaque).

[0194] Furthermore, the Fc domain (or fragment or variant thereof) can be derived from any immunoglobulin class including IgM, IgG, IgD, IgA, and IgE, as well as any immunoglobulin isotype including IgG1, IgG2, IgG3, and IgG4.

[0195] 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 that contain Fc domain sequences having specific modifications that lack certain effector functions and / or reduce immunogenicity. Many sequences of antibodies and genes encoding antibodies are published, and suitable Fc domain sequences (e.g., hinge, CH2, and / or CH3 sequences, or fragments or variants thereof) can be obtained from these sequences using techniques well recognized in the art.

[0196] In certain embodiments, the extended PK moiety is a serum albumin-binding protein such as those described in U.S. Patent Application No. 2005 / 0287153, U.S. Patent Application No. 2007 / 0003549, U.S. Patent Application No. 2007 / 0178082, U.S. Patent Application No. 2007 / 0269422, U.S. Patent Application No. 2010 / 0113339, International Publication No. 2009 / 083804, and International Publication No. 2009 / 133208, which are hereby incorporated by reference in their entirety. In certain embodiments, the extended PK moiety is transferrin, as disclosed in U.S. Patent No. 7,176,278 and U.S. Patent No. 8,158,579, which are hereby incorporated by reference in their entirety. In certain embodiments, the extended PK moiety is a serum immunoglobulin-binding protein such as those described in U.S. Patent Application No. 2007 / 0178082, which is hereby incorporated by reference in its entirety. In certain embodiments, the extended PK moiety is a fibronectin (Fn)-based scaffold domain protein that binds to serum albumin such as those described in U.S. Patent Application No. 2012 / 0094909, which is hereby incorporated by reference in its entirety. Methods for making fibronectin-based scaffold domain proteins are also disclosed in U.S. Patent Application No. 2012 / 0094909. A non-limiting example of an Fn3-based extended PK moiety is Fn3(HSA), i.e., an Fn3 protein that binds to human serum albumin.

[0197] In certain aspects, extended PK cytokines such as extended PK IL suitable for use according to the present disclosure can 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 moiety and an IL moiety such as IL2, IL7, or IL21) in a linear amino acid sequence of a polypeptide chain. For example, a peptide linker can be used to link an IL2 moiety to an HSA domain. In another embodiment, a peptide linker can be used to link an IL7 moiety to an HSA domain. In another embodiment, a peptide linker can be used to link an IL21 moiety to an HSA domain.

[0198] Linkers suitable for fusing an extended PK group to, for example, IL2, IL7 or IL21 are well known in the art. Exemplary linkers include glycine-serine polypeptide linkers, glycine-proline polypeptide linkers, and proline-alanine polypeptide linkers. In certain embodiments, the linker is a glycine-serine polypeptide linker, i.e., a peptide consisting of glycine and serine residues.

[0199] Antigen Peptide and protein antigens suitable for use according to the present disclosure, i.e., antigens or variants thereof, typically include peptides or proteins that contain epitopes for inducing an immune response. The peptide or protein or epitope can be derived from a target antigen, i.e., the antigen against which an immune response is to be elicited. For example, the peptide or protein antigen, or the epitope contained within the peptide or protein antigen, can be the target antigen or a fragment or variant of the target antigen.

[0200] The administered or administered nucleic acid, particularly the peptide and protein antigens encoded by RNA, i.e., the vaccine antigen, preferably results in the stimulation, priming, and / or expansion of T cells genetically modified to express CAR in the subject to whom the peptide or protein antigen or nucleic acid is administered. The stimulated, primed, and / or expanded T 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 terms "fragment of an antigen" or "variant of an antigen" mean an agent that results in the stimulation, priming, and / or expansion of CAR-engineered T cells, and the stimulated, primed, and / or expanded T cells target an antigen, i.e., a disease-associated antigen, particularly when presented by diseased cells, tissues, and / or organs. Thus, the vaccine antigen may correspond to or comprise a disease-associated antigen, may correspond to or comprise a fragment of a disease-associated antigen, or may correspond to or comprise an antigen homologous to a disease-associated antigen or a fragment thereof. When the 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 the CAR of the CAR-engineered T cells, or a sequence homologous to an epitope of the disease-associated antigen. Thus, according to the present disclosure, the 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. The "immunogenic fragment of an antigen" according to the present disclosure preferably relates to a fragment of an antigen that can stimulate, prime, and / or expand T cells carrying a CAR that binds to the antigen or cells expressing the antigen. The vaccine antigen is preferably capable of being expressed on the surface of cells such as antigen-presenting cells to provide a relevant epitope for binding by CAR-engineered T cells (similar to the disease-associated antigen). The vaccine antigen can be a recombinant antigen.

[0201] The term "immunologically equivalent" means that immunologically equivalent molecules, such as immunologically equivalent amino acid sequences, exhibit the same or essentially the same immunological properties and / or exert the same or essentially the same immunological effects, for example, with respect to the type of immunological action. In the context of the present disclosure, the term "immunologically equivalent" is preferably used with respect to the immunological effects or properties of an antigen or antigen variant used for immunization. For example, when an amino acid sequence induces an immune response having specificity to react with a reference amino acid sequence when exposed to the immune system of a subject, such as a T cell that binds to the reference amino acid sequence or a cell expressing the reference amino acid sequence, the amino acid sequence is immunologically equivalent to the reference amino acid sequence. Thus, a molecule that is immunologically equivalent to an antigen exhibits the same or essentially the same properties as the antigen targeted by T cells and / or exerts the same or essentially the same effects with respect to T cell stimulation, priming, and / or expansion.

[0202] The term "priming" refers to the process by which T cells first contact their specific antigen and trigger differentiation into effector T cells.

[0203] The term "clonal expansion" or "expansion" refers to the process by which a particular entity increases. In the context of the present disclosure, this 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 the antigen are amplified. Preferably, clonal expansion results in lymphocyte differentiation.

[0204] The term "antigen" relates to an agent that includes an epitope capable of eliciting an immune response. The term "antigen" particularly includes proteins and peptides. In one embodiment, the antigen is present on the surface of cells of the immune system, such as antigen-presenting cells like dendritic cells or macrophages. An antigen or a processing product thereof, such as a T cell epitope, is bound by a CAR molecule in one embodiment. Thus, the antigen or its processing product can specifically react with 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.

[0205] The term "disease-related antigen" is used in its broadest sense to refer to any antigen related to a disease. A disease-related antigen is a molecule that includes 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-related antigen or its epitope can be used for therapeutic purposes. A disease-related antigen can be related to an infection by a microorganism, typically a microbial antigen, or can be related to cancer, typically a tumor.

[0206] The term "tumor antigen" refers to components of cancer cells that can be derived from the cytoplasm, cell surface, and cell nucleus. In particular, this term refers to antigens that are 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 on non-cancer cells), and in some cases, are expressed only by cancer cells. Examples of tumor antigens include, without limitation, cell surface proteins of the claudin family such as p53, ART-4, BAGE, β-catenin / m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, 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, MUC1, 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.

[0207] The term "viral antigen" refers to any viral component that has antigenic properties, i.e., can induce an immune response in an individual. Viral antigens can be viral ribonucleoproteins or envelope proteins.

[0208] The term "bacterial antigen" refers to any bacterial component that has antigenic properties, i.e., can induce an immune response in an individual. Bacterial antigens can be derived from the cell wall or cytoplasmic membrane of bacteria.

[0209] The term "epitope" refers to a part or fragment of a molecule such as an antigen that is recognized by the immune system. For example, an epitope can be recognized by a T cell, a B cell, or an antibody. The epitope of an antigen can include contiguous or non - contiguous portions of the antigen and can be about 5 to about 100, such as 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 preferably be 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, the epitope is about 10 to about 25 amino acids in length. The term "epitope" includes T - cell epitopes.

[0210] The term "T - cell epitope" refers to a part or fragment of a protein that is recognized by a T cell when presented in association with an MHC molecule. The terms "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes that includes MHC class I and MHC class II molecules and is present in all vertebrates. MHC proteins or molecules are important for signal transduction between lymphocytes and antigen - presenting cells or diseased cells in an immune response. MHC proteins or molecules bind to peptide epitopes and present them for recognition by T - cell receptors on T cells. Proteins encoded by MHC are expressed on the surface of cells and present both self - antigens (peptide fragments from the cell itself) and non - self antigens (e.g., fragments of invading microorganisms) to T cells. In the case of class I MHC / peptide complexes, the bound peptide is typically about 8 to about 10 amino acids long, although longer or shorter peptides can also be effective. In the case of class II MHC / peptide complexes, the bound 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.

[0211] In one embodiment, the target antigen is a tumor antigen, and the vaccine antigen or a fragment thereof (e.g., an epitope) is derived from the tumor antigen. The tumor antigen can be a "standard" antigen that is generally known to be expressed in various cancers. The tumor antigen can also be a "neoantigen" that is specific to an individual's tumor and has not been previously recognized by the immune system. A neoantigen or neoepitope can result from one or more cancer-specific mutations in the genome of cancer cells that cause amino acid changes. When the tumor antigen is a neoantigen, the vaccine antigen preferably includes an epitope or fragment of the neoantigen that includes one or more amino acid changes.

[0212] Peptide and protein antigens can be 2 to 100 amino acids in length, including, for example, lengths of 5 amino acids, 10 amino acids, 15 amino acids, 20 amino acids, 25 amino acids, 30 amino acids, 35 amino acids, 40 amino acids, 45 amino acids, or 50 amino acids. In some embodiments, the peptide can be more than 50 amino acids. In some embodiments, the peptide can be more than 100 amino acids.

[0213] According to the present invention, the antigen or a variant thereof should be recognizable by a CAR. Preferably, when the antigen or a variant thereof is recognized by a CAR, in the presence of an appropriate co-stimulatory signal, it can induce the stimulation, priming and / or expansion of T cells carrying a CAR that recognizes the antigen or a variant thereof. In the context of embodiments of the present invention, the antigen or a variant thereof is preferably present on the surface of a cell, preferably an antigen-presenting cell. Recognition of an antigen on the surface of diseased cells can result in an immune response against the antigen (or the cell expressing the antigen).

[0214] According to various aspects of the present invention, the object is, preferably, to provide an immune response against cancer cells expressing tumor antigens such as CLDN6 or CLDN18.2, and to treat cancer diseases in which cells expressing tumor antigens such as CLDN6 or CLDN18.2 are involved. Preferably, the present invention includes the administration of CAR-engineered T cells targeting cancer cells expressing tumor antigens such as CLDN6 or CLDN18.2.

[0215] "Cell surface" is used according to its ordinary meaning in the art and thus includes the outer side of the cell accessible for binding by proteins and other molecules. An antigen is expressed on the surface of a cell if it is located on the surface of the cell and accessible, for example, for binding by an antigen-specific antibody added to the cell. In one embodiment, the antigen expressed on the surface of the cell is an endogenous membrane protein having an extracellular portion recognized by a CAR.

[0216] The term "extracellular portion" or "ectodomain" in the context of the present invention faces the extracellular space of the cell and is preferably accessible from the outside of the cell by binding to a molecule such as a protein that is accessible from the outside of the cell, for example, by binding to an antibody located outside the cell. Preferably, the term refers to one or more extracellular loops or domains or fragments thereof.

[0217] In one embodiment of all aspects of the present invention, the antigen is expressed in diseased cells such as cancer cells. In one embodiment, the antigen is expressed on the surface of diseased cells such as cancer cells. In one embodiment, the CAR binds to the extracellular domain of the antigen or its variant or an epitope within the extracellular domain. In one embodiment, the CAR binds to the native epitope of the antigen or its variant present on the surface of living cells. In one embodiment, the antigen is claudin, particularly claudin 6 or claudin 18.2, and the CAR binds to the first extracellular loop of the claudin. In one embodiment, the binding of the CAR, when expressed by a T cell and / or present on a T cell, to the antigen or its variant present on a cell such as an antigen-presenting cell results in the stimulation, priming and / or expansion of the T cell. In one embodiment, the binding of the CAR, when expressed by a T cell and / or present on a T cell, to the antigen present on diseased cells such as cancer cells results in the cytolysis and / or apoptosis of the diseased cells, and the T cell preferably releases cytotoxic factors such as perforin and granzyme.

[0218] Immune checkpoint inhibitor In certain embodiments, the immune checkpoint inhibitor is used in combination with other therapeutic agents described herein.

[0219] As used herein, "immune checkpoint" refers to co-stimulatory and inhibitory signals that regulate the magnitude and quality of T cell receptor recognition of an antigen. 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, which replaces CD28 binding, and CD80 or CD86. In certain embodiments, the inhibitory signal is the interaction between LAG3 and MHC class II molecules. In certain embodiments, the inhibitory signal is the interaction between TIM3 and galectin 9.

[0220] As used herein, "immune checkpoint inhibitor" refers to a molecule that completely or partially reduces, inhibits, blocks or modulates one or more checkpoint proteins. In certain embodiments, the immune checkpoint inhibitor prevents an inhibitory signal associated with an immune checkpoint. In certain embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that interferes with inhibitory signal transduction associated with an immune checkpoint. In certain embodiments, the immune checkpoint inhibitor is a small molecule that interferes with inhibitory signal transduction. In certain embodiments, the immune checkpoint inhibitor is an antibody, fragment thereof, or antibody mimetic that interferes with the interaction between checkpoint blockade proteins, e.g., an antibody or fragment thereof that interferes with the interaction between PD-1 and PD-L1. In certain embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that can prevent the interaction between CTLA-4 and CD80 or CD86. In certain embodiments, the 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. The checkpoint inhibitor can also be in the soluble form of the molecule (or variant thereof) itself, e.g., in the form of soluble PD-L1 or a PD-L1 fusion.

[0221] The "programmed death 1 (PD-1)" receptor refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is mainly expressed on previously activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. The term "PD-1" as used herein includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, as well as analogs having at least one common epitope with hPD-1.

[0222] "Programmed death ligand 1 (PD-L1)" is one of two cell surface glycoprotein ligands of PD-1 (the other being PD-L2) that, when bound to PD-1, downregulates T cell activation and cytokine secretion. As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, as well as analogs having at least one common epitope with hPD-L1.

[0223] "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 having at least one common epitope with hCTLA-4.

[0224] "Lymphocyte activation gene 3 (LAG3)" is an inhibitory receptor associated with the inhibition of lymphocyte activation 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 having at least one common epitope.

[0225] "T cell membrane protein 3 (TIM3)" is an inhibitory receptor involved in the inhibition of lymphocyte activation 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 having at least one common epitope.

[0226] The "B7 family" refers to inhibitory ligands of undefined receptors. The B7 family includes B7-H3 and B7-H4, both of which are upregulated in tumor cells and tumor infiltrating cells.

[0227] In certain embodiments, an immune checkpoint inhibitor suitable for use in the methods disclosed herein is an antagonist of an inhibitory signal, e.g., an antibody that targets 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.

[0228] In certain embodiments, the immune checkpoint inhibitor is an antibody or antigen-binding portion thereof that interferes with or inhibits signal transduction from an inhibitory immunoregulatory factor. In certain embodiments, the immune checkpoint inhibitor is a small molecule that interferes with or inhibits signal transduction from an inhibitory immunoregulatory factor.

[0229] In certain embodiments, the inhibitory immunoregulatory factor is a component of the PD-1 / PD-L1 signal transduction pathway. Accordingly, certain embodiments of the present disclosure provide for administration 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.

[0230] In certain embodiments, the inhibitory immunoregulatory factor is a component of the CTLA4 signal transduction pathway. Accordingly, certain embodiments of the present disclosure provide for administration to a subject an antibody or antigen-binding portion thereof that targets CTLA4 and interferes with its interaction with CD80 and CD86.

[0231] In certain embodiments, the inhibitory immunoregulatory factor is a component of the LAG3 (lymphocyte activation gene 3) signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administration to a subject of an antibody or antigen-binding portion thereof that targets LAG3 and interferes with its interaction with MHC class II molecules.

[0232] In certain embodiments, the inhibitory immunoregulatory factor is a component of the B7 family signaling pathway. In certain embodiments, the B7 family members are B7-H3 and B7-H4. Accordingly, certain embodiments of the present disclosure provide for administration to a subject of an antibody or antigen-binding portion thereof that targets B7-H3 or B7-H4. The B7 family does not have defined receptors, but these ligands are upregulated in tumor cells or tumor-infiltrating cells. Preclinical mouse models have shown that blockade of these ligands can enhance anti-tumor immunity.

[0233] In certain embodiments, the inhibitory immunoregulatory factor is a component of the TIM3 (T cell membrane protein 3) signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administration to a subject of an antibody or antigen-binding portion thereof that targets TIM3 and interferes with its interaction with galectin 9.

[0234] One of ordinary skill in the art will understand that other immune checkpoint targets may 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, by increased T cell proliferation, enhanced T cell activation, and / or increased cytokine production (e.g., IFN-γ, IL2).

[0235] According to the present disclosure, the term "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The term "antibody" includes monoclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and chimeric antibodies. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain is composed 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 hypervariable regions called complementarity determining regions (CDRs) with more conserved regions called framework regions (FRs) incorporated therein. Each VH and VL is composed of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of the 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 of the classical complement system (C1q).

[0236] Antibodies can be derived from various species including, but not limited to, mouse, rat, rabbit, guinea pig, and human.

[0237] Antibodies described herein include IgA such as IgA1 or IgA2, IgG1, IgG2, IgG3, IgG4, IgE, IgM, and IgD antibodies. In various embodiments, the antibody is an IgG1 antibody, more specifically IgG1, kappa or IgG1, lambda isotype (i.e., IgG1, κ, λ), IgG2a antibody (e.g., IgG2a, κ, λ), IgG2b antibody (e.g., IgG2b, κ, λ), IgG3 antibody (e.g., IgG3, κ, λ) or IgG4 antibody (e.g., IgG4, κ, λ).

[0238] The term "antigen-binding portion" (or simply "binding portion") of an antibody or "antigen-binding fragment" (or simply "binding fragment") of an antibody, or similar terms, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be carried out by fragments of a full-length antibody. Examples of binding fragments included within the term "antigen-binding portion" of an antibody are: (i) the Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH domains; (ii) the F(ab') 2(iii) an Fd fragment consisting of a VH domain and a CH domain; (iv) an Fv fragment consisting of a VL domain and a VH domain of one arm of an antibody; (v) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); (vi) an isolated complementarity determining region (CDR), and (vii) a combination of two or more isolated CDRs optionally linked by a synthetic linker. Further, the two domains of an Fv fragment, VL and VH, are encoded by separate genes, but they are linked by recombinant methods using a synthetic linker that enables them to pair to form a monovalent molecule (known as a single-chain Fv (scFv); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody. Further examples include (i) a binding domain polypeptide fused to an immunoglobulin hinge region polypeptide, (ii) an immunoglobulin heavy chain CH2 constant region fused to a hinge region, and (iii) an immunoglobulin heavy chain CH3 constant region fused to a CH2 constant region, which are binding domain immunoglobulin fusion proteins. The binding domain polypeptide can be a heavy chain variable region or a light chain variable region. The binding domain immunoglobulin fusion proteins are further disclosed in U.S. Patent Application No. 2003 / 0118592 and U.S. Patent Application No. 2003 / 0133939. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0239] RNA targeting According to the present disclosure, after administration of the RNA described herein, at least a portion of the RNA is delivered to the target cells. In one embodiment, at least a portion of the RNA is delivered to the cytosol of the target cells. In one embodiment, the RNA is translated by the target cells to produce the encoded peptide or protein.

[0240] Some aspects of the present disclosure include targeted delivery of RNA disclosed herein (e.g., RNA encoding a cytokine and RNA encoding an antigen or a variant thereof).

[0241] In one embodiment, the present disclosure includes targeting the lymphatic system, particularly secondary lymphoid organs, more specifically the spleen. When the RNA to be administered is RNA encoding an antigen or a variant thereof, it is particularly preferred to target the lymphatic system, particularly secondary lymphoid organs, more specifically the spleen.

[0242] In one embodiment, the target cells are spleen cells. In one embodiment, the target cells are antigen-presenting cells such as professional antigen-presenting cells in the spleen. In one embodiment, the target cells are dendritic cells of the spleen.

[0243] The "lymphatic system" is an important part of the immune system that is part of the circulatory system and includes a network of lymphatic vessels that carry lymph. The lymphatic system consists of lymphoid organs, a conducting network of lymphatic vessels, and circulating lymph. Primary or central lymphoid organs generate lymphocytes from immature progenitor cells. The thymus and bone marrow constitute primary lymphoid organs. Secondary or peripheral lymphoid organs, including lymph nodes and the spleen, maintain mature naive lymphocytes and initiate an adaptive immune response.

[0244] RNA can be delivered to the spleen by so-called lipoplex formulations in which the RNA binds to liposomes containing cationic lipids and optionally further lipids or helper lipids to form an injectable nanoparticle formulation. The liposomes can be obtained by injecting a solution of the lipids in ethanol into water or a suitable aqueous phase. The RNA lipoplex particles can be prepared by mixing the liposomes with the RNA. Spleen-targeted RNA lipoplex particles are described in International Publication No. WO 2013 / 143683, which is incorporated herein by reference. It has been found that RNA lipoplex particles having 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. Accordingly, 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 within the spleen. Accordingly, 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.

[0245] In the context of the present disclosure, the term "RNA lipoplex particles" relates to particles comprising lipids, particularly cationic lipids, and RNA. Electrostatic interactions between the positively charged liposomes and the negatively charged RNA result in complexation and the spontaneous formation of the RNA lipoplex particles. The positively charged liposomes can generally be synthesized using a cationic lipid such as DOTMA and a further lipid such as DOPE. In one embodiment, the RNA lipoplex particles are nanoparticles.

[0246] As used herein, "cationic lipid" refers to a lipid having a net positive charge. Cationic lipids bind negatively charged RNA to the lipid matrix by electrostatic interaction. Generally, cationic lipids have a lipophilic moiety such as a sterol, acyl or diacyl chain, and the head group of the lipid typically bears a positive charge. Examples of cationic lipids include, but are not limited to, 1,2-di-O-octadecyl-3-trimethylammonium propane (DOTMA), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); 1,2-dioleoyl-3-dimethylammonium propane (DODAP); 1,2-diacetyloxy-3-dimethylammonium propane; 1,2-dialkyloxy-3-dimethylammonium propane; dioctadecyldimethylammonium chloride (DODAC), 2,3-di(tetradecyloxy)propyl-(2-hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), and 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanaminium trifluoroacetate (DOSPA). DOTMA, DOTAP, DODAC, and DOSPA are preferred. In certain embodiments, the cationic lipid is DOTMA and / or DOTAP.

[0247] To adjust the overall ratio of positive to negative charges and the physical stability of the RNA lipoplex particles, additional lipids may be incorporated. In certain embodiments, the additional lipid is a neutral lipid. As used herein, "neutral lipid" refers to a lipid having a net charge of zero. 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.

[0248] In certain embodiments, the RNA lipoplex particles contain both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

[0249] In some embodiments, the molar ratio of at least one cationic lipid to at least one additional lipid is from about 10:0 to about 1:9, from about 4:1 to about 1:2, or from 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 at least one cationic lipid to at least one additional lipid is about 2:1.

[0250] In one embodiment, the RNA lipoplex particles described in this specification 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 in the range of about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.

[0251] The charge of the RNA lipoplex particles of the present disclosure is the sum of the charges present in at least one cationic lipid and the charges present in the RNA. The charge ratio is the ratio of the positive charges present in at least one cationic lipid to the negative charges present in the RNA. The charge ratio of the positive charges present in at least one cationic lipid to the negative charges present in the RNA is calculated by the following formula: Charge ratio = [(cationic lipid concentration (mol)) * (total number of positive charges in the cationic lipid)] / [(RNA concentration (mol)) * (total number of negative charges in the RNA)].

[0252] At physiological pH, the spleen-targeted RNA lipoplex particles described herein preferably have a net negative charge, such as a charge ratio of positive charge to negative charge of about 1.9:2 to about 1:2. In certain embodiments, the charge ratio of positive charge to negative charge 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.

[0253] Extended PK cytokines, particularly cytokines such as extended PK interleukins as described herein, can be delivered to a target organ or target tissue in a subject, which involves administering to the subject an RNA encoding the cytokine in a formulation for selective delivery of the RNA to the target organ or target tissue.

[0254] In one embodiment, the target organ is the lymphatic system, particularly secondary lymphoid organs, more specifically the spleen, and the target tissue is lymphatic tissue, particularly the tissue of secondary lymphoid organs, more specifically spleen tissue. Delivery of cytokines to such target tissues is particularly preferred when the presence of cytokines in this organ or tissue is desirable (e.g., to induce an immune response, particularly when cytokines are required during T cell priming, or for activation of resident immune cells), but it is not desirable for the cytokines to be present systemically, particularly in significant amounts (e.g., because the cytokines have systemic toxicity). Particularly preferred examples of suitable cytokines are those involved in T cell priming.

[0255] In another embodiment of delivery of cytokines to a target organ or target tissue of a subject, the target organ is the liver and the target tissue is liver tissue. Delivery of cytokines to such target tissues is particularly preferred when the presence of cytokines in this organ or tissue is desirable, and / or when it is desirable to express large amounts of cytokines, and / or when systemic presence of the cytokines, particularly in significant amounts, may be desirable or required.

[0256] In one embodiment, the RNA encoding the cytokine is administered in a formulation for targeting the liver. Such formulations are described herein. Examples of suitable cytokines include IL2, IL7 or IL21, fragments and variants thereof, and fusion proteins of these cytokines, fragments and variants such as the extended PK cytokines as described herein. Particularly preferred examples of suitable cytokines are cytokines involved in the proliferation and / or maintenance of T cells.

[0257] The RNA delivery system has intrinsic selectivity for the liver. This is related to lipid-based particles, cationic and neutral nanoparticles, particularly lipid nanoparticles such as liposomes, nanomicelles and lipophilic ligands of bioconjugates. Liver accumulation is caused by the discontinuous nature of the hepatic vasculature or lipid metabolism (liposomes and lipid or cholesterol conjugates).

[0258] For in vivo delivery of RNA to the liver, a drug delivery system can be used to transport the 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 the RNA under physiological conditions. Furthermore, the stealth properties provided by the polyplex nanomicelle surface composed of a high density of PEG paricles effectively avoid the host's immune defense.

[0259] Pharmaceutical composition The agents described herein can be administered in a pharmaceutical composition or medicament and can be administered in any suitable form of pharmaceutical composition.

[0260] In one embodiment of all aspects of the present invention, the components described herein, such as T cells genetically modified to express a CAR, a nucleic acid encoding a cytokine, or a nucleic acid encoding an antigen or a variant thereof, may be administered in a pharmaceutical composition that may contain, together or separately from each other, a pharmaceutically acceptable carrier and optionally one or more adjuvants, stabilizers, etc. In one embodiment, the pharmaceutical composition is for therapeutic or prophylactic treatment, for example, for use in the treatment or prevention of a disease involving an antigen, such as a cancer disease as described herein.

[0261] The term "pharmaceutical composition" preferably relates to a formulation containing a therapeutically effective agent together with a pharmaceutically acceptable carrier, diluent and / or excipient. The pharmaceutical composition is useful for treating, preventing or reducing the severity of a disease or disorder by administering the pharmaceutical composition to a subject. Pharmaceutical compositions are also known in the art as pharmaceutical formulations.

[0262] The pharmaceutical compositions of the present disclosure preferably contain one or more adjuvants or may be administered with one or more adjuvants. The term "adjuvant" relates to a compound that prolongs, enhances or accelerates an immune response. Adjuvants include a group of heterogeneous compounds such as oil emulsions (e.g., Freund's adjuvant), inorganic compounds (such as alum), bacterial products (such as Bordetella pertussis toxin), or immunostimulatory complexes. Examples of adjuvants include, without limitation, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines such as monokines, lymphokines, interleukins, chemokines. Chemokines can be IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IFNα, IFNγ, GM-CSF, LT-α. Further known adjuvants are aluminum hydroxide, Freund's adjuvant, or oils such as Montanide™ ISA51. Other suitable adjuvants for use in the present disclosure include lipopeptides such as Pam3Cys.

[0263] The pharmaceutical compositions according to the present disclosure are generally applied with a "pharmaceutically effective amount" and a "pharmaceutically acceptable formulation".

[0264] The term "pharmaceutically acceptable" refers to the non-toxicity of substances that do not interact with the action of the active ingredient of the pharmaceutical composition.

[0265] The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to the amount that, alone or in combination with additional dosages, achieves a desired response or a desired effect. In the case of the treatment of a particular disease, the desired response preferably relates to the inhibition of the progression of the disease. This includes slowing down the progression of the disease, particularly interrupting or reversing the progression of the disease. The desired response in the treatment of a disease can also be the delay or prevention of the onset of the said disease or the said condition. The effective amount of the compositions described herein depends on individual parameters of the patient including the condition being treated, the severity of the disease, age, physiological state, size and weight, the duration of the treatment, the type of concomitant treatment (if any), the particular route of administration as well as similar factors. Thus, the dosage of the compositions described herein can depend on such various parameters. If the patient's response is insufficient with the initial dosage, higher dosages (or different, effectively higher dosages achieved by a different, more local route of administration) can be used.

[0266] The pharmaceutical compositions of the present disclosure can include salts, buffers, 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.

[0267] Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, without limitation, benzalkonium chloride, chlorobutanol, parabens and thimerosal.

[0268] As used herein, the term "excipient" 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, without limitation, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

[0269] The term "diluent" relates to an agent that dilutes and / or thins. Further, the term "diluent" includes any one or more of a fluid, a liquid or solid suspension, and / or a mixed medium. Examples of suitable diluents include ethanol, glycerol, and water.

[0270] The term "carrier" refers to a component, which may be natural, synthetic, organic, or inorganic, with which the active ingredient is combined to facilitate, enhance, or enable the administration of a pharmaceutical composition. The carrier used herein may be one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a subject. Suitable carriers include, without limitation, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycol, hydrogenated naphthalene, and, in particular, biocompatible polylactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers. In one embodiment, the pharmaceutical composition of the present disclosure comprises isotonic saline.

[0271] 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. (A.R Gennaro edit. 1985).

[0272] Pharmaceutical carriers, excipients, or diluents can be selected with respect to the intended route of administration and standard pharmaceutical practice.

[0273] In one embodiment, the pharmaceutical composition described herein can be administered intravenously, intraarterially, subcutaneously, intradermally, or intramuscularly. In certain embodiments, the pharmaceutical composition is formulated for topical or systemic administration. Systemic administration can include enteral administration, including absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to administration by any method other than through the gastrointestinal tract, such as by intravenous injection. In a preferred embodiment, the pharmaceutical composition is formulated for systemic administration. In another preferred embodiment, the systemic administration is by intravenous administration.

[0274] In one embodiment of all aspects of the present invention, a nucleic acid encoding a cytokine or encoding an antigen or a variant thereof is administered systemically. In one embodiment of all aspects of the present invention, after systemic administration of a nucleic acid encoding an antigen or a variant thereof, expression of the antigen or its variant occurs in the spleen. In one embodiment of all aspects of the present invention, after systemic administration of a nucleic acid encoding an antigen or a variant thereof, expression of the antigen or its variant occurs 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 of all aspects of the present invention, after systemic administration of a nucleic acid encoding an antigen or a variant thereof, expression of the antigen or its variant in the lung and / or liver does not occur at all or essentially does not occur. In one embodiment of all aspects of the present invention, after systemic administration of a nucleic acid encoding an antigen or a variant thereof, the expression of the antigen or its variant in the spleen is at least 5-fold the expression level in the lung.

[0275] As used herein, the term "co-administration" means a method of administering different compounds or compositions (e.g., RNA encoding an interleukin and RNA encoding an antigen or a variant thereof) to the same patient simultaneously, essentially simultaneously, or sequentially. When the administrations are simultaneous, the different compounds or compositions need not be administered within the same composition.

[0276] Treatment The agents, compositions, and methods described herein can be used to treat a subject having a disease, such as a disease characterized by the presence of diseased cells that express an antigen. Particularly preferred diseases are cancer diseases. For example, if the antigen is derived from a virus, the agents, compositions, and methods can be useful for the treatment of viral diseases caused by said virus. If the antigen is a tumor antigen, the agents, compositions, and methods can be useful for the treatment of cancer diseases in which cancer cells express said tumor antigen.

[0277] In one embodiment, the present disclosure relates to a method for inducing an immune response in a subject. In an exemplary embodiment, the immune response is against cancer.

[0278] The term "disease" refers to an abnormal condition that affects an individual's body. A disease is often interpreted as a medical condition associated with specific symptoms and signs. A disease can be caused by external factors such as infections, or by internal dysfunctions such as autoimmune diseases. In humans, the term "disease" is often used more broadly to refer to a condition that causes pain, dysfunction, suffering, social problems, or death in the affected individual, or similar problems in people who come into contact with the individual. In this broader sense, a disease sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, deviant behaviors, and atypical changes in structure and function, although in other contexts and for other purposes these may be considered distinguishable categories. Since living with and suffering from many diseases can change a person's outlook on life and personality, diseases usually affect an individual not only physically but also emotionally.

[0279] In this context, the terms "treatment", "treating" or "therapeutic intervention" relate to the management and care of a subject aimed at combating a condition such as a disease or disorder. This term is intended to include any range of treatment for a given condition that a subject is suffering from, such as the administration of a therapeutically effective compound to alleviate symptoms or complications, to slow the progression of a disease, disorder or condition, to alleviate or reduce symptoms and complications, and / or to cure or eliminate a disease, disorder or condition, as well as to prevent a condition, where prevention should be understood as the management and care of an individual aimed at combating a disease, condition or disorder and includes the administration of an active compound to prevent the onset of symptoms or complications.

[0280] The term "therapeutic treatment" relates to any treatment that improves the health status of an individual and / or extends (increases) lifespan. The treatment can eliminate a disease in an individual, stop or delay the onset of a disease in an individual, inhibit or delay the onset of a 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 a disease.

[0281] The terms "preventive treatment" or "prophylactic treatment" relate to any treatment aimed at preventing the occurrence of a disease in an individual. The terms "preventive treatment" or "prophylactic treatment" are used interchangeably herein.

[0282] The terms "individual" and "subject" are used interchangeably herein. These refer to a human or another mammal (e.g., mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate) that may or is likely to contract a disease or disorder (e.g., cancer), and may or may not have the disease or disorder. In many embodiments, the individual is human. Unless otherwise specified, the terms "individual" and "subject" do not denote a particular age and thus include adults, the elderly, children, and newborns. In embodiments of the present disclosure, an "individual" or "subject" is a "patient".

[0283] The term "patient" means an individual or subject for treatment, particularly an individual or subject suffering from a disease.

[0284] In one embodiment of the present disclosure, the objective is to provide an immune response against disease cells expressing an antigen such as cancer cells expressing a tumor antigen, and to treat a disease such as a cancer disease in which cells expressing an antigen such as a tumor antigen are involved.

[0285] An immune response against an antigen can be induced, which can be therapeutic or partially or fully protective. The pharmaceutical compositions described herein are applicable for inducing or enhancing an immune response. Thus, the pharmaceutical compositions described herein are useful in the prophylactic and / or therapeutic treatment of diseases involving an antigen.

[0286] As used herein, "immune response" refers to an integrated bodily response to an antigen or to cells expressing an antigen, and refers to a cellular immune response and / or a humoral immune response. Cellular immune responses include, but are not limited to, cellular responses to cells expressing an antigen. Such cells may be characterized by expression of the antigen on the cell surface, or presentation of the antigen by class I or class II MHC molecules. The cellular response is associated with T lymphocytes, which play a central role by regulating the immune response, helper T cells (also referred to as CD4+ T cells), or killer cells that induce apoptosis in infected or cancer cells (also referred to as cytotoxic T cells, CD8+ T cells, or CTLs). In one embodiment, administration of the pharmaceutical compositions of the present disclosure includes stimulation of an anti-tumor CD8+ T cell response against cancer cells expressing one or more tumor antigens.

[0287] The present disclosure contemplates immune responses that can be defensive, prophylactic, preventive and / or therapeutic. As used herein, "inducing (or induction of) an immune response" may indicate that an immune response to a particular antigen did not exist prior to induction, or that there was a basal level of immune response to a particular antigen prior to induction and this was enhanced after induction. Thus, "inducing (or induction of) an immune response" includes "enhancing (or enhancement of) an immune response".

[0288] The term "immunotherapy" relates to the treatment of a disease or condition by inducing or enhancing an immune response.

[0289] The term "vaccination" or "immunization" represents the process of administering an antigen to an individual for the purpose of inducing an immune response, for example for therapeutic or prophylactic reasons.

[0290] In one embodiment, the present disclosure contemplates embodiments in which an RNA formulation, such as the RNA particles described herein, is administered.

[0291] Accordingly, the present disclosure relates to the RNAs described herein for use in the prophylactic and / or therapeutic treatment of antigen-involved diseases, preferably cancer diseases.

[0292] The term "macrophage" refers to a subgroup of phagocytic cells produced by the differentiation of monocytes. Macrophages activated by inflammation, immune cytokines or microbial products non-specifically engulf foreign pathogens within the macrophage and kill them by hydrolytic and oxidative attacks that result in the degradation of the pathogens. Peptides from the degraded proteins are displayed on the macrophage cell surface, which can be recognized by T cells, and can directly interact with antibodies on the B cell surface, leading to the activation of T cells and B cells and further stimulation of the immune response. Macrophages belong to the class of antigen-presenting cells. In one embodiment, the macrophage is a splenic macrophage.

[0293] The term "dendritic cell" (DC) refers to another subtype of phagocytic cells belonging to the class of antigen-presenting cells. In one embodiment, dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells initially transform into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation ability. Immature dendritic cells constantly sample the surrounding environment of pathogens such as viruses and bacteria. When they come into contact with presentable antigens, they are activated and become mature dendritic cells, and begin to migrate to the spleen or lymph nodes. Immature dendritic cells phagocytose pathogens, break down their proteins into small fragments, and when mature, present these fragments on the cell surface using MHC molecules. At the same time, they upregulate cell surface receptors that function as co-receptors for 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 induces dendritic cells to migrate through the bloodstream to the spleen or through the lymphatic system to the lymph nodes. Here, they function as antigen-presenting cells and, together with non-antigen-specific co-stimulatory signals, activate helper T cells, killer T cells, and B cells by presenting antigens. Thus, dendritic cells can actively induce T cell- or B cell-related immune responses. In one embodiment, the dendritic cells are splenic dendritic cells.

[0294] The term "antigen-presenting cell" (APC) is one of various cells that can display, acquire, and / or present at least one antigen or antigenic fragment on (or at) its cell surface. Antigen-presenting cells can be distinguished into professional antigen-presenting cells and non-professional antigen-presenting cells.

[0295] The term "professional antigen-presenting cell" relates to an antigen-presenting cell that constitutively expresses major histocompatibility complex class II (MHC class II) molecules necessary for interaction with naive T cells. When a T cell interacts with an MHC class II molecular complex on the membrane of an antigen-presenting cell, the antigen-presenting cell produces co-stimulatory molecules that induce activation of the T cell. Professional antigen-presenting cells include dendritic cells and macrophages.

[0296] The term "non-professional antigen-presenting cell" relates to an antigen-presenting cell that does not constitutively express MHC class II molecules but expresses them upon stimulation by certain cytokines such as interferon γ. Exemplary non-professional antigen-presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells, or vascular endothelial cells.

[0297] "Antigen processing" refers to the degradation of an antigen into processing products that are fragments of the antigen (e.g., degradation of a protein into peptides), and the association (e.g., by binding) of one or more of these fragments with MHC molecules for presentation to specific T cells by cells such as antigen-presenting cells.

[0298] The terms "disease involving an antigen", "disease involving cells expressing an antigen" or similar terms refer to any disease in which an antigen is involved, e.g., a disease characterized by the presence of an antigen. The disease can be an infectious disease, or a cancer disease, or simply cancer. As described above, the antigen can be a disease-related antigen such as a tumor-associated antigen, a viral antigen, or a bacterial antigen. Preferably, the disease involving an antigen is preferably a disease involving cells expressing the antigen on the cell surface.

[0299] The term "infectious disease" refers to any disease (e.g., the common cold) that can be transmitted from individual to individual or from organism to organism and is caused by a microbial agent. Infectious diseases are known in the art and include, for example, viral diseases, bacterial diseases, or parasitic diseases, which are caused by viruses, bacteria, and parasites, respectively. In this regard, infectious diseases can be, for example, hepatitis, sexually transmitted diseases (e.g., chlamydia or gonorrhea), tuberculosis, HIV / acquired immunodeficiency syndrome (AIDS), diphtheria, hepatitis B, hepatitis C, cholera, severe acute respiratory syndrome (SARS), avian influenza, and influenza.

[0300] The term "cancer disease" or "cancer" typically refers to or represents a physiological state of an individual characterized by disordered cell growth. Examples of cancers include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias. More specifically, examples of such cancers include bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma of the skin or eye, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, prostate cancer, cervical cancer, cancers of the genital and reproductive organs, Hodgkin's disease, esophageal cancer, small intestine cancer, cancers 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 cancer, spinal cord tumors, gliomas, meningiomas, and pituitary adenomas. The term "cancer" according to the present disclosure also includes cancer metastasis.

[0301] Combination strategies in cancer treatment may be desirable due to the resulting synergistic effects, which can be considerably more potent than the effects of single-agent therapy approaches. In one embodiment, the pharmaceutical composition is administered with an immunotherapeutic agent. As used herein, an "immunotherapeutic agent" relates to any agent that may be involved in the activation of a specific immune response and / or one or more immune effector functions. The present disclosure contemplates the use of antibodies as immunotherapeutic agents. Without wishing to be bound by theory, antibodies can achieve a therapeutic effect on cancer cells through various mechanisms, including inducing apoptosis, blocking components of signaling pathways, or inhibiting the growth of tumor cells. In certain embodiments, the antibody is a monoclonal antibody. Monoclonal antibodies can induce cell death via antibody-dependent cell-mediated cytotoxicity (ADCC) or bind to complement proteins to bring about direct cytotoxicity known as complement-dependent cytotoxicity (CDC). Non-limiting examples of anti-cancer antibodies and potential antibody targets (in parentheses) that can be used in combination with the present disclosure include abagovomab (CA-125), abciximab (CD41), adecatumumab (EpCAM), afucosylated anti-CD20 monoclonal antibody (CD20), alacizumab pegol (VEGFR2), altumomab pentetate (CEA), amatuximab (MORAb-009), anatumomab mafenatox (TAG-72),apolizumab (HLA-DR), arcitumomab (CEA), atezolizumab (PD-L1), bavituximab (phosphatidylserine), bectumomab (CD22), belimumab (BAFF), bevacizumab (VEGF-A), bivatuzumab mertansine (CD44 v6), blinatumomab (CD19), brentuximab vedotin (CD30TNFRSF8), Cantuzumab mertansine (MUC1 CanAg), Cantuzumab ravtansine (MUC1), Capromab pendetide (prostate cancer cells), Carlumab (CNT0888), Catumaxomab (EpCAM, CD3), Cetuximab (EGFR), Cituximab bogatox (EpCAM), Cixutumumab (IGF-1 receptor), Claudiximab (claudin), Clivatuzumab tetraxetan (MUC1), Conatumumab (TRAIL-R2), Dacetuzumab (CD40), Daratumumab (insulin-like growth factor I receptor), Denosumab (RANKL), Detumomab (B lymphoma cells), Dorzigomab (DR5), Ecrromeximab (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), flanvotumab (glycoprotein 75), fresolimumab (TGF-β), galiximab (CD80), ganitumab (IGF-I), gemtuzumab ozogamicin (CD33), gebotuzumab (ILΙβ), girenuximab (carbonic anhydrase 9 (CA-IX)), glembatumumab vedotin (GPNMB), ibritumomab tiuxetan (CD20), icrucumab (VEGFR-1), igovomab (CA-125), indatuximab ravtansine (SDC1), intetumumab (CD51), inotuzumab ozogamicin (CD22), ipilimumab (CD152), iratumumab (CD30), labeluximab (CEA), lexatumumab (TRAIL-R2), livibizumab (hepatitis B surface antigen), lintuzumab (CD33), lorvotuzumab mertansine (CD56), lucatumumab (CD40), lumiliximab (CD23), mapatumumab (TRAIL-R1), matuzumab (EGFR), mepolizumab (IL5), miratumumab (CD74), mitumomab (GD3 ganglioside), mogamulizumab (CCR4), moxetumomab pasudotox (CD22), necitumumab (C242 antigen), napumomab estafenatox (5T4), namatumumab (RON), nesitumumab (EGFR), nimotuzumab (EGFR), nivolumab (IgG4), ofatumumab (CD20), olaratumumab (PDGF-Ra) Omalizumab (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), purtuzumab (vimentin), racotumomab (N-glycolylneuraminic acid), radretumab (fibronectin extra domain B), ravivirumab (rabies virus glycoprotein), ramucirumab (VEGFR2), rilotumumab (HGF), rituximab (CD20), robatumumab (IGF-1 receptor), samalizumab (CD200), sibrotuzumab (FAP), siltuximab (IL6), tabalumab (BAFF), takatuzumab tetraxetan (alpha-fetoprotein), tapritumomab paptox (CD19), tenatumomab (tenascin C), teprotumumab (CD221), tisilimumab (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), volociximab (integrin α5β1), bovatumumab (tumor antigen CTAA 16.88), zalutumumab (EGFR), and zanolimumab (CD4) are included.

[0302] The citation of documents and tests referred to herein is not intended to constitute an admission that any of the foregoing is prior art relevant thereto. All statements as to the content of these documents are based on the information available to the applicant and do not constitute an admission as to the accuracy of the content of these documents.

[0303] The following description is presented to enable those skilled in the art to make and use various embodiments. The descriptions of specific devices, techniques, and applications are provided by way of example only. 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 should be accorded a scope consistent with the claims.

Example

[0304] Example: Method: Animal C57BL / 6BrdCrHsd-Tyr c The mice were purchased from Envigo Labs. Throughout the experiment, animals of the same age (8 - 10 weeks old) and sex (male or female) were used. The congenic C57Bl / 6-Thy1.1 mice were bred in the animal facility of BioNTech AG, Germany.

[0305] CAR construct / CAR T cell Using the γ-retroviral self-inactivating (SIN) vector pES.12-6, under the control of the internal eukaryotic promoter, the short intronless human elongation factor 1-α promoter (EFS-213 / +31), CLDN6-CAR-BBz-T2A-Luc-T2A-GFP was stably overexpressed in murine T cells. The vector backbone contains the MLV wild-type sequences of the R region and U5 region in the 5'- and 3'-LTRs as well as the packaging regions (ψ and ψ+). The enhancer element in the U3 region of the 3'-LTR was removed (including the CAAT box), and the TATA box sequence was mutated to prevent transcription initiation. A truncated form of the post-transcriptional regulatory element (PRE) of woodchuck hepatitis virus (WHV) was used to prevent the expression of unwanted viral proteins. CLDN6-CAR-BBz consists of the signal transduction peptide of human IgG (SEQ ID NO: 12), the heavy chain (V H)(SEQ ID NO: 13) and the light chain (V L )(SEQ ID NO: 15) with (G 4 S) 3 having a linker (SEQ ID NO: 14), and V L containing a single-chain Fv fragment of the claudin 6-specific antibody IMAB206 (Ganymed Pharmaceuticals) having a cysteine to serine substitution at position 46. The ScFv fragment is fused to the human CD8α hinge and transmembrane region (SEQ ID NO: 16), followed by fusion to the human 4-1BB (SEQ ID NO: 17) and human CD3ζ (Q14K) (SEQ ID NO: 18) signaling moieties. The CAR is linked to the active firefly luciferase (SEQ ID NO: 20) and eGFP (SEQ ID NO: 21) using the T2A ribosome skip element (SEQ ID NO: 19), enabling equimolar production of the indicated proteins in transduced T cells.

[0306] Preparation of CAR T cells for retroviral gene manipulation and adoptive T cell transfer Naïve C57Bl / 6-Thy1.1 + spleen cells were isolated and pre-activated with Dynabeads™ Mouse T-Activator CD3 / CD28 (Invitrogen) at a bead-to-T cell ratio of 1:1 in the presence of 5 ng / mL of recombinant human (rh) IL-7 and 5 ng / mL of rh IL-15 (Miltenyi Biotec). For transduction of mouse cells, MLV-E pseudotyped retroviral supernatant was loaded onto non-tissue culture-treated well plates coated with RetroNectin (2 μg / cm 2 ) according to the manufacturer's instructions (Takara Bio Inc., Otsu, Japan), and the cycles of virus loading and centrifugation (1,300 × g, 15 °C, 15 minutes) were repeated three times to increase binding. Twenty-four hours after pre-activation, 0.5 - 0.6 × 10 6 cells / cm 2It was spin - downed onto the virus - particle - coated well (300×g, 37 °C, 1 hour). After overnight culture, spin - down transduction was repeated using a newly virus - particle - coated plate. 72 hours after pre - activation, Dynabeads™ Mouse T Activator CD3 / CD28 were removed from the culture, and the cells were grown in the presence of 5 ng / mL of rhIL - 7 and 5 ng / mL of rhIL - 15. After Ficoll washing, the cells were washed twice with PBS to remove serum proteins and then prepared for adoptive cell transfer (ACT). A pES12.6 - based retroviral vector containing either the encoded OT1 - TCR or CLDN6 - CAR, and the separately expressed enhanced firefly luciferase (effLuc; Rabinovich et al. (2008) PNAS 105(38):14342 - 6) and eGFP (enhanced green fluorescent protein) reporter genes using the 2A splice element (Szymczak et al. (2004) Nat Biotechnol. 22(5):589 - 94) was used for transduction.

[0307] Production of in vitro transcribed (IVT) mRNA In vitro transcription of mRNA encoding cytokine-albumin fusion proteins was based on the pST4-T7-GG-TEV-MCS-FI-A30LA70 plasmid backbone and derived DNA constructs. These plasmid constructs contain the 5' leader sequence of tobacco etch virus (TEV), the 3' Fl element (F is a 136-nucleotide-long 3'-UTR fragment of the split amino-terminal enhancer mRNA, I is a 142-nucleotide-long fragment of the 12S RNA encoded by mitochondria, both identified in Homo sapiens; International Publication No. WO 2017 / 060314) and a 100-nucleotide poly(A) tail, with a linker included 70 nucleotides later. The cytokine and serum albumin coding sequences were derived from Mus musculus, and no changes were introduced into the resulting amino acid sequences (mouse (m) IL-2, SEQ ID NO: 5, mIL-7, SEQ ID NO: 6, and mIL-21, SEQ ID NO: 7). The encoded proteins are provided with an N-terminal signal peptide which is the native signal peptide of the N-terminal portion. Only the SP of the N-terminal portion was maintained, and for the further portion, only the mature portion (protein without SP) was encoded. The stop codon was maintained only for most of the C-terminal portion. The albumin portion and the cytokine portion in the construct were separated by a 30-nucleotide-long linker sequence encoding glycine and serine residues. The orientation of the albumin-cytokine fusion proteins used was as follows: albumin-linker-mIL2 (SEQ ID NOs: 8, 9, and 10 in sequence from the N-terminus to the C-terminus), mIL7-linker-albumin (SEQ ID NOs: 6, 9, and 11 in sequence from the N-terminus to the C-terminus), and mIL21-linker-albumin (SEQ ID NOs: 7, 9, and 11 in sequence from the N-terminus to the C-terminus). In vitro transcription of mRNA encoding the antigen was based on the pST1-T7-GG-hAg-MCS-2hBg-A30LA70 plasmid backbone and derived DNA constructs.These plasmid constructs, in addition to full-length human CLDN6 or the chicken ovalbumin epitope SIINFEKL (OvaI; as described in Kreiter et al (2008) J Immunol. 180(1):309-18 and further flanked by 3'Sec and 5'TM1 sequences), contain a 5' human α-globin, two consecutive 3' human β-globin UTRs and a 100 nucleotide poly(A) tail, and a linker 70 nucleotides later. As described in Holtkamp S. et al. (2006) Blood 108(13):4009-17, mRNAs encoding antigens and cytokines were generated by in vitro transcription. The latter was further modified by replacing normal nucleoside uridine with 1-methyl-pseudouridine. The resulting cytokine mRNAs were equipped with a cap1 structure, and double-stranded (dsRNA) molecules were depleted by cellulose purification. The purified mRNA was eluted with H. 2 O and stored at -80 °C until further use. In vitro transcription of all mRNA constructs described was performed at BioNTech RNA Pharmaceuticals GmbH.

[0308] Generation of IVT RNA (RNA (LIP) ) encoding liposome-formulated antigen Complexation of IVT RNA encoding antigen with liposomes was previously described in Kranz et al (2016) Nature 534(7607):396-401. A 1.3 to 2 charge ratio of cationic DOTMA to RNA was used. In addition to DOTMA, the lipid fraction contained the helper lipid DOPE at a molar ratio of 2:1 DOTMA per DOPE.

[0309] Mouse experiments 5×10 6 individuals of γ-retrovirally transduced CAR or TCR transgenic Thy1.1 + T cells were transferred into immunocompetent or moderately whole-body irradiated (2.5 Gy - XRAD320) C57BL / 6BrdCrHsd-Tyr cIt was intravenously (i.v.) transferred into one of the donor mice at 200 μL. Subsequently, at various time points after ACT, the mice were intravenously (i.v.) vaccinated with RNA encoding an antigen with an F12:RNA ratio of 1.3:2. (LIP) At the indicated time points, the mice were repeatedly treated with mRNA encoding a 1 μg nucleoside-modified mouse albumin-cytokine fusion protein formulated with TransIT (Mirrus) or buffer only. Peripheral blood collection and whole-body bioluminescence imaging were performed at the indicated time points.

[0310] In vivo luciferase imaging (BLI) The proliferation and distribution of CAR or TCR-effLuc-GFP transduced T cells were evaluated by in vivo bioluminescence imaging using an IVIS Lumina imaging system (Caliper Life Sciences). Briefly, at the indicated time points after adoptive transfer of the transduced T cells, an aqueous solution of D-luciferin (80 mg / kg body weight; Perkin Elmer) was injected i.p. Five minutes later, the emitted photons were quantified (integration time 1 minute, binning 8). In vivo bioluminescence in the region of interest (ROI) was quantified as total light flux (photons / second) using IVIS Living Image 4.0 software. The intensity of the transmitted light from luciferase-expressing cells within the animal was represented as a grayscale image, with black being the weakest and white to dark gray being the strongest bioluminescence signal. A grayscale reference image of the mouse was acquired under LED low-light illumination. The images were overlaid using Living Image 4.0 software.

[0311] Example 1: The selected pharmacokinetics-extended γ-chain cytokine (IL-2 / 7) results in repeated proliferation of CAR T cells in vivo upon antigen contact. Typically, a certain cytokine environment is required to maintain the persistence of T cells upon antigen contact. Gamma-chain cytokines, such as IL-2 and IL-7, have been shown to enhance T cell proliferation and survival (e.g., Blattman et al. (2003) Nat. Med. 9(5):540-7, Fry et al. (2001) Trends Immunol. 22(10):564-71, Bradley et al. (2005) Trends Immunol. 26(3):172-6, Jiang et al. (2005) Cytokine Growth Factor Rev. 16(4-5):513-33). However, the use of recombinant cytokines such as IL-2 has been limited by its short half-life and its dose-dependent toxicity (Vial et al. (1992) Drug Saf. 7(6):417-33). To overcome the limited cytokine support of adoptively transferred T cells, mRNA constructs encoding cytokine-albumin fusion proteins were developed and were actually able to significantly increase the serum half-life of the encoded cytokines in vivo upon systemic administration. The systemic availability of cytokine-albumin constructs is extended when they are encoded by nucleoside-modified mRNA.

[0312] Accordingly, the inventors posed the question of whether a combination of a TAA, such as RNA encoding CLDN6 (LIP) formulated in liposomes that selectively targets APCs in secondary lymphoid organs and supports the selected cytokine, can result in proper repetitive expansion and persistence of CAR-T cells in vivo.

[0313] To test this concept, CLDN6-CAR T cells transduced with γ-retrovirus were adoptively transferred into either moderately irradiated (2.5 Gy) mice or immunocompetent mice (Figure 1 and Figure 2, respectively). To visualize the in vivo proliferation and fate of these murine CLDN6-CAR T cells, the inventors utilized co-expression of luciferase and GFP reporters in a retroviral vector encoding the same CLDN6 CAR, separated by the viral T2A sequence (Figure 1A). Notably, surface expression and antigen specificity of CLDN6-CAR were not significantly affected by co-expression of luciferase and GFP in CAR-transduced murine T cells (data not shown).

[0314] Moderately irradiated (2.5 Gy with XRAD320) albino C57Bl / 6 mice were transplanted with 5×10 6 individual CLDN6-CAR reporter-transduced congenic Thy1.1 + murine bulk T cells (approximately 2.5×10 8 cells / kg body weight). RNA encoding 20 μg of CLDN6 or control RNA was formulated into spleen-targeted liposomes and injected intravenously into the mice 1 day after adoptive CAR-T transfer. Simultaneously with CLDN6 RNA (LIP) vaccination, mice were administered intraperitoneally with albumin-conjugated murine IL-2 and murine IL-7 encoding mRNA (1 μg / cytokine RNA) formulated with TransIT or mock control (buffer). The treatment was repeated 7 days later. Thereafter, at the indicated time points, CAR-T proliferation and biodistribution were followed in vivo by intraperitoneal administration of 1.66 mg of D-luciferin solution per mouse. Twenty-four hours after ACT, most CAR-T cells were already found in the spleen. In the absence of cytokines (mock), an approximately 21-fold increase in CAR-T cells (compared to day 1) was induced only by treatment with CLDN6-RNA (LIP) as detected by bioluminescence on day 4 after ACT. CLDN6-RNA (LIP)A second boost with IL-2 still resulted in a 15-fold higher luminescence intensity on day 11 compared to the baseline luminescence measured on day 1. The coadministration of albumin-fused IL-2 and IL-7-encoding mRNA formulated with TransIT significantly increased the proliferative capacity of CAR T cells. (LIP) A 75-fold expansion of CAR-T cells was achieved after treatment, and the second CLDN6 RNA (LIP) This effect was further improved by up to 114-fold with the addition of CLDN6-encoding RNA alone or in combination with the cytokine-albumin-encoding RNA (Figure 1C and Figure 1D). (LIP) This was observed in mice receiving CLDN6-CAR T cells after treatment with a control RNA encoding OvaI, either in the presence or absence of an RNA encoding the cytokine-albumin. (LIP) These data demonstrate that CAR-T cells can be successfully expanded in situ in a highly antigen-specific manner in moderately irradiated mice.

[0315] CAR T cells were then transduced with RNA encoding the respective antigen in the presence of RNA encoding the cytokine in moderately irradiated mice. (LIP) After demonstrating that lymphodepletion can be used to repeatedly expand in situ, we investigated whether this effect could also be achieved in immunocompetent hosts. However, lymphodepletion has several drawbacks, including well-known side effects and risks associated with chemotherapy, such as potential infection and sepsis (Brentjens et al. (2010) Mol Ther. 18(4): 666-8 and Robbins et al. (2015) Clin Cancer Res. 21(5): 1019-27). Furthermore, in the case of on-target and / or off-target toxicity, rapid expansion of adoptively transferred CAR-T cells can be lethal (Morgan et al. (2010) Mol Ther. 18(4): 843-51). To this end, we used CLDN6-CAR transduced mice Thy1.1 +Non-irradiated albino C57Bl / 6 mice transplanted with T cells were treated as described above (Figure 2A). The systemic presence of IL-2 and IL-7 during the first stimulation round, compared to the control group administered buffer (mock) instead of RNA encoding cytokine-albumin formulated with TransIT, showed that CLDN6-RNA (LIP) had no significant effect on the proliferation of CLDN6-CAR T cells after vaccination (Day 4: proliferation index: mock 192-fold and IL-2 / 7: 223-fold). However, in the absence of IL-2 / 7 cytokines, the CAR T cell population strongly contracted after the first CLDN6 RNA (LIP) mediated proliferation and could not re-proliferate a second time. Only in the presence of RNA encoding IL-2 / IL-7-albumin could CLDN6-CAR T cells repeatedly proliferate in immunocompetent mice and persist over several days (Day 11: proliferation index: mock: 0.5-fold and IL-2 / 7: 79-fold) (Figure 2B and Figure 2C).

[0316] These data strongly support the idea that direct controlled CAR-T cell proliferation in patients is achievable using RNA (LIP) technology, but for persistence, cells require a favorable cytokine environment such as IL-2 and IL-7, which can be achieved by administration of RNA encoding extended pharmacokinetic gamma chain cytokines.

[0317] Example 2: Optimal combination of cytokine albumin fusions during repeated proliferation of CAR T cells. Since several gamma chain cytokines actively support T cell survival and assist in the therapeutic effect of T cells in an antigen-specific manner (e.g., Markley et al. (2010) Blood 115(17):3508-19, He et al. (2006) J Transl Med. 4:24.), the inventors compared nucleoside-modified RNAs encoding mIL-2, mIL-7, mIL-21, and the combination of IL-2 / 7 and IL-2 / 21 from the perspective of promoting the in vivo supporting effect on the proliferation and persistence of CAR-modified T cells during repeated RNA (LIP) treatment.

[0318] In the same manner as described in Example 1, RNA encoding hCLDN6 formulated in liposomes or a control was vaccinated simultaneously with treatment with RNA encoding mouse albumin-binding mIL-2, mIL-7, mIL-21 (1 μg / cytokine RNA) or RNA encoding mouse albumin (Alb control) into moderately irradiated albino C57Bl / 6 mice transplanted with CLDN6-CAR reporter-transduced T cells. The antigen / cytokine cocktail was administered at 1-week intervals (Figure 3A). Bioluminescence intensity was analyzed at the peak of in vivo CAR T cell proliferation (usually reached 2 - 3 days after RNA-based treatment) (Figure 3B). Systemic presence of IL-7 and IL-21 alone resulted in a decrease in antigen-specific CART proliferation ability at the time of treatment compared to the albumin control. Co-treatment with IL-2 resulted in up to 164-fold CAR T cell proliferation compared to the baseline. However, in vivo CAR T cell accumulation could only be achieved when IL-2 RNA was co-administered with IL-7 (up to 214-fold increase after the third proliferation) or IL-21 (up to 141-fold increase after the third proliferation). In addition to the in vivo CAR T cell accumulation ability, the clinical success of adoptive tumor-reactive T cell therapy is also positively correlated with the persistence of those cells in vivo (Robbins et al. (2004) J Immunol. 173(12):7125 - 30, Huang et al. (2005) 28(3):258 - 67). Therefore, the inventors analyzed the contraction of CART T cells after three antigen-specific proliferations using bioluminescence in the presence of IL-7 alone or in combination with IL-2 (Figure 3C) or in the presence of IL-21 (Figure 3D). The CAR T cell population decreased after the third CLDN6 RNA when only albumin, IL-2 or IL-7 was present. (LIP) treatment. The co-treatment with IL-2 resulted in up to 164-fold CAR T cell proliferation compared to the baseline. However, in vivo CAR T cell accumulation could only be achieved when IL-2 RNA was co-administered with IL-7 (up to 214-fold increase after the third proliferation) or IL-21 (up to 141-fold increase after the third proliferation). In addition to the in vivo CAR T cell accumulation ability, the clinical success of adoptive tumor-reactive T cell therapy is also positively correlated with the persistence of those cells in vivo (Robbins et al. (2004) J Immunol. 173(12):7125 - 30, Huang et al. (2005) 28(3):258 - 67). Therefore, the inventors analyzed the contraction of CART T cells after three antigen-specific proliferations using bioluminescence in the presence of IL-7 alone or in combination with IL-2 (Figure 3C) or in the presence of IL-21 (Figure 3D). The CAR T cell population decreased after the third CLDN6 RNA (LIP)It contracted immediately after that. However, only the combination of IL-2 and IL-7 was able to enhance the deceleration of the contraction of CLDN6 CAR T cells after antigen removal (Figure 3C). This effect was even more prominent in IL-2 and IL-21-RNA co-treated mice (Figure 3D).

[0319] Overall, these results indicate that systemic administration of nucleoside-modified RNAs encoding IL-2 and involved with IL-7 and IL-21 can enhance highly antigen-dependent accumulation and long-term persistence of CAR T cells in vivo upon antigen-specific stimulation.

Claims

1. 1. A method for inducing an immune response in a subject, comprising: a. providing to the subject T cells genetically modified to express a chimeric antigen receptor (CAR); and b. administering IL2 or a polynucleotide encoding IL2 to said subject. The method includes:

2. 2. The method of claim 1, comprising administering IL2 or a polynucleotide encoding IL2 and an additional cytokine or a polynucleotide encoding an additional cytokine.

3. 3. The method of claim 2, wherein the additional cytokine is selected from the group consisting of IL7 and IL21.

4. The method of any one of claims 1 to 3, comprising administering IL2 or a polynucleotide encoding IL2 and IL7 or a polynucleotide encoding IL7.

5. The method of any one of claims 1 to 3, comprising administering a polynucleotide encoding L2 or IL2 and IL21 or a polynucleotide encoding IL21.

6. The method according to any one of claims 1 to 5, wherein the polynucleotide encoding IL2 is RNA and optionally the polynucleotide encoding a further cytokine is RNA.

7. 7. The method of any one of claims 1-6, wherein the T cells genetically modified to express a CAR are provided to the subject by administering the T cells genetically modified to express a CAR or by generating in the subject the T cells genetically modified to express a CAR.

8. 8. The method of any one of claims 1 to 7, further comprising administering to the subject an antigen or a variant thereof, or a polynucleotide encoding said antigen or variant, wherein said T cells genetically modified to express a CAR target said antigen, and wherein said immune response is an immune response against a target cell population or target tissue expressing said antigen.

9. The method of claim 8, wherein the polynucleotide encoding the antigen or variant is RNA.

10. 1. A method for inducing an immune response in a subject, comprising: a. providing to the subject T cells genetically modified to express a chimeric antigen receptor (CAR); and b. administering to said subject RNA encoding IL2. The method includes:

11. 11. The method of claim 10, comprising administering RNA encoding IL2 and RNA encoding an additional cytokine.

12. 12. The method of claim 11, wherein the additional cytokine is selected from the group consisting of IL7 and IL21.

13. The method of any one of claims 10 to 12, comprising administering RNA encoding IL2 and RNA encoding IL7.

14. The method of any one of claims 10 to 12, comprising administering RNA encoding IL2 and RNA encoding IL21.

15. 15. The method of any one of claims 10-14, wherein the T cells genetically modified to express a CAR are provided to the subject by administering the T cells genetically modified to express a CAR or by generating in the subject the T cells genetically modified to express a CAR.

16. 16. The method of any one of claims 10-15, further comprising administering to the subject RNA encoding an antigen or a variant thereof, wherein the T cells genetically modified to express a CAR target the antigen, and wherein the immune response is an immune response against a target cell population or tissue expressing the antigen.

17. The method of any one of claims 1 to 16, wherein the immune response is a T cell mediated immune response.

18. 1. A method for treating a subject having a disease, disorder, or condition associated with expression or up-regulation of an antigen, comprising: a. providing to the subject T cells genetically modified to express a chimeric antigen receptor (CAR) that targets the antigen; and b. administering IL2 or a polynucleotide encoding IL2 to said subject. The method includes:

19. 20. The method of claim 18, comprising administering IL2 or a polynucleotide encoding IL2 and an additional cytokine or a polynucleotide encoding an additional cytokine.

20. 20. The method of claim 19, wherein the additional cytokine is selected from the group consisting of IL7 and IL21.

21. The method of any one of claims 18 to 20, comprising administering IL2 or a polynucleotide encoding IL2 and IL7 or a polynucleotide encoding IL7.

22. The method of any one of claims 18 to 20, comprising administering a polynucleotide encoding L2 or IL2 and IL21 or a polynucleotide encoding IL21.

23. The method according to any one of claims 18 to 22, wherein the polynucleotide encoding IL2 is RNA and, optionally, the polynucleotide encoding a further cytokine is RNA.

24. 24. The method of any one of claims 18-23, wherein the T cells genetically modified to express a CAR are provided to the subject by administering the T cells genetically modified to express a CAR or by generating in the subject the T cells genetically modified to express a CAR.

25. The method of any one of claims 18 to 24, further comprising administering to the subject the antigen or variant thereof, or a polynucleotide encoding the antigen or variant.

26. 26. The method of claim 25, wherein the polynucleotide encoding the antigen or variant is RNA.

27. 1. A method for treating a subject having a disease, disorder, or condition associated with expression or up-regulation of an antigen, comprising: a. providing to the subject T cells genetically modified to express a chimeric antigen receptor (CAR) that targets the antigen; and b. administering to said subject RNA encoding IL2. The method includes:

28. 28. The method of claim 27, comprising administering RNA encoding IL2 and RNA encoding an additional cytokine.

29. 29. The method of claim 28, wherein the additional cytokine is selected from the group consisting of IL7 and IL21.

30. 30. The method of any one of claims 27 to 29, comprising administering RNA encoding IL2 and RNA encoding IL7.

31. The method of any one of claims 27 to 29, comprising administering RNA encoding IL2 and RNA encoding IL21.

32. 32. The method of any one of claims 27-31, wherein the T cells genetically modified to express a CAR are provided to the subject by administering the T cells genetically modified to express a CAR or by generating in the subject the T cells genetically modified to express a CAR.

33. The method of any one of claims 27 to 32, further comprising administering to the subject RNA encoding said antigen or a variant thereof.

34. The method of any one of claims 18 to 33, wherein the disease, disorder or condition is cancer and the antigen is a tumor associated antigen.

35. The method of any one of claims 1 to 34, wherein the IL2 is extended pharmacokinetic (PK) IL2.

36. 36. The method of claim 35, wherein the extended PK IL2 comprises a fusion protein.

37. 37. The method of claim 36, wherein the fusion protein comprises an IL2 portion and a portion selected from the group consisting of serum albumin, an immunoglobulin fragment, transferrin, Fn3, and variants thereof.

38. The method according to any one of claims 2 to 9, 11 to 17, 19 to 26 and 28 to 37, wherein said further cytokine, in particular IL7 or IL21, is an extended pharmacokinetic (PK) cytokine, in particular extended PK IL7 or extended PK IL21.

39. 39. The method of claim 38, wherein the extended PK cytokine, in particular extended PK IL7 or extended PK IL21, comprises a fusion protein.

40. 40. The method of claim 39, wherein the fusion protein comprises a portion of the further cytokine, in particular an IL7 portion or an IL21 portion, and a portion selected from the group consisting of serum albumin, an immunoglobulin fragment, transferrin, Fn3, and variants thereof.

41. 41. The method of any one of claims 37 to 40, wherein the serum albumin comprises mouse serum albumin or human serum albumin.

42. The method of any one of claims 37 to 41, wherein the immunoglobulin fragment comprises an immunoglobulin Fc domain.

43. A method for treating or preventing cancer in a subject according to any one of claims 1 to 42, wherein the antigen is a tumor-associated antigen.

44. a. T cells genetically modified to express a chimeric antigen receptor (CAR), and b. IL2 or a polynucleotide encoding IL2 13. A pharmaceutical formulation comprising:

45. 45. The pharmaceutical formulation of claim 44, comprising IL2 or a polynucleotide encoding IL2 and a further cytokine or a polynucleotide encoding a further cytokine.

46. 46. ​​The pharmaceutical preparation of claim 45, wherein the additional cytokine is selected from the group consisting of IL7 and IL21.

47. 47. The pharmaceutical formulation of any one of claims 44 to 46, comprising IL2 or a polynucleotide encoding IL2 and IL7 or a polynucleotide encoding IL7.

48. 47. The pharmaceutical formulation of any one of claims 44 to 46, comprising IL2 or a polynucleotide encoding IL2 and IL21 or a polynucleotide encoding IL21.

49. 49. The pharmaceutical formulation of any one of claims 44 to 48, wherein the polynucleotide encoding IL2 is RNA and optionally the polynucleotide encoding a further cytokine is RNA.

50. 50. The pharmaceutical formulation of any one of claims 44 to 49, further comprising an antigen or a variant thereof, or a polynucleotide encoding said antigen or variant, wherein said T cells genetically modified to express a CAR target said antigen.

51. 51. The pharmaceutical formulation of claim 50, wherein the polynucleotide encoding the antigen or variant is RNA.

52. The pharmaceutical preparation according to any one of claims 44 to 51, which is a kit.

53. 53. The pharmaceutical formulation of claim 52, comprising in separate containers the T cell genetically modified to express a CAR, the IL2 or the polynucleotide encoding IL2, optionally the additional cytokine or the polynucleotide encoding an additional cytokine, and optionally the antigen or variant thereof, or the polynucleotide encoding the antigen or variant.

54. 54. The pharmaceutical preparation of claim 52 or 53, further comprising instructions for using said pharmaceutical preparation for treating or preventing cancer, wherein said antigen is a tumor-associated antigen.

55. The pharmaceutical preparation according to any one of claims 44 to 51, which is a pharmaceutical composition.

56. 56. The pharmaceutical formulation of claim 55, wherein the pharmaceutical composition further comprises one or more pharma- ceutically acceptable carriers, diluents and / or excipients.

57. a. T cells genetically modified to express a chimeric antigen receptor (CAR), and b. RNA encoding IL2 13. A pharmaceutical formulation comprising:

58. 58. The pharmaceutical formulation of claim 57, comprising RNA encoding IL2 and RNA encoding a further cytokine.

59. 59. The pharmaceutical preparation of claim 58, wherein the additional cytokine is selected from the group consisting of IL7 and IL21.

60. 60. The pharmaceutical formulation of any one of claims 57 to 59, comprising RNA encoding IL2 and RNA encoding IL7.

61. 61. The pharmaceutical formulation of any one of claims 57 to 60, comprising RNA encoding IL2 and RNA encoding IL21.

62. 62. The pharmaceutical formulation of any one of claims 57 to 61, further comprising RNA encoding an antigen or a variant thereof, wherein said T cells genetically modified to express a CAR target said antigen.

63. The pharmaceutical preparation according to any one of claims 57 to 62, which is a kit.

64. 64. The pharmaceutical formulation of claim 63, comprising in separate containers the T cells genetically modified to express a CAR, the RNA encoding IL2, optionally the RNA encoding a further cytokine, and optionally the RNA encoding an antigen or a variant thereof.

65. 65. The pharmaceutical preparation of claim 63 or 64, further comprising instructions for using said pharmaceutical preparation to treat or prevent cancer, and wherein said antigen is a tumor-associated antigen.

66. The pharmaceutical preparation according to any one of claims 57 to 62, which is a pharmaceutical composition.

67. 67. The pharmaceutical formulation of claim 66, wherein the pharmaceutical composition further comprises one or more pharma- ceutically acceptable carriers, diluents and / or excipients.

68. 68. The pharmaceutical formulation of any one of claims 44 to 67, wherein the IL2 is an extended pharmacokinetic (PK) IL2.

69. 69. The pharmaceutical preparation of claim 68, wherein the extended PK IL2 comprises a fusion protein.

70. 70. The pharmaceutical formulation of claim 69, wherein the fusion protein comprises an IL2 portion and a portion selected from the group consisting of serum albumin, an immunoglobulin fragment, transferrin, Fn3, and variants thereof.

71. 71. The pharmaceutical formulation of any one of claims 45 to 56 and 58 to 70, wherein the further cytokine is an extended pharmacokinetic (PK) cytokine.

72. 72. The pharmaceutical formulation of claim 71, wherein the extended PK cytokine comprises a fusion protein.

73. 73. The pharmaceutical formulation of claim 72, wherein the fusion protein comprises a cytokine portion and a portion selected from the group consisting of serum albumin, an immunoglobulin fragment, transferrin, Fn3, and variants thereof.

74. 74. The pharmaceutical formulation of any one of claims 70 to 73, wherein the serum albumin comprises mouse serum albumin or human serum albumin.

75. 75. The pharmaceutical preparation of any one of claims 70 to 74, wherein the immunoglobulin fragment comprises an immunoglobulin Fc domain.

76. A pharmaceutical formulation according to any one of claims 44 to 75 for pharmaceutical use.

77. 77. The pharmaceutical formulation of claim 76, wherein the medical use comprises therapeutic or prophylactic treatment of a disease or disorder.

78. 78. The pharmaceutical preparation of any one of claims 44 to 77 for use in a method for treating or preventing cancer in a subject, wherein the antigen is a tumor associated antigen.

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