HK40060040A

HK40060040A - Treatment involving car-engineered t cells and cytokines

Info

Publication number: HK40060040A
Application number: HK62022047708.2A
Authority: HK
Inventors: U·沙欣; P·厄姆; B·伦斯特尔; K·赖因哈德
Original assignee: 生物技术细胞和基因治疗公司
Priority date: 2019-02-08
Filing date: 2020-02-06
Publication date: 2022-05-13

Description

Treatment involving CAR engineered T cells and cytokines

Technical Field

The present disclosure relates to methods and materials for enhancing the effect of T cells engineered to express Chimeric Antigen Receptors (CARs). These methods and materials are particularly useful for treating diseases characterized by diseased cells expressing the antigen to which the CAR is directed. In particular, the disclosure relates to a method comprising providing a subject with a T cell genetically modified to express a Chimeric Antigen Receptor (CAR) and administering to the subject IL2 or a polynucleotide encoding IL 2. The methods of the present disclosure may comprise administering IL2 or a polynucleotide encoding IL2 and a further cytokine or a polynucleotide encoding a further cytokine, wherein the further cytokine may be IL7 or IL 21. The subject can be provided with a T cell genetically modified to express a CAR by administering a T cell genetically modified to express a CAR or by generating a T cell genetically modified to express a CAR in the subject. The methods of the present disclosure may further comprise administering to the subject an antigen or variant thereof, or a polynucleotide encoding an antigen or variant thereof, wherein the T cell modified to express the CAR is targeted to the antigen. In a particularly preferred embodiment, the polynucleotide administered according to the present disclosure is RNA.

Background

The immune system plays an important role in cancer, autoimmunity, allergy and pathogen-related diseases. T cells play a central role in cell-mediated immunity in humans and animals. T cell recognition and binding of specific antigens is mediated by T Cell Receptors (TCRs) expressed on the surface of T cells. The TCR of a T cell is capable of interacting with an immunogenic peptide (epitope) that binds to a Major Histocompatibility Complex (MHC) molecule and is presented on the surface of a target cell. Specific binding of the TCR triggers a signaling cascade within the T cell, leading to proliferation and differentiation into mature effector T cells.

The diversity of TCRs is achieved by genetic rearrangement of different discrete segments of genes encoding different structural regions of the TCR. The TCR comprises 1 α chain and 1 β chain or 1 γ chain and 1 δ chain. The TCR alpha/beta chain includes an N-terminal highly polymorphic variable region and a constant region involved in antigen recognition. At the genetic level, these chains are divided into several regions, variable (V) regions, diversity (D) regions (only β -and δ -chains), junction (J) regions and constant (C) regions. During T cell differentiation, specific T cell receptor genes are generated by rearranging one V, one D (β and δ chains only), one J and one C region genes. The diversity of TCRs is further amplified by imprecise V- (D) -J rearrangements, in which random nucleotides are introduced and/or deleted at the recombination site. Since rearrangement of the TCR locus occurs in 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, including heterodimeric complexes of TCR α -and β -chains, co-receptor CD4 or CD8 and CD3 number transduction modules. The CD3 chain transmits activation signals within the cell, whereas the TCR α/β heterodimer is only responsible for antigen recognition.

Adoptive Cell Transfer (ACT) based immunotherapy can be broadly defined as a form of passive immunization, in which previously sensitized T cells are transferred to a non-immune recipient or autologous host after in vitro expansion from a low precursor frequency to a clinically relevant cell number. 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 Du-specific T cell lines or clones, M.E.et al. (2001) J.dInother.24, 363-373; yee, C.et al (2002) Proc.Natl.Acad.Sci.U.S.A. 99, 16168-16173). Adoptive T cell transfer has been demonstrated to be therapeutically active against human viral infections (e.g., CMV). While CMV infection and reactivation of endogenous latent viruses are controlled by the immune system of healthy individuals, it can lead to significant morbidity and mortality in immunocompromised individuals (such as transplant recipients or AIDS patients). Riddell and colleagues demonstrated that immunosuppressed patients reconstituted viral immunity by adoptive T cell therapy after transfer of CD8+ CMV-specific T cell clones from HLA matched CMV seropositive transplant donors (Riddell, S.R. (1992) Science 257, 238-. As an alternative, the transfer of polyclonal donor-derived CMV or EBV-specific T cell populations to transplant recipients resulted in an increase in persistence of the transferred T cells (Rooney, C.M.et al (1998) Blood 92, 1549-1371555; Peggs, K.S.et al (2003) Lancet 362, 1375-1377). For adoptive immunotherapy of melanoma, Rosenberg and colleagues established an ACT approach that relies on infusion of ex vivo expanded autologous Tumor Infiltrating Lymphocytes (TILs) isolated from resected tumors, in combination with non-myeloablative lymphoablative chemotherapy and high dose IL 2. A recently published clinical study showed that the objective response rate of metastatic melanoma patients receiving treatment was about 50% (Dudley, M.E.et al. (2005) J.Clin.Oncol.23: 2346-2357).

An alternative approach is adoptive transfer reprogramming during short ex vivo cultures to express autologous T cells with tumor-reactive immune receptors of defined specificity, followed by infusion into patients (Kershaw m.h.et al (2013) Nature Reviews Cancer 13(8): 525-41). This strategy makes ACT applicable to a variety of common malignancies even in the absence of tumor-reactive T cells in the patient. Since the antigen specificity of T cells is entirely dependent on the heterodimeric complex of TCR α -and β -chains, transferring cloned TCR genes into T cells offers the potential to redirect them to the antigen of interest. Thus, TCR gene therapy offers an attractive strategy for the development of antigen-specific immunotherapy with autologous lymphocytes as the therapeutic choice. The main advantage of TCR gene transfer is the generation of therapeutic amounts of antigen-specific T cells within a few days and the possibility to introduce specificities that are not present in the patient's endogenous TCR repertoire.

Several groups have demonstrated that TCR gene transfer is an attractive strategy to redirect 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). Rosenberg and his team were the first to demonstrate the feasibility of TCR gene therapy in clinical trials for treating malignant melanoma. Adoptive transfer of autologous lymphocytes transduced with melanoma/melanocyte antigen specific TCR retroviruses resulted in cancer regression in up to 30% of treated melanoma patients (Morgan, r.a.et al. (2006) Science 314, 126-. Meanwhile, clinical testing of TCR gene therapy also extends to cancers other than melanoma that target a number of different tumor antigens (Park, t.s.et al, (2011) Trends biotechnol.29, 550-557).

The potential of ACT is greatly expanded by the use of genetic engineering methods to insert specific antigen-targeting receptors into T cells. A Chimeric Antigen Receptor (CAR) is an antigen-targeting receptor comprising an intracellular T cell signaling domain fused to an extracellular antigen-binding moiety, most commonly a single-chain variable fragment (scFv) from a monoclonal antibody. CARs recognize cell surface antigens directly, independent of MHC-mediated presentation, allowing the use of a single receptor construct specific for any given antigen in all patients. The original CAR fused the antigen recognition domain to the CD3 ζ activation chain of the T Cell Receptor (TCR) complex. Subsequent CAR iterations included secondary costimulatory signals in tandem with CD3 ζ, including the intracellular domains from CD28 or various TNF receptor family molecules such as 4-1BB (CD137) and OX40(CD 134). Furthermore, the third generation receptor contains two costimulatory signals in addition to CD3 ζ, most commonly from CD28 and 4-1 BB. Second and third generation CARs significantly improved the anti-tumor efficacy, and in some cases induced complete remission in patients with advanced cancer.

The number of T cells transferred is generally 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 generating large numbers of T cells for adoptive T cell transfer remains a challenge. A significant increase in cell persistence can be achieved when the patient is subjected to a lymphocyte depletion preparation protocol prior to infusion of TIL or engineered recipient T cells. However, the transfer of large numbers of engineered T cells into empty hosts also carries the risk of serious adverse events if the targeted antigen is accidentally expressed in the relevant normal tissue. Therefore, it is desirable to transfer limited amounts of engineered T cells that can be expanded in patients after safety is demonstrated.

The inventors have found that CAR-T cells can be expanded in a subject by administering an RNA encoding IL2, optionally in combination with an RNA encoding other cytokines such as IL7 or IL21, and optionally using RNA vaccination to provide an antigen for stimulation of the CAR-T cells. The methods of the invention allow for only a small number of CAR-engineered T cells to be provided to a patient, and then the T cells are expanded in vivo.

Disclosure of Invention

The invention generally includes treating diseases by targeting cells that express antigens on the cell surface, such as diseased cells that express antigens on the cell surface, particularly cancer cells that express tumor antigens on the cell surface. The method provides selective eradication of cells expressing an antigen on their surface, thereby minimizing adverse effects on normal cells that do not express the antigen. The T cells are provided in a subject, for example, by administering T cells that are genetically modified to express a Chimeric Antigen Receptor (CAR) that targets the cells by binding to an antigen. IL2 or a nucleic acid encoding same is administered. In one embodiment, other cytokines, or nucleic acids encoding them, such as IL7 or IL21 are administered. In one embodiment, an antigen or variant thereof or nucleic acid encoding the same is administered to provide (optionally after expression of the nucleic acid by a suitable target cell) the antigen for stimulation, priming and/or expansion of T cells. T cells stimulated, primed and/or expanded in a patient are capable of recognizing cells expressing an antigen on the cell surface, such as diseased cells, thereby destroying the diseased cells. The methods of the invention may be considered to involve passive and active immunization. Treatments involving administration of T cells modified by genetic modification to express a CAR can be considered a form of passive immunization. Treatments involving administration of an antigen or variant thereof to stimulate a T cell-mediated immune response against a target cell population or tissue may be considered in the form of active immunization.

The immune response of the present disclosure is directed to a target cell population or target tissue in a mammal that expresses an antigen, and T cells genetically modified to express a Chimeric Antigen Receptor (CAR) target the antigen. The methods of the invention optionally further comprise administering the antigen or 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 a tumor cell or tumor tissue.

The methods and materials described herein are particularly effective if an RNA encoding IL2 linked to a pharmacokinetic modifying group (hereinafter referred to as "Pharmacokinetic (PK) extending IL 2") is administered, optionally in combination with an RNA encoding other cytokines linked to a pharmacokinetic modifying group, such as IL7 or IL21 (hereinafter referred to as "Pharmacokinetic (PK) extending cytokine"). The methods and materials described herein are particularly effective if RNA encoding IL2 for extended PK and/or RNA encoding cytokines for extended PK is targeted to the liver for systemic availability. Hepatocytes can be efficiently transfected and are capable of producing large amounts of protein. The RNA encoding the antigen is preferably targeted to the secondary lymphoid organs.

In one aspect, the invention provides a method of inducing an immune response in a subject comprising:

a. providing to the subject a T cell genetically modified to express a Chimeric Antigen Receptor (CAR), and

b. administering to the subject IL2 or a polynucleotide encoding IL 2.

In one embodiment, the method comprises administering IL2 or a polynucleotide encoding IL2 in combination with another cytokine or polynucleotide encoding another cytokine. In one embodiment, the additional cytokine is selected from IL7 and IL 21. In one embodiment, the method comprises administering IL2 or a polynucleotide encoding IL2 and IL7 or a polynucleotide encoding IL 7. In one embodiment, the method comprises administering IL2 or a polynucleotide encoding IL2 and IL21 or a polynucleotide encoding IL 21.

In one embodiment, the polynucleotide encoding IL2 is RNA, and optionally, the polynucleotide encoding the other cytokine is RNA.

In one embodiment, the subject is provided with a T cell genetically modified to express a CAR by administering a T cell genetically modified to express a CAR or by generating a T cell genetically modified to express a CAR in the subject.

In one embodiment, the method further comprises administering to the subject an antigen or variant thereof or a polynucleotide encoding the antigen or variant, wherein the genetic modification targets the antigen with a CAR-expressing T cell and the immune response is an immune response against a target cell population or a target tissue expressing the antigen. In one embodiment, the polynucleotide encoding an antigen or variant is RNA.

b. administering to the subject an RNA encoding IL 2.

In one embodiment, the method comprises administering an RNA encoding IL2 and an RNA encoding other cytokines. In one embodiment, the additional cytokine is selected from IL7 and IL 21. In one embodiment, the method comprises administering an RNA encoding IL2 and an RNA encoding IL 7. In one embodiment, the method comprises administering an RNA encoding IL2 and an RNA encoding IL 21.

In one embodiment, the method further comprises administering to the subject an RNA encoding an antigen or a variant thereof, wherein the genetic modification targets the antigen with a CAR-expressing T cell, and the immune response is an immune response against a target cell population or a target tissue expressing the antigen.

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

In one aspect, the invention provides a method of treating a subject having a disease, disorder or condition associated with expression or elevated expression of an antigen, the method comprising:

a. providing the subject with a T cell genetically modified to express a Chimeric Antigen Receptor (CAR) targeting the antigen, and

b. administering to the subject IL2 or a polynucleotide encoding IL 2.

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

b. administering to the subject an RNA encoding IL 2.

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

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

In one embodiment of all aspects, IL2 is extended Pharmacokinetic (PK) IL 2. In one embodiment, the PK extended 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.

In one embodiment of all aspects, the further cytokine, in particular IL7 or IL21, is a Pharmacokinetic (PK) extending cytokine, in particular PK extending IL7 or PK extending IL 21. In one embodiment, the PK-extending cytokine, in particular PK-extending IL7 or PK-extending IL21, comprises a fusion protein. In one embodiment, the fusion protein comprises portions of other cytokines, in particular an IL7 portion or an IL21 portion, and a portion selected from: serum albumin, immunoglobulin fragments, transferrin, Fn3, and variants thereof.

In one embodiment, the serum albumin comprises mouse serum albumin or human serum albumin. In one embodiment, the immunoglobulin fragment comprises an immunoglobulin Fc domain.

In one embodiment of all aspects, the method is a method for treating or preventing cancer in a subject, wherein the antigen is a tumor associated antigen.

In one aspect, the present invention provides a pharmaceutical product comprising:

a. t cells genetically modified to express a Chimeric Antigen Receptor (CAR), and

il2 or a polynucleotide encoding IL 2.

In one embodiment, the pharmaceutical product comprises IL2 or a polynucleotide encoding IL2 in combination with other cytokines or polynucleotides encoding other cytokines. In one embodiment, the additional cytokine is selected from IL7 and IL 21. In one embodiment, the pharmaceutical product comprises IL2 or a polynucleotide encoding IL2 and IL7 or a polynucleotide encoding IL 7. In one embodiment, the pharmaceutical product comprises IL2 or a polynucleotide encoding IL2 and IL21 or a polynucleotide encoding IL 21.

In one embodiment, the pharmaceutical product further comprises an antigen or variant thereof or a polynucleotide encoding the antigen or variant, wherein the T cell genetically modified to express a CAR is targeted to the antigen. In one embodiment, the polynucleotide encoding the antigen or variant is RNA.

In one embodiment, the pharmaceutical product is a kit.

In one embodiment, the pharmaceutical product comprises a T cell genetically modified to express a CAR, IL2, or a polynucleotide encoding IL2, optionally other cytokines or polynucleotides encoding other cytokines, and optionally an antigen or variant thereof or a polynucleotide encoding the antigen or variant, in separate containers.

In one embodiment, the pharmaceutical product further comprises instructions for use of the pharmaceutical product in the treatment or prevention of cancer, wherein the antigen is a tumor associated antigen.

In one embodiment, the pharmaceutical product is a pharmaceutical composition.

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

b. RNA encoding IL 2.

In one embodiment, the pharmaceutical product comprises an RNA encoding IL2 and an RNA encoding other cytokines. In one embodiment, the additional cytokine is selected from IL7 and IL 21. In one embodiment, the pharmaceutical product comprises RNA encoding IL2 and RNA encoding IL 7. In one embodiment, the pharmaceutical product comprises an RNA encoding IL2 and an RNA encoding IL 21.

In one embodiment, the pharmaceutical product further comprises an RNA encoding an antigen or variant thereof, wherein the T cell genetically modified to express the CAR is targeted to the antigen.

In one embodiment, the pharmaceutical product is a kit.

In one embodiment, the pharmaceutical product comprises a T cell genetically modified to express a CAR, an RNA encoding IL2, optionally an RNA encoding other cytokines, and optionally an RNA encoding an antigen or variant thereof, in separate containers.

In one embodiment, the pharmaceutical product is a pharmaceutical composition.

In one embodiment of all aspects, the additional cytokine is a prolonged Pharmacokinetic (PK) cytokine. In one embodiment, the PK extending cytokine comprises a fusion protein. In one embodiment, the fusion protein comprises a cytokine moiety and a moiety selected from the group consisting of: serum albumin, immunoglobulin fragments, transferrin, Fn3, and variants thereof.

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

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

In one aspect, the invention provides a pharmaceutical product as described herein for pharmaceutical use. In one embodiment, the pharmaceutical use comprises a therapeutic or prophylactic treatment of a disease or disorder.

In one aspect, the invention provides a pharmaceutical product as described herein, for use in a method of treating or preventing cancer in a subject, wherein the antigen is a tumor associated antigen.

In one aspect, the invention provides materials and compositions as described herein for use in the methods described herein.

In one aspect, the invention provides T cells genetically modified to express a Chimeric Antigen Receptor (CAR), which target an antigen, for use in the methods described herein.

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

In one aspect, the invention provides cytokines other than IL2, such as IL7 or IL21, or polynucleotides encoding same, for use in the methods described herein.

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

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

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.

Drawings

FIG. 1: mAll-mIL-2 and mIL-7-mAll enhanced in situ repeat antigen-specific amplification of genetically engineered CAR T cells in pre-conditioned mice. (A) To C57BL/6BrdCrHsd-Tyr irradiated with 2.5Gy (XRAD320)^cMice (n-2-3/group) were transplanted with 5x10 i.v⁶Individual CLDN6-CAR-BBz-Luc-GFP transduced C57Bl/6-thy1.1⁺T cells. One day later, mice were treated with mRNA lipoplex vaccination (20 μ g, i.v.) encoding hCLDN6 or OvaI (control RNA) followed by i.p. administration of nucleoside modified formulated RNAs encoding mab l-mIL-2 and mIL-7-mab (1 μ g/mRNA/mouse). Buffer was used as a mock control. After another 7 days, the treatment was repeated. Bioluminescence imaging (BLI) was performed to monitor amplification and persistence from day 1 (baseline) to day 15 post ACT. (B) Transgenic expression of adoptively transferred murine CAR-transduced T cells. Cells were stained with fluorochrome-conjugated antibodies against CD8 and CD4 and idiotypic specific antibodies against the scFv portion of CLDN6-CAR (anti-IMAB 206) and analyzed by flow cytometry. Left, gating on individual lymphocytes for cells; on the right, the cells are in CD8⁺The T cells are gated. (C) Use of antigen RNA in ACT_(LIP)Bioluminescence imaging of lateral mice at various time points following treatment with the indicated mRNA encoding albumin-cytokine. The achromatic image represents the light intensity (black, least intense) superimposed on the grayscale reference imageStrong; white to dark grey, most intense). (D) Amplification index (mean ± s.d.) for total flux calculated 4 and 11 days post ACT compared to baseline on day 1; ACT: adoptive T cell transfer; TBI: irradiating the whole body; BLI: bioluminescence imaging, Luc: an effective firefly luciferase; and (3) mAllb: mouse serum albumin; mIL-2: murine interleukin-2; mIL-7: murine interleukin-7.

FIG. 2: even in immunocompetent mice, mILs-mIL-2 and mIL-7-mILs prolong the persistence of antigen-specifically amplified CAR T cells in vivo. (A) Unirradiated C57BL/6BrdCrHsd-Tyr^cMice (n-2-3/group) received the same dose of CAR-transduced T cells and were treated as described in legend 1A-B. (B) Use of antigen RNA in ACT_(LIP)Bioluminescence imaging of lateral mice at various time points following treatment with the indicated mRNA encoding albumin-cytokine. The achromatic image represents the light intensity superimposed on the grey-scale reference image (black, least intense; white to dark grey, most intense). (C) Amplification index of total flux 4 days and 11 days post ACT compared to baseline at day 1 (mean ± s.d.); ACT: adoptive T cell transfer; BLI: bioluminescence imaging, Luc: an effective firefly luciferase; and (3) mAllb: mouse serum albumin; mIL-2: murine interleukin-2; mIL-7: murine interleukin-7.

FIG. 3: the presence of mIL-2 in combination with mIL-7-mIL or mIL-21-mIL results in the accumulation of in situ repeated antigen-specific expansion and prolonged persistence of genetically engineered CAR T cells in vivo. (A) 2.5Gy irradiated C57BL/6BrdCrHsd-Tyr^cMice (n-2-3/group) received the same dose of CAR transduced T cells and mRNA lipoplex vaccination encoding hCLDN6 or OvaI (control RNA) in combination with different nucleoside modified formulated cytokines (1 μ g of each individual mRNA used per animal) as described in figure 1A. (B) During 3 rounds of vaccination (imaging is usually at the indicated RNA)_(LIP)And 2-3 days after the cytokine RNA treatment round) to quantify and calculate the relative increase in bioluminescence of the treated mice. The amplification index was calculated as follows: total flux [ p/s ] for each round of amplification]Total flux [ p/s ] at day 1 Baseline post ACT](mean. + -. s.e.m.). (C-D) preparation of cytokine RNA modified at the indicated nucleosides(mean +/-s.e.m.) by using CLDN6-RNA_(LIP)Quantification of bioluminescence during and after the amplification wheel (round). Arrow indicates hLDN 6 RNA_(LIP)Vaccination and treatment with nucleoside-modified formulated cytokines (ribo-cytokines). ACT: adoptive T cell transfer; TBI: irradiating the whole body; BLI: bioluminescence imaging; luc: an effective firefly luciferase; and (3) mAllb: mouse serum albumin; mIL-2: murine interleukin-2, mIL-7: murine interleukin-7.

Detailed Description

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

Preferably, the terms used herein are defined as "multilingual vocabulary of biotechnological terms: (IUPAC recommendations) ", h.g.w.leuenberger, b.nagel, and H.Eds., Helvetica Chimica Acta, CH-4010Basel, Switzerland, (1995).

Unless otherwise indicated, the practice of the present disclosure employs conventional methods of chemistry, biochemistry, cell biology, immunology and recombinant DNA technology, which are explained in the literature of the art (see, e.g., Molecular Cloning: A Laboratory Manual,2nd Edition, J.Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

Hereinafter, elements of the present disclosure will be described. These elements are listed with particular embodiments, however, it should be understood that they may be combined in any manner and in any number to produce additional embodiments. The various described examples and embodiments should not be construed as limiting the disclosure to only the explicitly described embodiments. The description of the present disclosure should be understood to disclose and encompass embodiments combining the explicitly described embodiments with any number of the disclosed elements. Moreover, any arrangement or combination of the elements described is deemed to be disclosed by the specification unless otherwise indicated by the context.

The term "about" refers to about or nearly, and the context of values or ranges set forth herein in an embodiment refers to ± 20%, ± 10%, ± 5% or ± 3% of the recited or claimed value or range.

The use of the terms "a" and "an" and "the" and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the claims. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

The term "comprising" as used in the context of this document means that, unless explicitly stated otherwise, there may optionally be present other members in addition to the list member to which "comprising" is introduced. However, it is contemplated that as a specific embodiment of the disclosure, the term "comprising" encompasses the possibility that no other member is present, i.e., for the purposes of this embodiment, "comprising" is understood to have the following meaning: "consists of.

Throughout this specification, several documents are cited. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such disclosure by virtue of prior disclosure.

In the following, definitions will be provided that apply to all aspects of the present disclosure. Unless otherwise indicated, the following terms have the following meanings. Any undefined terms have art-recognized meanings.

According to the present disclosure, the term "peptide" includes oligopeptides and polypeptides, and refers to a substance comprising about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids 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, although the terms "peptide", "protein" and "polypeptide" are generally used herein as synonyms.

A "therapeutic protein" has a positive or beneficial effect on a disease condition or disease state in a subject when provided to the subject in a therapeutically effective amount. In one embodiment, the therapeutic protein has a curative or palliative nature and can be administered to improve, alleviate, reduce, reverse, delay the onset of, or reduce the severity of one or more symptoms of a disease or disorder. Therapeutic proteins may have prophylactic properties and may 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 intact proteins or peptides, which may also refer to therapeutically active fragments thereof. It may also include therapeutically active variants of the protein. Examples of therapeutically active proteins include, but are not limited to, cytokines.

"fragments" in reference to an amino acid sequence (peptide or protein) relate to a portion of the amino acid sequence, i.e. a sequence representing an amino acid sequence shortened at the N-terminus and/or the C-terminus. A shortened fragment at the C-terminus (N-terminal fragment) is available, for example, by translation of a truncated open reading frame that lacks the 3' end of the open reading frame. A shortened fragment at the N-terminus (C-terminal fragment) is available, for example, by translation of a truncated open reading frame lacking the 5' end of the open reading frame, so long as the truncated open reading frame contains a start codon for initiation of translation. Fragments of an amino acid sequence comprise, 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 comprise at least 6, in particular at least 8, at least 12, at least 15, at least 20, at least 30, at least 50 or at least 100 consecutive amino acids from the amino acid sequence.

For the purposes of the present disclosure, "variants" of an amino acid sequence (peptide or protein) include amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and/or amino acid substitution variants. The term "variant" especially includes fragments of the amino acid sequence.

Amino acid insertion variants comprise the insertion of a single or two or more amino acids into a particular amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted at specific positions in the amino acid sequence, although random insertions and appropriate screening of the resulting product are also possible. Amino acid addition variants comprise amino-terminal and/or carboxy-terminal fusions of one or more amino acids, e.g., 1,2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, e.g., the removal of 1,2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion may be at any position of the protein. Amino acid deletion variants comprising deletions at the N-terminus and/or C-terminus of the protein are also referred to as N-terminal and/or C-terminal truncation variants. Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Modifications at amino acid sequence positions that are not conserved between homologous proteins or peptides and/or substitutions of amino acids with other amino acids having similar properties are preferred. Preferably, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes involve substitution of one of a family of related amino acids in its side chain. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes collectively classified as aromatic amino acids.

Preferably, the degree of similarity, preferably the degree of identity, between a given amino acid sequence and an amino acid sequence that is a variant of said given amino acid sequence is at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Preferably, the degree of similarity or identity is given to 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 amino acid region over the entire length of the reference amino acid sequence. For example, if a reference amino acid sequence consists of 200 amino acids, it is preferred to give a degree of similarity or identity to at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, preferably consecutive amino acids. In a preferred embodiment, the degree of similarity or identity is given over the full length of the reference amino acid sequence. The alignment for determining sequence similarity, preferably sequence identity, can be accomplished using tools known in the art, preferably using optimal sequence alignment, e.g., using Align, using standard settings, preferably EMBOSS:needle, Matrix: Blosum62, Gap Open 10.0, Gap extended 0.5.

"sequence similarity" refers to the percentage of amino acids that are identical or represent conservative amino acid substitutions. "sequence identity" between two amino acid sequences means the percentage of amino acids that are identical between the sequences.

The term "percent identity" is intended to mean the percentage of amino acid residues that are identical between two sequences to be compared, obtained after optimal alignment, which percentage is purely statistical, and the differences between the two sequences are randomly distributed over their entire length. Sequence comparisons between two amino acid sequences are typically performed by comparing the sequences after they have been optimally aligned, either by segment or "comparison window" to identify and compare local regions of sequence similarity. In addition to manual, optimal alignment of sequences for comparison can be generated by: 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; similarity search methods by 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 in the Wisconsin Genetics software package (Genetics Computer Group,575Science Drive, Madison, Wis.).

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

According to the present disclosure, homologous amino acid sequences exhibit at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably at least 95%, at least 98 or at least 99% amino acid residue identity.

Amino acid sequence variants described herein can be readily prepared by those skilled in the art, for example, by recombinant DNA procedures. For example, procedures for preparing DNA sequences having substituted, added, inserted or deleted peptides or proteins are described in detail in Sambrook et al (1989). In addition, the peptides and amino acid variants described herein can be readily prepared by means of known peptide synthesis techniques, such as by solid phase synthesis and the like.

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

An amino acid sequence (peptide or protein) "derived from" a given amino acid sequence (peptide or protein) refers to the origin of the first amino acid sequence. Preferably, the amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, substantially identical, or homologous to the particular sequence or fragment thereof. An amino acid sequence derived from a particular amino acid sequence can be a variant of that particular sequence or a fragment thereof. For example, one of ordinary skill in the art will appreciate that antigens and cytokines suitable for use herein (e.g., IL2, IL7, or IL21) can be altered such that their sequences differ from the naturally occurring sequences or the native sequences from which they are derived, while retaining the desired activity of the native sequences.

T cells

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

Most T cells have a T Cell Receptor (TCR), which exists as a complex of several proteins. The actual T cell receptor comprises two separate peptide chains, which are produced by separate T cell receptor alpha and beta (TCR alpha and TCR beta) genes, and are referred to as alpha-and beta-TCR chains. γ δ T cells (gamma delta T cells) represent a small subset of T cells, with a unique T Cell Receptor (TCR) on their surface. However, in γ δ T cells, the TCR consists of one γ chain and one δ chain. This group of T cells was fewer than α β T cells (2% of the total number of T cells).

All T cells are derived from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells derived from hematopoietic stem cells are located in the thymus and are expanded by cell division to produce large quantities of immature thymocytes. The earliest thymocytes expressed neither CD4 nor CD8 and were therefore classified as double negative (CD4-CD8-) cells. As they develop, they become double positive thymocytes (CD4+ CD8+) and eventually mature into single positive (CD4+ CD 8-or CD4-CD8+) thymocytes, which are then released from the thymus into peripheral tissues.

The terms "T cell" and "T lymphocyte" are used interchangeably herein and include T helper 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 refer to a T cell that recognizes an antigen targeted by the T cell, particularly when presented on the surface of an antigen presenting cell or diseased cell (e.g., cancer cell), and preferably functions as an effector of the T cell. T cells are considered specific for an antigen if the cell kills a target cell that expresses the antigen. T cell specificity can be assessed using any of a variety of standard techniques, for example, in a chromium release assay or a proliferation assay. Alternatively, synthesis of lymphokines (e.g., interferon- γ) can be measured.

T helper cells assist in the immune process with other leukocytes, including B cell maturation to plasma cells and activation of cytotoxic T cells and macrophages, among other functions. These cells are also called CD4+ T cells because they express CD4 protein on their surface. Helper T cells are activated when peptide antigens are presented 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.

Cytotoxic T cells destroy virus-infected cells and tumor cells, and are also associated with transplant rejection. These cells are also called 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 is present on almost every cell surface of the body.

T cell mediated effector functions are included in helper T cells (CD4)⁺T cells) release cytokines and/or activate CD8⁺Lymphocytes (CTL) and/or B-cells, and in the case of CTLs, elimination of cells, i.e., cells characterized by expression of an antigen, e.g., by apoptosis or perforin-mediated lysis, production of cytokines (e.g., IFN- γ and TNF- α), and specific cytolytic killing of target cells expressing the antigen.

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

T cells can generally be prepared in vitro or ex vivo using standard procedures. For example, T cells can be isolated from bone marrow, peripheral blood, or fractions of bone marrow or peripheral blood of a mammal (e.g., a patient) using commercially available cell isolation systems. Alternatively, the T cells may be derived from related or unrelated humans, non-human animals, cell lines or cultures. The sample comprising T cells may be, for example, Peripheral Blood Mononuclear Cells (PBMCs).

T cells used in the invention may express endogenous T cell receptors or may lack expression of endogenous T cell receptors.

CAR

Nucleic acids such as (CAR-encoding RNA) can be introduced into T cells or other cells with lytic potential, particularly lymphoid cells.

According to the present disclosure, as described above, when present on a T cell, the CAR recognizes an antigen, such as on the surface of an antigen presenting cell or diseased cell (e.g., cancer cell), such that the T cell is stimulated, primed, and/or expanded or exerts effector functions.

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".

Preferably, the CAR is expressed on the surface of a cell.

According to the present invention, the term "CAR" (or "chimeric antigen receptor") relates to an artificial receptor comprising a single molecule or molecular complex that recognizes, i.e., binds, a target structure (e.g., an antigen) on a target cell, such as a cancer cell (e.g., bound to an antigen expressed on the surface of the target cell by an antigen binding domain), and can confer specificity on an immune effector cell (e.g., a T cell expressing the CAR on the surface of the cell). Such cells do not necessarily need to process and present antigens to recognize the target cell, but may preferably specifically recognize any antigen present on the target cell. Preferably, recognition of the target structure by the CAR results in activation of an 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 T cell receptors.

According to the invention, a CAR may generally comprise several domains. In one embodiment of all aspects of the invention, the CAR comprises an antigen binding domain, a transmembrane domain and a T cell signalling domain.

The binding domain recognizes and binds an antigen. In one embodiment, single chain variable fragments (scFv) derived from monoclonal antibodies are used as binding domains. Antigen recognition domains that may also be used include T Cell Receptor (TCR) alpha and beta single chains, and the like. In fact, almost any substance that binds a given target with high affinity can be used as an antigen recognition domain. In one embodiment of all aspects of the invention, the CAR comprises an antigen binding domain. In one embodiment, the ectodomain (exodomain) of the CAR comprises an antigen binding domain. In one embodiment, the antigen binding domain comprises a single chain variable fragment (scFv) of an antibody directed against an antigen. In one embodiment, the antigen binding domain comprises a heavy chain variable region (VH) of an immunoglobulin specific for an antigen (VH (antigen)) and a light chain variable region (VL) of an immunoglobulin specific for an antigen (VL (antigen)). In one embodiment, the heavy chain variable region (VH) and the corresponding light chain variable region (VL) are linked by a peptide linker, preferably a peptide linker comprising the amino acid sequence (GGGGS) 3.

In one embodiment of all aspects of the invention, the CAR comprises a transmembrane domain. In one embodiment, the transmembrane domain is a hydrophobic alpha helix across the membrane. In one embodiment, the transmembrane domain comprises a CD28 transmembrane domain or a fragment thereof.

The activation signaling domain (or T cell signaling domain) is used to activate the cytotoxic lymphocyte upon binding of the CAR to the antigen. The nature of the activation signaling domain is limited to its ability to induce activation of selected cytotoxic lymphocytes upon binding of the CAR to the antigen. Suitable activation signaling domains include the T cell CD3[ zeta ] chain and the Fc receptor [ gamma ]. Those skilled in the art will appreciate that sequence variants of these mentioned activation signaling domains, which have the same or similar activity as the domain it models, can be used without adversely affecting the present invention. Such variants will have at least about 80% sequence identity with the amino acid sequence of the domain from which they are derived.

In one embodiment, the T cell signaling domain is located intracellularly. In one embodiment, the T cell signaling domain comprises CD 3-zeta (zeta), preferably the endodomain of CD 3-zeta, optionally in combination with CD 28.

Another domain that may be present is a co-stimulatory domain. The co-stimulatory domain is used to enhance proliferation and survival of cytotoxic lymphocytes upon binding of the CAR to the targeting moiety. The properties of the co-stimulatory domain are limited to its ability to enhance cell proliferation and survival upon binding of the CAR to the targeting moiety. Suitable co-stimulatory domains include CD28, CD137(4-1BB), members of the Tumor Necrosis Factor (TNF) receptor family, CD134(OX40), TNFR receptor superfamily members, and CD278(ICOS), CD28 superfamily co-stimulatory molecules expressed on activated T cells. One skilled in the art will appreciate that sequence variants of these mentioned co-stimulatory domains can be used without adversely affecting the present invention, wherein the variants have the same or similar activity as the domain they model. Such variants will 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 invention, the CAR construct comprises two co-stimulatory domains. Although a particular combination includes all possible variants of the four mentioned domains, specific examples include CD28+ CD137(4-1BB) and CD28+ CD134(OX 40).

The CARs of the invention may together comprise the above domains in the form of a fusion protein. Such fusion proteins typically comprise a binding domain, one or more costimulatory domains, and an activation signaling domain linked in an N-terminal to C-terminal direction. However, the CARs of the invention are not limited to this arrangement and other arrangements are also acceptable and include a binding domain, an activation signaling domain, and one or more co-stimulatory domains. It will be appreciated that, because the binding domain must be able to freely bind antigen, placement of the binding domain in the fusion protein will typically effect display of the region outside the cell. In the same way, since the costimulatory and activation signaling domains are used to induce the activity and proliferation of cytotoxic lymphocytes, the fusion protein will typically display both domains inside the cell. The CAR may include other elements, such as a signal peptide to ensure proper export of the fusion protein to the cell surface, a transmembrane domain and a hinge domain (or spacer) to ensure that the fusion protein is maintained as an integral membrane protein, which confers flexibility to the binding domain and allows strong binding to the antigen.

In one embodiment of all aspects of the invention, the CAR comprises a signal peptide that directs nascent protein into the endoplasmic reticulum. In one embodiment, the signal peptide precedes the antigen binding domain.

In one embodiment of all aspects of the invention, the CAR comprises a spacer linking the antigen binding domain to the transmembrane domain. In one embodiment, the spacer allows the antigen binding domains to be oriented in different directions to facilitate antigen recognition. In one embodiment, the spacer comprises a hinge region from IgG 1.

In one embodiment of all aspects of the invention, the CAR comprises the structure:

NH 2-signal peptide-antigen binding domain-spacer-transmembrane domain-T cell signaling domain-COOH.

In one embodiment of all aspects of the invention, the CAR is preferably specific for the antigen it is targeting, particularly when present on the surface of a cell (e.g., a diseased cell or an antigen presenting cell).

In one embodiment of all aspects of the invention, the CAR may be expressed by and/or present on the surface of a T cell, preferably a cytotoxic T cell. In one embodiment, the T cell is reactive to an antigen targeted by the CAR.

The cells used in conjunction with the CAR system of the invention are preferably T cells, in particular cytotoxic lymphocytes, preferably selected from the group consisting of T cells, in particular cytotoxic T cells, Natural Killer (NK) cells and lymphokine-activated killer (LAK) cells. Upon activation, each of these cytotoxic lymphocytes triggers the destruction of the target cell. For example, cytotoxic T cells trigger destruction of target cells by either or both of the following means. First, after activation, T cells release cytotoxins such as perforin, granzyme and granulysin. Perforin and granulysin form pores in the target cell and granzymes enter the cell and trigger a caspase cascade in the cytoplasm, which induces apoptosis (programmed cell death). Second, apoptosis can be induced by Fas-Fas ligand interaction between T cells and target cells. Preferably, the cytotoxic lymphocytes are autologous cells, although allogeneic or allogeneic cells may be used.

Adoptive cell transfer therapy using T cells expressing chimeric antigen receptors is a promising anti-cancer therapy because CAR-modified T cells can be designed against almost any tumor antigen. For example, the patient's T cells can be genetically engineered (genetically modified) to express a CAR specific for an antigen on the patient's tumor cells, and then infused back into the patient.

According to the invention, the CAR may replace the function of a T cell receptor and may in particular confer reactivity, e.g. cytolytic activity, on a cell, such as a T cell. However, in contrast to T cell receptor binding to antigenic peptide-MHC complexes, CARs can bind to antigens, especially when expressed on the cell surface.

Various methods can be used to introduce the CAR construct into T cells, 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 that do not contain an integration element. Viral-based systems include the use of gamma-retroviruses and lentiviral vectors. Gamma-retroviruses are relatively easy to produce, transduce T cells efficiently and permanently, and have been initially proven safe from the standpoint of integrating primary human T cells. Lentiviral vectors are also capable of transducing T cells efficiently and permanently, but at a higher cost of manufacture. They may also be safer than retrovirus-based systems.

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

CAR T cells can be generated in vivo, and thus are nearly transient using T cell-targeting nanoparticles. For example, poly (β -amino ester) -based nanoparticles can be coupled with anti-CD 3e f (ab) fragments to bind CD3 on T cells. For this purpose, the anti-CD 3e f (ab) fragment may be covalently linked to polyglutamic acid (PGA). PGA surrounds a core of particles comprising nucleic acids and excess poly (β -amino ester) (PBAE) polymers and is attached thereto by charge interaction. Upon binding to T cells, these nanoparticles are endocytosed. The inclusion of peptides containing microtubule associated sequences (MTAS) and Nuclear Localization Signals (NLS) covalently linked to PBAE polymers, such as plasmid DNA encoding the anti-tumor antigen CAR, can target the T cell nucleus. The inclusion of a transposon flanking the CAR gene expression cassette and a separate plasmid encoding a highly active transposase may allow for efficient integration of the CAR vector into the chromosome. Such systems that allow the production of CAR T cells in vivo following nanoparticle infusion are described in Smith et al (2017) nat. nanotechnol.12: 813-820.

Another possibility is to deliberately place the CAR coding sequence at a specific locus using the CRISPR/Cas9 method. For example, an existing T Cell Receptor (TCR) can be knocked-in and the CAR placed under the dynamic regulatory control of an endogenous promoter, otherwise expression of the TCR would be attenuated; see, e.g., Eyquem et al (2017) Nature543: 113-.

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

In one embodiment of all aspects of the invention, the T cells or T cell progenitors are from the subject to be treated. In one embodiment of all aspects of the invention, the T cells or T cell progenitors are from a different subject than the subject to be treated.

In one embodiment of all aspects of the invention, the T cells may be autologous, allogeneic or syngeneic to the subject to be treated. T cells can be genetically modified in vitro to express Chimeric Antigen Receptors (CARs) that target antigens.

In one embodiment of all aspects of the invention, expression of an endogenous T cell receptor and/or an endogenous HLA of a T cell genetically modified to express the CAR is inactivated.

The term "autologous" is used to describe any substance derived from the same subject. For example, "autograft" refers to the transplantation of a tissue or organ from the same subject. Such procedures are advantageous because they overcome the immune barrier that can lead to rejection.

The term "allogeneic" is used to describe any substance from different individuals of the same species. When the genes at one or more loci are not identical, two or more individuals are said to be allogeneic with respect to each other.

The term "syngeneic" is used to describe any substance derived from individuals or tissues having the same genotype, i.e., the same inbred strain of the same egg twin or animal, or tissue thereof.

The term "heterologous" is used to describe a substance that is composed of a plurality of different elements. For example, transplantation of the bone marrow of one individual to a different individual constitutes allografting. A heterologous gene is a gene from a source other than the subject.

RNA

The term "polynucleotide" or "nucleic acid" as used herein is intended to include DNA and RNA, e.g., genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules. The nucleic acid may be single-stranded or double-stranded. RNA includes in vitro transcribed RNA, (IVT RNA) or synthetic RNA. According to the invention, the polynucleotide is preferably isolated.

The nucleic acid may be contained in a vector. The term "vector" as used herein includes any vector known to those skilled in the art, including a plasmid vector, a cosmid vector, a phage (e.g., lambda phage) vector, a viral vector (e.g., an adenovirus or baculovirus vector), or an artificial chromosome vector (e.g., a Bacterial Artificial Chromosome (BAC), a Yeast Artificial Chromosome (YAC), or a P1 Artificial Chromosome (PAC)). The vector includes an expression vector and a cloning vector. Expression vectors include plasmids as well as viral vectors, and typically contain the desired coding sequence and appropriate DNA sequences required for expression of an operably linked coding sequence in a particular host organism (e.g., bacteria, yeast, plant, insect, or mammalian) or in an in vitro expression system. Cloning vectors are commonly used to engineer and amplify a particular desired DNA fragment, and may lack the functional sequences required to express the desired DNA fragment.

In one embodiment of all aspects of the invention, the nucleic acid encoding the cytokine or encoding the antigen or variant thereof is expressed in cells of the subject being treated to provide the cytokine or antigen or variant thereof. In one embodiment of all aspects of the invention, the nucleic acid is transiently expressed in the mammalian cell. Thus, in one embodiment, the nucleic acid is not integrated into the genome of the cell. In one embodiment of all aspects of the invention, the nucleic acid is RNA, preferably in vitro transcribed RNA. In one embodiment of all aspects of the invention, the antigen or variant thereof is expressed on the surface of the cell.

In one embodiment of all aspects of the invention, the nucleic acid encoding the antigen or variant thereof is expressed in a mammalian cell to provide an antigen or variant thereof that is bound by a T cell genetically modified to express the CAR, said binding resulting in stimulation, priming and/or expansion of the T cell genetically modified to express the CAR.

According to the present invention, the term "expression" is used in its most general sense and includes the production of RNA and/or peptides or proteins, e.g. by transcription and/or translation. Expression may be transient or stable. According to the present invention, the term expression also includes "abnormal expression" or "abnormal expression".

According to the present invention, the term "nucleic acid encoding" refers to a nucleic acid, if present in a suitable environment, such as a cell, that can be expressed to produce the protein or peptide that it encodes.

The nucleic acids described herein may be recombinant and/or isolated molecules.

As used herein, "isolated molecule" refers to a molecule that is substantially free of other molecules, such as other cellular material.

The term "recombinant" in the context of the present invention means "prepared by genetic engineering". Preferably, a "recombinant subject" such as a recombinant cell in the context of the present invention is not naturally occurring.

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

The term "transfection" refers 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 uptake of nucleic acids by such cells, wherein the cells may be present in a subject, e.g., a patient. Thus, according to the present invention, the cells used to transfect the nucleic acids described herein may be present in vitro or in vivo, e.g., the cells may form part of an organ, tissue and/or organism of a patient. According to the invention, transfection may be transient or stable. For certain transfection applications, only transient expression of the transfected genetic material may be sufficient. Since the nucleic acid introduced during transfection is not normally integrated into the nuclear genome, the exogenous nucleic acid is diluted or degraded by mitosis. Cells that allow for free amplification of nucleic acids greatly reduce the dilution rate. If it is desired that the transfected nucleic acid actually remains in the genome of the cell and its daughter cells, stable transfection must be performed. The RNA can be transfected into cells to transiently express the protein it encodes.

In one embodiment of all aspects of the invention, the nucleic acid encoding the cytokine or encoding the antigen or variant thereof is formulated in a delivery vehicle (e.g., a particle). In one embodiment, the delivery vehicle comprises at least one lipid. In one embodiment, the at least one lipid comprises at least one cationic lipid. In one embodiment, the lipid forms a complex with and/or encapsulates the nucleic acid. In one embodiment, the lipid is contained in a vesicle encapsulating the nucleic acid. In one embodiment of all aspects of the invention, the nucleic acid is formulated in a liposome.

In the present disclosure, the term "RNA" relates to a nucleic acid molecule comprising ribonucleotide residues. In a preferred embodiment, the RNA contains all or most 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, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides. These changes may refer to the addition of non-nucleotide material to internal RNA nucleotides or to the ends of RNA. It is also contemplated herein that the nucleotides in the RNA can be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For the purposes of this disclosure, these altered RNAs are considered analogs of naturally occurring RNAs.

In certain embodiments of the present disclosure, the RNA is messenger RNA (mrna) associated with an RNA transcript encoding a peptide or protein. As established in the art, mRNAs typically comprise a5 'untranslated region (5' -UTR), a peptide coding region, and a3 'untranslated region (3' -UTR). In some embodiments, the RNA is produced by in vitro transcription or chemical synthesis. In one embodiment, mRNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid containing deoxyribonucleotides.

In one embodiment, the RNA is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription of an appropriate DNA template. The promoter controlling transcription may be any promoter of any RNA polymerase. DNA templates for in vitro transcription can be obtained by cloning nucleic acids, in particular cDNA, and introducing them into suitable vectors for in vitro transcription. cDNA can be obtained by reverse transcription of RNA.

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

In some embodiments, the RNA of the present disclosure comprises a 5' -cap. In one embodiment, the RNA of the present disclosure does not have an uncapped 5' -triphosphate. In one embodiment, the RNA may be modified with a 5' -cap analog. The term "5 '-cap" refers to the structure found at the 5' end of an mRNA molecule and typically consists of a guanosine nucleotide linked to the mRNA by a5 'to 5' triphosphate linkage. In one embodiment, the guanosine is methylated at position 7. Providing RNA with a 5' -cap or 5' -cap analog can be achieved by in vitro transcription, where the 5' -cap is co-transcribed into the RNA strand, or can be post-transcribed to the RNA using a capping enzyme.

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

In some embodiments, the RNA of the present disclosure comprises a 3' -poly (a) sequence. The term "poly (a) sequence" relates to a sequence of adenylate (a) residues, which is typically located at the 3' -end of an RNA molecule. In accordance with 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.

In the context of the present disclosure, the term "transcription" relates to the process of transcribing a genetic code in a DNA sequence into RNA. Subsequently, the RNA can be translated into a peptide or protein.

With respect to RNA, the terms "expression" or "translation" relate to the process in the nuclear sugar body by which an mRNA strand directs the assembly of amino acid sequences to make peptides or proteins.

According to the present disclosure, the term "RNA-encoded" refers to RNA, if present in a suitable environment, such as within a cell of a target tissue, that can direct the assembly of amino acids to produce the peptide or protein that it encodes during translation. In one embodiment, the RNA is capable of interacting with cellular translation machinery, allowing translation of peptides or proteins. The encoded peptide or protein may be produced intracellularly (e.g., in the cytoplasm and/or nucleus), may be secreted, or may be produced on the surface by the cell.

The terms "linked," "fused," or "fused" are used interchangeably herein. These terms refer to the joining together of two or more elements or components or domains.

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. PK-extending cytokines suitable for use herein, e.g., PK-extending Interleukins (IL), are stable in vivo and have their half-lives increased, e.g., by fusion with serum albumin (e.g., HSA or MSA), which is resistant to degradation and/or clearance or sequestration. The half-life can be determined in any manner known per se, for example by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art and may for example generally comprise the following steps: suitably administering to the subject a suitable dose of the amino acid sequence or compound; collecting blood or other samples from a subject at regular intervals; determining the level or concentration of an amino acid sequence or compound in the blood sample; and calculating from the (plot of) data thus obtained the time until the level or concentration of the amino acid sequence or compound is reduced by 50% compared to the initial level at the time of dosing. Further details are provided, for example, in standard manuals, such as Kenneth, A.et al, Chemical Stability of Pharmaceuticals: A Handbook for Pharmaceuticals and in Peters et al, pharmaceutical Analysis: A Practical Approach (1996). See also Gibaldi, m.et al, Pharmacokinetics,2nd rev.edition, Marcel Dekker (1982).

Cytokine

Cytokines are a small class of proteins (5-20 kDa) important in cell signaling. Their release affects the behavior of the cells surrounding 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 the terms overlap somewhat). Cytokines are produced by a 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 may be produced by more than one type of cell. Cytokines act through receptors and are particularly important in the immune system; cytokines regulate the balance between humoral and cellular immune responses, and they regulate the maturation, growth and reactivity of specific cell populations. Some cytokines enhance or inhibit the action of other cytokines in a complex manner.

IL2

Interleukin 2(IL2) is a cytokine that induces antigen-activated T cell proliferation and stimulates Natural Killer (NK) cells. The biological activity of IL2 is mediated by the multi-subunit IL2 receptor complex (IL2R) spanning three polypeptide subunits of the cell membrane: p55(IL2R α, α subunit, also known in humans as CD25), p75(IL2R β, β subunit, also known in humans as CD122) and p64(IL2R γ, γ subunit, also known in humans as CD 132). The response of T cells to IL2 depends on various factors including: (1) the concentration of IL 2; (2) the amount of cell surface IL2R molecules; (3) IL2 occupies an amount of IL2R (i.e., the affinity of the binding interaction between IL2 and IL2R (Smith, "Cell Growth Signal Transduction is quantitative" In Receptor Activation by antibodies, Cytokines, hormons, and Growth Factors 766:263-271, 1995)). IL2 IL2R complex is internalized upon ligand binding and the different components are differentially sorted. When administered as an intravenous (i.v.) bolus, IL2 has rapid systemic clearance (12.9 minutes half-life for the initial clearance phase followed by 85 minutes half-life for the slower clearance phase) (Konrad et al, Cancer Res.50: 2009-.

The results of systemic IL2 administration in cancer patients are far from ideal. Although 15-20% of patients respond objectively to high doses of IL2, most patients do not respond and many patients experience serious, life-threatening side effects including nausea, confusion, hypotension, and septic shock. The severe toxicity associated with high dose IL2 treatment was mainly attributed to the activity of Natural Killer (NK) cells. Attempts have been made to reduce serum concentrations by reducing the dose and adjusting the dosing regimen, and although less toxic, such treatments have also been less effective.

In certain embodiments, IL2 is linked to a pharmacokinetic modifying group in accordance with the present disclosure. The resulting molecule, hereinafter referred to as "extended Pharmacokinetic (PK) IL 2", has an extended circulating half-life relative to free IL 2. The extended circulating half-life of PK-extended IL2 allows serum IL2 concentrations in vivo to be maintained within a therapeutic range, potentially leading to enhanced activation of many types of immune cells, including T cells. Due to its favorable pharmacokinetic profile, the dosing frequency of IL2 with extended PK can be lower and last longer compared to unmodified IL 2.

According to the present disclosure, IL2 (optionally as part of PK-extending IL2) may be naturally occurring IL2 or a fragment or variant thereof. IL2 may be human IL2 and may be derived from any vertebrate, in particular 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, the IL2 or IL2 fragment or variant binds to a subunit of the IL2 receptor or IL2 receptor, such as the alpha subunit and/or the beta/gamma subunit.

In certain embodiments, the IL2 portion of PK-extended IL2 is human IL 2. In other embodiments, the IL2 portion of IL2 that extends PK is a fragment or variant of human IL 2.

In certain embodiments described herein, IL2 is fused to a heterologous polypeptide (i.e., a polypeptide that is not IL 2). The heterologous polypeptide may increase the circulating half-life of IL 2. As discussed in further detail below, the polypeptide that increases circulating half-life can be serum albumin, such as human (e.g., SEQ ID NO:4) or mouse (e.g., SEQ ID NO:8, 11) serum albumin.

IL7

IL7 is a hematopoietic growth factor secreted by stromal cells in 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 a cytokine important for B and T cell development. The IL7 cytokine and hepatocyte growth factor form heterodimers, which act as pro-pro (pre-pro) B cell growth stimulators. Mouse gene knockout studies have shown that IL7 plays an important role in lymphoid cell survival.

IL7 binds to the IL7 receptor, a heterodimer consisting of the IL7 receptor alpha and the common gamma chain receptor. Binding results in a signaling cascade important for T cell intra-thymic development and peripheral survival. Knockout mice genetically lacking the IL7 receptor exhibit thymus atrophy, arrest of T cell development in the double positive phase and severe lymphopenia. Administration of IL7 to mice resulted in an increase in recent thymus emigration cells, an increase in B cells and T cells, and an increase in recovery of T cells after administration of cyclophosphamide or after bone marrow transplantation.

In certain embodiments, IL7 is linked to a pharmacokinetic modifying group in accordance with the present disclosure. The resulting molecule, hereinafter referred to as "extended Pharmacokinetic (PK) IL 7", has an extended circulating half-life relative to free IL 7. The extended circulating half-life of PK-extending IL7 allows serum IL7 concentrations in vivo to be maintained within a therapeutic range, potentially leading to enhanced survival of many types of immune cells, including T cells. Due to its favorable pharmacokinetic profile, the dosing frequency of IL7 with extended PK can be lower and last longer compared to unmodified IL 7.

According to the present disclosure, IL7 (optionally as part of PK-extending IL 7) may be naturally occurring IL7 or a fragment or variant thereof. IL7 may be human IL7 and may be derived from any vertebrate, in particular any mammal. In one embodiment, IL7 comprises 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, the IL7 or IL7 fragment or variant binds to the IL7 receptor.

In certain embodiments, the IL7 portion of PK-extended IL7 is human IL 7. In other embodiments, the IL7 portion of IL7 that extends PK is a fragment or variant of human IL 7.

In certain embodiments described herein, IL7 is fused to a heterologous polypeptide (i.e., a polypeptide that is not IL 7). The heterologous polypeptide may increase the circulating half-life of IL 7. As discussed in further detail below, the polypeptide that increases circulating half-life can be serum albumin, such as human (e.g., SEQ ID NO:4) or mouse (e.g., SEQ ID NO:8, 11) serum albumin.

IL21

Interleukin-21 (IL21) is a cytokine that has potent regulatory effects 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. In addition, IL21 expression is up-regulated in the Th2 and Th17 subsets of T helper cells as well as T follicular cells. In addition, IL21 in NK T cells in expression, regulation of these cells function. Interleukin 21 is also produced by Hodgkin Lymphoma (HL) cancer cells.

IL21 receptor (IL21R) is expressed on the surface of T, B and NK cells. IL21R is structurally similar to receptors for other type I cytokines (e.g., IL2 or IL-15), and requires dimerization with a common gamma chain (yc) to bind IL-21. When bound to IL21, the IL21 receptor acts through the Jak/STAT pathway, using Jak1 and Jak3 and STAT3 homodimers to activate its target genes.

In certain embodiments, IL21 is linked to a pharmacokinetic modifying group in accordance with the present disclosure. The resulting molecule, hereinafter referred to as "extended Pharmacokinetic (PK) IL 21", has an extended circulating half-life relative to free IL 21. The extended circulating half-life of PK-extended IL21 allows serum IL21 concentrations in vivo to be maintained within a therapeutic range, potentially leading to enhanced activation of many types of immune cells, including T cells. Due to its favorable pharmacokinetic profile, prolonged PK of IL21 may be administered less frequently and for a longer duration than unmodified IL 21.

According to the present disclosure, IL21 (optionally as part of PK-extending IL21) may be naturally occurring IL21 or a fragment or variant thereof. IL21 may be human IL21 and may be derived from any vertebrate, in particular any mammal. In one embodiment, IL21 comprises 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, the IL21 or IL21 fragment or variant binds to the IL21 receptor.

In certain embodiments, the IL21 portion of PK-extended IL21 is human IL 21. In other embodiments, the IL21 portion of IL21 that extends PK is a fragment or variant of human IL 21.

In certain embodiments described herein, IL21 is fused to a heterologous polypeptide (i.e., a polypeptide that is not IL 21). The heterologous polypeptide may increase the circulating half-life of IL 21. As discussed in further detail below, the polypeptide that increases circulating half-life can be serum albumin, such as human (e.g., SEQ ID NO:4) or mouse (e.g., SEQ ID NO:8, 11) serum albumin.

PK-extending groups

Cytokines, e.g., interleukins described herein, such as IL2, IL7, or IL21, may be fused to the PK extending group, thereby increasing circulating half-life. Non-limiting examples of groups that extend PK are described below. It is understood that other PK groups that increase the circulating half-life of cytokines or variants thereof are also suitable for use in the present disclosure. In certain embodiments, the PK-extending group is a serum albumin domain (e.g., mouse serum albumin, human serum albumin).

The term "PK" as used herein is an acronym for "pharmacokinetics" and encompasses the properties of a compound, including, for example, absorption, distribution, metabolism, and elimination in a subject. As used herein, a "PK-extending group" refers to a protein, peptide or moiety that increases the circulating half-life of a biologically active molecule when fused to or administered with the biologically active molecule. Examples of PK extending groups include serum albumin (e.g., HSA), Fc or Fc fragments and variants thereof, transferrin and variants thereof, and Human Serum Albumin (HSA) binders (as disclosed in U.S. publication nos. 2005/0287153 and 2007/0003549). Other exemplary PK extending groups 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, "PK-extending cytokine" refers to a cytokine moiety combined with a PK-extending moiety. In one embodiment, the PK-extending cytokine is a fusion protein, wherein the cytokine moiety is linked or fused to a PK-extending moiety. As used herein, "PK-extended IL" refers to an Interleukin (IL) moiety in combination with a PK-extending group. In one embodiment, the PK-extended IL is a fusion protein, wherein the IL moiety is linked or fused to the PK-extending group. An exemplary fusion protein is the HSA/IL2 fusion protein, wherein the IL2 portion is fused to HSA. Another exemplary fusion protein is an HSA/IL7 fusion protein, wherein the IL7 portion is fused to HSA. Another exemplary fusion protein is an HSA/IL21 fusion protein in which the IL21 moiety is fused to HSA.

In certain embodiments, the serum half-life of the PK-extending cytokine is increased relative to the cytokine alone (i.e., the cytokine is not fused to the PK-extending moiety). In certain embodiments, the serum half-life of the PK-extending cytokine is at least 20, 40, 60, 80, 100, 120, 150, 180, 200, 400, 600, 800, or 1000% longer relative to the serum half-life of the cytokine alone. In certain embodiments, the serum half-life of the PK-extending 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 of the serum half-life of the cytokine alone. In certain embodiments, the PK-extending cytokine has a serum half-life of 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.

In certain embodiments, the PK-extending group comprises serum albumin or a fragment thereof, or a variant of serum albumin or a fragment thereof (all of which are encompassed by the term "albumin" for purposes of this disclosure). The polypeptides described herein may be fused to albumin (or fragments or variants thereof) to form an albumin fusion protein. Such albumin fusion proteins are described in U.S. publication No. 20070048282.

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 protein, such as a therapeutic protein, in particular IL2, IL7, or IL21 (or a fragment or variant thereof). Albumin fusion proteins can be produced by translation of a nucleic acid in which a polynucleotide encoding a therapeutic protein is linked in-frame to a polynucleotide encoding albumin. The therapeutic protein and albumin, once part of the albumin fusion protein, may each be referred to as a "portion," "region," or "portion" of the albumin fusion protein (e.g., "therapeutic protein portion" or "albumin portion"). In a particularly 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 a host cell, e.g., a cell of a target organ to which RNA is administered (e.g., a hepatocyte), and secreted into the circulation. Processing of nascent albumin fusion proteins that occurs in the secretory pathway of host cells for expression of RNA can include, but is not limited to, signal peptide cleavage; disulfide bond formation; folding correctly; carbohydrate addition and processing (e.g., N-and O-linked glycosylation); specific proteolytic cleavage; and/or assembly into a multimeric protein. The albumin fusion protein is preferably encoded by RNA in unprocessed form, which has a signal peptide, in particular at its N-terminus, and is preferably present in processed form after secretion by the cell, wherein in particular the signal peptide has been cleaved off. In a most preferred embodiment, "processed form of an albumin fusion protein" refers to an albumin fusion protein product that undergoes N-terminal signal peptide cleavage, also referred to herein as a "mature albumin fusion protein".

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

As used herein, "albumin" refers collectively to albumin or an amino acid sequence, or a fragment or variant of albumin, having one or more functional activities (e.g., biological activities) of albumin. In particular, "albumin" refers to human albumin or fragments or variants thereof, especially the mature form of human albumin, or albumin or fragments thereof from other vertebrates, or variants of these molecules. The albumin may be from any vertebrate, in particular any mammal, such as a human, bovine, ovine or porcine. Non-mammalian albumins include, but are not limited to, chicken and salmon. The albumin portion of the albumin fusion protein can be from a different animal than the therapeutic protein portion.

In certain embodiments, the albumin is Human Serum Albumin (HSA), or a fragment or variant thereof, such as those disclosed below: US 5,876,969, WO 2011/124718, WO 2013/075066, and WO 2011/0514789.

The terms Human Serum Albumin (HSA) and Human Albumin (HA) are used interchangeably herein. The terms "albumin" and "serum albumin" are broader and encompass human serum albumin (and fragments and variants thereof) as well as albumins of other species (and fragments and variants thereof).

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

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

Generally, the length of the albumin fragment or variant is at least 100 amino acids, preferably at least 150 amino acids.

In accordance with the present disclosure, the albumin may be a naturally occurring albumin or a fragment or variant thereof. The albumin may be human albumin and may be derived from any vertebrate, in particular any mammal. In one embodiment, 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.

Preferably, the albumin fusion protein comprises albumin as the N-terminal portion and a therapeutic protein as the C-terminal portion. Alternatively, albumin fusion proteins comprising albumin as the C-terminal portion and a therapeutic protein as the N-terminal portion may also be used. In other embodiments, the albumin fusion protein has a therapeutic protein fused to the N-terminus and C-terminus of albumin. In a preferred embodiment, the therapeutic protein fused at the N and C termini is the same therapeutic protein. In another preferred embodiment, the therapeutic protein fused at the N and C termini is a different therapeutic protein. In one embodiment, different therapeutic proteins may be used to treat or prevent the same or related diseases, disorders, or conditions. In one embodiment, the different therapeutic proteins are all cytokines.

In one embodiment, the therapeutic protein is linked to the albumin through a peptide linker. Linker peptides between fusion moieties may provide greater physical separation between the moieties, thereby maximizing accessibility of the therapeutic protein moiety, for example, for binding to its cognate receptor. The linker peptide may be composed of amino acids, making it flexible or more rigid. The linker sequence may be cleaved by proteases or chemically.

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

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

In certain embodiments, the PK-extending group comprises an Fc domain or fragment thereof or a variant of an Fc domain or fragment thereof (all of which are encompassed by the term "Fc domain" for the purposes of this disclosure). The Fc domain does not contain a variable region that binds to an antigen. Suitable Fc domains for use in the present disclosure may be obtained from a number of 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 to be understood that the Fc domain may be derived from another mammalian species, including, for example, an immunoglobulin of a rodent (e.g., mouse, rat, rabbit, guinea pig) or non-human primate (e.g., chimpanzee, macaque) species.

Furthermore, the Fc domain (or fragment or variant thereof) may be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG 4.

Various Fc domain gene sequences (e.g., mouse and human constant region gene sequences) are available in publicly available deposits. The constant region domain comprising an Fc domain sequence may be selected that lacks a particular effector function and/or has a particular modification that reduces immunogenicity. Many antibodies and sequences of antibody-encoding genes have been disclosed, 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 art-recognized techniques.

In certain embodiments, the PK-extending group is a serum albumin binding protein, such as those disclosed in: US2005/0287153, US2007/0003549, US2007/0178082, US2007/0269422, US2010/0113339, WO2009/083804 and WO2009/133208, which are incorporated herein by reference in their entirety. In certain embodiments, the PK-extending group is transferrin, such as those disclosed in US 7,176,278 and US 8,158,579 (incorporated herein by reference in their entirety). In certain embodiments, the PK-extending group is a serum immunoglobulin-binding protein, such as those disclosed in US2007/0178082 (incorporated herein by reference in its entirety). In certain embodiments, the PK extending group is a fibronectin (Fn) -based scaffold domain protein that binds serum albumin, such as those disclosed in US2012/0094909 (incorporated herein by reference in its entirety). Methods of making fibronectin based scaffold domain proteins are also disclosed in US 2012/0094909. A non-limiting example of a PK extending group based on Fn3 is Fn3(HSA), i.e., the Fn3 protein that binds human serum albumin.

In certain aspects, PK-extending cytokines, e.g., PK-extending IL, suitable for use according to the present disclosure may employ one or more peptide linkers. The term "peptide linker" as used herein refers to a peptide or polypeptide sequence that connects two or more domains (e.g., a PK-extending portion and an IL portion, such as IL2, IL7, or IL21) in a linear amino acid sequence of a polypeptide chain. For example, a peptide linker may be used to link the IL2 portion to the HSA domain. In another embodiment, a peptide linker may be used to link the IL7 portion to the HSA domain. In another embodiment, a peptide linker may be used to link the IL21 portion to the HSA domain.

Linkers suitable for fusing PK extending groups to e.g. 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.

Antigens

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

Peptide and protein antigens, particularly RNA, i.e., vaccine antigens, administered or encoded by nucleic acids, preferably result in stimulation, priming and/or expansion of T cells genetically modified to express the CAR in a subject administered the peptide or protein antigen or nucleic acid. The stimulated, primed and/or expanded T cells are preferably directed against a target antigen, in particular a target antigen expressed by diseased cells, tissues and/or organs, i.e. a disease-associated antigen. Thus, a vaccine antigen may comprise a disease-associated antigen, or a fragment or variant thereof. In one embodiment, such a fragment or variant is immunologically equivalent to a disease-associated antigen. In the context of the present disclosure, the term "fragment of an antigen" or "variant of an antigen" refers to a substance that causes stimulation, priming and/or expansion of CAR-engineered T cells, which target the antigen, i.e., a disease-associated antigen, particularly when presented by a diseased cell, tissue and/or organ. Thus, a vaccine antigen may correspond to or may comprise a disease-associated antigen, may correspond to or may comprise a fragment of a disease-associated antigen, or may correspond to or may comprise an antigen that is homologous to a disease-associated antigen or a fragment thereof. If the vaccine antigen comprises a fragment of a disease-associated antigen or an amino acid sequence that is homologous to a fragment of a disease-associated antigen, the fragment or amino acid sequence may comprise an epitope of the disease-associated antigen to which the CAR of the CAR-engineered T cell is targeted, or a sequence that is homologous to an epitope of the disease-associated antigen. Thus, according to the present disclosure, a vaccine antigen can comprise an immunogenic fragment of a disease-associated antigen or an amino acid sequence that is homologous to an immunogenic fragment of a disease-associated antigen. An "immunogenic fragment of an antigen" of the present disclosure preferably relates to an antigenic fragment capable of stimulating, priming and/or expanding T cells carrying a CAR that binds to the antigen or antigen-expressing cells. Preferably, vaccine antigens (similar to disease-associated antigens) can be expressed on the surface of cells (e.g., antigen presenting cells) to provide relevant epitopes that are bound by CAR-engineered T cells. The vaccine antigen may be a recombinant antigen.

The term "immunologically equivalent" means an immunologically equivalent molecule, e.g., an immunologically equivalent amino acid sequence, that exhibits the same or substantially the same immunological properties and/or exerts the same or substantially the same immunological effect, e.g., with respect to the type of immunological effect. In the context of the present disclosure, the term "immunological equivalence" is preferably used with respect to the immunological effect or nature of the antigen or antigen variant used for immunization. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if it induces an immune response having specificity for reacting with the reference amino acid sequence when exposed to the immune system of the subject, e.g., a T cell that binds to the reference amino acid sequence or a cell that expresses the reference amino acid sequence. Thus, a molecule immunologically equivalent to an antigen exhibits the same or substantially the same properties and/or performs the same or substantially the same function as the antigen targeted by the T cell with respect to T cell stimulation, priming and/or expansion.

The term "priming" refers to the process by which a T cell first contacts its specific antigen and results in differentiation into effector T cells.

The term "clonal amplification" or "amplification" refers to a process in which a particular entity is multiplied. In the context of the present disclosure, the term is preferably used in the context of an immune response, wherein lymphocytes are stimulated, proliferated by an antigen, and specific lymphocytes recognizing the antigen are expanded. Preferably, clonal expansion results in differentiation of lymphocytes.

The term "antigen" relates to a substance comprising an epitope against which an immune response can be generated. The term "antigen" includes, inter alia, proteins and peptides. In one embodiment, the antigen is present on the surface of a cell of the immune system, e.g., the surface of an antigen presenting cell, such as a dendritic cell or macrophage. In one embodiment, the antigen or a processed product thereof (e.g., a T cell epitope) is bound by a CAR molecule. Therefore, the antigen or its processed product can specifically react with T lymphocytes (T cells). In one embodiment, the antigen is a disease-associated antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, and the epitope is derived from such an antigen.

The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule comprising an epitope that stimulates the immune system of the host to generate a cellular antigen-specific immune response and/or a humoral antibody response against the disease. Thus, the disease-associated antigen or epitope thereof may be used for therapeutic purposes. The disease-associated antigen may be associated with a microbial infection, typically a microbial antigen, or with a cancer, typically a tumour.

The term "tumor antigen" refers to a component of a cancer cell, which may be derived from the cytoplasm, cell surface and nucleus. In particular, it refers to those antigens that are produced intracellularly or as tumor cell surface antigens. Tumor antigens are typically preferentially expressed by cancer cells (e.g., they are expressed at higher levels in cancer cells than non-cancer cells), and in some cases, they are expressed only by cancer cells. Examples of tumor antigens include, but are not limited to, p53, ART-4, BAGE, beta-catenin/m, Bcr-abL CAMEL, CAP-1, CASP-8, CDC27/m, CDK4/m, CEA, cell surface proteins of the tight-junction protein family, such as CLAUDI N-6, CLAUDIN-18.2 and CLAUDIN-12, c-MYC, CT, Cyp-B, DAM, ELF2 HASE 3, ETV6-AML1, G250, GAGE, GnT V, Gap, HAGE, HER-2/neu, HPV-E7, HPV-E6, MAG-2, hTERT (or hTERT), LAGE, MAGE LR/FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-5838, MAGE 3926, MAGE-3946, MAGE-3958, MAGE-A, MAGE-11, MAGE-A-3, MAGE-A, MAGE-3, MAGE-A, MAGE-3, MAGE-3, MAGE-3, MAGE-3, MAGE, MAGE-A11 or MAGE-A12, MAGE-B, MAGE-C, MART-1/Melan-A, MC1R, myosin/m, MUC1, MUM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, pl90 Small BCR-abL, Pml/RARa, PRAME, protease 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/INT2, TPTE, WT, and WT-1.

The term "viral antigen" refers to any viral component having antigenic properties (i.e., capable of eliciting an immune response in an individual). The viral antigen may be a viral ribonucleoprotein or an envelope protein.

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

The term "epitope" refers to a portion or fragment of a molecule (e.g., an antigen) that is recognized by the immune system. For example, the epitope may be recognized by a T cell, B cell, or antibody. An epitope of an antigen may comprise a continuous or discontinuous portion of the antigen and may be from about 5 to about 100 amino acids in length, for example from about 5 to about 50, more preferably from about 8 to about 30, most preferably from about 10 to about 25 amino acids, e.g. an epitope may 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.

The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in the context of MHC molecules. The term "major histocompatibility complex" and the abbreviation "MHC" include both MHC class I and MHC class II molecules and refer to the gene complex present in all vertebrates. MHC proteins or molecules are important for signaling between lymphocytes and antigen presenting or diseased cells in an immune response, where they bind peptide epitopes and present them for recognition by T cell receptors on T cells. MHC-encoded proteins are expressed on the cell surface and display 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 MHC class I/peptide complexes, the binding peptides are typically about 8 to about 10 amino acids in length, although longer or shorter peptides may be effective. In the case of MHC class II/peptide complexes, the length of the bound peptide is typically from about 10 to about 25 amino acids, particularly from about 13 to about 18 amino acids, although longer and shorter peptides may be effective.

In one embodiment, the target antigen is a tumor antigen, and the vaccine antigen or fragment thereof (e.g., epitope) is derived from the tumor antigen. The tumor antigen may be a "standard" antigen, which is generally known to be expressed in various cancers. Tumor antigens may also be "neoantigens" that are specific to an individual's tumor and have not been previously recognized by the immune system. The neoantigen or neoepitope may be caused by one or more cancer-specific mutations in the genome of the cancer cell, resulting in amino acid changes. If the tumor antigen is a neoantigen, the vaccine antigen preferably comprises an epitope or fragment of said neoantigen, said neoantigen comprising one or more amino acid changes.

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

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

According to various aspects of the present invention, it is preferred that the object is to provide an immune response against cancer cells expressing a tumor antigen (e.g. CLDN6 or CLDN18.2) and to treat cancer diseases involving cells expressing a tumor antigen (e.g. CLDN6 or CLDN 18). Preferably, the invention relates to the administration of CAR-engineered T cells targeted to cancer cells expressing a tumor antigen (e.g. CLDN6 or CLDN 18.2).

"cell surface" is used according to its usual meaning in the art and thus includes the exterior of a cell which is susceptible to 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 can be brought into close binding by, for example, an antigen-specific antibody added to the cell. In one embodiment, the antigen expressed on the cell surface is an integral membrane protein, having an extracellular portion that is recognized by the CAR.

In the context of the present invention, the term "extracellular portion" or "ectodomain" refers to a portion of a molecule (e.g. a protein) that faces the extracellular space of a cell and is preferably accessible from the outside of said cell, e.g. by a binding molecule, such as an antibody, located outside the cell. Preferably, the term refers to one or more extracellular loops or domains or fragments thereof.

In one embodiment of all aspects of the invention, the antigen is expressed in a diseased cell (e.g., a cancer cell). In one embodiment, the antigen is expressed on the surface of a diseased cell (e.g., a cancer cell). In one embodiment, the CAR binds to an epitope in the extracellular domain or extracellular domain of the antigen or variant thereof. In one embodiment, the CAR binds to a native epitope of an antigen or variant thereof present on the surface of a living cell. In some embodiments, the antigen is a tight junction protein, particularly tight junction protein 6 or tight junction protein 18.2, and the CAR binds to the first extracellular loop of the tight junction protein. In one embodiment, when expressed by and/or present on a T cell, binding of the CAR to an antigen or variant thereof present on a cell, e.g., an antigen presenting cell, results in stimulation, priming and/or expansion of the T cell. In one embodiment, binding of the CAR to an antigen present on a diseased cell (e.g., a cancer cell), when expressed by and/or present on a T cell, results in cytolysis and/or apoptosis of the diseased cell, wherein the T cell preferably releases cytotoxic factors such as perforin and granzyme.

Immunity check point inhibitor

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

As used herein, "immunoassay" refers to co-stimulatory and inhibitory signals that modulate the amplitude and quality of T cell receptor recognition of antigens. In certain embodiments, the immunoassay 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 an interaction between CTLA-4 and CD80 or CD86 in place of CD28 binding. 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.

As used herein, "immune checkpoint inhibitor" refers to a molecule that reduces, inhibits, interferes with, or modulates, in whole or in part, one or more checkpoint proteins. In certain embodiments, the immune checkpoint inhibitor prevents inhibitory signals associated with the immune checkpoint. In certain embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that disrupts inhibitory signaling associated with the immune checkpoint. In certain embodiments, the immune checkpoint inhibitor is a small molecule that disrupts inhibitory signaling. In certain embodiments, the immune checkpoint inhibitor is an antibody, fragment thereof, or antibody mimetic that prevents interaction between checkpoint blocking proteins, e.g., an antibody, fragment thereof, or antibody mimetic that prevents interaction between PD-1 and PD-L1. In certain embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that prevents an interaction between CTLA-4 and CD80 or CD 86. In certain embodiments, the immune checkpoint inhibitor is an antibody or fragment thereof that prevents the interaction between LAG3 and its ligand or TIM-3 and its ligand. Checkpoint inhibitors may also be soluble forms of the molecule (or variants thereof) itself, for example, soluble PD-L1 or PD-L1 fusion proteins.

The "programmed death-1 (PD-1)" receptor refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is expressed predominantly 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 variants, isoforms, and species homologs of human PD-1(hPD-1), hPD-1, and analogs having at least one common epitope with hPD-1.

"programmed death ligand 1 (PD-L1)" is one of two cell surface glycoprotein ligands of PD-1 (the other is PD-L2), which upon binding to PD-1 down-regulates T cell activation and cytokine secretion. The term "PD-L1" as used herein includes variants, isoforms and species homologs of human PD-L1(hPD-L1), hPD-L1, and analogs having at least one common epitope with hPD-L1.

"cytotoxic T lymphocyte-associated antigen-4 (CTLA-4)" is a T cell surface molecule and is a member of the immunoglobulin superfamily. This protein down regulates the immune system by binding to CD80 and CD 86. The term "CTLA-4" as used herein includes human CTLA-4(hCTLA-4), variants, isoforms (isofom) and species homologs of hCTLA-4, and analogs having at least one common epitope with hCTLA-4.

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

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

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

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, e.g., PD-1, PD-L1, CTLA-4, LAG3, B7-H3, B7-H4, or TIM 3. These ligands and receptors are reviewed in Pardol, D.Nature.12: 252-one 264,2012.

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

In certain embodiments, the inhibitory immunomodulator is a component of the PD-1/PD-L1 signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administering to a subject an antibody, or antigen-binding portion thereof, that disrupts the interaction between the PD-1 receptor and its ligand PD-L1. Antibodies that bind to PD-1 and disrupt the interaction between PD-1 and its ligand PD-L1 are known in the art. In certain embodiments, the antibody, or 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.

In certain embodiments, the inhibitory immunomodulatory agent is a component of the CTLA4 signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administering to a subject an antibody or antigen-binding portion thereof that targets CTLA4 and disrupts its interaction with CD80 and CD 86.

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

In certain embodiments, the inhibitory immunomodulatory agent is a component of a 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 administering to a subject an antibody, or antigen-binding portion thereof, targeting B7-H3 or H4. The B7 family does not have any defined receptors, but these ligands are upregulated on tumor cells or tumor infiltrating cells. Preclinical mouse models suggest that blocking these ligands can enhance anti-tumor immunity.

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

It will be understood by those of ordinary skill in the art that other immune checkpoint targets may also be targeted by antagonists or antibodies, so long as the targeting results in stimulation of an immune response, e.g., an anti-tumor immune response, as reflected in, e.g., increased T cell proliferation, enhanced T cell activation, and/or increased cytokine (e.g., IFN- γ, IL2) production.

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 comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions may be further subdivided into regions of hypervariability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FRs). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The variable regions of the heavy and light chains comprise binding domains that interact with an antigen. The constant region of an antibody may 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 (Clq).

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

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

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 term refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been demonstrated that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH domains; (ii) f (ab')₂A fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bond in the hinge region; (iii) fd fragment, consisting of VH and CH domains; (iv) (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al, (1989) Nature 341:544-546) consisting of the VH domain; (vi) (vii) an isolated Complementarity Determining Region (CDR), and (vii) a combination of two or more isolated CDRs, which may optionally be joined by a synthetic linker. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by different genes, they can be joined by a synthetic linker using recombinant methods, which enables them to be prepared as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al (1988) Science 242: 423-426;and Huston et al (1988) Proc. Natl. Acad. Sci. USA 85: 5879-. Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. Further examples are binding domain immunoglobulin fusion proteins comprising (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. The binding domain polypeptide may be a heavy chain variable region or a light chain variable region. Binding domain immunoglobulin fusion proteins are further disclosed in US 2003/0118592 and US 2003/0133939. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

RNA targeting

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

Some aspects of the disclosure relate to targeted delivery of the RNAs disclosed herein (e.g., RNA encoding a cytokine and RNA encoding an antigen or variant thereof).

In one embodiment, the present disclosure relates to targeting the lymphatic system, particularly the secondary lymphoid organs, more particularly the spleen. If the RNA administered is an RNA which encodes an antigen or a variant thereof, it is particularly preferred to target the lymphatic system, in particular the secondary lymphoid organs, more particularly the spleen.

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

The "lymphatic system" is part of the circulatory system and is an important part of the immune system, including the lymphatic network that carries lymph. The lymphatic system is composed of lymphatic organs, lymphatic conduction networks, and circulating lymph. Primary or central lymphoid organs produce lymphocytes from immature progenitor cells. The thymus and bone marrow constitute the primary lymphoid organs. Secondary or peripheral lymphoid organs, including lymph nodes and spleen, maintain mature naive lymphocytes and initiate an adaptive immune response.

RNA can be delivered to the spleen by so-called lipoplex formulations, wherein the RNA is combined with liposomes comprising cationic lipids and optionally additional or helper lipids to form an injectable nanoparticle formulation. Liposomes can be obtained by injecting an ethanol solution of the lipids into water or a suitable aqueous phase. RNA lipoplex particles can be prepared by mixing liposomes with RNA. Spleen-targeted RNA lipoplex particles are described in WO 2013/143683, which is incorporated herein by reference. It has been found that RNA lipoplex particles having a net negative charge are useful for preferentially targeting spleen tissue or spleen cells, such as antigen presenting cells, in particular dendritic cells. Thus, following administration of RNA lipoplex particles, RNA accumulation and/or RNA expression occurs in the spleen. Thus, the RNA lipoplex particles of the present disclosure can be used to express RNA in the spleen. In one embodiment, no or substantially no RNA accumulation and/or RNA expression occurs in the lung and/or liver following administration of the RNA lipoplex particles. In one embodiment, RNA accumulation and/or RNA expression occurs in antigen presenting cells, e.g., professional antigen presenting cells in the spleen, following administration of the RNA lipoplex particles. Thus, the RNA lipoplex particles of the present disclosure may be used to express RNA in such antigen presenting cells. In one embodiment, the antigen presenting cell is a dendritic cell and/or macrophage.

In the context of the present disclosure, the term "RNA lipoplex particle" relates to a particle comprising lipids, in particular cationic lipids and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA lead to the complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes can generally be synthesized using cationic lipids (e.g., DOTMA) and additional lipids (e.g., DOPE). In one embodiment, the RNA lipoplex particle is a nanoparticle.

As used herein, "cationic lipid" refers to a lipid having a net positive charge. Cationic lipids bind negatively charged RNA to the lipid matrix through electrostatic interactions. Typically, cationic lipids have lipophilic moieties, such as sterol, acyl, or diacyl chains, and the head of the lipid typically carries a positive charge. Examples of cationic lipids include, but are not limited to, 1, 2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), dimethyldioctadecylammonium (DDAB), 1, 2-dioleoyl-3-trimethylammonium propane (DOTAP), 1, 2-dioleoyl-3-dimethylammonium propane (DODAP), 1, 2-diacyloxy-3-dimethylammonium propane, 1, 2-dialkoxy-3-dimethylammonium propane, dioctadecyldimethylammonium chloride (DODAC), 2, 3-ditetradecyloxy) propyl- (2-hydroxyethyl) -dimethylammonium (2,3-di (tetracoxy) propyl- (2-hydroxyethyi) -dimethylammonium (2,3-di (tetracoxy) propyloxy), 1, 2-dimyristoyl-sn-glycero-3-ethylphosphonic acid choline (DMEPC), l, 2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1, 2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DORIE) and 2, 3-dioleoyloxy-N- [2 (spermine carboxamide) ethyl ] -N, N-dimethyl-l-trimethylammonium trifluoroacetate (DOSPA). DOTMA, DOTAP, DODAC and DOSPA are preferred. In particular embodiments, the cationic lipid is DOTMA and/or DOTAP.

Additional lipids may be added to adjust the overall positive-negative charge ratio and physical stability of the RNA lipoplex particles. In certain embodiments, the additional lipid is a neutral lipid. As used herein, "neutral lipid" refers to a lipid 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 particular embodiments, the additional lipid is DOPE, cholesterol, and/or DOPC.

In certain embodiments, the RNA lipoplex particle comprises a cationic lipid and an additional lipid. In exemplary embodiments, the cationic lipid is DOTMA, and the additional lipid is DOPE.

In some embodiments, the molar ratio of the at least one cationic lipid to the 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 particular embodiments, the molar ratio may 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 exemplary embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2: 1.

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

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

The spleen-targeting RNA lipoplex particles described herein preferably have a net negative charge at physiological pH, e.g., a charge ratio of positive to negative charge of about 1.9:2 to about 1:2. In particular embodiments, the ratio of positive to negative charge in the RNA lipoplex particle 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 at physiological pH.

Cytokines, e.g., PK-extending cytokines, particularly PK-extending interleukins, such as those described herein, may be delivered to a target organ or tissue of a subject, including administering to the subject an RNA encoding the cytokine in a formulation for preferential delivery of the RNA to the target organ or tissue.

In an embodiment, the target organ is a tissue of the lymphatic system, in particular a tissue of a secondary lymphatic organ, more particularly a spleen. Delivery of cytokines to such target tissues is preferred, particularly if the presence of cytokines in the organ or tissue is desired (e.g., for inducing an immune response, particularly during T cell priming or for activating cytokines required by resident immune cells), while the systemic presence, particularly in large amounts, of cytokines is not desired (e.g., because cytokines are systemically toxic). Particularly preferred examples of suitable cytokines are cytokines involved in T cell priming.

In another embodiment of delivering the cytokine to a target organ or target tissue of a subject, the target organ is a liver and the target tissue is a liver tissue. It is preferred to deliver the cytokine to such a target tissue, in particular if the cytokine is desired to be present in this organ or tissue, and/or if it is desired to express a large amount of cytokine, and/or if it is desired or required to have the cytokine present systemically, in particular in large amounts.

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 PK extending cytokines, e.g., those described herein. Particularly preferred examples of suitable cytokines are cytokines involved in T cell proliferation and/or maintenance.

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

To deliver RNA to the liver in vivo, a drug delivery system can be used to transport RNA into the liver by preventing its degradation. For example, polyplex nanomicelles composed of a poly (ethylene glycol) (PEG) coated surface and an mRNA-containing core are useful systems because nanomicelles provide excellent in vivo stability to RNA under physiological conditions. In addition, the stealth properties provided by polyplex nanomicelle surfaces comprising a dense PEG palisade (palisade) effectively evade host immune defenses.

Pharmaceutical composition

The agents described herein can be administered in a pharmaceutical composition or medicament, and can be administered in any suitable pharmaceutical composition.

In one embodiment of all aspects of the invention, the components described herein, e.g., T cells genetically modified to express a CAR, a cytokine-encoding nucleic acid, or an antigen or variant thereof, may be administered together or separately from each other in a pharmaceutical composition, which may comprise a pharmaceutically acceptable carrier, and may optionally comprise one or more adjuvants, stabilizers, and the like. In one embodiment, the pharmaceutical composition is for use in therapeutic or prophylactic treatment, e.g., for the treatment or prevention of a disease in which an antigen is involved, such as a cancer disease, e.g., those described herein.

The term "pharmaceutical composition" relates to a formulation comprising a therapeutically effective substance, preferably comprising a pharmaceutically acceptable carrier, diluent and/or excipient. The pharmaceutical composition is 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.

The pharmaceutical compositions of the present disclosure preferably comprise, or may be administered with, one or more adjuvants. The term "adjuvant" relates to compounds that prolong, enhance or accelerate the immune response. Adjuvant(s)Agents include a heterogeneous group of compounds such as oil emulsions (e.g., freund's adjuvant), mineral compounds (e.g., alum), bacterial products (e.g., Bordetella pertussis toxins), or immunostimulatory complexes. Examples of adjuvants include, but are not limited to, LPS, GP96, CpG oligodeoxynucleotides, growth factors and cytokines, such as monokines, lymphokines, interleukins, chemokines. The chemokine can be IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IFN alpha, IFN gamma, GM-CSF, LT-a. Other known adjuvants are aluminium hydroxide, Freund's adjuvant or oils, for exampleISA 51. Other suitable adjuvants for use in the present disclosure include lipopeptides, such as Pam3 Cys.

The pharmaceutical compositions of the present disclosure are generally applied in "pharmaceutically effective amounts" and "pharmaceutically acceptable formulations".

The term "pharmaceutically acceptable" refers to the non-toxicity of materials that do not interact with the active ingredients of a pharmaceutical composition.

The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount that alone or in combination with other dosages achieves a desired response or desired effect. In the case of treatment of a particular disease, the desired response preferably involves inhibition of the disease process. This includes slowing the progression of the disease, in particular interrupting or reversing the progression of the disease. The desired response in the treatment of a disease may also be to delay the onset of the disease or the disease condition or to prevent the onset of the disease or the disease condition. An effective amount of a composition described herein will depend on the disease condition to be treated, the severity of the disease, individual parameters of the patient including age, physiological condition, size and weight, duration of treatment, type of concomitant therapy (if any), specific route of administration, and the like. Thus, the dosage of administration of the compositions described herein may depend on a variety of such parameters. In the event that the response of the patient is insufficient at the initial dose, a higher dose may be used (or an effective higher dose may be achieved by a different, more local route of administration).

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

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

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

The term "diluent" relates to a diluent (diluting agent) and/or a diluent (diluting agent). Further, the term "diluent" includes any one or more of a fluid, liquid or solid suspension, and/or a mixing medium. Examples of suitable diluents include ethanol, glycerol and water.

The term "carrier" refers to a component which may be natural, synthetic, organic, inorganic, in which an active ingredient is incorporated to facilitate, enhance or enable 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, but are not limited to, sterile water, Ringer's solution, Ringer's lactic acid solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes, especially biocompatible lactide polymers, lactide/glycolide copolymers, or polyoxyethylene/polyoxypropylene copolymers. In one embodiment, the pharmaceutical composition of the present disclosure comprises isotonic saline.

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).

The pharmaceutical carrier, excipient or diluent may be selected according to the intended route of administration and standard pharmaceutical practice.

In one embodiment, the pharmaceutical compositions described herein may be administered intravenously, intraarterially, subcutaneously, intradermally, or intramuscularly. In certain embodiments, the pharmaceutical composition is formulated for topical administration or systemic administration. Systemic administration may include enteral administration, which involves absorption through the gastrointestinal tract, or parenteral administration. As used herein, "parenteral administration" refers to administration in any manner other than through the gastrointestinal tract, for example, by intravenous injection. In a preferred embodiment, the pharmaceutical composition is formulated for systemic administration. In another preferred embodiment, systemic administration is by intravenous administration.

In one embodiment of all aspects of the invention, the nucleic acid encoding the cytokine or encoding the antigen or variant thereof is administered systemically. In one embodiment of all aspects of the invention, the antigen or variant thereof is expressed in the spleen following systemic administration of a nucleic acid encoding the antigen or variant thereof. In one embodiment of all aspects of the invention, the antigen or variant thereof is expressed in an antigen presenting cell, preferably a professional antigen presenting cell, upon systemic administration of a nucleic acid encoding the antigen or variant thereof. In one embodiment, the antigen presenting cell is selected from the group consisting of a dendritic cell, a macrophage, and a B cell. In one embodiment of all aspects of the invention, there is no or substantially no expression of the antigen or variant thereof in the lung and/or liver following systemic administration of the nucleic acid encoding the antigen or variant thereof. In one embodiment of all aspects of the invention, the amount of expression of the antigen or variant thereof in the spleen is at least 5-fold greater than the amount of expression in the lung following systemic administration of the nucleic acid encoding the antigen or variant thereof.

The term "co-administration" as used herein refers to the process of administering different compounds or compositions (e.g., an RNA encoding an interleukin and an RNA encoding an antigen or variant thereof) simultaneously, substantially simultaneously, or sequentially to the same patient. If administered simultaneously, the different compounds or compositions need not be administered in the same composition.

Treatment of

The materials, compositions, and methods described herein are useful for treating a subject having a disease, e.g., 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 materials, compositions and methods may be used to treat viral diseases caused by the virus. If the antigen is a tumor antigen, the materials, compositions and methods can be used to treat cancer diseases, wherein cancer cells express the tumor antigen.

In one embodiment, the present disclosure relates to a method of inducing an immune response in a subject. In exemplary embodiments, the immune response is directed against cancer.

The term "disease" refers to an abnormal condition affecting the body of an individual. A disease is generally interpreted as a medical condition associated with specific symptoms and indications. The disease may be caused initially by exogenous factors, such as infectious diseases, but also by internal dysfunctions, such as autoimmune diseases. In humans, "disease" is generally used more broadly to refer to any condition that causes pain, dysfunction, distress, social problems or death in the afflicted individual, or causes similar problems to individuals in contact with the individual. In this broader sense it sometimes includes injuries, disabilities, disorders, syndromes, infections, isolated symptoms, abnormal behavior, and atypical changes in structure and function, while in other cases and for other purposes these may be considered distinguishable categories. Diseases often affect not only the body of an individual but also the mood, as infections and life in many diseases change one's opinion of life and one's personality.

In the present context, the terms "treatment", "treating" or "therapeutic intervention" relate to the management and care of a subject for the purpose of combating a disease condition, such as a disease or disorder. The term is intended to include the full spectrum of treatments for a given condition from which a subject suffers, such as the administration of therapeutically effective compounds to alleviate symptoms or complications, delay the progression of a disease, disorder, or condition, alleviate or alleviate symptoms and complications, and/or cure or eliminate a disease, disorder, or condition, as well as the prevention of a condition, where prevention is understood to be the management and care of an individual for the purpose of combating a disease, disorder, or condition, and includes the administration of active compounds to prevent the onset of symptoms or complications.

The term "therapeutic treatment" relates to any treatment that improves a health condition and/or extends (increases) the longevity of an individual. The treatment can eliminate the disease in the subject, arrest or slow the progression of the disease in the subject, inhibit or slow the progression of the disease in the subject, reduce the frequency or severity of symptoms in the subject, and/or reduce relapse in a subject currently or previously having the disease.

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

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

The term "patient" refers to an individual or subject undergoing treatment, particularly an individual or subject suffering from a disease.

In one embodiment of the disclosure, it is an object to provide an immune response against diseased cells expressing an antigen (e.g., cancer cells expressing a tumor antigen), and to treat diseases (e.g., cancer diseases) involving cells expressing an antigen (e.g., a tumor antigen).

An immune response to the antigen can be elicited, which can be therapeutic, or can be partially or fully protective. The pharmaceutical composition described herein, is suitable for inducing or enhancing an immune response. Accordingly, the pharmaceutical compositions described herein may be used for the prophylactic and/or therapeutic treatment of diseases involving antigens.

As used herein, "immune response" refers to the body's overall bodily response to an antigen or antigen-expressing cell, and refers to a cellular immune response and/or a humoral immune response. Cellular immune responses include, but are not limited to, cellular responses against cells expressing an antigen. Such cells may be characterized by expressing the antigen on their cell surface or by presenting the antigen through MHC class I or class II molecules. The cellular response is associated with T lymphocytes, which can be classified as helper T cells (also known as CD4+ T cells), which exert a central role by modulating the immune response or killing cells (also known as cytotoxic T cells, CD8+ T cells, or CTLs), inducing apoptosis of infected or cancer cells. In one embodiment, administration of the pharmaceutical composition of the present disclosure involves stimulating an anti-tumor CD8+ T cell response against cancer cells expressing one or more tumor antigens.

The present disclosure contemplates that protective, prophylactic and/or therapeutic immune responses may be considered. As used herein, "inducing an immune response" may mean that there is no immune response to a particular antigen prior to induction, or may mean that there is a basal level of immune response to a particular antigen prior to induction, which is enhanced after induction. Thus, "inducing an immune response" includes "enhancing an immune response".

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

The term "vaccination" or "immunization" describes the process of administering an antigen to an individual with the aim of inducing an immune response, e.g. for therapeutic or prophylactic reasons.

In one embodiment, the present disclosure contemplates embodiments wherein an RNA formulation, such as an RNA particle described herein, is administered.

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

The term "macrophage" refers to a subpopulation of phagocytes that result from monocyte differentiation. Macrophages, activated by inflammation, immune cytokines or microbial products, nonspecifically phagocytose and kill foreign pathogens within the macrophage through hydrolytic and oxidative attack, resulting in pathogen degradation. Peptides of the degraded proteins are displayed on the macrophage surface where they can be recognized by T cells and can interact directly with antibodies on the B cell surface, resulting in T and B cell activation, further stimulating the immune response. Macrophages belong to the class of antigen presenting cells. In one embodiment, the macrophage is a spleen macrophage.

The term "dendritic cell" (DC) refers to another subset of phagocytic cells that belong to the class of antigen presenting cells. In one embodiment, the dendritic cells are derived from hematopoietic bone marrow progenitor cells. These progenitor cells are initially transformed into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation potential. Immature dendritic cells are continuously sampled in the surrounding environment for pathogens such as viruses and bacteria. Once they are contacted with presentable antigens, they are activated into mature dendritic cells and begin to migrate to the spleen or lymph nodes. Immature dendritic cells phagocytose pathogens and degrade their proteins into small fragments, and after maturation present these fragments on their cell surface using MHC molecules. At the same time, they upregulate cell surface receptors that act as co-receptors in T cell activation, such as CD80, CD86, and CD40, greatly enhancing their ability to activate T cells. They also up-regulate CCR7, a chemoattractant receptor, and induce dendritic cells to reach the spleen through the bloodstream or lymph nodes through the lymphatic system. Here, they act as antigen presenting cells and activate helper and killer T cells as well as B cells by presenting them with antigen as well as non-antigen specific co-stimulatory signals. Thus, dendritic cells can actively induce an immune response associated with T cells or B cells. In one embodiment, the dendritic cells are splenic dendritic cells.

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

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

The term "non-professional antigen presenting cell" relates to an antigen presenting cell that is not constitutively expressed but expresses MHC class II molecules under stimulation by certain cytokines (e.g., 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.

"antigen processing" refers to the degradation of an antigen into a processed product, which is a fragment of the antigen (e.g., the degradation of a protein into a peptide) and the interaction of one or more of these fragments with an MHC (e.g., by binding) for presentation by a cell, e.g., a cell that presents the antigen to a particular T cell.

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

The term "infectious disease" refers to any disease that can be transmitted from individual to individual or from organism to organism and is caused by microbial matter (e.g., the common cold). 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, the infectious disease may 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 disease syndrome (SARS), avian influenza, and influenza.

The term "cancer disease" or "cancer" refers to or describes a physiological condition of an individual that is generally characterized by unregulated cell growth. Examples of cancer include, but are not limited to, tumors, 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, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, cancer of the sexual and reproductive organs, hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the bladder, cancer of the kidney, cancer of the renal cell, cancer of the renal pelvis, tumors of the Central Nervous System (CNS), cancer of the neuroectodermal, spinal axis, glioma, meningioma and pituitary adenoma. The term "cancer" according to the present disclosure also includes cancer metastasis.

Due to the resulting synergistic effect, combination strategies in cancer treatment may be desirable, which may be more influential than monotherapy approaches. In one embodiment, the pharmaceutical composition is administered with an immunotherapeutic agent. As used herein, "immunotherapeutic agent" relates to any substance that may be involved in activating a particular immune response and/or immune effector function. The present disclosure contemplates the use of antibodies as immunotherapeutic agents. Without wishing to be bound by theory, antibodies can achieve therapeutic effects against cancer cells through various mechanisms, including inducing apoptosis, blocking components of signal transduction pathways, or inhibiting proliferation 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 complement proteins, resulting in direct cytotoxicity, known as complement-dependent cytotoxicity (CDC). Non-limiting examples of anti-cancer antibodies and potential antibody targets (in parentheses) that can be used in conjunction with the present disclosure include: abavomab (Abagomomab) (CA-125), Abciximab (Abciximab) (CD41), Addimizumab (Adecatumumab) (EpCAM), Avermezumab (Aftuzumab) (CD20), Pego-Alatezumab (Alacizumab pegol) (VEGFR2), pentostatin (Altumomab pentate) (CEA), Amitumumab (Amatuximab) (MOAB-009), MAAMATUMOBUzumab (Anatuzumab mafenadox) (TAG-72), Apogliomab (HLA-ApoDR), Arcitmomab (Arcitmomab) (CEA), Alitumumab (Atezolizumab) (Atezab) (PD-L1), Baviximab (Baviximab) (phosphatidylserine), tuzumab (Bectumomab) (CD22), Betuluzumab (Betuluzumab) (Betuzumab-6778), Betezomib (Betulizumab) (CD 6719), Betulizumab (Betulumab) (PD-L1), Bavizumab (Betuzumab) (Betuximab-R-D-78), Betuzumab (Betuzumab) (CD-E-D-3), Betuzumab (Betub-D-E (E-D-E (E-D-E-D-E-D-E-D-, Mocantuzumab (Cantuzumab mertansin) (mucin) CanAg), Rakanotuzumab (Cantuzumab ravtansine) (MUC1), Carocumab pentosan (Capromumab pendend) (prostate cancer cells), Carlumab (Carlumab) (CNT0888), Katuzumab (Catuxomab) (EpCAM, CD3), Cetuximab (Cetuximab) (EGFR), Posituzumab (Cituzumab bogatuzumab) (CAM), Cetuzumab (Cixutuzumab) (IGF-1 receptor), Claudinmab (claudin-tight junction protein), Tatan-Clituzumab (Clivatuzumab tetataxe) (DeMUC 1), Comatuzumab (Conatuzumab) (TRAUmab-R2), Datuzumab (CD40), Suloxuzumab (Clivab tetuzumab) (Delimum-R5), Haemolizumab (Ginkyux-D-, Eperythromumab (Edbecolomab) (EpCAM), Epotuzumab (Elotuzumab) (SLAMF7), Epitamiumab (Enavatuzumab) (PDL192), Entuximab (Ensituzumab) (NPC-1C), Epratuzumab (Epratuzumab) (CD22), Ertuzumab (Ertuxomab) (HER2/neu, CD3), Elatatuzumab (Etarazeumab) (integrin. alpha. v. beta.3), Fatuzumab (Farletumab) (folate receptor 1), FBTA05(CD20), Filatuzumab (Filatuzumab) (SCH 900105), Figituzumab (Figituzumab) (IGF-1 receptor), Fluatuzumab (Flankuzumab) (glycoprotein 75), Nonellomab (Epaluzumab) (TGF-beta.TGF-9), Geigizumab (Geigit II), Gerituzumab (Geigin II) (IGF-9), Gerituzumab (Gerituzumab) (TGF-75), Gerituzumab (Gerituximab (Gejix) (TGF-III) (IGF-9), Gerituzumab (Gerituximab (Gejic-III) (TGF-III) (IGF-III), Gerituximab (Gejic-9 (Gerituximab (Gejic-III) (TGF-III), Gerituximab (Gejic-II), Gerituximab (Gettuzumab), Gejic-II), Gettuzumab) (TGF-III (Gettuzumab) (GLC-III) (TGF-III (Gettuzumab), Gettuzumab (TGF-III (Gettuzumab), or Gettuzumab (TGF-III), or (G-III (TGF-II), or Gettuzumab (G-III), or a (Gettuzumab), or a (Gettuyx (Gettuzumab), or a (Gettuzumab (Gettuyi) (glycoprotein (Gettuy), or Gettuzumab (Gettuyx (Gettuzumab (Gettuy), or a (Gettuyx (Gettuy), or a (Gettuzumab) or a (Gettuy), or a) or a (Gettuyx (Gettuy), or a) or a (Gettuyi), or Gettuyx (Gettuy), or Gettuyi), or a) or a (Gettuyx (Gettuyi), or Gettuyx (Gettuyx) or Gettuyx (Gettuyi), or Gettuyx (Gettuyx) or, Glembitumumab vedotin (GPNMB), Ibritumomab tiuxetan (CD20), eculizumab (Ibrucumab) (VEGFR-1), Igomoma (CA-125), Rituximab (Intuximab ravtansine) (SDC1), Rituzumab (Intitumumab) (CD51), Rituzumab (Inotuzumab) (CD22), Ipilimumab (Iililimumab) (CD 152), Rituzumab (Iratumab) (CD30), Labestuzumab (Labetuzumab) (CEA), Lexatuzumab (Lexatuzumab) (TRAIL-R2), Rituzumab (Libivimab) (hepatitis B surface antigen), Rituzumab (Lintuzumab) (CD33), Rituzumab-483 (Leumazumab) (TRAIL-R-3625), Luitumumab (Luitumumab) (CD 3625), Luitumumab (Luitumumab) (CD 5), Luitumumab (Luitumumab) (CD 865) (CD 8655), Rituzumab) (Libivumab (Lituzumab) (hepatitis B surface antigen), Lutuzumab) (Lintuzumab) (TRAUtuzumab) (TRAIL-11) (CD33), Lutuzumab) (Leutuzumab) (TRAIL-R-E) (Leutuzumab) (Leu) (CD 3625), Lutuzumab) (Leutuzumab) (L) (Leutuzumab) (Leu) (L) (Leu) (L-R-11) (L) (C) (L) (C) (L) (R) 2), L) (, Mitomumab (Mitumomab) (GD3 ganglioside), moglobizumab (Mogamulizumab) (CCR4), Moxetumomab pasudotox (CD22), tanacetumab (Nacolomab tafentox) (C242 antigen), eto-natamycin (napumomab estafenatox) (5T4), namatumab (ron), Nimotuzumab (necrozumab) (EGFR), Nimotuzumab (Nimotuzumab) (EGFR), nituzumab (Nimotuzumab) (EGFR), Nivolumab (Nivolumab) (IgG4), Ofatumumab (Ofatumumab) (CD20), olarumab (olamab) (obatuzumab) (PDGF-R a), onatuzumab (Onartuzumab) (human scatter factor receptor kinase), pertuzumab (montomozumab (cgumatomab), eputatum (epertuzumab) (EGFR-1), pemutab (pembrormutab) (EGFR-3526), pemutab (pembrotuzumab) (EGFR-3645), pemutab (pemutab) (EGFR/11), pemutab (r antigen), pemutab (pemutatum) (EGFR/3645 (pemutab) (EGFR), pemutab) (p, pemutab (r) and (r α), pemutab) (EGFR (r) and (r (C) 2 (r α), wherein C) are included in, and (e) and (e) and (e) are included in the like, Pritumumab (Pritumumab) (vimentin), Ratumomab (Racotumomab) (N-glycolylneuraminic acid), Rituzumab (Raretuzumab) (fibronectin extra domain-B), Rituzumab (Rafivirumab) (rabies glycoprotein), Ramuirumab (Ramucirumab) (VEGFR2), Rituzumab (Ritumumab) (HGF), Rituximab (Rituximab) (CD20), Rituzumab (Robaumumab) (IGF-1 receptor), Samacitumumab (CD200), Sirocuzumab (Sibrotuzumab) (FAP), Setuximab (Sirtuzumab) (IL6), Tabeumab (BALUMAb) (BAFF), Tatuzumab (Tatuzumab) (Tetuzumab), Tratan (alpha-tetatan), TRATUMOMb (TRAIL) (TRAIL-19), and Trautumab (TAbazumab) (CTLA-3), Trautumab (TAbazumab) (CTLA-2), and Trautumumab (CTLA) (CTLA-4 (Tatuzumab) (CTLA) (TAB-4 (Tatuzumab-E-4), and Trautumab (Tatuzumab) (TRATUMOX-E, CTLA), TNX-650(IL13), Tositumomab (Tositumomab) (CD20), Trastuzumab (Trastuzumab) (HER2/neu), TRBS07(GD2), Tremelimumab (Tremelimumab) (CTLA-4), simon interleukin mab (Tucotuzumab celloulkin) (EpCAM), Ublituximab (MS4a1), Urelumab (4-1BB), volaximab (Volociximab) (integrin α 5 β 1), volitumomab (volitumomab) (tumor antigen cta 16.88), Zalutumumab (zalutumumumab) (EGFR), and zanolimab (CD 4).

Citation of documents and research herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the contents of these documents are based on the information available to the applicants and do not constitute any admission as to the correctness of the contents of these documents.

The following description is presented to enable any person skilled in the art to make and use various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications to the examples described herein will be readily apparent to those of ordinary skill 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 various embodiments. Thus, the various embodiments are not intended to be limited to the examples described and illustrated herein, but are to be accorded the scope consistent with the claims.

Examples

Method

Animal(s) production

C57BL/6BrdCrHsd-Tyr^cMice were purchased from Envigo Labs. Age (8-10 weeks old) and sex (male or female) matched animals were used throughout the experiment. Syngeneic C57Bl/6-Thy1.1 mice were bred in animal facilities from BioNTech AG, Germany.

CAR construct/CAR T cells

Gamma-retroviral self-inactivating (SIN) vector pes.12-6 was used to stably overexpress CLDN6-CAR-BBz-T2A-Luc-T2A-GFP in murine T cells under the control of an internal eukaryotic promoter, a short intron-free form of the human elongation factor 1-alpha promoter (EFS-213/+ 31). The vector backbone contains the MLV wild-type sequence of the R-and U5-regions at the 5 'and 3' -LTRs and the packaging region (psi and psi +). Enhancer elements (including CAAT-Box) in the U3 region of the 3' -LTR were eliminated, and the TATA-Box sequence was mutated to prevent transcriptional initiation. Truncated forms of the post-transcriptional regulatory element (PRE) of Woodchuck Hepatitis Virus (WHV) are used to prevent expression of unwanted viral proteins. CLDN6-CAR-BBz comprises the signal peptide of human IgG (SEQ ID NO:12), the single-chain Fv fragment of the Claudin 6-specific antibody IMAB206 (secreted Pharmaceuticals), in the heavy chain (V)_H) (SEQ ID NO:13) and light chain (V)_L) (SEQ ID NO:15) having (G) between₄S)₃Linker (SEQ ID NO:14) and at V_LWith a cysteine to serine substitution at position 46. The ScFv fragment was fused to the human CD8 α hinge and transmembrane region (SEQ ID NO:16) followed by human 4-1BB (SEQ ID NO:17) and human CD3 ζ (Q14K) (SEQ ID NO: 18) signaling portions. The CAR was linked to a T2A ribosome-hopping element (SEQ ID NO:19) to an efficient firefly luciferase (SEQ ID NO:20) and eGFP (SEQ ID NO:21) to enable equimolar production of the indicated proteins in transduced T cells.

Retroviral gene manipulation and production of CAR T cells for adoptive T cell transfer

Using Dynabeads^TMMouse T-Activator CD3/CD28, at a bead to T cell ratio of 1:1 (Invitrogen), in the presence of 5ng/mL recombinant human (rh) IL-7 and 5ng/mL rh IL-15(Miltenyi Biotec), isolated and pre-activated naiveC57Bl/6-Thy1.1⁺The spleen cell of (3). For transduction of murine cells, MLV-E pseudotype retrovirus supernatant was loaded into RetroNectin (2. mu.g/cm) according to the manufacturer's instructions (Takara Bio Inc., Otsu, Japan)²) Coated non-tissue culture treated well plates, in which the virus was loaded and centrifuged (1,300 Xg)15 ℃,15 min) was repeated 3 times to increase binding. 24h after pre-activation, 0.5-0.6x10^6 cells/cm²Centrifugation (300Xg, 37 ℃, 1h) was carried out into wells coated with viral particles. After overnight incubation, spin-down transduction was repeated with plates coated with fresh virus particles. Dynabeads were removed from the culture 72h after pre-activation^TMMouse T-Activator CD3/CD28, and expanded cells in the presence of 5ng/mL rh IL-7 and 5ng/mL rh IL-15. After Ficoll washing, the cells were washed twice with PBS to remove serum proteins and then prepared for Adoptive Cell Transfer (ACT). pES12.6-based retroviral vectors contain OT1-TCR or CLDN6-CAR encoded and enhanced firefly luciferase (effLuc; Rabinovich et al (2008) PNAS 105(38):14342-6) and eGFP (enhanced green fluorescent protein) reporter genes, which are expressed for transduction using the 2A splice element (Szymczak et al (2004) Nat Biotechnol.22(5):589-94), respectively.

Production of In Vitro Transcribed (IVT) mRNA

In vitro transcription of mRNA encoding cytokine-albumin fusion protein was based on the pST4-T7-GG-TEV-MCS-FI-A30LA70 plasmid backbone and derived DNA constructs. These plasmid constructs comprise the 5' leader sequence of Tobacco Etch Virus (TEV), the 3' Fl element (where F is the amino terminal cleavage enhancer of a 136 nucleotide long 3' -UTR fragment, mRNA and I are fragments of 142 nucleotide long mitochondrially encoded 12S RNA, all identified in humans; WO 2017/060314) and a 100 nucleotide poly (A) tail with a linker after 70 nucleotides. The cytokine and albumin coding sequences were derived from mouse (Mus musculus) and NO changes were introduced in the amino acid sequences produced (mouse (m) IL-2, SEQ ID NO: 5; mIL-7, SEQ ID NO: 6; and mIL-21, SEQ ID NO: 7). The encoded protein is equipped with an N-terminal Signal Peptide (SP), which is the native SP of the N-terminal portion. Only the N-terminal part of the SP is retained, and for the other part, only the mature part (SP-free protein) is encoded. The stop codon remains only in the most C-terminal part. The albumin and cytokine portions of the construct are separated by a linker sequence of 30 nucleotides in length encoding glycine and serine residues. The orientation of the albumin-cytokine fusion protein used was as follows: Albumin-linker-mIL 2 (N to C terminal continuous SE)Q ID NO:8, 9 and 10), mIL 7-linker-albumin (SEQ ID NO:6, 9 and 11 contiguous from N to C terminus) and mIL 21-linker-albumin (SEQ ID NO:7, 9 and 11 contiguous from N to 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, except for the full length human CLDN6 or chicken ovalbumin epitope SIINFEKL (OvaI; additionally flanked by 3 'Sec and 5' TM 1-sequences as described in Kreiter et al (2008) J Immunol.180(1): 309-18), comprise 5 'human alpha-globin, two consecutive 3' human beta-globin UTR and a 100 nucleotide poly (A) tail, with a linker after 70 nucleotides. mRNA encoding antigens and cytokines are produced by in vitro transcription as described by Holtkamp S.et al (2006) Blood 108(13): 4009-17. The latter is additionally modified by substituting the normal nucleoside uridine with 1-methyl-pseudouridine. The cytokine mRNA thus produced was equipped with Cap1 structure and double-stranded (dsRNA) molecules were removed by cellulose purification. Purified mRNA in H₂Eluted in O and stored at-80 ℃ until further use. In vitro transcription of all described mRNA constructs was carried out in BioNTech RNA Pharmaceuticals GmbH.

Liposomal formulated IVT RNA (RNA) encoding antigens_(LIP)) Generation of

Complexation of antigen-encoding IVT RNA with liposomes was previously described in Kranz et al (2016) Nature 534(7607): 396-401. The charge ratio of cation DOTMA and RNA used was 1.3:2. In addition to DOTMA, the lipid fraction does comprise the helper lipid DOPE of DOTMA/DOPE in a molar ratio of 2: 1.

Mouse experiment

Thy1.1 of 5x10^6 gamma-retrovirus transduced CAR or TCR homologs⁺T cells were transferred intravenously (i.v.) at 200. mu.L to immunologically active or moderately whole body irradiated (2.5 Gy-XRAD 320) C57BL/6BrdCrHsd-Tyr^cIn donor mice. Subsequently, at various time points after ACT, antigen-encoding RNA was used in a ratio of F12: RNA of 1.3:2_(LIP)Mice were vaccinated intravenously (i.v.). At the indicated time points, the mice were treated repeatedly with 1. mu.g of nucleoside-modified mRNA encoding murine albumin-cytokine fusion protein formulated in TransIT (Mirrus) or buffer onlyMice. Peripheral blood donations and whole-body bioluminescence imaging were performed at the indicated time points.

In vivo luciferase imaging (BLI)

The amplification and distribution of CAR or TCR-effLuc-GFP transduced T cells was assessed by in vivo bioluminescence imaging using the IVIS Lumina imaging System (Caliper Life Sciences). Briefly, at the indicated time points after adoptive transfer of transduced T cells, aqueous D-fluorescein solution (80mg/kg body weight; Perkin Elmer) was injected i.p. After 5min, the emitted photons were quantified (integration time 1min, pixels merged to 8). In vivo bioluminescence in the region of interest (ROI) was quantified as total flux (photons/sec) using IVIS Living Image 4.0 software. The transmitted light intensity from cells expressing luciferase in animals is represented as a gray scale image, where black is the lowest intensity and white to dark gray is the strongest bioluminescent signal. And obtaining a gray reference image of the mouse under the low-light illumination of the LED. Images were overlaid using Living Image 4.0 software.

Example 1: selected extended pharmacokinetic gamma chain cytokines (IL-2/7) lead to repeated expansion of CAR T cells in vivo upon antigen contact

In general, the presence of a cytokine environment is required to maintain the persistence of T cells upon antigen exposure. 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 is limited by its short half-life and 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 indeed the serum half-life of the encoded cytokines in vivo could be significantly increased following systemic administration. When cytokine-albumin constructs are encoded on nucleoside-modified mrnas, their systemic availability is extended.

Thus, it is possible to provideWe focused on combinations of liposome-formulated TAAs (e.g., RNA (RNA) encoding CLDN6_(LIP)) (which selectively targets APCs in secondary lymphoid organs) and selection of cytokines support whether CAR-T cells can be caused to sufficiently repeat expansion and persistence in vivo.

To test this concept, gamma-retrovirus transduced CLDN6-CAR T cells were adoptively transferred to moderately irradiated (2.5Gy) or immunocompetent mice (fig. 1 and fig. 2, respectively). To visualize in vivo the amplification and fate of those murine CLDN6-CAR T cells, we utilized co-expression of luciferase and GFP reporter genes on the same retroviral vector encoding CLDN6 CAR but separated by the viral T2A sequence (fig. 1A). Notably, co-expression of luciferase and GFP in CAR-transduced murine T cells did not significantly affect the surface expression and antigen specificity of CLDN6-CAR (data not shown).

Moderately irradiated (2.5Gy, XRAD320) albino C57Bl/6 mice were transplanted with 5x10⁶Large number of homologous Thy1.1 s transduced by CLDN 6-CAR-reporter genes⁺Murine T cells (about 2.5X 10)⁸Individual cells/kg body weight). 20 μ g of RNA encoding CLDN6 or a control was formulated into spleen targeting liposomes and injected intravenously into mice 1 day after adoptive CAR-T transfer. Concomitant CLDN6 RNA_(LIP)-Vaccination, mice received intraperitoneally mRNA encoding albumin-conjugated murine IL-2 and murine IL-7 or mock control (buffer) formulated in TransIT (1 μ g/cytokine RNA). The treatment was repeated after 7 days. CAR-T amplification and biodistribution was followed in vivo by intraperitoneal administration of 1.66mg of D-fluorescein solution per mouse at the indicated time points. At 24 hours post ACT, most CAR-T cells have been found in the spleen. Detected in bioluminescence at day 4 post ACT, by CLDN6-RNA alone, in the absence of cytokines (mock)_(LIP)Treatment induced an approximately 21-fold increase in CAR-T cells (compared to day 1). CLDN6-RNA compared to baseline luminescence measured on day 1_(LIP)Still produces a 15-fold higher luminous intensity at day 11. When mRNA encoding albumin-fused IL-2 and IL-7 formulated in TransIT were co-administered, the expansion capacity of CAR T cells was significantly increased. CAR-T cells RNA in first time_(LIP)Can be realized after processing75 fold amplification and a second time CLDN6 RNA_(LIP)Treatment was even improved by 114-fold (FIG. 1C)&D) In that respect In mice receiving CLDN6-CAR T cells, CLDN 6-encoding RNA alone was used_(LIP)Or in combination with cytokine-albumin encoding RNA, but receiving a control RNA encoding ovaI_(LIP)This effect was not observed in the respective control group, whether or not the cytokine-albumin-encoding RNA was present. These data indicate that CAR-T cells can be successfully expanded in situ in a highly antigen-specific manner in moderately irradiated mice.

In the case where it is demonstrated that RNA encoding cytokines is present, RNA encoding the respective antigens may be used_(LIP)After repeated expansion of CAR T cells in situ in moderately irradiated mice, we investigated whether this effect could also be achieved in immunocompetent hosts. Lymphoablation, however, has several disadvantages, including well-known side effects and risks associated with chemotherapy, such as potential infection and sepsis (Bretjens et al (2010) Mol ther.18(4):666-8&Robbins et al, (2015) Clin Cancer Res.21(5): 1019-27). Furthermore, rapid expansion of adoptively transferred CAR-T cells can be fatal in the case of on-target and/or off-target toxicity (Morgan et al (2010) Mol Ther.18(4): 843-51). For this purpose CLDN6-CAR transduced murine thy1.1⁺T cell-transplanted unirradiated albino C57Bl/6 mice were treated as described above (FIG. 2A). IL-2 and IL-7 are present systemically during the first round of stimulation versus CLDN6-RNA as compared to a control group receiving buffer (mock) rather than TransIT formulated RNA encoding cytokine-albumin_(LIP)After vaccination, the expansion of CLDN6-CAR T cells was not significantly affected (day 4: expansion index: mock 192-fold and IL-2/7: 223-fold). However, in the absence of IL-2/7 cytokine, CAR T cell population was at the first CLDN6 RNA_(LIP)The mediated amplification is strongly contracted and cannot be amplified again. CLDN6-CAR T cells were repeatedly expanded in immunocompetent mice for several days only in the presence of RNA encoding IL-2/IL-7-albumin (day 11: expansion index: mock: 0.5-fold and IL-2/7: 79-fold) (fig. 2B + C).

These data strongly support the use of RNA_(LIP)Technology to control CAR-T cell expansion directly in patients is a viable idea, but for persistence, cells require favorable cytokine environments, such as IL-2 and IL-7, which can be achieved by administering RNA encoding extended pharmacokinetic gamma chain cytokines.

Example 2: optimal combination of cytokine albumin fusion during repeated CAR T cell expansion

Since several gamma-chain cytokines actively support T-cell survival and therapeutic efficacy 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.), we compared nucleoside-modified RNAs encoding mIL-2, mIL-7, mIL-21, and a combination of IL-2/7 and IL-2/21 in promoting CAR-modified T-cells in vivo against repeat RNA_(LIP)Supporting effect of proliferation and persistence of treatment.

Moderately irradiated albino C57Bl/6 mice transplanted with CLDN 6-CAR-reporter transduced T cells were inoculated with liposomal formulated RNA vaccine encoding hCLDN6 or control, while treated with murine albumin-conjugated mIL-2, mIL-7 encoding RNA, mIL-21 encoding mRNA, or murine albumin encoding RNA (Alb control) formulated in TransIT (1 μ g/cytokine RNA) in a similar manner as described in example 1. Antigen/cytokine cocktail (cocktail) was administered at weekly intervals (fig. 3A). Bioluminescence intensity was analyzed when CAR T cell in vivo expansion peaked (typically reached after 2-3 days post RNA-based treatment) (fig. 3B). The systemic presence of IL-7 and IL-21 alone resulted in repeated RNA compared to the albumin control_(LIP)Antigen-specific CART amplification capacity decreased after treatment. IL-2 combined treatment resulted in up to 164-fold expansion of CAR T cells compared to baseline. However, in vivo accumulation of CAR T cells can only be achieved when IL-2RNA is co-administered with IL-7 (up to a 214-fold increase after 3 rd round expansion) or IL-21 (up to a 141-fold increase after 3 rd round expansion), respectively. In addition to the ability of CAR T cells to accumulate in vivo, the clinical success of adoptive transfer of 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). Thus, we analyzed the contraction of CART T cells after 3 rounds of antigen-specific expansion using bioluminescence in the presence of IL-7 (fig. 3C) or IL-21 (fig. 3D), alone or in combination with IL-2, whereas the CAR T cell population was at CLDN6 RNA 3 times when albumin, IL-2 or IL-7 were present alone_(LIP)And shortly thereafter contracts. However, only the combination of IL-2 and IL-7 increased the slowed contraction of CLDN6 CAR T cells following antigen withdrawal (fig. 3C). This effect was more pronounced in mice co-treated with IL-2 and IL-21-RNA (FIG. 3D).

Overall, these results indicate that systemic administration of nucleoside-modified RNA encoding IL-2, in combination with IL-7 and IL-21, can increase the highly antigen-dependent accumulation of CAR T cells under antigen-specific stimulation and prolonged persistence of CAR T cells in vivo.

Sequence listing

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Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu

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Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu

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Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His

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Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg

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Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg

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Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro

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Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp

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Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser

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Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His

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Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser

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Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala

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Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg

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Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr

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Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu

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Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro

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Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu

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Phe Phe Arg Lys His Val Cys Asp Asp Thr Lys Glu Ala Ala Phe Leu

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Arg His Leu Ile Asp Ile Val Glu Gln Leu Lys Ile Tyr Glu Asn Asp

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Leu Asp Pro Glu Leu Leu Ser Ala Pro Gln Asp Val Lys Gly His Cys

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Asn Pro Gly Asn Asn Lys Thr Phe Ile Ile Asp Leu Val Ala Gln Leu

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Leu Ser

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His Arg Tyr Asn Asp Leu Gly Glu Gln His Phe Lys Gly Leu Val Leu

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Ile Ala Phe Ser Gln Tyr Leu Gln Lys Cys Ser Tyr Asp Glu His Ala

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Lys Leu Val Gln Glu Val Thr Asp Phe Ala Lys Thr Cys Val Ala Asp

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Glu Ser Ala Ala Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp

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Lys Leu Cys Ala Ile Pro Asn Leu Arg Glu Asn Tyr Gly Glu Leu Ala

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Asp Cys Cys Thr Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln

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His Lys Asp Asp Asn Pro Ser Leu Pro Pro Phe Glu Arg Pro Glu Ala

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Glu Ala Met Cys Thr Ser Phe Lys Glu Asn Pro Thr Thr Phe Met Gly

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His Tyr Leu His Glu Val Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro

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Glu Leu Leu Tyr Tyr Ala Glu Gln Tyr Asn Glu Ile Leu Thr Gln Cys

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Cys Ala Glu Ala Asp Lys Glu Ser Cys Leu Thr Pro Lys Leu Asp Gly

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Val Lys Glu Lys Ala Leu Val Ser Ser Val Arg Gln Arg Met Lys Cys

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Ser Ser Met Gln Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val

225 230 235 240

Ala Arg Leu Ser Gln Thr Phe Pro Asn Ala Asp Phe Ala Glu Ile Thr

245 250 255

Lys Leu Ala Thr Asp Leu Thr Lys Val Asn Lys Glu Cys Cys His Gly

260 265 270

Asp Leu Leu Glu Cys Ala Asp Asp Arg Ala Glu Leu Ala Lys Tyr Met

275 280 285

Cys Glu Asn Gln Ala Thr Ile Ser Ser Lys Leu Gln Thr Cys Cys Asp

290 295 300

Lys Pro Leu Leu Lys Lys Ala His Cys Leu Ser Glu Val Glu His Asp

305 310 315 320

Thr Met Pro Ala Asp Leu Pro Ala Ile Ala Ala Asp Phe Val Glu Asp

325 330 335

Gln Glu Val Cys Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly

340 345 350

Thr Phe Leu Tyr Glu Tyr Ser Arg Arg His Pro Asp Tyr Ser Val Ser

355 360 365

Leu Leu Leu Arg Leu Ala Lys Lys Tyr Glu Ala Thr Leu Glu Lys Cys

370 375 380

Cys Ala Glu Ala Asn Pro Pro Ala Cys Tyr Gly Thr Val Leu Ala Glu

385 390 395 400

Phe Gln Pro Leu Val Glu Glu Pro Lys Asn Leu Val Lys Thr Asn Cys

405 410 415

Asp Leu Tyr Glu Lys Leu Gly Glu Tyr Gly Phe Gln Asn Ala Ile Leu

420 425 430

Val Arg Tyr Thr Gln Lys Ala Pro Gln Val Ser Thr Pro Thr Leu Val

435 440 445

Glu Ala Ala Arg Asn Leu Gly Arg Val Gly Thr Lys Cys Cys Thr Leu

450 455 460

Pro Glu Asp Gln Arg Leu Pro Cys Val Glu Asp Tyr Leu Ser Ala Ile

465 470 475 480

Leu Asn Arg Val Cys Leu Leu His Glu Lys Thr Pro Val Ser Glu His

485 490 495

Val Thr Lys Cys Cys Ser Gly Ser Leu Val Glu Arg Arg Pro Cys Phe

500 505 510

Ser Ala Leu Thr Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Lys Ala

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Glu Thr Phe Thr Phe His Ser Asp Ile Cys Thr Leu Pro Glu Lys Glu

530 535 540

Lys Gln Ile Lys Lys Gln Thr Ala Leu Ala Glu Leu Val Lys His Lys

545 550 555 560

Pro Lys Ala Thr Ala Glu Gln Leu Lys Thr Val Met Asp Asp Phe Ala

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Gln Phe Leu Asp Thr Cys Cys Lys Ala Ala Asp Lys Asp Thr Cys Phe

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Ser Thr Glu Gly Pro Asn Leu Val Thr Arg Cys Lys Asp Ala Leu Ala

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Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

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Ala Pro Thr Ser Ser Ser Thr Ser Ser Ser Thr Ala Glu Ala Gln Gln

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Met Asp Leu Gln Glu Leu Leu Ser Arg Met Glu Asn Tyr Arg Asn Leu

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Lys Leu Pro Arg Met Leu Thr Phe Lys Phe Tyr Leu Pro Lys Gln Ala

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Arg His Val Leu Asp Leu Thr Gln Ser Lys Ser Phe Gln Leu Glu Asp

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Ala Glu Asn Phe Ile Ser Asn Ile Arg Val Thr Val Val Lys Leu Lys

100 105 110

Gly Ser Asp Asn Thr Phe Glu Cys Gln Phe Asp Asp Glu Ser Ala Thr

115 120 125

Val Val Asp Phe Leu Arg Arg Trp Ile Ala Phe Cys Gln Ser Ile Ile

130 135 140

Ser Thr Ser Pro Gln

145

<210> 11

<211> 584

<212> PRT

<213> little mouse (Mus musculus)

<400> 11

Glu Ala His Lys Ser Glu Ile Ala His Arg Tyr Asn Asp Leu Gly Glu

1 5 10 15

Gln His Phe Lys Gly Leu Val Leu Ile Ala Phe Ser Gln Tyr Leu Gln

20 25 30

Lys Cys Ser Tyr Asp Glu His Ala Lys Leu Val Gln Glu Val Thr Asp

35 40 45

Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Ala Asn Cys Asp Lys

50 55 60

Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Ala Ile Pro Asn Leu

65 70 75 80

Arg Glu Asn Tyr Gly Glu Leu Ala Asp Cys Cys Thr Lys Gln Glu Pro

85 90 95

Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Ser Leu

100 105 110

Pro Pro Phe Glu Arg Pro Glu Ala Glu Ala Met Cys Thr Ser Phe Lys

115 120 125

Glu Asn Pro Thr Thr Phe Met Gly His Tyr Leu His Glu Val Ala Arg

130 135 140

Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Tyr Tyr Ala Glu Gln

145 150 155 160

Tyr Asn Glu Ile Leu Thr Gln Cys Cys Ala Glu Ala Asp Lys Glu Ser

165 170 175

Cys Leu Thr Pro Lys Leu Asp Gly Val Lys Glu Lys Ala Leu Val Ser

180 185 190

Ser Val Arg Gln Arg Met Lys Cys Ser Ser Met Gln Lys Phe Gly Glu

195 200 205

Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Thr Phe Pro

210 215 220

Asn Ala Asp Phe Ala Glu Ile Thr Lys Leu Ala Thr Asp Leu Thr Lys

225 230 235 240

Val Asn Lys Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp

245 250 255

Arg Ala Glu Leu Ala Lys Tyr Met Cys Glu Asn Gln Ala Thr Ile Ser

260 265 270

Ser Lys Leu Gln Thr Cys Cys Asp Lys Pro Leu Leu Lys Lys Ala His

275 280 285

Cys Leu Ser Glu Val Glu His Asp Thr Met Pro Ala Asp Leu Pro Ala

290 295 300

Ile Ala Ala Asp Phe Val Glu Asp Gln Glu Val Cys Lys Asn Tyr Ala

305 310 315 320

Glu Ala Lys Asp Val Phe Leu Gly Thr Phe Leu Tyr Glu Tyr Ser Arg

325 330 335

Arg His Pro Asp Tyr Ser Val Ser Leu Leu Leu Arg Leu Ala Lys Lys

340 345 350

Tyr Glu Ala Thr Leu Glu Lys Cys Cys Ala Glu Ala Asn Pro Pro Ala

355 360 365

Cys Tyr Gly Thr Val Leu Ala Glu Phe Gln Pro Leu Val Glu Glu Pro

370 375 380

Lys Asn Leu Val Lys Thr Asn Cys Asp Leu Tyr Glu Lys Leu Gly Glu

385 390 395 400

Tyr Gly Phe Gln Asn Ala Ile Leu Val Arg Tyr Thr Gln Lys Ala Pro

405 410 415

Gln Val Ser Thr Pro Thr Leu Val Glu Ala Ala Arg Asn Leu Gly Arg

420 425 430

Val Gly Thr Lys Cys Cys Thr Leu Pro Glu Asp Gln Arg Leu Pro Cys

435 440 445

Val Glu Asp Tyr Leu Ser Ala Ile Leu Asn Arg Val Cys Leu Leu His

450 455 460

Glu Lys Thr Pro Val Ser Glu His Val Thr Lys Cys Cys Ser Gly Ser

465 470 475 480

Leu Val Glu Arg Arg Pro Cys Phe Ser Ala Leu Thr Val Asp Glu Thr

485 490 495

Tyr Val Pro Lys Glu Phe Lys Ala Glu Thr Phe Thr Phe His Ser Asp

500 505 510

Ile Cys Thr Leu Pro Glu Lys Glu Lys Gln Ile Lys Lys Gln Thr Ala

515 520 525

Leu Ala Glu Leu Val Lys His Lys Pro Lys Ala Thr Ala Glu Gln Leu

530 535 540

Lys Thr Val Met Asp Asp Phe Ala Gln Phe Leu Asp Thr Cys Cys Lys

545 550 555 560

Ala Ala Asp Lys Asp Thr Cys Phe Ser Thr Glu Gly Pro Asn Leu Val

565 570 575

Thr Arg Cys Lys Asp Ala Leu Ala

580

<210> 12

<211> 19

<212> PRT

<213> Artificial sequence

<220>

<223> Signal peptide

<400> 12

Met Asp Trp Ile Trp Arg Ile Leu Phe Leu Val Gly Ala Ala Thr Gly

1 5 10 15

Ala His Ser

<210> 13

<211> 117

<212> PRT

<213> Artificial sequence

<220>

<223> heavy chain variable region

<400> 13

Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala

1 5 10 15

Ser Met Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Gly Tyr

20 25 30

Thr Met Asn Trp Val Lys Gln Ser His Gly Lys Asn Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Tyr Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Leu Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asp Tyr Gly Phe Val Leu Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

<210> 14

<211> 15

<212> PRT

<213> Artificial sequence

<220>

<223> GS linker

<400> 14

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

1 5 10 15

<210> 15

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> light chain variable region

<400> 15

Asp Ile Val Leu Thr Gln Ser Pro Ser Ile Met Ser Val Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Lys Leu Ser Ile Tyr

35 40 45

Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Arg

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Ala Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Ser Asn Tyr Pro Pro Trp

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ser Asp Pro Ala

100 105 110

<210> 16

<211> 69

<212> PRT

<213> Artificial sequence

<220>

<223> human CD8 hinge and transmembrane domain

<400> 16

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

35 40 45

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

50 55 60

Ile Thr Leu Tyr Cys

65

<210> 17

<211> 42

<212> PRT

<213> Artificial sequence

<220>

<223> human 4-1BB signaling domain

<400> 17

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 18

<211> 112

<212> PRT

<213> Artificial sequence

<220>

<223> human CD3zeta signaling domain

<400> 18

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 19

<211> 21

<212> PRT

<213> Artificial sequence

<220>

<223> T2A element

<400> 19

Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu

1 5 10 15

Glu Asn Pro Gly Pro

20

<210> 20

<211> 550

<212> PRT

<213> Artificial sequence

<220>

<223> efficient firefly luciferase

<400> 20

Met Glu Asp Ala Lys Asn Ile Lys Lys Gly Pro Ala Pro Phe Tyr Pro

1 5 10 15

Leu Glu Asp Gly Thr Ala Gly Glu Gln Leu His Lys Ala Met Lys Arg

20 25 30

Tyr Ala Leu Val Pro Gly Thr Ile Ala Phe Thr Asp Ala His Ile Glu

35 40 45

Val Asp Ile Thr Tyr Ala Glu Tyr Phe Glu Met Ser Val Arg Leu Ala

50 55 60

Glu Ala Met Lys Arg Tyr Gly Leu Asn Thr Asn His Arg Ile Val Val

65 70 75 80

Cys Ser Glu Asn Ser Leu Gln Phe Phe Met Pro Val Leu Gly Ala Leu

85 90 95

Phe Ile Gly Val Ala Val Ala Pro Ala Asn Asp Ile Tyr Asn Glu Arg

100 105 110

Glu Leu Leu Asn Ser Met Gly Ile Ser Gln Pro Thr Val Val Phe Val

115 120 125

Ser Lys Lys Gly Leu Gln Lys Ile Leu Asn Val Gln Lys Lys Leu Pro

130 135 140

Ile Ile Gln Lys Ile Ile Ile Met Asp Ser Lys Thr Asp Tyr Gln Gly

145 150 155 160

Phe Gln Ser Met Tyr Thr Phe Val Thr Ser His Leu Pro Pro Gly Phe

165 170 175

Asn Glu Tyr Asp Phe Val Pro Glu Ser Phe Asp Arg Asp Lys Thr Ile

180 185 190

Ala Leu Ile Met Asn Ser Ser Gly Ser Thr Gly Leu Pro Lys Gly Val

195 200 205

Ala Leu Pro His Arg Thr Ala Cys Val Arg Phe Ser His Ala Arg Asp

210 215 220

Pro Ile Phe Gly Asn Gln Ile Ile Pro Asp Thr Ala Ile Leu Ser Val

225 230 235 240

Val Pro Phe His His Gly Phe Gly Met Phe Thr Thr Leu Gly Tyr Leu

245 250 255

Ile Cys Gly Phe Arg Val Val Leu Met Tyr Arg Phe Glu Glu Glu Leu

260 265 270

Phe Leu Arg Ser Leu Gln Asp Tyr Lys Ile Gln Ser Ala Leu Leu Val

275 280 285

Pro Thr Leu Phe Ser Phe Phe Ala Lys Ser Thr Leu Ile Asp Lys Tyr

290 295 300

Asp Leu Ser Asn Leu His Glu Ile Ala Ser Gly Gly Ala Pro Leu Ser

305 310 315 320

Lys Glu Val Gly Glu Ala Val Ala Lys Arg Phe His Leu Pro Gly Ile

325 330 335

Arg Gln Gly Tyr Gly Leu Thr Glu Thr Thr Ser Ala Ile Leu Ile Thr

340 345 350

Pro Glu Gly Asp Asp Lys Pro Gly Ala Val Gly Lys Val Val Pro Phe

355 360 365

Phe Glu Ala Lys Val Val Asp Leu Asp Thr Gly Lys Thr Leu Gly Val

370 375 380

Asn Gln Arg Gly Glu Leu Cys Val Arg Gly Pro Met Ile Met Ser Gly

385 390 395 400

Tyr Val Asn Asn Pro Glu Ala Thr Asn Ala Leu Ile Asp Lys Asp Gly

405 410 415

Trp Leu His Ser Gly Asp Ile Ala Tyr Trp Asp Glu Asp Glu His Phe

420 425 430

Phe Ile Val Asp Arg Leu Lys Ser Leu Ile Lys Tyr Lys Gly Tyr Gln

435 440 445

Val Ala Pro Ala Glu Leu Glu Ser Ile Leu Leu Gln His Pro Asn Ile

450 455 460

Phe Asp Ala Gly Val Ala Gly Leu Pro Asp Asp Asp Ala Gly Glu Leu

465 470 475 480

Pro Ala Ala Val Val Val Leu Glu His Gly Lys Thr Met Thr Glu Lys

485 490 495

Glu Ile Val Asp Tyr Val Ala Ser Gln Val Thr Thr Ala Lys Lys Leu

500 505 510

Arg Gly Gly Val Val Phe Val Asp Glu Val Pro Lys Gly Leu Thr Gly

515 520 525

Lys Leu Asp Ala Arg Lys Ile Arg Glu Ile Leu Ile Lys Ala Lys Lys

530 535 540

Gly Gly Lys Ile Ala Val

545 550

<210> 21

<211> 239

<212> PRT

<213> Artificial sequence

<220>

<223> eGFP

<400> 21

Met Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu

1 5 10 15

Val Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly

20 25 30

Glu Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile

35 40 45

Cys Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr

50 55 60

Leu Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys

65 70 75 80

Gln His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu

85 90 95

Arg Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu

100 105 110

Val Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly

115 120 125

Ile Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr

130 135 140

Asn Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn

145 150 155 160

Gly Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser

165 170 175

Val Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly

180 185 190

Pro Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu

195 200 205

Ser Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe

210 215 220

Val Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys

225 230 235

<210> 22

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> epitope

<400> 22

Ser Ile Ile Asn Phe Glu Lys Leu

1 5

Claims

1. A method of inducing an immune response in a subject, comprising:

b. administering to the subject IL2 or a polynucleotide encoding IL 2.

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

3. The method of claim 2, wherein the other cytokine is selected from the group consisting of IL7 and IL 21.

4. The method of any one of claims 1-3, comprising administering IL2 or a polynucleotide encoding IL2 and IL7 or a polynucleotide encoding IL 7.

5. The method of any one of claims 1-3, comprising administering IL2 or a polynucleotide encoding IL2 and IL21 or a polynucleotide encoding IL 21.

6. The method of any one of claims 1-5, wherein the polynucleotide encoding IL2 is RNA, and optionally, the polynucleotide encoding the other cytokine is RNA.

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

8. The method of any one of claims 1-7, further comprising administering to the subject an antigen or variant thereof or a polynucleotide encoding the antigen or variant, wherein the genetic modification targets the antigen with CAR-expressing T cells and the immune response is an immune response against a target cell population or target tissue that expresses the antigen.

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

10. A method of inducing an immune response in a subject, comprising:

b. administering to the subject an RNA encoding IL 2.

11. The method of claim 10, comprising administering an RNA encoding IL2 and an RNA encoding other cytokines.

12. The method of claim 11, wherein the other cytokine is selected from the group consisting of IL7 and IL 21.

13. The method of any one of claims 10-12, comprising administering an RNA encoding IL2 and an RNA encoding IL 7.

14. The method of any one of claims 10-12, comprising administering an RNA encoding IL2 and an RNA encoding IL 21.

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

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

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

18. A method of treating a subject having a disease, disorder or condition associated with expression or elevated expression of an antigen, comprising:

b. administering to the subject IL2 or a polynucleotide encoding IL 2.

19. The method of claim 18, comprising administering IL2 or a polynucleotide encoding IL2 in combination with other cytokines or polynucleotides encoding other cytokines.

20. The method of claim 19, wherein the other cytokine is selected from the group consisting of IL7 and IL 21.

21. The method of any one of claims 18-20, comprising administering IL2 or a polynucleotide encoding IL2 and IL7 or a polynucleotide encoding IL 7.

22. The method of any one of claims 18-20, comprising administering IL2 or a polynucleotide encoding IL2 and IL21 or a polynucleotide encoding IL 21.

23. The method of any one of claims 18-22, wherein the polynucleotide encoding IL2 is RNA, and optionally, the polynucleotide encoding the other cytokine is RNA.

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

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

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

27. A method of treating a subject having a disease, disorder or condition associated with expression or elevated expression of an antigen, comprising:

b. administering to the subject an RNA encoding IL 2.

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

29. The method of claim 28, wherein the other cytokine is selected from the group consisting of IL7 and IL 21.

30. The method of any one of claims 27-29, comprising administering an RNA encoding IL2 and an RNA encoding IL 7.

31. The method of any one of claims 27-29, comprising administering an RNA encoding IL2 and an RNA encoding IL 21.

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

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

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

35. The method of any one of claims 1-34, wherein IL2 is extended Pharmacokinetic (PK) IL 2.

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

37. The method of claim 36, wherein 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.

38. The method of any one of claims 2-9, 11-17, 19-26 and 28-37, wherein the other cytokine, in particular IL7 or IL21, is a Pharmacokinetic (PK) extending cytokine, in particular PK extending IL7 or PK extending IL 21.

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

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

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

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

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

44. A pharmaceutical product, comprising:

il2 or a polynucleotide encoding IL 2.

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

46. The pharmaceutical product of claim 45 wherein the additional cytokine is selected from the group consisting of IL7 and IL 21.

47. The pharmaceutical product of any one of claims 44-46, comprising IL2 or a polynucleotide encoding IL2 and IL7 or a polynucleotide encoding IL 7.

48. The pharmaceutical product of any one of claims 44-46, comprising IL2 or a polynucleotide encoding IL2 and IL21 or a polynucleotide encoding IL 21.

49. The pharmaceutical product of any one of claims 44-48, wherein the polynucleotide encoding IL2 is RNA, and optionally, the polynucleotide encoding the additional cytokine is RNA.

50. The pharmaceutical product of any one of claims 44-49, further comprising an antigen or variant thereof or a polynucleotide encoding the antigen or variant, wherein the T cell genetically modified to express the CAR is targeted to the antigen.

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

52. The pharmaceutical product of any one of claims 44-51 which is a kit.

53. The pharmaceutical preparation of claim 52, which comprises a T cell genetically modified to express a CAR, IL2, or a polynucleotide encoding IL2, optionally other cytokines or polynucleotides encoding other cytokines, and optionally the antigen or variant thereof or a polynucleotide encoding the antigen or variant, in separate containers.

54. The pharmaceutical product of claim 52 or 53, further comprising instructions for use of the pharmaceutical product in the treatment or prevention of cancer, wherein the antigen is a tumor associated antigen.

55. A pharmaceutical product according to any one of claims 44 to 51 which is a pharmaceutical composition.

56. The pharmaceutical product of claim 55, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, and/or excipients.

57. A pharmaceutical product, comprising:

b. RNA encoding IL 2.

58. The pharmaceutical preparation of claim 57, which comprises RNA encoding IL2 and RNA encoding other cytokines.

59. The pharmaceutical product of claim 58, wherein the additional cytokine is selected from the group consisting of IL7 and IL 21.

60. The pharmaceutical product of any one of claims 57-59, comprising an RNA encoding IL2 and an RNA encoding IL 7.

61. The pharmaceutical product of any one of claims 57-60, comprising an RNA encoding IL2 and an RNA encoding IL 21.

62. The pharmaceutical product of any one of claims 57-61, further comprising an RNA encoding an antigen or a variant thereof, wherein the T cells genetically modified to express the CAR are targeted to the antigen.

63. The pharmaceutical product of any one of claims 57-62, which is a kit.

64. The pharmaceutical preparation of claim 63, comprising a T cell genetically modified to express a CAR, an RNA encoding IL2, optionally an RNA encoding other cytokines, and optionally an RNA encoding an antigen or a variant thereof, in separate containers.

65. The pharmaceutical product of claim 63 or 64, further comprising instructions for use of the pharmaceutical product in the treatment or prevention of cancer, wherein the antigen is a tumor associated antigen.

66. A pharmaceutical product according to any one of claims 57 to 62 which is a pharmaceutical composition.

67. The pharmaceutical product of claim 66, wherein the pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers, diluents, and/or excipients.

68. The pharmaceutical product of any one of claims 44-67, wherein IL2 is extended Pharmacokinetic (PK) IL 2.

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

70. The pharmaceutical product of claim 69, wherein 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.

71. The pharmaceutical product of any one of claims 45-56 and 58-70, wherein the additional cytokine is a prolonged Pharmacokinetic (PK) cytokine.

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

73. The pharmaceutical product of claim 72, wherein the fusion protein comprises a cytokine moiety and a moiety selected from the group consisting of: serum albumin, immunoglobulin fragments, transferrin, Fn3, and variants thereof.

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

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

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

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

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