In vitro and in vivo gene delivery to immune effector cells using nanoparticles functionalized with designed ankyrin repeat proteins (darpins)

Abstract

Provided are therapies comprising immune effector cells, such as T cells, engineered to express an antigen receptor, such as a T cell receptor or a chimeric antigen receptor.SOLUTION: It is demonstrated that antigen receptor-engineered immune effector cells can be generated in vitro / ex vivo as well as in vitro by delivering a nucleic acid encoding an antigen receptor for genetic modification to a cell using a particle comprising the nucleic acid and a targeting molecule for targeting the immune effector cell, wherein the targeting molecule is a designed ankyrin repeat protein (DARPin). In particular, DARPins are provided that are high affinity binders for CD8 binding to CD8 receptors on human and non-human primate (NHP) cells. Nanoparticles functionalized with CD8 targeting DARPins (CD8 - DARPins) can deliver genes exclusively and specifically to human CD8 + T cells in vitro and in vivo.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure generally relates to therapies involving immune effector cells, such as T cells, engineered to express an antigen receptor, such as a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In one embodiment, the immune effector cells are genetically modified to express the antigen receptor. Such genetic modification can be performed ex vivo or in vitro, and the immune effector cells can then be administered to a subject in need of treatment, or can be performed in vivo in a subject in need of treatment. These methods are particularly useful for treating cancers characterized by disease cells expressing an antigen against which the antigen receptor is directed. It is demonstrated herein that such antigen receptor-engineered immune effector cells can be generated in vitro / ex vivo and in vitro by delivering a nucleic acid encoding the antigen receptor for genetic modification to cells using particles containing the nucleic acid and a targeting molecule for targeting the immune effector cells, where the targeting molecule is a designed ankyrin repeat protein (DARPin). In particular, DARPins are described herein as high-affinity binders for CD8 binding to the CD8 receptor on human and non-human primate (NHP) cells. Nanoparticles functionalized with CD8-targeting DARPins (CD8-DARPins) bind to human CD8 in vitro and in vivo. +Genes can be delivered exclusively and specifically to T cells. Antigen receptor-engineered immune effector cells can be provided to a subject by administering antigen receptor-engineered immune effector cells or by generating antigen receptor-engineered immune effector cells in the subject. In one embodiment, the antigen receptor-engineered immune effector cells are generated in the subject to be treated. Furthermore, the target antigen for the antigen receptor can be provided to the subject by administering to the subject the antigen targeted by the antigen receptor, a polynucleotide encoding the antigen, or a cell expressing the antigen. The antigen targeted by the antigen receptor can include a naturally occurring antigen or a variant thereof, or a fragment of a naturally occurring antigen or a variant thereof. In a particularly preferred embodiment, the polynucleotide encoding the antigen is RNA. The methods and medicaments described herein are particularly useful for treating diseases characterized by diseased cells expressing an antigen receptor or an antigen against which the antigen receptor-engineered immune effector cells are directed. [Background technology]

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

[0003] Adoptive cell transfer (ACT)-based immunotherapy can be broadly defined as a form of passive immunization with previously primed T cells that are expanded ex vivo from low precursor frequencies to clinically relevant cell numbers and then transferred into a non-immune recipient or autologous host. Cell types that have been used in ACT experiments include lymphokine-activated killer (LAK) cells (Mule, JJ et al. (1984) Science 225, 1487-1489; Rosenberg, SA et al. (1985) N. Engl. J. Med. 313, 1485-1492), tumor-infiltrating lymphocytes (TILs) (Rosenberg, SA et al. (1994) J. Natl. Cancer Inst. 86, 1159-1166), donor lymphocytes after hematopoietic stem cell transplantation (HSCT), and tumor-specific T cell lines or clones (Dudley, ME et al. (2001) J. Immunother. 24, 363-373; Yee, C. et al. (2002) Proc. Natl. Acad. Sci. USA 99, 16168-16173). An alternative approach is the adoptive transfer of autologous T cells reprogrammed to express tumor-reactive immune receptors of defined specificity during short-term ex vivo culture, followed by reinfusion into the patient (Kershaw MH et al. (2013) Nature Reviews Cancer 13(8):525-41). This strategy makes ACT applicable to a variety of common malignancies, even when tumor-reactive T cells are not present in the patient. For example, adoptive transfer of chimeric antigen receptor-modified T cells (CAR T cells) is being investigated in a wide range of clinical trials worldwide (Figure 1, left). Chimeric antigen receptors (CARs) are a type of antigen-targeting receptor consisting of 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 directly recognize cell surface antigens, independent of MHC-mediated presentation, allowing the use of a single receptor construct specific for a given antigen in every patient.Typically, CARs fuse an antigen-recognition domain to the CD3ζ activation chain of the T cell receptor (TCR) complex, and in parallel with CD3ζ, contain secondary costimulatory signals containing intracellular domains from various TNF receptor family molecules, such as CD28 or 4-1BB (CD137) and OX40 (CD134). CARs have dramatically improved antitumor efficacy and demonstrated remarkable clinical efficacy, especially in patients with hematological malignancies (Hartmann, J. et al. EMBO Mol. Med. 9, 1183-1197 (2017)). Recently, two CAR-T cell therapies have been approved by the FDA and EMA for the treatment of B-cell acute lymphoblastic leukemia (Kymriah®) and diffuse large B-cell lymphoma (Yescarta®) (Zheng, P. et al. Drug. Discov. Today 6, 1175-1182 (2018)). However, in the case of solid tumors, adoptive transfer of T cells has so far shown limited efficacy and requires improvement (Newick, K. et al. Annu. Rev. Med. 68, 139-152 (2017)).

[0004] Recently, receptor-targeted lentiviral vectors (LVs) have been shown to enable selective gene transfer to specific types of lymphocytes in vivo. LVs are pseudotyped with a single-chain variable fragment (scFv) against a receptor on the desired target cell, and another component of the envelope protein mediates membrane fusion with the target cell upon binding. This technology reduces the generation of CAR T cells to a single in vivo transduction process. Unfortunately, the production of such LVs remains a laborious and cost-intensive process, but it avoids the need for apheresis and the ex vivo handling of the patient's own T cells. Another strategy is to create lipid- or polymer-based artificial delivery systems (non-viral vectors) that can mimic LV function (Figure 1, right). Such nanoparticles (NPs) need to be functionalized by displaying / attaching a targeting ligand to their surface to mediate receptor-specific binding. The targeting ligand can be derived from a parent antibody, e.g., an scFv. In contrast to LVs, binding of the targeting ligand to its receptor is required to induce receptor-mediated endocytosis and trafficking to enable NP uptake. Indeed, receptor-targeted LVs are designed not to mediate endocytosis to avoid endosomal transport and lysis. On the other hand, there are NP variants (mainly polymer- or lipid-based) called polyplexes (PLX, upper panel in Figure 2) or lipid NPs (LNP, lower panel in Figure 2) that have the potential for endosomal escape. To fully mimic retroviral vectors and thus enable genome manipulation via NP-mediated gene delivery, the cargo must consist of a gene editing tool such as CRISPR / Cas9 (or related) or a transposon system such as Sleeping Beauty or Piggybag. Nevertheless, delivery of mRNA is also an option for inducing transient expression of therapeutic receptors such as CARs or T cell receptors (TCRs). Indeed, initial studies have recently demonstrated that NPs can generate CAR T cells in vivo.Again, the initial efficiency of this process is very low, and only CD19 can be targeted as long as circulating B cells present a small number of target cells to stimulate and expand in vivo generated CAR T cells. Recently, we developed a CAR vaccine concept (CARVac) based on nanoparticle-mediated delivery of mRNA for in vivo presentation of CAR antigens on professional antigen-presenting cells to induce in vivo expansion of CAR T cells (Figure 3). This technology not only enables efficient treatment of non-hematologic tumors with CAR T cells, but also overcomes the obstacle of low efficiency in in vivo generation of CAR T cells, since CARVac can expand low numbers of CAR T cells to therapeutically sufficient levels. Furthermore, the entire concept can be transferred to other immune receptors, such as TCRs.

[0005] Taken together, this approach could facilitate new ways of genetically engineering a patient's own T cells, potentially shifting the whole concept from personalized medicine to off-the-shelf treatments in the future. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Mule, JJet al.(1984)Science 225,1487-1489;Rosenberg,SAet al.(1985)N.Engl.J.Med.313,1485-1492 [Non-patent document 2] Rosenberg, SA et al. (1994) J. Natl. Cancer Inst. 86, 1159-1166 [Non-patent document 3] Dudley, ME et al. (2001) J. Immunother. 24, 363-373 [Non-patent document 4] Yee, C. et al. (2002) Proc. Natl. Acad. Sci. USA 99, 16168-16173 [Non-patent document 5] Kershaw MH et al. (2013) Nature Reviews Cancer 13(8):525-41 [Non-patent document 6] Hartmann,J.et al.EMBO Mol.Med.9,1183-1197(2017) [Non-Patent Document 7] Zheng,P.et al.Drug.Discov.Today 6,1175-1182(2018) [Non-patent document 8] Newick,K.et al.Annu.Rev.Med.68,139-152(2017) Summary of the Invention [Problem to be solved by the invention]

[0007] Despite its success, the overall in vivo transduction efficiency of CD8-specific scFv-LVs was quite low. Therefore, strategies to improve gene delivery to immune effector cells, especially in vivo, are needed. Such gene delivery may be useful for targeting cytotoxic T cells, which is particularly important for the further development of in vivo CAR T cell generation.

[0008] We describe here novel high-affinity binders, which are ankyrin repeat protein (DARPin)-based molecules designed to target surface antigens on immune effector cells. Specifically, we describe high-affinity binders for CD8 composed of DARPins selected to bind to the CD8 receptor on human and non-human primate (NHP) cells. These binders were identified by ribosome display screening of a DARPin library using recombinant human CD8 followed by receptor binding analysis on primary lymphocytes. Various NPs were then genetically engineered to target human CD8 in vitro and in vivo. +They were functionalized by various coupling strategies with CD8-targeted DARPins (CD8-DARPins) that deliver exclusively and specifically to T cells. Functionalization of particles carrying cargo for genetic modification of immune effector cells with the binders described herein results in the specific delivery of the cargo to immune effector cells and the modification of immune effector cells. [Means for solving the problem]

[0009] The present invention generally encompasses the treatment of disease by targeting cells, such as diseased cells, that express antigens, such as tumor antigens. Target cells may express antigens on the cell surface for recognition by a chimeric antigen receptor (CAR) or in the context of MHC for recognition by a T cell receptor (TCR). The method provides selective eradication of such cells expressing the antigen, thereby minimizing adverse effects on normal cells that do not express the antigen. Immune effector cells genetically modified to express a chimeric antigen receptor (CAR) or T cell receptor (TCR) that targets cells via binding to the antigen (or its processing product) are provided to a subject, such as by administering the genetically modified immune effector cells to the subject or by generating genetically modified immune effector cells in the subject. Genetic modification is achieved using particles containing a nucleic acid encoding the antigen receptor for genetic modification and a targeting molecule for targeting the immune effector cells, where the targeting molecule is a designed ankyrin repeat protein (DARPin). The particles can deliver nucleic acids to cells in vitro / ex vivo as well as in vivo. A vaccine antigen, which may be a disease-associated antigen or a variant thereof (e.g., a peptide or protein comprising an epitope of the disease-associated antigen), a nucleic acid encoding the same, or cells expressing the antigen, may be administered to provide the antigen for stimulation, priming, and / or expansion of immune effector cells (optionally after expression of the nucleic acid by appropriate target cells). The stimulated, primed, and / or expanded immune effector cells in the patient can recognize and eradicate disease cells expressing the antigen. In one embodiment, the immune effector cells are CD8 +In one embodiment, the targeting molecule described herein is a CD8 T cell. + It binds to the CD8 receptor on T cells. In one embodiment, the immune effector cells are directed against a tumor or cancer. In one embodiment, the target cell population or target tissue is a tumor cell or tumor tissue, particularly of a solid tumor. In one embodiment, the target antigen is a tumor antigen.

[0010] The methods and medicaments described herein are particularly useful for treating diseases characterized by disease cells expressing antigens against which immune effector cells are directed. In one aspect, chimeric antigen receptor (CAR)-based immune effector cells have binding specificity for vaccine antigens and disease-associated antigens when present on antigen-presenting cells and disease cells, respectively. In one embodiment, T cell receptor (TCR)-based immune effector cells have binding specificity for processed products of vaccine antigens and disease-associated antigens when presented on antigen-presenting cells and disease cells, respectively. CARs are molecules that combine specificity for a desired antigen (e.g., a tumor antigen), preferably antibody-based, with a T cell receptor activating intracellular domain to generate chimeric proteins that exhibit specific cellular immune activity (e.g., specific anti-tumor cellular immune activity). Preferably, the cells stably express antigen receptors on their surface and can be genetically modified to confer novel antigen specificity, which may be MHC-independent. In one embodiment, immune effector cells from either the subject to be treated or a different subject are administered to the subject to be treated. The administered immune effector cells can be genetically modified ex vivo before administration, or can be genetically modified in vivo in the subject after administration to express the antigen receptor described herein. In one embodiment, the immune effector cells are endogenous to the subject being treated (and therefore not administered to the subject being treated) and are genetically modified in vivo in the subject to express the antigen receptor described herein. Thus, the immune effector cells can be genetically modified ex vivo or in vivo to express the antigen receptor. Thus, such genetic modification with the antigen receptor can be performed in vitro, after which the immune effector cells can be administered to the subject in need of treatment, or can be performed in vivo in the subject in need of treatment. In one aspect, the present invention generally encompasses the treatment of disease by targeting diseased cells, particularly cells that express an antigen, such as cancer cells that express a tumor antigen. The target cells can express the antigen on the cell surface or display a processed product of the antigen. In one embodiment, the antigen is a tumor-associated antigen, and the disease is cancer.Such treatment provides selective eradication of cells expressing the antigen, thereby minimizing adverse effects on normal cells that do not express the antigen. In one embodiment, a vaccine antigen, a polynucleotide encoding it, or cells expressing the vaccine antigen are administered to provide the antigen for stimulation, priming, and / or expansion of immune effector cells genetically modified to express an antigen receptor (optionally after expression of the polynucleotide by appropriate target cells), where the immune effector cells target the antigen or its processing products, and the immune response is directed against a target cell population or tissue expressing the antigen. In one embodiment, the polynucleotide encoding the vaccine antigen is RNA. Immune effector cells, such as T cells, stimulated, primed, and / or expanded in the patient can recognize cells expressing the antigen, resulting in eradication of disease cells. In one embodiment, the RNA encoding the vaccine antigen is targeted to secondary lymphoid organs.

[0011] In one aspect, the invention relates to a method for preparing immune effector cells genetically modified to express an antigen receptor, the method comprising contacting the immune effector cells with particles comprising a nucleic acid encoding the antigen receptor and a targeting molecule for targeting the immune effector cells, wherein the targeting molecule is an ankyrin repeat protein.

[0012] In one embodiment, contacting an immune effector cell with the particle delivers the nucleic acid to the immune effector cell.

[0013] In one embodiment, the genetically modified immune effector cells are present in vivo or in vitro. In one embodiment, the genetically modified immune effector cells are present in vivo. In one embodiment, the genetically modified immune effector cells are present in vivo in a subject, and the method comprises administering the particles to the subject.

[0014] In a further aspect, the present invention provides a method for treating a subject, comprising: (i) preparing in vitro immune effector cells genetically modified to express an antigen receptor, using a method comprising contacting the immune effector cells with particles comprising a nucleic acid encoding the antigen receptor and a targeting molecule for targeting the immune effector cells, wherein the targeting molecule is an ankyrin repeat protein; and (ii) administering immune effector cells genetically modified to express an antigen receptor to the subject. The present invention relates to a method comprising:

[0015] In one embodiment, contacting an immune effector cell with the particle delivers the nucleic acid to the immune effector cell.

[0016] In a further aspect, the present invention provides a method for treating a subject, comprising: The present invention relates to a method comprising administering to a subject particles comprising a nucleic acid encoding an antigen receptor and a targeting molecule for targeting immune effector cells, wherein the targeting molecule is an ankyrin repeat protein.

[0017] In one embodiment, the particles deliver the nucleic acid to immune effector cells of a subject.

[0018] In one embodiment, immune effector cells that are genetically modified to express an antigen receptor are generated in a subject by delivering the nucleic acid to the immune effector cells.

[0019] In one embodiment, the method described herein is a method for inducing an immune response in a subject. In one embodiment, the immune response is a T cell-mediated immune response. In one embodiment, the immune response is an immune response against a target cell population or target tissue that expresses an antigen. In one embodiment, the target cell population or target tissue is a cancer cell or cancer tissue. In one embodiment, the cancer cell or cancer tissue is a solid tumor.

[0020] In a further aspect, the present invention provides a method for treating a subject having a disease, disorder, or condition associated with expression or up-regulation of an antigen, comprising: (i) preparing in vitro immune effector cells genetically modified to express an antigen receptor that targets an antigen associated with a disease, disorder, or condition, or a cell expressing an antigen associated with a disease, disorder, or condition, using a method comprising contacting the immune effector cells with particles comprising a nucleic acid encoding the antigen receptor and a targeting molecule for targeting the immune effector cells, wherein the targeting molecule is an ankyrin repeat protein; and (ii) administering immune effector cells genetically modified to express an antigen receptor to the subject. The present invention relates to a method comprising:

[0021] In one embodiment, contacting an immune effector cell with the particle delivers the nucleic acid to the immune effector cell.

[0022] In a further aspect, the present invention provides a method for treating a subject having a disease, disorder, or condition associated with expression or up-regulation of an antigen, comprising: administering to a subject particles comprising a nucleic acid encoding an antigen receptor that targets an antigen associated with a disease, disorder, or condition or a cell that expresses an antigen associated with a disease, disorder, or condition, and a targeting molecule for targeting immune effector cells, the targeting molecule being an ankyrin repeat protein. The present invention relates to a method comprising:

[0023] In one embodiment, the particles deliver the nucleic acid to immune effector cells of a subject.

[0024] In one embodiment, immune effector cells that are genetically modified to express an antigen receptor are generated in a subject by delivering the nucleic acid to the immune effector cells.

[0025] In one embodiment, the disease, disorder or condition is cancer and the antigen associated with the disease, disorder or condition is a tumor antigen, hi one embodiment, the disease, disorder or condition is a solid tumor.

[0026] In one embodiment, the method described herein is a method for treating or preventing cancer in a subject. In one embodiment, the cancer is a solid cancer. In one embodiment, the cancer is associated with expression or increased expression of a tumor antigen targeted by an antigen receptor.

[0027] In one embodiment, the method described herein further comprises administering to a subject an antigen targeted by an antigen receptor, a polynucleotide encoding the antigen, or a host cell genetically modified to express the antigen. In one embodiment, the polynucleotide is RNA. In one embodiment, the host cell comprises a polynucleotide encoding the antigen.

[0028] In one embodiment of all aspects described herein, the antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0029] In one embodiment of all aspects described herein, the nucleic acid is RNA.

[0030] In one embodiment of all aspects described herein, the nucleic acid is DNA.

[0031] In one embodiment of all aspects described herein, the genetic modification is transient or stable.

[0032] In one embodiment of all aspects described herein, genetic modification is carried out by virus-based method, transposon-based method or gene editing-based method.In one embodiment, gene editing-based method comprises CRISPR-based gene editing.

[0033] In one embodiment of all aspects described herein, the particle is a non-viral particle.In one embodiment of all aspects described herein, the particle is a lipid-based and / or polymer-based particle.In one embodiment of all aspects described herein, the particle is a nanoparticle.

[0034] In one embodiment of all aspects described herein, the particles are functionalized with targeting molecules on their surfaces. In one embodiment of all aspects described herein, the particles are functionalized with targeting molecules by linking the targeting molecules to at least one particle-forming component.

[0035] In one embodiment of all aspects described herein, the immune effector cells are T cells. In one embodiment of all aspects described herein, the immune effector cells are CD8+ T cells.

[0036] In one embodiment of all aspects described herein, the targeting molecule targets CD8.

[0037] In one embodiment of all aspects described herein, the targeting molecule comprises a repeating consensus sequence: NX1X2DX3X4X5X6TPX7HLX8X9X 10 X 11 X 12 HX 13 X 14 IVX 15 VLLKX 16 X 17 X 18 DX 19 a repeat module including where: X1 is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V; X2 is any amino acid, X3 is any amino acid, X4 is any amino acid, X5 is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably G; X6 is any amino acid, X7 is any amino acid, preferably an amino acid selected from the group consisting of A, C, F, G, H, I, K, L, M, R, T, V, W, Y, more preferably L; X8 is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably A or V; X9 is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably A; X 10 is any amino acid, X 11 is any amino acid, X 12 is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably G; X 13 is any amino acid, preferably L, X 14 is any amino acid, preferably an amino acid selected from the group consisting of D, E, H, K and R, more preferably E; X 15 is any amino acid, preferably D or E, X 16 is any amino acid, X 17 is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably an amino acid selected from the group consisting of A, G and S, more preferably G; X 18 is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably A; X 19 is any amino acid, preferably an amino acid selected from the group consisting of I, L and V, more preferably V.

[0038] In one embodiment of all aspects described herein, the targeting molecule comprises a repeating consensus sequence: NX1X2DX3X4GX6TPLHLX8X9X 10 X 11 GHX 13 X 14 IVX15 VLLKX 16 GADV a repeat module including where: X1 is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V; X2 is any amino acid, X3 is any amino acid, X4 is any amino acid, X6 is any amino acid, X8 is A or V, X9 is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably A; X 10 is any amino acid, X 11 is any amino acid, X 13 is any amino acid, preferably L, X 14 is any amino acid, preferably an amino acid selected from the group consisting of D, E, H, K and R, more preferably E; X 15 is any amino acid, preferably D or E, X 16 is any amino acid.

[0039] In one embodiment of all aspects described herein, the targeting molecule comprises a repeating consensus sequence: NX1X2DX3X4GX6TPLHLX8AX 10 X 11 GHLEIVX 15 VLLKX 16 GADV a repeat module including where: X1 is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V; X2 is any amino acid, X3 is any amino acid, X4 is any amino acid, X6 is any amino acid, X8 is A or V, X 10 is any amino acid, X 11 is any amino acid, X 15 is D or E, X 16 is any amino acid.

[0040] In one embodiment of all aspects described herein, the targeting molecule comprises at least two repeat modules, which may be identical or different, each comprising a repeat consensus sequence.

[0041] In one embodiment of all aspects described herein, the targeting molecule comprises 2 to 20 repeat modules, which may be identical or different, each comprising a repeat consensus sequence.

[0042] In one embodiment of all aspects described herein, the targeting molecule comprises three repeat modules, which may be identical or different, each comprising a repeat consensus sequence.

[0043] In one embodiment of all aspects described herein, the targeting molecule comprises three repeat modules, wherein: The first repeat module of the targeting molecule has the consensus sequence: NAX2DX3X4GX6TPLHLX8AWHGHLEIVX 15 VLLKX 16 GADV, Including, The second repeat module of the targeting molecule has the consensus sequence: NAX2DX3X4GX6TPLHLAAX 10 X 11 GHLEIVEVLLKX 16 GADV, and The third repeat module of the targeting molecule has the consensus sequence: NX1X2DX3X4GX6TPLHLAAX 10 X 11 GHLEIVEVLLKX 16 GADV, Including, where: X1 is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V; X2 is any amino acid, X3 is any amino acid, X4 is any amino acid, X6 is any amino acid, X8 is A or V, X 10 is any amino acid, X 11 is any amino acid, X 15 is D or E, X 16 is any amino acid, preferably an amino acid selected from the group consisting of Y, H and N.

[0044] In one embodiment of all aspects described herein, the targeting molecule comprises at least one repeat module comprising a sequence selected from the group of repeat modules 1, 2 and 3 of SEQ ID NOs: 1-28, respectively, as shown in Figure 5.

[0045] In one embodiment of all aspects described herein, the targeting molecule comprises three repeat modules, wherein repeat module 1 is selected from the group of repeat module 1 of SEQ ID NOs: 1-28 as shown in Figure 5, repeat module 2 is selected from the group of repeat module 2 of SEQ ID NOs: 1-28 as shown in Figure 5 and repeat module 3 is selected from the group of repeat module 3 of SEQ ID NOs: 1-28 as shown in Figure 5.

[0046] In one embodiment of all aspects described herein, the targeting molecule comprises three repeat modules, wherein repeat module 1, repeat module 2 and repeat module 3 are repeat module 1, repeat module 2 and repeat module 3 of sequences selected from the group consisting of SEQ ID NOs: 1 to 28 as shown in Figure 5.

[0047] In one embodiment of all aspects described herein, the repeat module is present in the repeat domain.

[0048] In one embodiment of all aspects described herein, the repeat domain further comprises an N-terminal and / or a C-terminal capping module.

[0049] In one embodiment of all aspects described herein, the targeting molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1-28, or positions 29-127 thereof.

[0050] In a further aspect, the present invention relates to molecules comprising ankyrin repeat proteins that target immune effector cells.

[0051] In one embodiment, the immune effector cells are T cells. In one embodiment, the immune effector cells are CD8+ T cells.

[0052] In one embodiment, the molecule targets CD8.

[0053] In one embodiment, the ankyrin repeat protein comprises the repeat consensus sequence: NX1X2DX3X4X5X6TPX7HLX8X9X 10 X 11 X 12 HX 13 X 14 IVX 15 VLLKX 16 X 17 X 18 DX 19 a repeat module including where: X1 is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V; X2 is any amino acid, X3 is any amino acid, X4 is any amino acid, X5 is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably G; X6 is any amino acid, X7 is any amino acid, preferably an amino acid selected from the group consisting of A, C, F, G, H, I, K, L, M, R, T, V, W, Y, more preferably L; X8 is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably A or V; X9 is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably A; X 10 is any amino acid, X 11 is any amino acid, X 12 is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably G; X 13 is any amino acid, preferably L, X 14 is any amino acid, preferably an amino acid selected from the group consisting of D, E, H, K and R, more preferably E; X 15 is any amino acid, preferably D or E, X 16 is any amino acid, X 17 is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably an amino acid selected from the group consisting of A, G and S, more preferably G; X 18 is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably A; X 19 is any amino acid, preferably an amino acid selected from the group consisting of I, L and V, more preferably V.

[0054] In one embodiment, the ankyrin repeat protein comprises the repeat consensus sequence: NX1X2DX3X4GX6TPLHLX8X9X 10 X 11 GHX 13 X 14 IVX 15 VLLKX 16 GADV a repeat module including where: X1 is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V; X2 is any amino acid, X3 is any amino acid, X4 is any amino acid, X6 is any amino acid, X8 is A or V, X9 is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably A; X 10 is any amino acid, X 11 is any amino acid, X 13 is any amino acid, preferably L, X 14 is any amino acid, preferably an amino acid selected from the group consisting of D, E, H, K and R, more preferably E; X 15 is any amino acid, preferably D or E, X 16 is any amino acid.

[0055] In one embodiment, the ankyrin repeat protein comprises the repeat consensus sequence: NX1X2DX3X4GX6TPLHLX8AX 10 X 11 GHLEIVX 15 VLLKX 16 GADV a repeat module including where: X1 is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V; X2 is any amino acid, X3 is any amino acid, X4 is any amino acid, X6 is any amino acid, X8 is A or V, X 10 is any amino acid, X 11 is any amino acid, X 15 is D or E, X 16 is any amino acid.

[0056] In one embodiment, the ankyrin repeat protein comprises at least two repeat modules, which may be identical or different, each comprising a repeat consensus sequence.

[0057] In one embodiment, the ankyrin repeat protein comprises 2 to 20 repeat modules, which may be identical or different, each containing a repeated consensus sequence.

[0058] In one embodiment, the ankyrin repeat protein comprises three repeat modules, which may be identical or different, each containing a repeated consensus sequence.

[0059] In one embodiment the ankyrin repeat protein comprises three repeat modules, wherein: The first repeat module of the targeting molecule has the consensus sequence: NAX2DX3X4GX6TPLHLX8AWHGHLEIVX 15 VLLKX 16 GADV, Including, The second repeat module of the targeting molecule has the consensus sequence: NAX2DX3X4GX6TPLHLAAX 10 X 11 GHLEIVEVLLKX 16 GADV, and The third repeat module of the targeting molecule has the consensus sequence: NX1X2DX3X4GX6TPLHLAAX 10 X 11 GHLEIVEVLLKX 16 GADV, Including, where: X1 is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V; X2 is any amino acid, X3 is any amino acid, X4 is any amino acid, X6 is any amino acid, X8 is A or V, X 10 is any amino acid, X 11 is any amino acid, X 15 is D or E, X 16 is any amino acid, preferably an amino acid selected from the group consisting of Y, H and N.

[0060] In one embodiment, the ankyrin repeat protein comprises at least one repeat module comprising a sequence selected from the group of repeat modules 1, 2 and 3 of SEQ ID NOs: 1 to 28, respectively, as shown in Figure 5.

[0061] In one embodiment the ankyrin repeat protein comprises three repeat modules, wherein repeat module 1 is selected from the group of repeat module 1 of SEQ ID NOs: 1-28 as shown in Figure 5, repeat module 2 is selected from the group of repeat module 2 of SEQ ID NOs: 1-28 as shown in Figure 5 and repeat module 3 is selected from the group of repeat module 3 of SEQ ID NOs: 1-28 as shown in Figure 5.

[0062] In one embodiment, the ankyrin repeat protein comprises three repeat modules, wherein repeat module 1, repeat module 2 and repeat module 3 are repeat module 1, repeat module 2 and repeat module 3 of a sequence selected from the group consisting of SEQ ID NOs: 1 to 28 as shown in Figure 5.

[0063] In one embodiment, the repeat module is present in the repeat domain.

[0064] In one embodiment, the repeat domain further comprises an N-terminal and / or a C-terminal capping module.

[0065] In one embodiment, the ankyrin repeat protein comprises a sequence selected from the group consisting of SEQ ID NOs: 1 to 28, or positions 29 to 127 thereof.

[0066] In one embodiment, the molecule further comprises another peptide or protein moiety, optionally fused to the ankyrin repeat protein.

[0067] In one embodiment, the molecule is a polypeptide compound.

[0068] In one embodiment, the molecule further comprises a lipid or lipid-like component or another non-peptide component.

[0069] In a further aspect, the invention relates to nucleic acids encoding the molecules described herein.

[0070] In a further aspect, the invention relates to host cells comprising the nucleic acids described herein, optionally expressing the molecules.

[0071] In a further aspect, the present invention relates to particles comprising the molecules described herein.

[0072] In one aspect, the particle further comprises a nucleic acid encoding an antigen receptor. In one aspect, the antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In one embodiment, the antigen is associated with a disease, disorder, or condition. In one embodiment, the antigen is a tumor-associated antigen.

[0073] In one embodiment, the nucleic acid is RNA. In one embodiment, the nucleic acid is DNA.

[0074] In one embodiment, the particle is a non-viral particle. In one embodiment, the particle is a lipid-based and / or polymer-based particle. In one embodiment, the particle is a nanoparticle.

[0075] In one embodiment, the particles are functionalized with ankyrin repeat proteins on their surface. In one embodiment, the particles are functionalized with ankyrin repeat proteins by linking the ankyrin repeat proteins to at least one particle-forming component.

[0076] In a further aspect, the invention relates to a composition comprising a molecule as described herein, a particle as described herein, or a plurality thereof.

[0077] In a further aspect, the present invention relates to a pharmaceutical composition comprising a molecule as described herein, a particle as described herein, or a plurality thereof.

[0078] In a further aspect, the invention relates to a kit comprising a molecule described herein, a nucleic acid described herein, a host cell described herein, a particle described herein, a composition described herein, or a pharmaceutical composition described herein.

[0079] In one embodiment, the kit further comprises instructions for using the kit in the methods described herein.

[0080] In a further aspect, the present invention relates to a particle or a plurality of particles as described herein for use in the methods described herein.

[0081] In a further aspect, the invention relates to the agents and compositions described herein, e.g., targeting molecules, particles, nucleic acids encoding antigen receptors, and / or antigens, polynucleotides encoding antigens, or host cells genetically modified to express antigens, for therapeutic use, particularly for use in the methods described herein.

[0082] Other features and advantages of the invention will become apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0083] [Figure 1] Comparison of classical and in vivo CAR T cell therapy [Figure 2] Gene delivery devices: PLX and LNP [Figure 3] Vaccine Concept (CARVac) [Figure 4A]CD8-specific binding of DARPins. (A) To identify DARPins that specifically bind to CD8, crude E. coli lysates of 94 DARPin clones were analyzed for binding to Molt4.8 cells expressing CD8αα and J67S8ab cells expressing CD8αβ. (B, C) Thirty-one CD8-darpin clones that bound equally to CD8 homodimers and heterodimers were then tested in binding assays on primary human PBMCs (B) and NHP PBMCs (C) by flow cytometry. Bar graphs show binding to CD8+ and CD8- PBMCs for each DARPin. The dotted line indicates the two-fold change above background, which was used as the threshold for classifying DARPins as specific binders when observed on CD8+ but not CD8- cells. The arrow indicates the DARPin selected for further analysis. (*) DARPin 5SE11 was analyzed in a separate binding assay using PBMCs from a different NHP donor. [Figure 4B] CD8-specific binding of DARPins. (A) To identify DARPins that specifically bind to CD8, crude E. coli lysates of 94 DARPin clones were analyzed for binding to Molt4.8 cells expressing CD8αα and J67S8ab cells expressing CD8αβ. (B, C) Thirty-one CD8-darpin clones that bound equally to CD8 homodimers and heterodimers were then tested in binding assays on primary human PBMCs (B) and NHP PBMCs (C) by flow cytometry. Bar graphs show binding to CD8+ and CD8- PBMCs for each DARPin. The dotted line indicates the two-fold change above background, which was used as the threshold for classifying DARPins as specific binders when observed on CD8+ but not CD8- cells. The arrow indicates the DARPin selected for further analysis. (*) DARPin 5SE11 was analyzed in a separate binding assay using PBMCs from a different NHP donor. [Figure 5A] Alignment of CD8-specific DARPin sequences. An alignment of 28 DARPin sequences is shown. [Figure 5B] Alignment of CD8-specific DARPin sequences. An alignment of 28 DARPin sequences is shown. [Figure 6] Analytical SDS-PAGE of H6-HA-63H6-Cys, H6-HA-63H6-E10, and H6-HA-63H6-E20 DARPins after purification and IMAC. A total of 5 μg of protein was applied to SDS-PAGE under reducing (+β-mercaptoethanol) and non-reducing (-β-mercaptoethanol) conditions. [Figure 7] Specific binding of H6-HA-63H6-Cys, H6-HA-63H6-E10, and H6-HA-63H6-E20 to human CD8+ T cells. For flow cytometry analysis, human PBMCs from three different donors were stained with anti-CD3-FITC antibody (clone SK-7, BD Bioscience) and anti-CD4-BV421 antibody (clone Okt04, BioLegend). Binding of DARPin to CD8 was detected with anti-his-APC antibody. For data evaluation, the mean fluorescence intensity (MFI) of the APC signal on CD8+ T cells was calculated. [Figure 8A]CD8-specific transfection with DARPin-functionalized LNPs. Conjugation of CD8-DARPin to LNPs requires covalent attachment to PEG-lipids. To enable click chemistry, a terminal cysteine ​​(LNP-Mal, the counterpart of the maleimide terminal group on the PEG-lipid) was introduced into two selected CD8-DARPin clones (63H6 and 63A4). The constructs were then produced in E. coli and purified. (A) Native PAGE of free DARPin, LNP-Mal alone, and DARPin plus LNP-Mal and LNPs bearing a terminal azide (LNP-N3, a negative control) was performed. (B) DLS data of LNPs bound and unbound to CD8-DARPin (gray bars indicate diameters, and crosses indicate PDI). (C, D) CD8-DARPin-modified LNPs encapsulating luciferase-mRNA were tested for transfection efficiency in CD8+ and CD8- Jurkat cell lines (C) and human Pan T cells (E), and luciferase expression was assessed 16 hours after administration of 300 ng of RNA formulated in LNPs per 1 x 106 cells. LNPs with an irrelevant terminal group (N3) or no DARPin conjugated were used as controls. [Figure 8C]CD8-specific transfection with DARPin-functionalized LNPs. Conjugation of CD8-DARPin to LNPs requires covalent attachment to PEG-lipids. To enable click chemistry, a terminal cysteine ​​(LNP-Mal, the counterpart of the maleimide terminal group on the PEG-lipid) was introduced into two selected CD8-DARPin clones (63H6 and 63A4). The constructs were then produced in E. coli and purified. (A) Native PAGE of free DARPin, LNP-Mal alone, and DARPin plus LNP-Mal and LNPs bearing a terminal azide (LNP-N3, a negative control) was performed. (B) DLS data of LNPs bound and unbound to CD8-DARPin (gray bars indicate diameters, and crosses indicate PDI). (C, D) CD8-DARPin-modified LNPs encapsulating luciferase-mRNA were tested for transfection efficiency in CD8+ and CD8- Jurkat cell lines (C) and human Pan T cells (E), and luciferase expression was assessed 16 hours after administration of 300 ng of RNA formulated in LNPs per 1 x 106 cells. LNPs with an irrelevant terminal group (N3) or no DARPin conjugated were used as controls. [Figure 9A]CD8-Specific Transfection with DARPin-Functionalized PLX. Binding of CD8-DARPin to PLX requires electrostatic attraction between the cationic PLX core and an anionic moiety linked to the targeting ligand. As an alternative to coupling to synthetic polyglutamic acid (PGA) via reactive ester chemistry as previously described (Smith et al., 2017), we recombinantly generated the CD8-specific DARPin clone 63H6 bearing an E20 tag. (A) Agarose gel electrophoresis showing bands for free DARPin-E20, PLX core alone, and DARPin + PLX core at different w / w ratios. (B) DLS data for core PLX and DARPin-modified PLX (gray bars indicate diameters, crosses indicate PDI). (C) Zeta potential of core PLX and DARPin-modified PLX. (D, E) The transfection ability of CD8-DARPin-modified PLX loaded with luciferase-mRNA and Thy1.1-mRNA (50 / 50) was tested in CD8- and CD8+ Jurkat cell lines. (F, G) CD8-DARPin-modified PLX loaded with luciferase-mRNA and Thy1.1-mRNA (50 / 50) was tested in human pan T cells with additional viability testing and flow cytometry analysis, including parallel evaluation of CD4+ and CD8+ T cells. Assays were performed 16 h after administration of 330 ng (Jurkat cells) or 50 ng (primary T cells) of RNA formulated in PLX per 1 x 106 cells. [Figure 9B]CD8-Specific Transfection with DARPin-Functionalized PLX. Binding of CD8-DARPin to PLX requires electrostatic attraction between the cationic PLX core and an anionic moiety linked to the targeting ligand. As an alternative to coupling to synthetic polyglutamic acid (PGA) via reactive ester chemistry as previously described (Smith et al., 2017), we recombinantly generated the CD8-specific DARPin clone 63H6 bearing an E20 tag. (A) Agarose gel electrophoresis showing bands for free DARPin-E20, PLX core alone, and DARPin + PLX core at different w / w ratios. (B) DLS data for core PLX and DARPin-modified PLX (gray bars indicate diameters, crosses indicate PDI). (C) Zeta potential of core PLX and DARPin-modified PLX. (D, E) The transfection ability of CD8-DARPin-modified PLX loaded with luciferase-mRNA and Thy1.1-mRNA (50 / 50) was tested in CD8- and CD8+ Jurkat cell lines. (F, G) CD8-DARPin-modified PLX loaded with luciferase-mRNA and Thy1.1-mRNA (50 / 50) was tested in human pan T cells with additional viability testing and flow cytometry analysis, including parallel evaluation of CD4+ and CD8+ T cells. Assays were performed 16 h after administration of 330 ng (Jurkat cells) or 50 ng (primary T cells) of RNA formulated in PLX per 1 x 106 cells. [Figure 9D]CD8-Specific Transfection with DARPin-Functionalized PLX. Binding of CD8-DARPin to PLX requires electrostatic attraction between the cationic PLX core and an anionic moiety linked to the targeting ligand. As an alternative to coupling to synthetic polyglutamic acid (PGA) via reactive ester chemistry as previously described (Smith et al., 2017), we recombinantly generated the CD8-specific DARPin clone 63H6 bearing an E20 tag. (A) Agarose gel electrophoresis showing bands for free DARPin-E20, PLX core alone, and DARPin + PLX core at different w / w ratios. (B) DLS data for core PLX and DARPin-modified PLX (gray bars indicate diameters, crosses indicate PDI). (C) Zeta potential of core PLX and DARPin-modified PLX. (D, E) The transfection ability of CD8-DARPin-modified PLX loaded with luciferase-mRNA and Thy1.1-mRNA (50 / 50) was tested in CD8- and CD8+ Jurkat cell lines. (F, G) CD8-DARPin-modified PLX loaded with luciferase-mRNA and Thy1.1-mRNA (50 / 50) was tested in human pan T cells with additional viability testing and flow cytometry analysis, including parallel evaluation of CD4+ and CD8+ T cells. Assays were performed 16 h after administration of 330 ng (Jurkat cells) or 50 ng (primary T cells) of RNA formulated in PLX per 1 x 106 cells. [Figure 9E]CD8-Specific Transfection with DARPin-Functionalized PLX. Binding of CD8-DARPin to PLX requires electrostatic attraction between the cationic PLX core and an anionic moiety linked to the targeting ligand. As an alternative to coupling to synthetic polyglutamic acid (PGA) via reactive ester chemistry as previously described (Smith et al., 2017), we recombinantly generated the CD8-specific DARPin clone 63H6 bearing an E20 tag. (A) Agarose gel electrophoresis showing bands for free DARPin-E20, PLX core alone, and DARPin + PLX core at different w / w ratios. (B) DLS data for core PLX and DARPin-modified PLX (gray bars indicate diameters, crosses indicate PDI). (C) Zeta potential of core PLX and DARPin-modified PLX. (D, E) The transfection ability of CD8-DARPin-modified PLX loaded with luciferase-mRNA and Thy1.1-mRNA (50 / 50) was tested in CD8- and CD8+ Jurkat cell lines. (F, G) CD8-DARPin-modified PLX loaded with luciferase-mRNA and Thy1.1-mRNA (50 / 50) was tested in human pan T cells with additional viability testing and flow cytometry analysis, including parallel evaluation of CD4+ and CD8+ T cells. Assays were performed 16 h after administration of 330 ng (Jurkat cells) or 50 ng (primary T cells) of RNA formulated in PLX per 1 x 106 cells. [Figure 9F]CD8-Specific Transfection with DARPin-Functionalized PLX. Binding of CD8-DARPin to PLX requires electrostatic attraction between the cationic PLX core and an anionic moiety linked to the targeting ligand. As an alternative to coupling to synthetic polyglutamic acid (PGA) via reactive ester chemistry as previously described (Smith et al., 2017), we recombinantly generated the CD8-specific DARPin clone 63H6 bearing an E20 tag. (A) Agarose gel electrophoresis showing bands for free DARPin-E20, PLX core alone, and DARPin + PLX core at different w / w ratios. (B) DLS data for core PLX and DARPin-modified PLX (gray bars indicate diameters, crosses indicate PDI). (C) Zeta potential of core PLX and DARPin-modified PLX. (D, E) The transfection ability of CD8-DARPin-modified PLX loaded with luciferase-mRNA and Thy1.1-mRNA (50 / 50) was tested in CD8- and CD8+ Jurkat cell lines. (F, G) CD8-DARPin-modified PLX loaded with luciferase-mRNA and Thy1.1-mRNA (50 / 50) was tested in human pan T cells with additional viability testing and flow cytometry analysis, including parallel evaluation of CD4+ and CD8+ T cells. Assays were performed 16 h after administration of 330 ng (Jurkat cells) or 50 ng (primary T cells) of RNA formulated in PLX per 1 x 106 cells. [Figure 9G]CD8-Specific Transfection with DARPin-Functionalized PLX. Binding of CD8-DARPin to PLX requires electrostatic attraction between the cationic PLX core and an anionic moiety linked to the targeting ligand. As an alternative to coupling to synthetic polyglutamic acid (PGA) via reactive ester chemistry as previously described (Smith et al., 2017), we recombinantly generated the CD8-specific DARPin clone 63H6 bearing an E20 tag. (A) Agarose gel electrophoresis showing bands for free DARPin-E20, PLX core alone, and DARPin + PLX core at different w / w ratios. (B) DLS data for core PLX and DARPin-modified PLX (gray bars indicate diameters, crosses indicate PDI). (C) Zeta potential of core PLX and DARPin-modified PLX. (D, E) The transfection ability of CD8-DARPin-modified PLX loaded with luciferase-mRNA and Thy1.1-mRNA (50 / 50) was tested in CD8- and CD8+ Jurkat cell lines. (F, G) CD8-DARPin-modified PLX loaded with luciferase-mRNA and Thy1.1-mRNA (50 / 50) was tested in human pan T cells with additional viability testing and flow cytometry analysis, including parallel evaluation of CD4+ and CD8+ T cells. Assays were performed 16 h after administration of 330 ng (Jurkat cells) or 50 ng (primary T cells) of RNA formulated in PLX per 1 x 106 cells. [Figure 10] CD8-specific transfection with DARPin-modified LNPs in vivo. Immunodeficient mice were transplanted with human PBMCs and treated 21 days later with 20 μg of mRNA (luciferase and Thy1.1, 50 / 50) encapsulated in either non-functionalized or CD8-DARPin-modified LNPs. LNPs were functionalized via a cysteine / maleimide reaction. One day after LNP administration, luciferase signals were detected by in situ bioluminescence imaging (A), and Thy1.1 expression was assessed by flow cytometry analysis of peripheral blood (B). [Figure 11]Functionalized nanoparticles as vehicles for delivering mixed RNA / DNA cargo. CD8+ T cells were isolated from peripheral blood of healthy donors and treated with 50 ng of mixed cargo (improved YFP-encoding minicircle DNA and Thy1.1 mRNA, 50 / 50) encapsulated in either unfunctionalized or CD8-DARPinE20-modified PLX per 1 x 10 target cells. One day after PLX administration, expression of the gene of interest was assessed by flow cytometry, and cells were activated with CD3 / CD28 beads to achieve proliferation. Five days after treatment, cells were again assessed by flow cytometry. DETAILED DESCRIPTION OF THE INVENTION

[0084] Although the present disclosure will be described in detail below, it should be understood that the disclosure is not limited to the specific methodology, protocols and reagents described herein, which may vary.It should also be understood that the terms used herein are only intended to describe specific embodiments and are not intended to limit the scope of the present disclosure, which is limited only by the scope of the appended claims.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0085] Preferably, the terms used herein are defined as set forth in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0086] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, immunology, and recombinant DNA techniques as described in the art (see, e.g., Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989).

[0087] The elements of the present disclosure are described below. Although these elements are listed with specific embodiments, it should be understood that they may be combined in any manner and in any number to create further embodiments. The various described examples and embodiments should not be construed as limiting the disclosure to only the embodiments explicitly described. This description should be understood to disclose and encompass embodiments combining the explicitly described embodiments with any number of the disclosed elements. Furthermore, any permutation and combination of all described elements should be considered disclosed by this description unless the context dictates otherwise.

[0088] The term "about" means approximately or near, and in the context of numerical values ​​or ranges described herein, means, in one embodiment, ±20%, ±10%, ±5%, or ±3% of the recited or claimed numerical value or range.

[0089] As used in the context of describing the present disclosure (particularly in the context of the claims), the terms "a," "an," "the," and similar references should be construed to encompass both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "etc.") provided herein is intended merely to better explain the disclosure and does not impose limitations on the claims. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0090] Unless otherwise specified, the term "comprises" is used in the context of this document to indicate that additional members may optionally be present in addition to the members of the list introduced by "comprises." However, it is contemplated as a specific embodiment of the present disclosure that the term "comprises" encompasses the possibility that additional members are not present, i.e., for the purposes of this embodiment, "comprises" should be understood to have the meaning of "consisting of."

[0091] Several documents are cited throughout the text of this specification. Each document cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the present disclosure is not entitled to antedate such disclosure.

[0092] The following provides definitions that apply to all aspects of this disclosure. The following terms have the following meanings unless otherwise indicated. Terms not defined have their art-wide accepted meanings.

[0093] definition As used herein, terms such as "reduce," "diminish," "inhibit," or "impair" relate to an overall decrease or ability to cause an overall decrease in levels, e.g., binding levels, preferably by 5% or more, 10% or more, 20% or more, more preferably 50% or more, and most preferably 75% or more.

[0094] Terms such as "increase," "enhance," or "exceed" preferably relate to an increase or enhancement of at least about 10%, preferably at least 20%, preferably at least 30%, more preferably at least 40%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 100%, at least 200%, at least 500%, or even more.

[0095] The term "plurality" with respect to an object refers to a collection of a particular number of said objects. In certain embodiments, the term refers to a collection of 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 , 10 14 , 10 15 , 10 16 , 10 17 , 10 18 , 10 19 , 10 20 , 10 21 , 10 22 , or 10 23 This refers to the above groups.

[0096] Amino acids are the building blocks that form peptides, polypeptides, and proteins. Below are the abbreviations and one-letter codes used for amino acids:

[0097] [Table 1]

[0098] According to this disclosure, the term "peptide" includes oligopeptides and polypeptides and refers to a substance comprising about 2 or more, about 3 or more, about 4 or more, about 6 or more, about 8 or more, about 10 or more, about 13 or more, about 16 or more, about 20 or more, and up to about 50, about 100, or about 150 consecutive amino acids joined together by peptide bonds. The terms "protein" or "polypeptide" refer to large peptides, particularly peptides having at least about 151 amino acids, although the terms "peptide," "protein," and "polypeptide" are generally used synonymously herein.

[0099] A "therapeutic protein" when provided to a subject in a therapeutically effective amount has a positive or beneficial effect on the subject's condition or pathology. In one embodiment, a therapeutic protein has curative or palliative properties and can be administered to improve, alleviate, relieve, reverse, delay the onset, or reduce the severity of one or more symptoms of a disease or disorder. A therapeutic protein can have prophylactic properties and can be used to delay the onset of a disease or reduce the severity of such a disease or pathological condition. The term "therapeutic protein" includes whole proteins or peptides and can also refer to therapeutically active fragments thereof. It can also include therapeutically active variants of proteins. Examples of therapeutically active proteins include, but are not limited to, antigens and cytokines for vaccination.

[0100] With respect to an amino acid sequence (peptide or protein), the term "fragment" refers to a portion of the amino acid sequence, i.e., a sequence representing an amino acid sequence truncated at the N-terminus and / or C-terminus. A C-terminally truncated fragment (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3' end of the open reading frame. An N-terminally truncated fragment (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5' end of the open reading frame, as long as the truncated open reading frame contains an initiation codon that serves to initiate translation. A fragment of an amino acid sequence comprises, for example, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the amino acid residues from the amino acid sequence. A fragment of an amino acid sequence preferably comprises at least 6, particularly at least 8, at least 12, at least 15, at least 20, at least 30, at least 50, or at least 100 consecutive amino acids from the amino acid sequence.

[0101] As used herein, "variant" or "variant protein" or "variant polypeptide" refers to a protein that differs from a wild-type protein by at least one amino acid modification. The parent polypeptide can be a naturally occurring or wild-type (WT) polypeptide, or can be a modified version of a wild-type polypeptide. Preferably, the variant polypeptide has at least one amino acid modification compared to the parent polypeptide, e.g., 1 to about 20 amino acid modifications compared to the parent polypeptide, preferably 1 to about 10 or 1 to about 5 amino acid modifications.

[0102] As used herein, "parent polypeptide," "parent protein," "precursor polypeptide," or "precursor protein" refers to an unmodified polypeptide that is subsequently modified to produce a variant. A parent polypeptide can be a wild-type polypeptide, or a variant or engineered version of a wild-type polypeptide.

[0103] As used herein, "wild-type" or "WT" or "native" refers to an amino acid sequence found in nature, including allelic variations. A wild-type protein or polypeptide has an amino acid sequence that has not been intentionally modified.

[0104] For purposes of this disclosure, a "variant" of an amino acid sequence (peptide, protein, or polypeptide) includes amino acid insertion variants, amino acid addition variants, amino acid deletion variants, and / or amino acid substitution variants. The term "variant" includes all splice variants, post-translationally modified variants, conformational variants, isoform variants, and species homologs, particularly those naturally expressed by cells. The term "variant" particularly includes fragments of an amino acid sequence.

[0105] Amino acid insertion variants include the insertion of one or more amino acids into a specific amino acid sequence. In the case of amino acid sequence variants with insertions, one or more amino acid residues are inserted at specific sites in the amino acid sequence, although random insertion with appropriate screening of the resulting product is also possible. Amino acid addition variants include amino- and / or carboxy-terminal fusions of one or more amino acids, for example, 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence, for example, the removal of 1, 2, 3, 5, 10, 20, 30, 50, or more amino acids. The deletion may occur at any position in the protein. Amino acid deletion variants containing deletions at the N- and / or C-termini of a protein are also referred to as N- and / or C-terminal truncation variants. Amino acid substitution variants are characterized by the removal of at least one residue in the sequence and the insertion of another residue in its place. Modifications at positions in the amino acid sequence that are not conserved between homologous proteins or peptides and / or substitutions of amino acids with other amino acids with similar properties are preferred. Preferably, the amino acid changes in peptide and protein variants are conservative amino acid changes, i.e., substitutions of similarly charged or uncharged amino acids. Conservative amino acid changes include substitutions of members of a family of amino acids whose side chains are related. Naturally occurring amino acids are generally divided into four families: acidic (aspartic acid, glutamic acid), basic (lysine, arginine, histidine), nonpolar (alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), and uncharged polar (glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine) amino acids. Phenylalanine, tryptophan, and tyrosine are sometimes classified together as aromatic amino acids. In one embodiment, conservative amino acid substitutions include substitutions within the following groups: Glycine, Alanine; valine, isoleucine, leucine; Aspartic acid, glutamic acid; Asparagine, glutamine; Serine, threonine; lysine, arginine; and Phenylalanine, tyrosine.

[0106] Preferably, the degree of similarity, preferably identity, between a given amino acid sequence and an amino acid sequence that is a variant of said given amino acid sequence is at least about 60%, 65%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. The degree of similarity or identity is preferably given over an amino acid region that is at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or about 100% of the entire length of the reference amino acid sequence. For example, if the reference amino acid sequence consists of 200 amino acids, the degree of similarity or identity is preferably given for at least about 20, at least about 40, at least about 60, at least about 80, at least about 100, at least about 120, at least about 140, at least about 160, at least about 180, or about 200 amino acids, preferably consecutive amino acids. In a preferred embodiment, the degree of similarity or identity is given for the entire length of the reference amino acid sequence. Alignment to determine sequence similarity, preferably sequence identity, can be performed using tools known in the art, preferably using optimal sequence alignment, for example, using Align, with standard settings, preferably EMBOSS::Needle, matrix:Blosum62, gap open 10.0, gap extension 0.5.

[0107] "Sequence similarity" indicates the percentage of amino acids that are identical or represent conservative amino acid substitutions. "Sequence identity" between two amino acid sequences indicates the percentage of amino acids that are identical between those sequences.

[0108] The term "percent identity" is intended to indicate the percentage of amino acid residues that are identical between the two sequences being compared after optimal alignment, and this percentage is purely statistical, with the differences between the two sequences being randomly distributed over their entire length. Sequence comparison between two amino acid sequences is usually carried out by comparing these sequences after optimal alignment, said comparison being carried out segment by segment or "comparison window" to identify and compare local regions of sequence similarity. In addition to being created manually, optimal alignment of sequences for comparison can be achieved by the local homology algorithm of Smith and Waterman, 1981, Ads App. Math. 2, 482, by the local homology algorithm of Neddleman and Wunsch, 1970, J. Mol. Biol. 48, 443, by the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or by computer programs using these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N, and TFASTA from the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, Wis.).

[0109] The percent identity is calculated by determining the number of identical positions between the two sequences being compared, dividing this number by the number of positions being compared, and multiplying the result by 100 to obtain the percent identity between the two sequences.

[0110] Homologous amino acid sequences, according to the present disclosure, exhibit an identity of at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, preferably at least 95%, at least 98, or at least 99% of the amino acid residues.

[0111] The amino acid sequence variants described herein can be readily prepared by those skilled in the art, for example, by recombinant DNA manipulation. The manipulation of DNA sequences to prepare peptides or proteins with substitutions, additions, insertions, or deletions is described in detail, for example, in Sambrook et al. (1989). Furthermore, the peptides and amino acid variants described herein can be readily prepared using known peptide synthesis techniques, for example, by solid-phase synthesis and similar methods.

[0112] In one embodiment, a fragment or variant of an amino acid sequence (peptide or protein) is preferably a "functional fragment" or "functional variant." The term "functional fragment" or "functional variant" of an amino acid sequence refers to any fragment or variant that exhibits one or more functional properties identical or similar to those of the amino acid sequence from which it is derived, i.e., is functionally equivalent. With respect to antigens, one specific function is one or more immunostimulatory activities exhibited by the amino acid sequence from which the fragment or variant is derived and / or binding to the receptor(s) to which the amino acid sequence from which the fragment or variant is derived binds. As used herein, the term "functional fragment" or "functional variant" particularly refers to a variant molecule or sequence that contains an amino acid sequence that has been altered by one or more amino acids compared to the amino acid sequence of a parent molecule or sequence and still performs one or more functions of the parent molecule or sequence, for example, is capable of binding to a target molecule. In one embodiment, alterations in the amino acid sequence of the parent molecule or sequence do not significantly affect or change the binding properties of the molecule or sequence. In different embodiments, binding of the functional fragment or functional variant may be reduced but still significant, for example, the binding of the functional variant may be at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of that of the parent molecule or sequence, however, in other embodiments, binding of the functional fragment or functional variant may be enhanced compared to the parent molecule or sequence.

[0113] An amino acid sequence (peptide, protein, or polypeptide) "derived from" a specified amino acid sequence (peptide, protein, or polypeptide) refers to the origin of the initial amino acid sequence. Preferably, an amino acid sequence derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical, or homologous to the particular sequence, or a fragment thereof. An amino acid sequence derived from a particular amino acid sequence may be a variant of the particular sequence, or a fragment thereof. For example, it will be understood by those skilled in the art that antigens suitable for use herein can be modified to differ in sequence from the naturally occurring or native sequence from which they are derived, while retaining the desired activity of the native sequence.

[0114] As used herein, "instructional material" or "instructions" includes publications, records, drawings, or any other medium of expression that can be used to communicate the usefulness of the compositions and methods of the present invention. The instructional material of the kits of the present invention may, for example, be affixed to a container containing the composition of the present invention or may be shipped together with a container containing the composition. Alternatively, the instructional material may be shipped separately from the container, with the intention that the instructional material and the composition be used in conjunction with each other by the recipient.

[0115] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally occurring in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as a host cell.

[0116] The term "recombinant" in the context of the present invention means "produced through genetic engineering." Preferably, a "recombinant," such as a recombinant cell, in the context of the present invention does not occur in nature.

[0117] As used herein, the term "naturally occurring" refers to the fact that an entity can be found in nature. For example, a peptide or nucleic acid that is present in an organism (including viruses), can be isolated from a natural source, and has not been intentionally modified by humans in a laboratory is naturally occurring.

[0118] " Lentivirus " as used herein refers to a genus of Retroviridae.Lentivirus is unique among retroviruses in that it can infect non-dividing cells; they can deliver significant amounts of genetic information to the DNA of host cells, so they are one of the most efficient methods of gene delivery vectors.HIV, SIV and FIV are all examples of lentivirus.Vector derived from lentivirus provides a means to achieve significant levels of gene transfer in vivo.

[0119] As used herein, the term "specifically binds" refers to a molecule, such as an antibody or CAR, that recognizes a specific antigen but does not substantially recognize or bind other molecules in a sample or subject. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. However, such species cross-reactivity does not, in itself, change the antibody's classification as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross-reactivity does not, in itself, change the antibody's classification as specific. In some cases, the terms "specific binding" or "specifically binds" can be used to refer to the interaction of an antibody, protein, or peptide with a second chemical species, where the interaction is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) in the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than proteins in general. If an antibody is specific for epitope "A," in a reaction involving labeled "A" and an antibody, the presence of a molecule containing epitope A (or free, unlabeled A) reduces the amount of labeled A that binds to the antibody.

[0120] The term "genetic modification" includes the transfection of cells with nucleic acids. The term "transfection" refers to the introduction of nucleic acids, particularly RNA, into cells. For purposes of the present invention, the term "transfection" also includes the introduction of nucleic acids into cells or the uptake of nucleic acids by such cells, which may be present in a subject, e.g., a patient. Thus, according to the present invention, cells for transfection with nucleic acids as described herein can be present in vitro or in vivo, e.g., the cells can form part of a patient's organ, tissue, and / or organism. According to the present invention, transfection can be transient or stable. In some applications of transfection, it is sufficient for the transfected genetic material to be expressed only transiently. RNA can be transfected into cells to transiently express its encoded protein. Nucleic acids introduced during transfection are usually not integrated into the nuclear genome, so the foreign nucleic acid is diluted or degraded by mitosis. Cells that allow episomal amplification of nucleic acids significantly reduce the dilution rate. If it is desired that the transfected nucleic acid actually remain in the genome of the cell and its daughter cells, stable transfection must occur. Such stable transfection can occur when the nucleic acid introduced during the transfection process is integrated into the nuclear genome, and can be achieved, for example, by using a viral-based or transposon-based system for transfection. Generally, cells genetically modified to express an antigen receptor are stably transfected with a nucleic acid encoding the antigen receptor, while generally, a nucleic acid encoding the antigen is transiently transfected into the cells.

[0121] immune effector cells Cells used in connection with the present invention and into which nucleic acids (DNA or RNA) encoding antigen receptors can be introduced include, in particular, immune effector cells, such as lymphoid cells, with lytic potential, preferably T cells, and in particular, cytotoxic lymphocytes, preferably selected from cytotoxic T cells, natural killer (NK) cells, and lymphokine-activated killer (LAK) cells. Upon activation, these cytotoxic lymphocytes cause the destruction of target cells. For example, cytotoxic T cells cause the destruction of target cells by one or both of the following means: First, upon activation, T cells release cytotoxins such as perforin, granzymes, and granulysin. Perforin and granulysin create pores in the target cell, and granzymes enter the cell and trigger a cytoplasmic caspase cascade that induces apoptosis (programmed cell death) of the cell. Second, apoptosis can be induced via Fas-Fas ligand interaction between T cells and target cells. Cells used in connection with the present invention are preferably autologous cells, although xenogeneic or allogeneic cells can also be used.

[0122] In the context of the present invention, the term "effector function" includes any function mediated by a component of the immune system that results in the inhibition of tumor growth and / or tumorigenesis, including, for example, the killing of diseased cells, such as tumor cells, or the suppression of tumor dissemination and metastasis. Preferably, the effector function in the context of the present invention is a T cell-mediated effector function. Such a function is mediated by helper T cells (CD4 + T cells), cytokine release and / or CD8 + It involves the activation of lymphocytes (CTLs) and / or B cells, and in the case of CTLs, the elimination of cells, i.e., cells characterized by expression of the antigen, e.g., via apoptosis or perforin-mediated cytolysis, the production of cytokines such as IFN-γ and TNF-α, and the specific cytolytic killing of target cells expressing the antigen.

[0123] The term "immune effector cells" or "immunoreactive cells" in the context of the present invention relates to cells that exert effector functions during an immune response. In one embodiment, "immune effector cells" are capable of binding to antigens, such as antigens presented in association with MHC on cells or expressed on the surface of cells, and mediating an immune response. For example, immune effector cells include T cells (cytotoxic T cells, helper T cells, tumor-infiltrating T cells), B cells, natural killer cells, neutrophils, macrophages, and dendritic cells. Preferably, in the context of the present invention, "immune effector cells" are T cells, preferably CD4 + and / or CD8 + T cells, most preferably CD8 + According to the present invention, the term "immune effector cells" also includes cells that can mature into immune cells (such as T cells, especially T helper cells, or cytolytic T cells) upon appropriate stimulation. Immune effector cells are CD34 + They include hematopoietic stem cells, immature and mature T cells, and immature and mature B cells. The differentiation of T cell precursors into cytolytic T cells resembles the clonal selection of the immune system upon exposure to antigen.

[0124] Preferably, "immune effector cells" recognize antigens with some degree of specificity, especially when presented in the context of MHC or present on the surface of diseased cells such as cancer cells. Preferably, said recognition enables the cells that recognize the antigen to be responsive or reactive. The cells are called helper T cells (CD4 + T cells), such responsiveness or reactivity may be mediated by the release of cytokines and / or CD8 +The activation of lymphocytes (CTLs) and / or B cells may be included. When the cells are CTLs, such responsiveness or reactivity may include the elimination of the cells, i.e., cells characterized by antigen expression, via, for example, apoptosis or perforin-mediated cytolysis. According to the present invention, CTL responsiveness may include sustained calcium flux, cell division, production of cytokines such as IFN-γ and TNF-α, upregulation of activation markers such as CD44 and CD69, and specific cytolytic killing of antigen-expressing target cells. CTL responsiveness may also be determined using an artificial reporter that accurately indicates CTL responsiveness. Such CTLs that recognize and are responsive or reactive to an antigen are also referred to herein as "antigen-responsive CTLs."

[0125] In one embodiment, the genetically modified immune effector cells are immune effector cells that express a CAR. In one embodiment, the genetically modified immune effector cells are immune effector cells that express a TCR.

[0126] Immune effector cells used in accordance with the present invention may express an endogenous antigen receptor, such as a T cell receptor or a B cell receptor, or may lack expression of an endogenous antigen receptor.

[0127] "Lymphoid cells" are cells or precursors of such cells that can generate immune responses, such as cellular immune responses, optionally after appropriate modification, for example, after introduction of antigen receptors such as TCRs or CARs, and include lymphocytes, preferably T lymphocytes, lymphoblasts, and plasma cells. Lymphoid cells can be immune effector cells as described herein. Preferred lymphoid cells are T cells that can be modified to express antigen receptors on their cell surface. In one embodiment, lymphoid cells lack endogenous expression of T cell receptors.

[0128] The terms "T cell" and "T lymphocyte" are used interchangeably herein and refer to T helper cells (CD4 + Cytotoxic T cells (CTL, CD8+ The term "antigen-specific T cells" or similar terms refers to T cells that recognize the antigen targeted by the T cell and preferably exert T cell effector functions. A T cell is considered specific for an antigen if it kills a target cell that expresses the antigen. The specificity of a T cell can be assessed using any of a variety of standard techniques, for example, in a chromium release assay or proliferation assay. Alternatively, the synthesis of lymphokines (such as interferon-γ) can be measured.

[0129] T cells belong to a group of white blood cells known as lymphocytes and play a central role in cell-mediated immunity. They can be distinguished from other types of lymphocytes, such as B cells and natural killer cells, by the presence of a special receptor on their cell surface called the T cell receptor (TCR). The thymus is the primary organ responsible for the maturation of T cells. Several different subsets of T cells have been discovered, each with distinct functions.

[0130] T helper cells assist other white blood cells in immunological processes, including, among other functions, the maturation of B cells into plasma cells and the activation of cytotoxic T cells and macrophages. These cells express the CD4 glycoprotein on their surface and are therefore CD4 + Also known as T cells, helper T cells are activated when presented with peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that regulate or support the active immune response.

[0131] Cytotoxic T cells destroy virus-infected and tumor cells and are also involved in transplant rejection. These cells express the CD8 glycoprotein on their surface and are therefore CD8 + Also known as T cells, these cells recognize their targets by binding to antigens associated with MHC class I, which are present on the surface of almost every cell in the body.

[0132] "Regulatory T cells" or "Tregs" are a subpopulation of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease. Tregs are immunosuppressive and generally suppress or downregulate the induction and proliferation of effector T cells. Tregs express the biomarkers CD4, FoxP3, and CD25.

[0133] As used herein, the term "naive T cells" refers to mature T cells that, unlike activated or memory T cells, have never encountered their cognate antigen in the periphery. Naive T cells are generally characterized by surface expression of L-selectin (CD62L), the absence of activation markers CD25, CD44, or CD69, and the absence of the memory CD45RO isoform.

[0134] As used herein, the term "memory T cells" refers to a subgroup or subpopulation of T cells that previously encountered and responded to their cognate antigen. Upon a second encounter with the antigen, memory T cells can be regenerated to mount a faster and stronger immune response than the first time the immune system responded to the antigen. Memory T cells are CD4 + or CD8 + and typically express CD45RO.

[0135] According to the present invention, the term "T cells" also includes cells that can mature into T cells upon appropriate stimulation.

[0136] The majority of T cells have a T cell receptor (TCR) that exists as a complex of several proteins. The actual T cell receptor is produced by independent T cell receptor alpha and beta (TCRα and TCRβ) genes and consists of two distinct peptide chains called the α- and β-TCR chains. γδ T cells (gamma delta T cells) are a small subset of T cells that have a different T cell receptor (TCR) on their surface. However, in γδ T cells, the TCR consists of one γ chain and one δ chain. This population of T cells is much smaller than αβ T cells (2% of all T cells).

[0137] All T cells originate from hematopoietic stem cells in the bone marrow. Hematopoietic progenitor cells derived from hematopoietic stem cells reside in the thymus and expand by cell division to generate a large population of immature thymocytes. The earliest thymocytes express neither CD4 nor CD8 and are therefore double-negative (CD4 - CD8 - ) cells. As development progresses, they become double-positive thymocytes (CD4 + CD8 + ) and eventually become single positive (CD4 + CD8 - or CD4 - CD8 + ) mature into thymocytes, which are then released from the thymus into peripheral tissues.

[0138] T cells can generally be prepared in vitro or ex vivo using standard procedures.For example, T cells can be isolated from the bone marrow, peripheral blood, or bone marrow or peripheral blood fraction of mammals such as patients using commercially available cell separation systems.Alternatively, T cells can be derived from related or unrelated humans, non-human animals, cell lines, or cultures.The sample containing T cells can be, for example, peripheral blood mononuclear cells (PBMCs).

[0139] As used herein, the term "NK cells" or "natural killer cells" refers to a subset of peripheral blood lymphocytes defined by expression of CD56 or CD16 and the absence of a T cell receptor. As provided herein, NK cells can also be differentiated from stem or progenitor cells.

[0140] Genetic modification to express antigen receptors The cells described herein, such as immune effector cells, are genetically modified ex vivo / in vitro or in vivo in the subject being treated so that they express an antigen receptor, such as a chimeric antigen receptor (CAR) or a T cell receptor (TCR)-binding antigen, or its processing product, particularly when present on or presented by target cells, such as antigen-presenting cells or diseased cells. In one embodiment, the modification to express the antigen receptor is performed ex vivo / in vitro. The modified cells can then be administered to the patient. In one embodiment, the modification to express the antigen receptor is performed in vivo. The cells may be endogenous cells of the patient or may have been administered to the patient.

[0141] Chimeric Antigen Receptor Adoptive cell transfer therapy using CAR-engineered T cells expressing chimeric antigen receptors is a promising anticancer treatment because CAR-modified T cells can be engineered to target virtually any tumor antigen. For example, a patient's T cells can be genetically engineered (modified) to express a CAR that specifically targets an antigen on the patient's tumor cells, and then infused back into the patient.

[0142] According to the present invention, the term "CAR" (or "chimeric antigen receptor") is synonymous with the terms "chimeric T cell receptor" and "artificial T cell receptor" and relates to an artificial receptor comprising a single molecule or complex of molecules that can recognize, i.e., bind to, a target structure (e.g., an antigen) on a target cell, such as a cancer cell (e.g., by binding of an antigen-binding domain to an antigen expressed on the surface of the target cell), and confer specificity to an immune effector cell, such as a T cell, that expresses the CAR on its cell surface. Such cells do not necessarily require antigen processing and presentation for target cell recognition, but rather can preferably specifically recognize any antigen present on the target cell. Preferably, recognition of the target structure by a CAR results in activation of an immune effector cell that expresses the CAR. A CAR can comprise one or more protein units comprising one or more domains described herein. The term "CAR" does not include T cell receptors.

[0143] CARs generally contain a target-specific binding element, also referred to as an antigen-binding portion or antigen-binding domain, which is part of the extracellular domain of the CAR. The antigen-binding domain recognizes a ligand that acts as a cell surface marker on target cells associated with a particular disease state. Specifically, the CARs of the present invention target antigens, such as tumor antigens, on diseased cells, such as tumor cells.

[0144] In one embodiment, the binding domain of the CAR specifically binds to an antigen. In one embodiment, the antigen to which the binding domain in the CAR binds is expressed on cancer cells (tumor antigen). In one embodiment, the antigen is expressed on the surface of cancer cells. In one embodiment, the binding domain binds to the extracellular domain of the antigen or an epitope of the extracellular domain. In one embodiment, the binding domain binds to a natural epitope of the antigen present on the surface of living cells.

[0145] In one embodiment of the present invention, the antigen-binding domain comprises a variable region (VH) of an immunoglobulin heavy chain having specificity for an antigen and a variable region (VL) of an immunoglobulin light chain having specificity for an antigen. In one embodiment, the immunoglobulin is an antibody. In one embodiment, the heavy chain variable region (VH) and the corresponding light chain variable region (VL) are linked by a peptide linker. Preferably, part of the antigen-binding portion in the CAR is an scFv.

[0146] CAR is designed to include a transmembrane domain fused to the extracellular domain of CAR.In one embodiment, the transmembrane domain is not naturally associated with one of the domains in CAR.In one embodiment, the transmembrane domain is naturally associated with one of the domains in CAR.In one embodiment, the transmembrane domain is modified by amino acid substitution to prevent such domain from binding to the transmembrane domain of the same or different surface membrane protein, thereby minimizing interaction with other members of the receptor complex.The transmembrane domain can be derived from either natural or synthetic sources.When the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. The transmembrane domains particularly useful in the present invention may be derived from (i.e., comprise at least one or more of) the α, β, or ζ chains of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domain may be synthetic, in which case it contains primarily hydrophobic residues such as leucine and valine. Preferably, triplets of phenylalanine, tryptophan, and valine are found at each end of the synthetic transmembrane domain.

[0147] In some cases, the CAR of the present invention comprises a hinge domain that forms the link between the transmembrane domain and the extracellular domain.

[0148] The cytoplasmic domain or other intracellular signaling domain of a CAR is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is placed. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits an effector function signal and instructs the cell to perform a specialized function. While the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used in place of the intact chain, as long as it transmits the effector function signal. Thus, the term intracellular signaling domain is meant to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0149] It is known that signals generated solely through the TCR are insufficient for full activation of T cells, and that secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences) and those that act antigen-independently to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).

[0150] In one embodiment, the CAR comprises a primary cytoplasmic signaling sequence derived from CD3ζ. Additionally, the cytoplasmic domain of the CAR may comprise a CD3ζ signaling domain combined with a costimulatory signaling region.

[0151] The identity of the costimulatory domain is limited only by its ability to enhance cell proliferation and survival upon binding of the targeting moiety by the CAR. Suitable costimulatory domains include CD28, CD137 (4-1BB), a member of the tumor necrosis factor receptor (TNFR) superfamily, CD134 (OX40), a member of the TNFR superfamily of receptors, and CD278 (ICOS), a costimulatory molecule of the CD28 superfamily expressed on activated T cells. Those skilled in the art will understand that sequence variants of these described costimulatory domains can be used without adversely affecting the present invention if they have the same or similar activity as the domain they are modeled after. Such variants have at least about 80% sequence identity with the amino acid sequence of the domain from which they are derived. In some embodiments of the present invention, the CAR construct comprises two costimulatory domains. Specific combinations include all possible variations of the four described domains, with specific examples including CD28+CD137(4-1BB) and CD28+CD134(OX40).

[0152] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other in random or specified order. Optionally, a short oligopeptide or polypeptide linker, preferably 2-10 amino acids in length, can form the linkage. A glycine-serine doublet provides a particularly suitable linker.

[0153] In one embodiment, the CAR comprises a signal peptide that targets the nascent protein to the endoplasmic reticulum. In one embodiment, the signal peptide precedes the antigen-binding domain. In one embodiment, the signal peptide is derived from an immunoglobulin, such as IgG.

[0154] CAR can comprise the above domains together in the form of a fusion protein.Such fusion protein generally comprises an antigen binding domain, one or more costimulatory domains, and an activation signaling domain linked in the direction from N-terminus to C-terminus.However, the CAR of the present invention is not limited to this arrangement, and other arrangements are also acceptable, including a binding domain, a signaling domain, and one or more costimulatory domains.Because the binding domain must be able to freely bind to the antigen, it is understood that the arrangement of the binding domain in the fusion protein is generally an arrangement that achieves the display of the domain outside the cell.Similarly, because the costimulatory and signaling domains function to induce the activity and proliferation of cytotoxic lymphocytes, fusion protein generally displays these two domains inside the cell.

[0155] In one embodiment, the CAR molecule is i) a target antigen (e.g., CLDN6 or CLDN18.2) binding domain; ii) a transmembrane domain; and iii) an intracellular domain containing the 4-1BB costimulatory domain and the CD3ζ signaling domain Includes.

[0156] In one embodiment, the antigen binding domain comprises an scFv. In one embodiment, the transmembrane domain is selected from the group consisting of the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19 , IL2Rβ, IL2Rγ, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CDlld, ITGAE, CD103, ITGAL, CDlla, LFA-1, ITGAM, CDllb, ITGAX, CDllc, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAMl(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRT The antigen-binding domain comprises a transmembrane domain of a protein selected from the group consisting of AM, Ly9 (CD229), CD160 (BY55), PSGL1, CDIOO (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and NKG2C, or a functional variant thereof. In one embodiment, the transmembrane domain comprises a CD8α transmembrane domain. In one embodiment, the antigen-binding domain is linked to the transmembrane domain by a hinge domain. In one embodiment, the hinge domain is a CD8α hinge domain.

[0157] In one embodiment, the CAR molecule of the invention comprises: i) a target antigen-binding domain; ii) CD8α hinge domain; iii) the CD8α transmembrane domain; and iv) an intracellular domain containing the 4-1BB costimulatory domain and the CD3ζ signaling domain Includes.

[0158] The term "antibody" includes immunoglobulins comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged in the following order from amino terminus to carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain the binding domains that interact with antigens. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Antibodies bind, preferably specifically, to antigens. Antibodies can be intact immunoglobulins derived from natural or recombinant sources, or can be immunoreactive portions or fragments of intact immunoglobulins. Antibodies are typically tetramers of immunoglobulin molecules. Antibodies in the present invention can exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, Fv, Fab and F(ab')2, as well as single-chain antibodies and humanized antibodies (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0159] Antibodies expressed by B cells are sometimes called BCRs (B cell receptors) or antigen receptors. The five members of this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody present in bodily secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response in most subjects. It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses and is important for defense against bacteria and viruses. IgD is an immunoglobulin whose antibody function is unknown but which can function as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by triggering the release of mediators from mast cells and basophils upon exposure to allergens.

[0160] The term "antibody fragment" refers to a portion of an intact antibody, typically comprising the antigen-determining variable region of the intact antibody.

[0161] Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multispecific antibodies formed from antibody fragments.

[0162] As used herein, "antibody heavy chain" refers to the larger of the two polypeptide chains present in antibody molecules in their native conformations.

[0163] As used herein, "antibody light chain" refers to the smaller of the two polypeptide chains present in antibody molecules in their native conformation; kappa light chain and lambda light chain refer to the two major antibody light chain isotypes.

[0164] According to the present disclosure, a CAR recognizes an antigen, such as on the surface of an antigen-presenting cell or a diseased cell, such as a cancer cell, such that when present on a T cell, the T cell is stimulated and / or expanded or performs an effector function, as described above.

[0165] Genetic modification of immune effector cells Immune effector cells, especially CD8 + The particles described herein functionalized with DARPins described herein for specific targeting of T cells can be used ex vivo / in vitro or in vivo to deliver nucleic acids encoding antigen receptors to immune effector cells, such as T cells, to produce cells genetically modified to express the antigen receptor. Such genetic modifications include non-viral DNA transfection, non-viral RNA transfection (e.g., mRNA transfection), transposon-based systems, and viral-based systems. Non-viral DNA transfection has a lower risk of insertional mutagenesis. Transposon-based systems can integrate transgenes more efficiently than plasmids that do not contain integration elements. Viral-based systems include the use of gamma-retroviruses and lentiviral vectors. Gamma-retroviruses are relatively easy to produce, efficiently and persistently transduce T cells, and have been preliminarily proven to be safe in terms of integration into primary human T cells. Lentiviral vectors also efficiently and persistently transduce T cells, but are more expensive to produce. They are also potentially safer than retroviral-based systems.

[0166] In one embodiment of all aspects of the invention, T cells or T cell precursors are transfected with a nucleic acid encoding an antigen receptor either ex vivo or in vivo. In one embodiment, a combination of ex vivo and in vivo transfection may be used. In one embodiment of all aspects of the invention, the T cells or T cell precursors are derived from a subject to be treated. In one embodiment of all aspects of the invention, the T cells or T cell precursors are derived from a subject different from the subject to be treated.

[0167] In one embodiment of the present invention, CAR T cells can be generated in vivo, and therefore almost instantly, using particles such as the nanoparticles described herein that target T cells. For example, lipid and / or polymer-based nanoparticles can be coupled to a CD8-specific DARPin to bind to CD8 on T cells. Upon binding to T cells, these nanoparticles are endocytosed. Their contents, such as nucleic acids encoding antigen receptors, such as plasmid DNA encoding anti-tumor antigen CARs, can be targeted to the T cell nucleus because they contain, for example, peptides containing microtubule-associated sequences (MTAS) and nuclear localization signals (NLS). The inclusion of a transposon flanking the nucleic acid encoding the antigen receptor, such as a CAR gene expression cassette, and a separate nucleic acid, such as a plasmid, encoding a hyperactive transposase can enable efficient integration of the nucleic acid encoding the antigen receptor, such as a CAR vector, into the chromosome.

[0168] Another possibility is to use CRISPR / Cas9 technology to deliberately place an antigen receptor coding sequence, such as a CAR coding sequence, at a specific genetic locus, for example, knocking out an existing T cell receptor (TCR) while knocking in the CAR and placing it under the dynamic regulatory control of an endogenous promoter that would otherwise silence TCR expression.

[0169] Therefore, in addition to nucleic acids encoding antigen receptors, the particles described herein can also deliver gene editing tools such as CRISPR / Cas9 (or related) or transposon systems such as Sleeping Beauty or Piggybag as cargo. Such tools for genome integration / editing (e.g., transposases, gene editing tools such as CRISPR / Cas9) can be delivered as proteins or encoding nucleic acids (DNA or RNA). Nevertheless, delivery of mRNA is also an option for inducing transient expression of antigen receptors such as CARs or T cell receptors (TCRs).

[0170] In one embodiment of all aspects of the invention, cells genetically modified to express an antigen receptor are stably or transiently transfected with a nucleic acid encoding the antigen receptor, such that the nucleic acid encoding the antigen receptor is either integrated or not integrated into the genome of the cell.

[0171] In one embodiment of all aspects of the invention, the cells genetically modified to express an antigen receptor are inactivated with respect to expression of endogenous T cell receptors and / or endogenous HLA.

[0172] In one embodiment of all aspects of the present invention, the cells described herein can be autologous, allogeneic, or syngeneic to the subject being treated. In one embodiment, the present disclosure contemplates the removal of cells from the patient and subsequent re-delivery of the cells to the patient. In one embodiment, the present disclosure does not contemplate the removal of cells from the patient. In the latter case, all steps of genetic modification of the cells are performed in vivo.

[0173] The term "autologous" is used to refer to something derived from the same subject. For example, "autologous transplantation" refers to the transplantation of tissue or organs derived from the same subject. Such procedures are advantageous because they overcome immunological barriers that would otherwise result in rejection.

[0174] The term "allogeneic" is used to describe something that is derived from different individuals of the same species. Two or more individuals are said to be allogeneic to one another if the genes at one or more loci are not identical.

[0175] The term "syngeneic" is used to describe individuals or tissues that have the same genotype, i.e., derived from identical twins or the same inbred strain of animals, or tissues thereof.

[0176] The term "xenogeneic" is used to describe something that is made up of multiple dissimilar elements. As an example, transferring bone marrow from one individual to another constitutes a xenogeneic transplant. A xenogeneic gene is a gene that originates from a source other than the subject.

[0177] Nucleic acid containing particles In the context of the present disclosure, the term "particle" refers to a structured entity formed by a molecule or molecular complex. In one embodiment, the term "particle" refers to a micro- or nano-sized structure, such as a micro- or nano-sized compact structure dispersed in a medium. In one embodiment, the particle is a nucleic acid-containing particle, such as a particle containing DNA, RNA, or a mixture thereof.

[0178] Electrostatic interactions between positively charged molecules such as polymers and lipids and negatively charged nucleic acids are involved in particle formation. This leads to complexation and spontaneous formation of nucleic acid particles. In one embodiment, the nucleic acid particles are nanoparticles.

[0179] As used in this disclosure, "nanoparticles" refers to particles having an average diameter suitable for parenteral administration.

[0180] "Nucleic acid particles" can be used to deliver nucleic acids to a desired target site (e.g., a cell, tissue, organ, etc.). Nucleic acid particles can be formed from at least one cationic or cationically ionizable lipid or lipid-like substance, such as DOTAP, at least one cationic polymer, such as protamine, or a mixture thereof, and nucleic acid. Nucleic acid particles include lipid nanoparticle (LNP)-based and lipoplex (LPX)-based formulations.

[0181] Without intending to be bound by any theory, it is believed that cationic or cationically ionizable lipids or lipid-like materials and cationic polymers form aggregates with nucleic acids, which aggregates result in colloidally stable particles.

[0182] In one embodiment, the particles described herein further comprise at least one lipid or lipid-like substance other than a cationic or cationically ionizable lipid or lipid-like substance, at least one polymer other than a cationic polymer, or a mixture thereof.

[0183] In some embodiments, nucleic acid particles comprise multiple types of nucleic acid molecules, and the molecular parameters of the nucleic acid molecules can be similar or different from one another, such as with respect to molar mass or basic structural elements such as molecular structure, capping, coding regions, or other features. The nucleic acid particles described herein, in one embodiment, can have an average diameter ranging from about 30 nm to about 1000 nm, from about 50 nm to about 800 nm, from about 70 nm to about 600 nm, from about 90 nm to about 400 nm, or from about 100 nm to about 300 nm.

[0184] For example, the nucleic acid particles described herein, produced by the processes described herein, exhibit a polydispersity index of less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less. By way of example, the nucleic acid particles can exhibit a polydispersity index ranging from about 0.1 to about 0.3 or from about 0.2 to about 0.3.

[0185] The nucleic acid particles described herein can be prepared using a wide variety of methods, which may include obtaining a colloid from at least one cationic or cationically ionizable lipid or lipid-like material and / or at least one cationic polymer, and mixing the colloid with nucleic acid to obtain the nucleic acid particles.

[0186] The term "colloid" as used herein refers to a type of homogeneous mixture in which dispersed particles do not settle. The insoluble particles in the mixture are microscopic and have a particle size between 1 and 1000 nanometers. The mixture may be referred to as a colloid or a colloidal suspension. The term "colloid" may refer only to the particles in the mixture and not to the suspension as a whole.

[0187] For the preparation of colloids containing at least one cationic or cationically ionizable lipid or lipid-like substance and / or at least one cationic polymer, methods conventionally used to prepare liposome vesicles and appropriately adapted for use are applicable herein. The most commonly used methods for preparing liposome vesicles share the following basic steps: (i) dissolving lipids in an organic solvent, (ii) drying the resulting solution, and (iii) hydrating the dried lipids (using various aqueous media).

[0188] In the film hydration method, lipids are first dissolved in a suitable organic solvent and dried to obtain a thin film at the bottom of a flask. The resulting lipid film is hydrated with a suitable aqueous medium to obtain a liposome dispersion. Further miniaturization steps may also be included.

[0189] Reverse phase evaporation is an alternative method to membrane hydration for preparing liposome vesicles, involving the formation of a water-in-oil emulsion between an aqueous phase and a lipid-containing organic phase. Brief sonication of this mixture is necessary to homogenize the system. Removal of the organic phase under reduced pressure results in a milky gel that then transforms into a liposome suspension.

[0190] Other methods that have organic solvent-free properties may also be used in accordance with the present disclosure to prepare colloids.

[0191] LNPs typically consist of four components: ionizable cationic lipids, phospholipids, cholesterol, and polyethylene glycol (PEG) lipids. Each component is responsible for payload protection and enables effective intracellular delivery. LNPs can be prepared by rapidly mixing lipids dissolved in ethanol with nucleic acid in an aqueous buffer.

[0192] The term "mean diameter" refers to the average hydrodynamic diameter of particles measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, resulting in the so-called Z dimension, which has a length dimension. 平均 , and the dimensionless polydispersity index (PI) (Koppel, D., J. Chem. Phys. 57, 1972, pp. 4814-4820, ISO 13321). Here, the "average diameter", "diameter" or "size" of a particle is this Z 平均 Used synonymously with the value of

[0193] The "polydispersity index", as mentioned in the definition of "mean diameter", is preferably calculated based on dynamic light scattering measurements by so-called cumulant analysis. Under certain prerequisites, it can be considered as a measure of the size distribution of the nanoparticle ensemble.

[0194] Various types of nucleic acid-containing particles have previously been described as suitable for delivery of nucleic acids in particulate form (e.g., Kaczmarek, JC et al., 2017, Genome Medicine 9, 60). In the case of non-viral nucleic acid delivery vehicles, nanoparticle encapsulation of nucleic acids can physically protect the nucleic acid from degradation and, depending on the specific chemical properties, aid in cellular uptake and endosomal escape.

[0195] The present disclosure describes a particle comprising nucleic acid, at least one cationic or ionizable lipid or lipid-like substance, and / or at least one cationic polymer that is associated with nucleic acid to form nucleic acid particle, and a composition comprising such particle.Nucleic acid particle can comprise nucleic acid that is complexed to particle in various forms by non-covalent interaction.The particle described herein is not a virus particle, particularly an infectious virus particle, i.e., they cannot infect cells with virus.

[0196] Suitable cationic or cationically ionizable lipids or lipid-like substances and cationic polymers form nucleic acid particles and are included in the term "particle-forming component" or "particle-forming agent." The term "particle-forming component" or "particle-forming agent" refers to any component that associates with nucleic acid to form a nucleic acid particle. Such components include any component that can be part of a nucleic acid particle.

[0197] cationic polymer Polymers are commonly used materials for nanoparticle-based delivery, given their high chemical flexibility. Cationic polymers are typically used to electrostatically condense negatively charged nucleic acids into nanoparticles. These positively charged groups often consist of amines that change protonation state in the pH range of 5.5 to 7.5, which is thought to lead to an ionic imbalance that results in endosomal rupture. Polymers such as poly-L-lysine, polyamidoamine, protamine, and polyethyleneimine, as well as naturally occurring polymers such as chitosan, have all been applied to nucleic acid delivery and are suitable as cationic polymers herein. Furthermore, some researchers have synthesized polymers specifically for nucleic acid delivery. Poly(β-amino esters), in particular, are widely used in nucleic acid delivery due to their ease of synthesis and biodegradability. Such synthetic polymers are also suitable as cationic polymers herein.

[0198] As used herein, the term "polymer" is given its usual meaning, i.e., a molecular structure comprising one or more repeating units (monomers) linked by covalent bonds. The repeating units may all be identical, or in some cases, multiple types of repeating units may exist within a polymer. In some cases, the polymer is biologically derived, i.e., a biopolymer such as a protein. In some cases, additional moieties, such as targeting moieties as described herein, may also be present in the polymer.

[0199] When multiple types of repeating units are present in a polymer, the polymer is said to be a "copolymer." It should be understood that a polymer as used herein may be a copolymer. The repeating units forming a copolymer may be arranged in any manner. For example, the repeating units may be arranged in a random order, an alternating order, or as a "block" copolymer, i.e., a copolymer comprising one or more regions each comprising a first repeating unit (e.g., a first block) and one or more regions each comprising a second repeating unit (e.g., a second block), etc. A block copolymer may have two (diblock copolymer), three (triblock copolymer), or more distinct blocks.

[0200] In certain embodiments, the polymer is biocompatible. Biocompatible polymers are typically polymers that do not cause significant cell death at moderate concentrations. In certain embodiments, the biocompatible polymer is biodegradable, i.e., the polymer can be chemically and / or biologically degraded in a physiological environment, such as within the body.

[0201] In certain embodiments, the polymer may be protamine or a polyalkyleneimine, particularly protamine.

[0202] The term "protamine" refers to any of a variety of relatively low molecular weight, strongly basic proteins that are rich in arginine and are found in the sperm cells of various animals (such as fish) in place of somatic histones, particularly in association with DNA. In particular, the term "protamine" refers to a protein found in fish sperm that is strongly basic, soluble in water, does not coagulate with heat, and produces primarily arginine upon hydrolysis. In purified form, they are used to neutralize the anticoagulant effect of heparin in long-acting formulations of insulin.

[0203] In accordance with the present disclosure, the term "protamine" as used herein is intended to include any protamine amino acid sequence and fragments thereof obtained or derived from natural or biological sources, and multimeric forms of said amino acid sequence or fragments thereof, as well as artificial, specifically designed for a particular purpose (synthetic) polypeptides that cannot be isolated from natural or biological sources.

[0204] In one embodiment, the polyalkyleneimine comprises polyethyleneimine and / or polypropyleneimine, preferably polyethyleneimine. A preferred polyalkyleneimine is polyethyleneimine (PEI). The average molecular weight of PEI is preferably 0.75×10 2 ~10 7 Da, preferably 1000 to 10 5 Da, more preferably 10,000 to 40,000 Da, more preferably 15,000 to 30,000 Da, and even more preferably 20,000 to 25,000 Da.

[0205] According to the present disclosure, linear polyalkyleneimines such as linear polyethyleneimine (PEI) are preferred.

[0206] Cationic polymers (including polycationic polymers) contemplated for use herein include any cationic polymer that can electrostatically bind to nucleic acids. In one embodiment, cationic polymers contemplated for use herein include any cationic polymer with which nucleic acids can associate, for example, by forming a complex with the nucleic acid or by forming vesicles in which the nucleic acid is entrapped or encapsulated.

[0207] The particles described herein may also include polymers other than cationic polymers, i.e., non-cationic polymers and / or anionic polymers. Collectively, anionic and neutral polymers are referred to herein as non-cationic polymers.

[0208] Lipids and lipid-like substances The terms "lipid" and "lipid-like substance" are broadly defined herein as molecules containing one or more hydrophobic moieties or groups and, optionally, one or more hydrophilic moieties or groups. Molecules containing both hydrophobic and hydrophilic moieties are often referred to as amphiphiles. Lipids are typically poorly soluble in water. In aqueous environments, their amphiphilic nature allows them to self-assemble into organized structures and various phases. One of these phases consists of lipid bilayers, such as those found in vesicles, multilamellar / unilamellar liposomes, or membranes in aqueous environments. Hydrophobicity can be imparted by the inclusion of nonpolar groups, including, but not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups, and such groups substituted with one or more aromatic, alicyclic, or heterocyclic groups. Hydrophilic groups can include polar and / or charged groups, including carbohydrates, phosphate groups, carboxylate groups, sulfate groups, amino groups, sulfhydryl groups, nitro groups, hydroxyl groups, and other similar groups.

[0209] As used herein, the term "amphiphilic" refers to a molecule having both polar and non-polar portions. Amphiphilic compounds often have a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non-polar portion is insoluble in water. Furthermore, the polar portion can have either a formal positive or a formal negative charge. Alternatively, the polar portion can have both a formal positive and a formal negative charge and can be a zwitterion or an internal salt. For purposes of this disclosure, an amphiphilic compound can be, but is not limited to, one or more natural or non-natural lipids and lipid-like compounds.

[0210] The terms "lipid-like substance," "lipid-like compound," or "lipid-like molecule" refer to substances that are structurally and / or functionally related to lipids but cannot be considered lipids in the strict sense. For example, this term includes compounds that can form amphiphilic layers such as those found in vesicles, multilamellar / unilamellar liposomes, or membranes in aqueous environments, and includes surfactants or synthetic compounds with both hydrophilic and hydrophobic moieties. Generally speaking, this term refers to molecules that contain hydrophilic and hydrophobic moieties with different structural organizations that may or may not resemble the structure of lipids. As used herein, the term "lipid" should be interpreted to encompass both lipids and lipid-like substances, unless otherwise indicated herein or clearly contradicted by the context.

[0211] Specific examples of amphiphilic compounds that can be included in the amphiphilic layer include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

[0212] In certain embodiments, the amphiphilic compound is a lipid. The term "lipid" refers to a group of organic compounds characterized by being insoluble in water but soluble in many organic solvents. Generally, lipids can be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, glycolipids, polyketides (derived from the condensation of ketoacyl subunits), sterol lipids, and prenol lipids (derived from the condensation of isoprene subunits). The term "lipid" is sometimes used as a synonym for fat, but fat is a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including triglycerides, diglycerides, monoglycerides, and phospholipids), as well as sterol-containing metabolites such as cholesterol.

[0213] Fatty acids, or fatty acid residues, are a diverse group of molecules made up of a hydrocarbon chain terminating in a carboxylic acid group; this arrangement gives the molecule a polar, hydrophilic end and a nonpolar, hydrophobic end that is insoluble in water. The carbon chain, typically 4 to 24 carbons long, can be saturated or unsaturated and can be bonded to functional groups including oxygen, halogens, nitrogen, and sulfur. When fatty acids contain double bonds, there is the possibility of cis or trans geometric isomerism, which significantly affects the molecular configuration. Cis double bonds cause bending of the fatty acid chain, an effect that can be compounded with more double bonds within the fatty acid chain. Other major lipid classes within the fatty acid category are fatty acid esters and fatty acid amides.

[0214] Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the most well-known of which are fatty acid triesters of glycerol, called triglycerides. The term "triacylglycerol" is sometimes used synonymously with "triglyceride." In these compounds, each of the three hydroxyl groups of glycerol is typically esterified with a different fatty acid. A further subclass of glycerolipids is represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via glycosidic bonds.

[0215] Glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core attached by ester bonds to two fatty acid-derived "tails" and by a phosphate ester bond to a "head" group. Examples of glycerophospholipids, commonly referred to as phospholipids (although sphingomyelin is also classified as a phospholipid), are phosphatidylcholine (PC, also known as GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn), and phosphatidylserine (PS or GPSer).

[0216] Sphingolipids are a complex family of compounds that share a common structural feature: a sphingoid base backbone. The predominant sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with amide-linked fatty acids. The fatty acids are typically saturated or monounsaturated and have chain lengths of 16–26 carbon atoms. The predominant sphingophospholipid in mammals is sphingomyelin (ceramide phosphocholine), while insects contain primarily ceramide phosphoethanolamine, and fungi have phytoceramide phosphoinositol and mannose-containing head groups. Glycosphingolipids are a diverse family of molecules composed of one or more sugar residues attached to a sphingoid base via glycosidic bonds. Examples of these are simple and complex glycosphingolipids such as cerebrosides and gangliosides.

[0217] Sterol lipids, such as cholesterol and its derivatives, or tocopherol and its derivatives, are important components of membrane lipids, along with glycerophospholipids and sphingomyelins.

[0218] Glycolipids are compounds in which fatty acids are directly attached to a sugar backbone, forming structures compatible with membrane bilayers. In glycolipids, monosaccharides replace the glycerol backbone present in glycerolipids and glycerophospholipids. The best-known glycolipid is the acylated glucosamine precursor of the lipid A component of the lipopolysaccharide of Gram-negative bacteria. A typical lipid A molecule is a disaccharide of glucosamine derivatized with as many as seven fatty acyl chains. The minimal lipopolysaccharide required for growth in Escherichia coli is Kdo2-lipid A, a hexaacylated disaccharide of glucosamine glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.

[0219] Polyketides are synthesized by the polymerization of acetyl and propionyl subunits by classical enzymes as well as by iterative and multimodular enzymes that share mechanistic features with fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal, and marine sources, and possess great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.

[0220] According to the present disclosure, lipids and lipid-like substances can be cationic, anionic, or neutral. Neutral lipids or lipid-like substances exist in an uncharged or neutral zwitterionic form at a selected pH.

[0221] Cationic or cationic ionizable lipids or lipid-like substances The nucleic acid particles described herein comprise at least one cationic or cationically ionizable lipid or lipid-like substance as particle-forming agent.The cationic or cationically ionizable lipid or lipid-like substance contemplated for use herein comprises any cationic or cationically ionizable lipid or lipid-like substance that can electrostatically bind to nucleic acid.In one embodiment, the cationic or cationically ionizable lipid or lipid-like substance contemplated for use herein can be associated with nucleic acid, for example, by forming a complex with nucleic acid or by forming a vesicle in which nucleic acid is enclosed or encapsulated.

[0222] As used herein, "cationic lipid" or "cationic lipid-like substance" refers to a lipid or lipid-like substance that has a net positive charge.Cationic lipid or lipid-like substance binds to negatively charged nucleic acid through electrostatic interaction.Generally, cationic lipids have a lipophilic moiety such as a sterol, an acyl chain, a diacyl chain or more acyl chains, and the head group of the lipid typically carries a positive charge.

[0223] In certain embodiments, the cationic lipid or lipid-like substance has a net positive charge only at a certain pH, particularly an acidic pH, but preferably has no net positive charge, preferably has no charge, i.e., is neutral at a different, preferably higher, pH, such as physiological pH. This ionizable behavior is thought to enhance efficacy by aiding endosomal escape and reducing toxicity, compared to particles that remain cationic at physiological pH.

[0224] For the purposes of this disclosure, such "cationically ionizable" lipids or lipid-like substances are included in the term "cationic lipids or lipid-like substances," unless the context indicates otherwise.

[0225] In one embodiment, the cationic or cationically ionizable lipid or lipid-like substance comprises a head group that includes at least one nitrogen atom (N) that is positively charged or capable of being protonated.

[0226] Examples of cationic lipids include 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA), 3-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium propane (DODAP); 1,2-diacyloxy-3-di Methylammonium propane; 1,2-dialkyloxy-3-dimethylammonium propane; dioctadecyldimethylammonium chloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2-hydroxyethyl)dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), l,2-dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyl Oxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE), and 2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), dioctadecylamidoglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3- beta-oxybutane-4-oxy)-1-(cis,cis-9,12-octadecadienooxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1-(cis,cis-9',12'-octadecadienooxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N-Dimethylpropylamine (DLinDAP), 1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-K-XTC2-DMA), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3] -dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9-tetradecenyloxy)-1-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N, N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (GAP-DMRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)-1-propanaminium bromide (βAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propan-1-aminium (DOBAQ), 2 -({8-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium propane (DMDAP), 1,2-dipalmitoyl-3-dimethylammonium propane (DPDAP), N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarboxamido)ethyl]-3,4-Di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N-dimethylpropan-1-aminium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide (DMORIE), di((Z)-non-2-en-1-yl)8,8'-((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl- 2,3-Bis(dodecyloxy)propan-1-amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-amine (DMDMA), di((Z)-non-2-en-1-yl)-9-((4-(dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2-dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)ethyl]-amino}-ethylamino)propionamide (Lipidoid 98N, 12 -5), 1-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2-hydroxydodecyl)amino]ethyl]piperazin-1-yl]ethyl]amino]dodecan-2-ol (lipidoid C12-200). DOTAP, DODMA, DOTMA, DODAC, and DOSPA are preferred. In certain embodiments, at least one cationic lipid is DOTAP.

[0227] In some embodiments, the cationic lipid may comprise from about 10 mol% to about 100 mol%, from about 20 mol% to about 100 mol%, from about 30 mol% to about 100 mol%, from about 40 mol% to about 100 mol%, or from about 50 mol% to about 100 mol% of the total lipid present in the particle.

[0228] Additional lipids or lipid-like substances The particles described herein can also contain lipids or lipid-like substances other than cationic or cationically ionizable lipids or lipid-like substances, i.e., non-cationic lipids or lipid-like substances (including non-cationically ionizable lipids or lipid-like substances).Collectively, anionic and neutral lipids or lipid-like substances are referred to herein as non-cationic lipids or lipid-like substances.In addition to ionizable / cationic lipids or lipid-like substances, the formulation of nucleic acid particles can be optimized by adding other hydrophobic moieties such as cholesterol and lipids, thereby improving the stability of particles and the effectiveness of nucleic acid delivery.

[0229] Additional lipids or lipid-like substances may be incorporated, which may or may not affect the overall charge of the nucleic acid particles. In certain embodiments, the additional lipids or lipid-like substances are non-cationic lipids or lipid-like substances. Non-cationic lipids may include, for example, one or more anionic lipids and / or neutral lipids. As used herein, "neutral lipid" refers to any of a number of lipid species that exist in an uncharged or neutral zwitterionic form at a selected pH. In preferred embodiments, the additional lipid comprises one of the following neutral lipid components: (1) phospholipid, (2) cholesterol or a derivative thereof, or (3) a mixture of phospholipid and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, tocopherol and their derivatives, and mixtures thereof.

[0230] Specific phospholipids that can be used include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, or sphingomyelin, among others, diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), diacylphosphatidylcholine (D ... PC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphosphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC) and phosphatidylethanolamines, particularly diacylphosphatidylethanolamines such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoyl-phosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroyl-phosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), and further phosphatidylethanolamine lipids with various hydrophobic chains.

[0231] In certain preferred embodiments, the additional lipid is DSPC, or DSPC and cholesterol.

[0232] In certain embodiments, the nucleic acid particle comprises both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTAP and the additional lipid is DSPC or DSPC and cholesterol.

[0233] Without wishing to be bound by theory, the amount of at least one cationic lipid relative to the amount of at least one additional lipid can affect important nucleic acid particle properties, such as charge, particle size, stability, tissue selectivity, and nucleic acid biological activity. Thus, in some embodiments, the molar ratio of at least one cationic lipid to at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1.

[0234] In some embodiments, non-cationic lipids, particularly neutral lipids (e.g., one or more phospholipids and / or cholesterol), may comprise from about 0 mol% to about 90 mol%, from about 0 mol% to about 80 mol%, from about 0 mol% to about 70 mol%, from about 0 mol% to about 60 mol%, or from about 0 mol% to about 50 mol% of the total lipid present in the particle.

[0235] target molecule One or more of the particle-forming components described herein, such as polymers, lipids and / or lipid-like substances, may be used to target the particles to immune effector cells, particularly CD8 + It may comprise or be functionalized with one or more DARPins that target T cells, such as T cells. The DARPins may be conjugated, in particular covalently or non-covalently bound, or linked to any particle-forming component, such as a lipid, lipid-like substance, or polymer.

[0236] Provided herein are DARPins that, in particular, when fused to nucleic acid particle components such as lipids or proteins, specifically bind to CD8 and exhibit increased transfection of CD8+ T cells in vitro and in vivo compared to particles not functionalized with a DARPin.

[0237] CD8 is the primary marker for the cytotoxic subset of T lymphocytes. It is a type I single-pass transmembrane protein expressed as a disulfide-linked homo- or heterodimeric molecule on the surface of immune cells. The CD8 heterodimer, consisting of the CD8α and CD8β chains, is expressed exclusively on the surface of immature CD4+CD8+ double-positive thymocytes and mature peripheral cytotoxic αβ T cells. The homodimer, consisting of two CD8α chains, is expressed on a much broader range of immune cells. In addition to classical cytotoxic αβ T cells and thymocytes, it is found on natural killer T (NKT) cells, a subset of dendritic cells (DCs), and a subpopulation of natural killer (NK) cells. Both CD8αβ and CD8αα can mediate MHC-I binding; however, the heterodimeric form is more prevalent on the surface of MHC-I-restricted cytotoxic T cells. Notably, the CD8ββ homodimer does not exist in nature.

[0238] The term "DARPin" refers to a designed ankyrin repeat protein. DARPins are based on naturally occurring ankyrin repeat proteins but contain one or more amino acid mutations that can affect, for example, their binding affinity to target molecules, their cell surface expression, etc. DARPins preferably contain two to three ankyrin repeat modules flanked by N- and C-capping repeats. Each ankyrin repeat module contains approximately 33 amino acid residues.

[0239] Ankyrin repeat proteins were identified in 1987 through sequence comparisons between four such proteins in Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. Breeden and Nasmyth reported multiple copies of a repeating unit of approximately 33 residues in the sequences of swi6p, cddOp, Notch, and lin-12 (Breeden et al., Nature 329, 651-654 (1987)). The subsequent discovery of 24 copies of this repeating unit in the ankyrin protein led to the designation of this repeat as an ankyrin repeat (Lux et al., Nature 344, 36-42 (1990)). This repeat unit has subsequently been identified in hundreds of proteins from various organisms and viruses (Bork, Proteins 17(4), 363-74 (1993)). These proteins are located in the nucleus, cytoplasm, or extracellular space. This is consistent with the fact that the ankyrin repeat domains of these proteins are independent of disulfide bridges and therefore independent of the oxidative state of the environment. The number of repeat units per protein varies from two to more than 20. The tertiary structure of ankyrin repeat units shares a characteristic fold consisting of a β-hairpin followed by two antiparallel α-helices, ending with a loop connecting the repeat unit to the next repeat unit (Sedgwick and Smerdon, Trends Biochem Sci. 24(8), 311-6 (1999)). Domains constructed from ankyrin repeat units are formed by stacking the repeat units into an extended, curved structure. Proteins containing ankyrin repeat domains often contain additional domains. While the latter domains have variable functions, the function of the ankyrin repeat domain is most often binding to other proteins. Analysis of the repeat units of these proteins reveals that the target interaction residues are primarily found in the exposed portions of the β-hairpin and the first α-helix.These target interaction residues therefore form a large contact surface on the ankyrin repeat domain, which is exposed on a framework constructed from stacked units of α-helix 1, α-helix 2, and loops.

[0240] DARPins that bind to specific targets can be identified by screening combinatorial libraries of DARPins and selecting those with desired binding properties to the target. Such screening methods are described, for example, in Muench et al., Molecular Therapy, 16(4), 686-693, 2011. For example, target-specific DARPins can be selected from diverse libraries using ribosome display or phage display methods.

[0241] The term "repeat protein" refers to a (poly)peptide / protein comprising one or more repeat domains. In one embodiment, the repeat protein comprises up to four repeat domains. In one embodiment, the repeat protein comprises up to three repeat domains. In one embodiment, the repeat protein comprises up to two repeat domains. In a most preferred embodiment, the repeat protein comprises one repeat domain.

[0242] Repeat proteins may comprise additional non-repetitive protein domains such as (poly)peptide tags, enzymes that may allow detection of the repeat protein (e.g. alkaline phosphatase), or moieties that can be used for targeting (such as immunoglobulins or fragments thereof) and / or as effector molecules.

[0243] The term "(poly)peptide tag" refers to an amino acid sequence attached to a (poly)peptide / protein, said amino acid sequence being useful for purifying, detecting or targeting said (poly)peptide / protein. Such (poly)peptide tags can be small polypeptide sequences, e.g., His n, myc, FLAG, or Strep tags. All these (poly)peptide tags are well known in the art.

[0244] The individual domains of the repeat proteins can be linked to each other directly or via a (poly)peptide linker. The term "(poly)peptide linker" refers to an amino acid sequence capable of linking two protein domains. Such linkers include, for example, glycine-serine linkers of various lengths and are known to those skilled in the art.

[0245] The term "repeat domain" refers to a protein domain comprising two or more consecutive repeat units (modules). In one embodiment, said repeat units are structural units with the same or similar fold, preferably tightly stacked to form a coiled-coil structure, preferably with a bound hydrophobic core.

[0246] The term "structural unit" refers to a locally ordered portion of a (poly)peptide formed by three-dimensional interactions between two or more segments of secondary structure that are in close proximity to each other along the (poly)peptide chain. Such structural units comprise structural motifs.

[0247] The term "structural motif" refers to the three-dimensional arrangement of secondary structural elements present in at least one structural unit. Structural motifs are well known to those skilled in the art. The structural units may not be able to obtain a defined three-dimensional arrangement by themselves; however, their sequential arrangement as repeat modules within a repeat domain may lead to the mutual stabilization of adjacent units, resulting in a coiled-coil structure.

[0248] The term "repeat module" refers to a repeated amino acid sequence of a repeat protein, which is derived from a repeat unit of a naturally occurring protein. Each repeat module comprised in a repeat domain is derived from one or more repeat units of a naturally occurring repeat protein, such as the ankyrin repeat protein family.

[0249] The term "set of repeat modules" refers to the total number of repeat modules present in a repeat domain. Such a "set of repeat modules" present in a repeat domain may comprise two or more consecutive repeat modules, and may contain only one type of repeat module in two or more copies, or two or more different types of modules, each present in one or more copies. Such a set of repeat modules, for example comprising three repeat modules, may comprise, consecutively from N- to C-terminus, repeat module 1, repeat module 2 and repeat module 3, as shown, for example, in Figure 5. Repeat module 1 as shown in Figure 5 preferably comprises amino acids 29-61. Repeat module 2 as shown in Figure 5 preferably comprises amino acids 62-94. Repeat module 3 as shown in Figure 5 preferably comprises amino acids 95-127.

[0250] Different repeat domains may have the same number of repeat modules per repeat domain or may have different numbers of repeat modules per repeat domain.

[0251] Preferably, the repeat modules comprised in the set are homologous repeat modules. In the context of the present invention, the term "homologous repeat module" refers to repeat modules in which more than 70% of the framework residues of said repeat modules are homologous. Preferably, more than 80% of the framework residues of said repeat modules are homologous. Most preferably, more than 90% of the framework residues of said repeat modules are homologous. Computer programs for determining the percentage of homology between polypeptides, such as Fasta, Blast or Gap, are known to those skilled in the art.

[0252] The term "repeat unit" refers to an amino acid sequence that contains one or more naturally occurring protein sequence motifs, said "repeat unit" being found in multiple copies and exhibiting a defined folding topology common to all said motifs that determines the folding of the protein. Such a repeat unit contains framework residues and interacting residues.

[0253] An example of such a repeat unit is an ankyrin repeat unit. A naturally occurring protein comprising two or more such repeat units is referred to as a "naturally occurring repeat protein." The amino acid sequences of the individual repeat units of a repeat protein may have a significant number of mutations, substitutions, additions and / or deletions when compared with each other, while still substantially retaining the general pattern or motif of the repeat units.

[0254] The term "repeat sequence motif" or "repeat consensus sequence" refers to an amino acid sequence deduced from one or more repeat units. Such a repeat sequence motif comprises a framework residue position and a target interaction residue position. The framework residue position corresponds to the framework residue position of the repeat unit. The target interaction residue position corresponds to the target interaction residue position of the repeat unit. Such a repeat sequence motif comprises fixed positions and randomized positions. The term "fixed position" refers to an amino acid position within a repeat sequence motif, where the position is set to a specific amino acid. In many cases, such fixed positions correspond to framework residue positions.

[0255] The term "randomized position" refers to an amino acid position within a repeat motif where two or more amino acids are allowed at that amino acid position. Often, such randomized positions correspond to the positions of target-target interaction residues. However, some positions of framework residues may also be randomized.

[0256] The term "folding topology" refers to the tertiary structure of said repeat units. The folding topology is determined by stretches of amino acids forming at least part of alpha helices or beta sheets, or by stretches of amino acids forming linear polypeptides or loops, or by any combination of alpha helices, beta sheets and / or linear polypeptides / loops.

[0257] The term "series" refers to an arrangement in which said modules are arranged in tandem.

[0258] Repeat proteins have at least 2, often 6 or more, 10 or more, or 20 or more repeat units, usually about 2 to 6. In most cases, repeat proteins are structural and / or adhesive proteins and are found in prokaryotes and eukaryotes, including vertebrates and invertebrates.

[0259] In most cases, the repeat units show a high degree of sequence identity (same amino acid residues at corresponding positions) or sequence similarity (amino acid residues that are different but have similar physicochemical properties), and some of the amino acid residues may be key residues that are highly conserved in different repeat units found in naturally occurring proteins.

[0260] However, a high degree of sequence variability is possible through insertions and / or deletions and / or substitutions of amino acids between the different repeat units found in naturally occurring proteins, so long as the common folding topology is maintained.

[0261] The term "framework residue" relates to amino acid residues of a repeat unit or corresponding amino acid residues of a repeat module that contribute to the folding topology, i.e. contribute to the folding of said repeat unit (or module) or contribute to interactions with neighboring units (or modules). Such contributions can be interactions with other residues in the repeat unit (module) or influence on the conformation of the polypeptide backbone found in alpha helices or beta sheets, or on amino acid stretches that form linear polypeptides or loops.

[0262] The term "target interacting residue" refers to an amino acid residue of a repeat unit or a corresponding amino acid residue of a repeat module that contributes to the interaction with the target substance. Such contribution can be a direct interaction with the target substance or an influence on other directly interacting residues, e.g. by stabilizing the conformation of the (poly)peptide of said repeat unit (module) to enable or enhance the interaction of said directly interacting residue with said target.

[0263] A "target" can be an individual molecule, such as a nucleic acid molecule, a (poly)peptide protein, a carbohydrate, or any other naturally occurring molecule containing any portion of such an individual molecule, or a complex of two or more such molecules. A target can in particular be a molecule on an immune effector cell, in particular CD8.

[0264] In one embodiment, the repeat modules are directly linked. In the context of the present invention, the term "directly linked" refers to repeat modules that are arranged as direct repeats in the repeat protein without any intervening amino acid sequences.

[0265] In another embodiment, the repeat modules are linked by (poly)peptide linkers. Thus, the repeat modules may be indirectly linked via (poly)peptide linkers as intervening sequences separating the individual modules. An "intervening sequence" may be any amino acid sequence that allows linking the individual modules without interfering with the folding topology or stacking of the modules. Preferably, said intervening sequence is a short (poly)peptide linker of less than 10, even more preferably less than 5 amino acid residues.

[0266] In one embodiment, the repeat protein further comprises an N- and / or C-terminal capping module having an amino acid sequence different from any one of said repeat modules. The term "capping module" refers to a polypeptide fused to the N- or C-terminal repeat module of a repeat domain, said capping module forming tight tertiary interactions with said repeat module, thereby providing a cap that shields the hydrophobic core of said repeat module from solvent on the side not in contact with consecutive repeat modules.

[0267] Said N-terminal and / or C-terminal capping modules may be or may be derived from capping units or other domains found in naturally occurring repeat proteins adjacent to the repeat units.

[0268] The term "capping unit" refers to a naturally occurring folded (poly)peptide, which defines a specific structural unit fused N- or C-terminally to a repeating unit, with which it forms tight tertiary interactions, thereby providing a cap that shields the hydrophobic core of the repeating unit from solvent on one side. Such capping units may have sequence similarity to the repeating sequence motif.

[0269] nucleic acid As used herein, the term "polynucleotide" or "nucleic acid" is intended to include DNA and RNA, such as genomic DNA, cDNA, mRNA, recombinantly produced molecules, and chemically synthesized molecules. Nucleic acids can be single-stranded or double-stranded. RNA includes in vitro transcribed RNA (IVT RNA) or synthetic RNA. According to the present invention, polynucleotides are preferably isolated.

[0270] The nucleic acid may be contained in a vector. As used herein, the term "vector" includes any vector known to those skilled in the art, including plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retrovirus, adenovirus, or baculovirus vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or P1 artificial chromosomes (PAC). Such vectors include expression vectors and cloning vectors. Expression vectors include plasmids and viral vectors and generally contain a desired coding sequence and appropriate DNA sequences required for expression of the operably linked coding sequence in a specific host organism (e.g., bacteria, yeast, plants, insects, or mammals) or in an in vitro expression system. Cloning vectors are generally used to manipulate and amplify specific desired DNA fragments and may lack functional sequences required for expression of the desired DNA fragment.

[0271] In one embodiment of all aspects of the invention, a nucleic acid, such as a nucleic acid encoding an antigen receptor or a nucleic acid encoding a vaccine antigen, is expressed in the cells of the subject to be treated to provide the antigen receptor or vaccine antigen. In one embodiment of all aspects of the invention, the nucleic acid is transiently expressed in the cells of the subject. 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, expression of the antigen occurs at the cell surface. In one embodiment of all aspects of the invention, expression of the vaccine antigen occurs at the cell surface. In one embodiment of all aspects of the invention, the vaccine antigen is expressed and presented in the context of MHC.

[0272] In one embodiment of all aspects of the invention, a nucleic acid encoding a vaccine antigen is expressed in cells, such as antigen-presenting cells, of the subject to be treated to provide the vaccine antigen for binding by immune effector cells genetically modified to express an antigen receptor, said binding resulting in stimulation, priming and / or expansion of the immune effector cells genetically modified to express the antigen receptor.

[0273] The nucleic acids described herein can be recombinant and / or isolated molecules.

[0274] In this disclosure, the term "RNA" refers to a nucleic acid molecule containing ribonucleotide residues. In preferred embodiments, RNA contains all or most of the ribonucleotide residues. As used herein, "ribonucleotide" refers to a nucleotide having a hydroxyl group at the 2' position of a β-D-ribofuranosyl group. RNA includes, but is not limited to, double-stranded RNA, single-stranded RNA, isolated RNA such as partially purified RNA, essentially pure RNA, synthetic RNA, recombinantly produced RNA, and modified RNA that differs from naturally occurring RNA by the addition, deletion, substitution, and / or modification of one or more nucleotides. Such modifications may refer to the addition of non-nucleotide material to internal RNA nucleotides or to the termini (either or both) of the RNA. It is also contemplated herein that the nucleotides in RNA may be non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. In this disclosure, these modified RNAs are considered analogs of naturally occurring RNA.

[0275] In certain embodiments of the present disclosure, the RNA is messenger RNA (mRNA), which refers to an RNA transcript encoding a peptide or protein. As established in the art, mRNA generally comprises a 5' untranslated region (5'-UTR), a peptide coding region, and a 3' untranslated region (3'-UTR). In some embodiments, the RNA is produced by in vitro transcription or chemical synthesis. In one embodiment, the mRNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid comprising deoxyribonucleotides.

[0276] In one embodiment, the RNA is in vitro transcribed RNA (IVT-RNA), which can be obtained by in vitro transcription of a suitable DNA template. The promoter for controlling transcription can be any promoter for any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning a nucleic acid, particularly a cDNA, and introducing it into a suitable vector for in vitro transcription. The cDNA can be obtained by reverse transcription of RNA.

[0277] In one embodiment, the RNA may have modified ribonucleotides. Examples of modified ribonucleotides include, but are not limited to, 5-methylcytidine, pseudouridine, and / or 1-methylpseudouridine.

[0278] In some embodiments, the RNA of the present disclosure includes a 5' cap. In one embodiment, the RNA of the present disclosure does not have an uncapped 5'-triphosphate. In one embodiment, the RNA may be modified with a 5' cap analog. The term "5' cap" refers to the structure found at the 5' end of an mRNA molecule and generally consists of a guanosine nucleotide linked to the mRNA by a 5'-5' triphosphate bond. In one embodiment, the guanosine is methylated at position 7. Providing an RNA with a 5' cap or 5' cap analog can be achieved by in vitro transcription, in which the 5' cap is co-transcriptionally expressed on the RNA strand, or can be attached to the RNA post-transcriptionally using a capping enzyme.

[0279] In some embodiments, the building block cap for RNA is m2 7,3'-O Gppp(m1 2'-O ) ApG (sometimes m2 7,3'O G(5')ppp(5')m 2'-O ApG), which has the following structure:

[0280] [ka]

[0281] It has.

[0282] The following are RNA and m2 7,3'O G(5')ppp(5')m 2'-O An exemplary Cap1 RNA containing ApG is:

[0283] [ka]

[0284] Below is another exemplary Cap1 RNA (no cap analog):

[0285] [ka]

[0286] In some embodiments, the RNA has, in one embodiment, the structure:

[0287] [ka]

[0288] The anti-reverse cap of the cap analogue (ARCA cap (m2 7,3'O G(5')ppp(5')G)) is modified with a "cap 0" structure.

[0289] The following are RNA and m2 7,3'O An exemplary Cap 0 RNA containing G(5')ppp(5')G is:

[0290] [ka]

[0291] In some embodiments, the "cap 0" structure has the structure:

[0292] [ka]

[0293] Cap analogue β-S-ARCA (m2 7,2'O G(5')ppSp(5')G).

[0294] The following is β-S-ARCA(m2 7,2'O An exemplary capped RNA is a capped RNA comprising: G(5')ppSp(5')G) and RNA:

[0295] [ka]

[0296] A particularly preferred cap is the 5' cap m2 7,2'O Contains G(5')ppSp(5')G.

[0297] In some embodiments, an RNA according to the present disclosure comprises a 5'-UTR and / or a 3'-UTR. The term "untranslated region" or "UTR" refers to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or a corresponding region in an RNA molecule, such as an mRNA molecule. The untranslated region (UTR) can be located 5' (upstream) of the open reading frame (5'-UTR) and / or 3' (downstream) of the open reading frame (3'-UTR). If present, the 5'-UTR is located at the 5' end upstream of the start codon of the protein-coding region. If present, the 5'-UTR is downstream of the 5' cap (if present), e.g., directly adjacent to the 5' cap. If present, the 3'-UTR is located at the 3' end downstream of the stop codon of the protein-coding region, although the term "3'-UTR" preferably does not include a poly(A) tail. Thus, the 3'-UTR is upstream of the poly(A) sequence (if present), e.g., directly adjacent to the poly(A) sequence.

[0298] In some embodiments, the RNA according to the present disclosure comprises a 3'-poly(A) sequence.

[0299] As used herein, the term "poly A tail" or "poly A sequence" refers to a continuous or intermittent sequence of adenylate residues typically located at the 3' end of an RNA molecule. Poly A tails or poly A sequences are known to those skilled in the art and may follow the 3'-UTR of the RNA described herein. A continuous poly A tail is characterized by consecutive adenylate residues. Continuous poly A tails are typical in nature. The RNAs disclosed herein may have a poly A tail attached to the free 3' end of the RNA by a template-independent RNA polymerase after transcription, or a poly A tail encoded by DNA and transcribed by a template-dependent RNA polymerase.

[0300] Poly(A) tails of approximately 120 A nucleotides have been demonstrated to have beneficial effects on the levels of RNA in transfected eukaryotic cells and on the levels of proteins translated from open reading frames located 5' upstream of the poly(A) tail (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017).

[0301] The poly-A tail can be of any length. In some embodiments, the poly-A tail comprises, consists essentially of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, particularly about 120 A nucleotides. In this context, "consisting essentially of" means that most of the nucleotides in the poly-A tail, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the number of nucleotides in the poly-A tail, are A nucleotides, while allowing for the remaining nucleotides to be nucleotides other than A nucleotides, such as U nucleotides (uridylic acid), G nucleotides (guanylic acid), or C nucleotides (cytidylic acid). In this context, "consisting of" means that all nucleotides of the poly A tail, i.e., 100% of the number of nucleotides in the poly A tail, are A nucleotides. The term "A nucleotide" or "A" refers to adenylic acid.

[0302] In some embodiments, the poly(A) tail is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template containing repeated dT nucleotides (deoxythymidylic acid) in the strand complementary to the coding strand. The DNA sequence encoding the poly(A) tail (coding strand) is referred to as a poly(A) cassette.

[0303] In some embodiments, the poly(A) cassette present in the coding strand of DNA consists essentially of dA nucleotides but is interrupted by random sequences of four nucleotides (dA, dC, dG, and dT). Such random sequences can be 5-50, 10-30, or 10-20 nucleotides in length. Such cassettes are disclosed in International Publication No. 2016 / 005324, which is incorporated herein by reference. Any poly(A) cassette disclosed in International Publication No. 2016 / 005324 can be used in the present invention. Poly(A) cassettes consisting essentially of dA nucleotides but interrupted by random sequences in which the four nucleotides (dA, dC, dG, and dT) are evenly distributed and have a length of, for example, 5-50 nucleotides, have been shown to confer sustained propagation of plasmid DNA in E. coli at the DNA level and, at the RNA level, are still associated with beneficial properties related to supporting RNA stability and translation efficiency. Consequently, in some embodiments, the poly-A tails included in the RNA molecules described herein consist essentially of A nucleotides, but are interrupted by random sequences of four nucleotides (A, C, G, U). Such random sequences can be 5-50, 10-30, or 10-20 nucleotides in length.

[0304] In some embodiments, no nucleotides other than A nucleotides are adjacent to the polyA tail at its 3' end, i.e., the polyA tail is not masked or followed by a nucleotide other than A at its 3' end.

[0305] In some embodiments, the polyA tail may comprise at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the polyA tail may consist essentially of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the polyA tail may consist of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, the polyA tail comprises at least 100 nucleotides. In some embodiments, the polyA tail comprises about 150 nucleotides. In some embodiments, the polyA tail comprises about 120 nucleotides.

[0306] According to the present disclosure, vaccine antigens are preferably administered as single-stranded 5'-capped mRNAs that are translated into their respective proteins upon entry into antigen-presenting cells (APCs). Preferably, the RNAs contain structural elements (5'-cap, 5'-UTR, 3'-UTR, poly(A) tail) optimized for maximum RNA efficacy in terms of stability and translation efficiency.

[0307] In one embodiment, β-S-ARCA (D1) is used as a specific capping structure at the 5' end of the RNA. In one embodiment, the 5'-UTR sequence is derived from human α-globin mRNA. In one embodiment, two repeated 3'-UTRs derived from human β-globin mRNA are placed between the coding sequence and the poly(A) tail to ensure higher maximum protein levels and long-term persistence of the mRNA. In one embodiment, a poly(A) tail measuring 110 nucleotides in length is used, consisting of a stretch of 30 adenosine residues followed by a 10-nucleotide linker sequence and another 70 adenosine residues. This poly(A) tail sequence was designed to increase RNA stability and translation efficiency in dendritic cells.

[0308] The RNA is preferably administered as lipoplex particles, preferably comprising DOTMA and DOPE, as further described below. Such particles are preferably administered systemically, particularly intravenously.

[0309] In the context of the present disclosure, the term "transcription" refers to the process by which the genetic code in a DNA sequence is transcribed into RNA, which can then be translated into peptides or proteins.

[0310] With respect to RNA, the terms "expression" or "translation" refer to the process in a cell's ribosomes by which a chain of mRNA directs the assembly of a sequence of amino acids to make a peptide or protein.

[0311] "Encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties that result therefrom. Thus, a gene encodes a protein when transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be said to encode the protein or other product of that gene or cDNA.

[0312] According to the present disclosure, the term "RNA-encoded" means that the RNA, when present in the appropriate environment, such as within a cell of a target tissue, is capable of directing the assembly of amino acids to produce the peptide or protein it encodes during the translation process. In one embodiment, the RNA is capable of interacting with the cellular translation machinery to enable translation of the peptide or protein. The cell may produce the encoded peptide or protein intracellularly (e.g., in the cytoplasm and / or nucleus), may secrete the encoded peptide or protein, or may produce it on its surface.

[0313] As used herein, "endogenous" refers to any substance that is produced from or within an organism, cell, tissue, or system.

[0314] As used herein, the term "exogenous" refers to any substance that is introduced into or produced outside of an organism, cell, tissue, or system.

[0315] The term "expression" as used herein is defined as the transcription and / or translation of a particular nucleotide sequence. Expression can be transient or stable. According to the present invention, the term expression also includes "ectopic expression" or "abnormal expression."

[0316] As used herein, the terms "linked," "fused," or "fusion" are used interchangeably and refer to the joining of two or more elements or components or domains.

[0317] cytokines The methods described herein can include providing one or more cytokines to a subject, for example, by administering to the subject one or more cytokines, polynucleotides encoding one or more cytokines, or host cells expressing one or more cytokines.

[0318] As used herein, the term "cytokine" includes naturally occurring cytokines and functional variants thereof, including fragments of naturally occurring cytokines and variants thereof. One particularly preferred cytokine is IL2.

[0319] Cytokines are a category of small proteins (approximately 5–20 kDa) that are important in cell signaling. Their release affects the behavior of surrounding cells. Cytokines, as immunomodulators, participate in autocrine, paracrine, and endocrine signaling. Cytokines include chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors, but generally do not include hormones or growth factors (despite some overlap in terminology). Cytokines are produced by a wide range of cells, including immune cells such as macrophages, B lymphocytes, T lymphocytes, and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. A given cytokine can be produced by multiple cell types. Cytokines act through receptors and are particularly important in the immune system; they regulate the balance between humoral and cellular immune responses and regulate the maturation, growth, and responsiveness of specific cell populations. Some cytokines enhance or inhibit the actions of other cytokines in complex ways.

[0320] IL2 Interleukin-2 (IL2) is a cytokine that induces proliferation of antigen-activated T cells and stimulates natural killer (NK) cells. IL2's biological activity is mediated through the multisubunit IL2 receptor complex (IL2R), which contains three membrane-spanning polypeptide subunits: p55 (IL2Rα, alpha subunit, also known as CD25 in humans), p75 (IL2Rβ, beta subunit, also known as CD122 in humans), and p64 ​​(IL2Rγ, gamma subunit, also known as CD132 in humans). T cell responses to IL2 depend on various factors, including (1) the concentration of IL2; (2) the number of IL2R molecules on the cell surface; and (3) the number of IL2Rs occupied by IL2 (i.e., the affinity of the binding interaction between IL2 and IL2R) (Smith, “Cell Growth Signal Transduction is Quantal” Receptor Activation by Antigens, Cytokines, Hormones, and Growth Factors 766:263-271, 1995). The IL2:IL2R complex is internalized upon ligand binding, and various components undergo differential sorting. When administered as an intravenous (iv) bolus, IL2 has rapid systemic clearance (an initial clearance phase with a half-life of 12.9 minutes, followed by a slower clearance phase with a half-life of 85 minutes) (Konrad et al., Cancer Res. 50:2009-2017, 1990).

[0321] In eukaryotic cells, human IL2 is synthesized as a 153-amino acid precursor polypeptide, from which 20 amino acids are removed to generate the mature, secreted form. Recombinant human IL2 has been produced in E. coli, insect cells, and mammalian COS cells.

[0322] According to the present disclosure, IL2 (optionally as part of extended PK IL2) can be naturally occurring IL2 or a fragment or variant thereof. The IL2 can be human IL2 and can be derived from any vertebrate, particularly any mammal.

[0323] extended PK group The cytokine polypeptides described herein can be prepared as fusion or chimeric polypeptides comprising a cytokine moiety and a heterologous polypeptide (i.e., a polypeptide that is not a cytokine or a variant thereof). The resulting molecules, hereafter referred to as "extended pharmacokinetic (PK) cytokines," have an extended circulating half-life compared to the free cytokine. The extended circulating half-life of extended PK cytokines allows in vivo serum cytokine concentrations to be maintained within the therapeutic range, potentially resulting in enhanced activation of many types of immune cells, including T cells. Due to their favorable pharmacokinetic profile, extended PK cytokines can be administered less frequently and for longer periods of time compared to unmodified cytokines.

[0324] As used herein, "half-life" refers to the time required for the serum or plasma concentration of a compound, such as a peptide or protein, to decrease by 50% in vivo, for example, due to degradation and / or clearance or sequestration by natural mechanisms. Extended PK cytokines, such as extended PK interleukins (ILs), suitable for use herein are stabilized in vivo, and their half-life is increased, for example, by fusion to serum albumin (e.g., HSA or MSA), which resists degradation and / or clearance or sequestration. Half-life can be determined by any method known per se, such as by pharmacokinetic analysis. Suitable techniques will be apparent to those skilled in the art and may, for example, generally include the steps of administering an appropriate dose of an amino acid sequence or compound to a subject; collecting blood or other samples from the subject at regular intervals; determining the level or concentration of the amino acid sequence or compound in the blood samples; and calculating, from a plot of the data thus obtained, the time until the level or concentration of the amino acid sequence or compound decreases by 50% compared to the initial level at the time of administration. Further details are provided in standard handbooks such as, for example, Kenneth, A. et al., Chemical Stability of Pharmaceuticals: A Handbook for Pharmacists and Peters et al., Pharmacokinetic Analysis: A Practical Approach (1996). See also Gibaldi, M. et al., Pharmacokinetics, 2nd Rev. Edition, Marcel Dekker (1982).

[0325] Cytokines can be fused to an extended PK group that increases their circulating half-life. Non-limiting examples of extended PK groups are described below. It should be understood that other PK groups that increase the circulating half-life of cytokines or their variants are also applicable to the present disclosure. In certain embodiments, the extended PK group is a serum albumin domain (e.g., mouse serum albumin, human serum albumin).

[0326] As used herein, the term "PK" is an acronym for "pharmacokinetics" and encompasses the properties of a compound, including, by way of example, absorption, distribution, metabolism, and excretion by a subject. As used herein, an "extended PK group" refers to a protein, peptide, or moiety that, when fused to or administered together with a biologically active molecule, increases the circulating half-life of the biologically active molecule. Examples of extended PK groups include serum albumin (e.g., HSA), immunoglobulin Fc or Fc fragments and variants thereof, transferrin and variants thereof, and human serum albumin (HSA) binders (disclosed in U.S. Patent Application Publication Nos. 2005 / 0287153 and 2007 / 0003549). Other exemplary extended PK groups are disclosed in Kontermann, Expert Opin Biol Ther, 2016 Jul;16(7):903-15, the entire contents of which are incorporated herein by reference. As used herein, an "extended PK cytokine" refers to a cytokine moiety combined with an extended PK group. In one embodiment, the extended PK cytokine is a fusion protein in which the cytokine portion is linked or fused to an extended PK group. As used herein, "extended PK IL" refers to an interleukin (IL) portion (including an IL variant portion) combined with an extended PK group. In one embodiment, the extended PK IL is a fusion protein in which the IL portion is linked or fused to an extended PK group. An exemplary fusion protein is an HSA / IL2 fusion in which the IL2 portion is fused to HSA.

[0327] In certain embodiments, the serum half-life of the extended PK cytokine is increased compared to the cytokine alone (i.e., the cytokine not fused to an extended PK group). In certain embodiments, the serum half-life of the extended PK cytokine is at least 20, 40, 60, 80, 100, 120, 150, 180, 200, 400, 600, 800, or 1000% longer than the serum half-life of the cytokine alone. In certain embodiments, the serum half-life of the extended PK cytokine is at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 10-fold, 12-fold, 13-fold, 15-fold, 17-fold, 20-fold, 22-fold, 25-fold, 27-fold, 30-fold, 35-fold, 40-fold, or 50-fold longer than the serum half-life of the cytokine alone. In certain embodiments, the serum half-life of the extended PK cytokine is at least 10 hours, 15 hours, 20 hours, 25 hours, 30 hours, 35 hours, 40 hours, 50 hours, 60 hours, 70 hours, 80 hours, 90 hours, 100 hours, 110 hours, 120 hours, 130 hours, 135 hours, 140 hours, 150 hours, 160 hours, or 200 hours.

[0328] In certain embodiments, the extended PK group comprises serum albumin or a fragment thereof, or a variant of serum albumin or a fragment thereof (all of which are included in the term "albumin" for purposes of this disclosure). The polypeptides described herein can be fused to albumin (or a fragment or variant thereof) to form an albumin fusion protein. Such albumin fusion proteins are described in U.S. Patent Application Publication No. 20070048282.

[0329] As used herein, "albumin fusion protein" refers to a protein formed by the fusion of at least one molecule of albumin (or a fragment or variant thereof) with at least one molecule of a protein, such as a Therapeutic protein, particularly IL2 (or a variant thereof). Albumin fusion proteins can be produced by translation of a nucleic acid in which a polynucleotide encoding a Therapeutic protein is joined in-frame with a polynucleotide encoding albumin. Once part of the albumin fusion protein, the Therapeutic protein and albumin can be referred to as a "portion," "region," or "moiety," respectively, of the albumin fusion protein (e.g., a "Therapeutic protein portion" or an "albumin protein portion"). In a highly preferred embodiment, the albumin fusion protein comprises at least one molecule of a Therapeutic protein (including, but not limited to, the mature form of a Therapeutic protein) and at least one molecule of albumin (including, but not limited to, the mature form of albumin). In one embodiment, the albumin fusion protein is processed by host cells, such as hepatocytes, in the target organ of the administered RNA and secreted into the circulation. Processing of the nascent albumin fusion protein in the secretory pathway of the host cell used to express the RNA may include, but is not limited to, signal peptide cleavage, disulfide bond formation, proper folding, carbohydrate addition and processing (e.g., N-linked and O-linked glycosylation), specific proteolytic cleavage, and / or assembly into a multimeric protein. The albumin fusion protein is preferably encoded by RNA in an unprocessed form, particularly with a signal peptide at its N-terminus, and, following secretion by the cell, preferably exists in a processed form, particularly with the signal peptide cleaved. In the most preferred embodiment, the "processed form of the albumin fusion protein" refers to the albumin fusion protein product that has undergone N-terminal signal peptide cleavage, also referred to herein as the "mature albumin fusion protein."

[0330] In preferred embodiments, albumin fusion proteins containing a therapeutic protein have higher plasma stability compared to the plasma stability of the same therapeutic protein when not fused to albumin. Plasma stability typically refers to the period from when a therapeutic protein is administered in vivo and transported into the bloodstream, until the therapeutic protein is degraded and removed from the bloodstream to organs such as the kidneys or liver, and finally, when the therapeutic protein is removed from the body. Plasma stability is calculated in terms of the half-life of the therapeutic protein in the bloodstream. The half-life of a therapeutic protein in the bloodstream can be easily determined by common assays known in the art.

[0331] As used herein, "albumin" collectively refers to an albumin protein or amino acid sequence, or an albumin fragment or variant, having one or more functional activities (e.g., biological activities) of albumin. In particular, "albumin" refers to human albumin or a fragment or variant thereof, particularly the mature form of human albumin, or albumin or a fragment thereof from another vertebrate, or a variant of these molecules. Albumin can be derived from any vertebrate, particularly any mammal, such as human, mouse, cow, sheep, or pig. Non-mammalian albumins include, but are not limited to, hen and salmon. The albumin portion of the albumin fusion protein can be derived from a different animal than the therapeutic protein portion.

[0332] In certain embodiments, the albumin is human serum albumin (HSA), or a fragment or variant thereof, such as those disclosed in U.S. Pat. No. 5,876,969, WO 2011 / 124718, WO 2013 / 075066, and WO 2011 / 0514789.

[0333] The terms human serum albumin (HSA) and human albumin (HA) are used interchangeably herein. The terms "albumin" and "serum albumin" are broader and encompass human serum albumin (and fragments and variants thereof) as well as albumins (and fragments and variants thereof) from other species.

[0334] As used herein, a fragment of albumin sufficient to prolong the therapeutic activity or plasma stability of a Therapeutic protein refers to a fragment of albumin of sufficient length or structure to stabilize or prolong the therapeutic activity or plasma stability of the protein, such that the plasma stability of the Therapeutic protein portion of the albumin fusion protein is extended or expanded compared to its plasma stability in the unfused state.

[0335] The albumin portion of the albumin fusion protein may comprise the full length of the albumin sequence, or may comprise one or more fragments thereof that can stabilize or extend therapeutic activity or plasma stability. Such fragments may be 10 or more amino acids in length, or may comprise approximately 15, 20, 25, 30, 50, or more consecutive amino acids from the albumin sequence, or may comprise part or all of a particular domain of albumin. For example, one or more fragments of HSA spanning the first two immunoglobulin-like domains may be used. In a preferred embodiment, the HSA fragment is the mature form of HSA.

[0336] Generally speaking, an albumin fragment or variant is at least 100 amino acids in length, preferably at least 150 amino acids in length.

[0337] According to the present disclosure, the albumin can be naturally occurring albumin or a fragment or variant thereof. The albumin can be human albumin and can be derived from any vertebrate, particularly any mammal.

[0338] 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.

[0339] In one embodiment, the therapeutic protein(s) are linked to albumin via a peptide linker(s). A linker peptide between the fusion moieties can provide greater physical separation between the moieties, thus maximizing the accessibility of the therapeutic protein moiety to bind to, for example, its cognate receptor. The linker peptide can be composed of amino acids so that it is flexible or more rigid. The linker sequence can be cleavable by protease or chemically.

[0340] As used herein, the term "Fc region" refers to the portion of a native immunoglobulin formed by the Fc domains (or Fc portions) of each of the two heavy chains of the native immunoglobulin. As used herein, the term "Fc domain" refers to a portion or fragment of a single immunoglobulin (Ig) heavy chain in which the Fc domain does not include an Fv domain. In certain embodiments, the Fc domain begins at the hinge region immediately upstream of the papain cleavage site and ends at the C-terminus of the antibody. Thus, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CH3 domain. In certain embodiments, an Fc domain comprises at least one of a hinge (e.g., upper, middle, and / or lower hinge region), a CH2 domain, a CH3 domain, a CH4 domain, or a variant, portion, or fragment thereof. In certain embodiments, an Fc domain comprises a complete Fc domain (i.e., a hinge domain, a CH2 domain, and a CH3 domain). In certain embodiments, an Fc domain comprises a hinge domain (or portion thereof) fused to a CH3 domain (or portion thereof). In certain embodiments, an Fc domain comprises a CH2 domain (or a portion thereof) fused to a CH3 domain (or a portion thereof). In certain embodiments, an Fc domain consists of a CH3 domain or a portion thereof. In certain embodiments, an Fc domain consists of a hinge domain (or a portion thereof) and a CH3 domain (or a portion thereof). In certain embodiments, an Fc domain consists of a CH2 domain (or a portion thereof) and a CH3 domain. In certain embodiments, an Fc domain consists of a hinge domain (or a portion thereof) and a CH2 domain (or a portion thereof). In certain embodiments, an Fc domain lacks at least a portion of the CH2 domain (e.g., all or a portion of the CH2 domain). An Fc domain, as used herein, generally refers to a polypeptide comprising all or a portion of the Fc domain of an immunoglobulin heavy chain. This includes, but is not limited to, polypeptides comprising the entire CH1, hinge, CH2, and / or CH3 domains, as well as fragments of such peptides, e.g., comprising only the hinge, CH2, and CH3 domains.The Fc domain can be derived from any species and / or any subtype of immunoglobulin, including, but not limited to, human IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies. Fc domains encompass natural Fc and Fc variant molecules. As described herein, those skilled in the art will understand that any Fc domain can be modified such that its amino acid sequence differs from that of a naturally occurring Fc domain of an immunoglobulin molecule. In certain embodiments, the Fc domain has reduced effector function (e.g., FcγR binding).

[0341] The Fc domain of the polypeptide described herein can be derived from different immunoglobulin molecules.For example, the Fc domain of the polypeptide can include a CH2 and / or CH3 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule.In another example, the Fc domain can include a chimeric hinge region derived in part from an IgG1 molecule and in part from an IgG3 molecule.In another example, the Fc domain can include a chimeric hinge derived in part from an IgG1 molecule and in part from an IgG4 molecule.

[0342] In certain embodiments, the extended PK group comprises an Fc domain or a fragment thereof, or a variant of an Fc domain or a fragment thereof (all of which are encompassed by the term "Fc domain" for purposes of this disclosure). The Fc domain does not comprise the variable region that binds to the antigen. Fc domains suitable for use in the present disclosure can be obtained from several different sources. In certain embodiments, the Fc domain is derived from a human immunoglobulin. In certain embodiments, the Fc domain is derived from a human IgG1 constant region. However, it is understood that the Fc domain can be derived from an immunoglobulin of another mammalian species, including, for example, a rodent species (e.g., mouse, rat, rabbit, guinea pig) or a non-human primate species (e.g., chimpanzee, macaque).

[0343] Furthermore, the Fc domain (or a fragment or variant thereof) can be derived from any immunoglobulin class, including IgM, IgG, IgD, IgA, and IgE, and any immunoglobulin isotype, including IgG1, IgG2, IgG3, and IgG4.

[0344] Various Fc domain gene sequences (e.g., mouse and human constant region gene sequences) are available in the form of publicly accessible deposits. Constant region domains can be selected, including Fc domain sequences that lack specific effector functions and / or have specific modifications that reduce immunogenicity. Many sequences of antibodies and antibody-encoding genes have been published, and appropriate Fc domain sequences (e.g., hinge, CH2, and / or CH3 sequences, or fragments or variants thereof) can be derived from these sequences using techniques widely recognized in the art.

[0345] In certain embodiments, the extended PK group is a serum albumin binding protein, such as those described in U.S. Patent Application Nos. 2005 / 0287153, 2007 / 0003549, 2007 / 0178082, 2007 / 0269422, 2010 / 0113339, WO 2009 / 083804, and WO 2009 / 133208, which are incorporated by reference in their entireties. In certain embodiments, the extended PK group is transferrin, as disclosed in U.S. Patent Nos. 7,176,278 and 8,158,579, which are incorporated by reference in their entireties. In certain embodiments, the extended PK group is a serum immunoglobulin binding protein, such as those disclosed in US Patent Application No. 2007 / 0178082, US Patent Application No. 2014 / 0220017, and US Patent Application No. 2017 / 0145062, the entire contents of which are incorporated herein by reference. In certain embodiments, the extended PK group is a fibronectin (Fn)-based scaffold domain protein that binds to serum albumin, such as those disclosed in US Patent Application No. 2012 / 0094909, the entire contents of which are incorporated herein by reference. A method for producing a fibronectin-based scaffold domain protein is also disclosed in US Patent Application No. 2012 / 0094909. A non-limiting example of an Fn3-based extended PK group is Fn3 (HSA), i.e., an Fn3 protein that binds to human serum albumin.

[0346] In certain embodiments, an extended PK cytokine suitable for use according to the present disclosure may use one or more peptide linkers. As used herein, the term "peptide linker" refers to a peptide or polypeptide sequence that links two or more domains (e.g., an extended PK portion and an IL portion, such as IL2) in the linear amino acid sequence of a polypeptide chain. For example, a peptide linker may be used to link the cytokine portion to the HSA domain.

[0347] Linkers suitable for fusing an extended PK group to, for example, IL2 are well known in the art. Exemplary linkers include a glycine-serine polypeptide linker, a glycine-proline polypeptide linker, and a proline-alanine polypeptide linker. In certain embodiments, the linker is a glycine-serine polypeptide linker, i.e., a peptide consisting of glycine and serine residues.

[0348] In addition to, or instead of, the heterologous polypeptides described above, the cytokine variant polypeptides described herein can include a sequence encoding a "marker" or "reporter." Examples of marker or reporter genes include β-lactamase, chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA), aminoglycoside phosphotransferase, dihydrofolate reductase (DHFR), hygromycin B phosphotransferase (HPH), thymidine kinase (TK), β-galactosidase, and xanthine guanine phosphoribosyltransferase (XGPRT).

[0349] antigen The methods described herein may further comprise contacting immune effector cells, particularly immune effector cells that express an antigen receptor, e.g., immune effector cells genetically engineered to express an antigen receptor, with a cognate antigen molecule (also referred to herein as an "antigen targeted by the antigen receptor," "vaccine antigen," or simply "antigen") in the subject to be treated, wherein the antigen molecule or a processed product thereof, e.g., a fragment thereof, binds to the antigen receptor, such as a TCR or CAR, carried by the immune effector cell. In one embodiment, the cognate antigen molecule comprises an antigen or fragment thereof, or a variant of said antigen or fragment, expressed by a target cell targeted by the immune effector cell.

[0350] Thus, the methods described herein include administering a cognate antigen molecule, a nucleic acid encoding the same, or a cell expressing the cognate antigen molecule to a subject. In one embodiment, the nucleic acid encoding the cognate antigen molecule is expressed in the subject's cells to provide the cognate antigen molecule. In one embodiment, expression of the cognate antigen molecule occurs on the cell surface. In one embodiment, the nucleic acid encoding the cognate antigen molecule is transiently expressed in the subject's cells. In one embodiment, the nucleic acid encoding the cognate antigen molecule is RNA. In one embodiment, the cognate antigen molecule or the nucleic acid encoding the same is administered systemically. In one embodiment, systemic administration of the nucleic acid encoding the cognate antigen molecule results in expression of the nucleic acid encoding the cognate antigen molecule in the spleen. In one embodiment, systemic administration of the nucleic acid encoding the cognate antigen molecule results in expression of the nucleic acid encoding the cognate antigen molecule in antigen-presenting cells, preferably professional antigen-presenting cells. In one embodiment, the antigen-presenting cells are selected from the group consisting of dendritic cells, macrophages, and B cells. In one embodiment, systemic administration of the nucleic acid encoding the cognate antigen molecule results in no or essentially no expression of the nucleic acid encoding the cognate antigen molecule in the lung and / or liver. In one embodiment, following systemic administration of nucleic acid encoding a cognate antigen molecule, expression of nucleic acid encoding a cognate antigen molecule in the spleen is at least 5-fold higher than expression in the lung.

[0351] The peptide and protein antigens, i.e., vaccine antigens, provided to a subject according to the present invention (by administering either the peptide and protein antigens, or nucleic acids, particularly RNA, encoding the peptide and protein antigens, or cells expressing the peptide and protein antigens) preferably result in the stimulation, priming, and / or expansion of immune effector cells in the subject receiving the peptide or protein antigen, nucleic acid, or cells. The stimulated, primed, and / or expanded immune effector cells are preferably directed against a target antigen, particularly a target antigen expressed by diseased cells, tissues, and / or organs, i.e., a disease-associated antigen. Thus, the vaccine antigen may comprise a disease-associated antigen, or a fragment or variant thereof. In one embodiment, such a fragment or variant is immunologically equivalent to the disease-associated antigen. In the context of the present disclosure, the term "antigen fragment" or "antigen variant" refers to an agent that results in the stimulation, priming, and / or expansion of immune effector cells, where the stimulated, primed, and / or expanded immune effector cells target the antigen, i.e., the disease-associated antigen, particularly when presented by diseased cells, tissues, and / or organs. Thus, a vaccine antigen may correspond to or comprise a disease-associated antigen, may correspond to or comprise a fragment of a disease-associated antigen, or may correspond to or comprise an antigen homologous to a disease-associated antigen or its fragment. When a vaccine antigen comprises a fragment of a disease-associated antigen or an amino acid sequence homologous to a fragment of a disease-associated antigen, said fragment or amino acid sequence may comprise an epitope of the disease-associated antigen targeted by an antigen receptor of an immune effector cell or a sequence homologous to an epitope of the disease-associated antigen. Thus, according to the present disclosure, a vaccine antigen may comprise an immunogenic fragment of a disease-associated antigen or an amino acid sequence homologous to an immunogenic fragment of a disease-associated antigen. An "immunogenic fragment of an antigen" according to the present disclosure preferably relates to a fragment of an antigen that is capable of stimulating, priming, and / or expanding immune effector cells bearing an antigen receptor that binds to the antigen or cells expressing the antigen.Preferably, the vaccine antigen (similar to the disease-associated antigen) provides a relevant epitope for binding by an antigen-binding domain present on an immune effector cell. In one embodiment, the vaccine antigen (similar to the disease-associated antigen) is expressed on the surface of a cell, such as an antigen-presenting cell, to provide a relevant epitope for binding by the immune effector cell. In one embodiment, the vaccine antigen (similar to the disease-associated antigen) is expressed on the surface of a cell, such as an antigen-presenting cell, in the context of an MHC to provide a relevant epitope for binding by the immune effector cell. The vaccine antigen may be a recombinant antigen.

[0352] In one embodiment of all aspects of the invention, a nucleic acid encoding a vaccine antigen is expressed in cells of a subject to provide the antigen or its processing product for binding by an antigen receptor expressed by immune effector cells, said binding resulting in stimulation, priming and / or expansion of the immune effector cells.

[0353] The term "immunologically equivalent" means that an immunologically equivalent molecule, such as an immunologically equivalent amino acid sequence, exhibits the same or essentially the same immunological properties and / or exerts the same or essentially the same immunological effect, e.g., with respect to the type of immunological action. In the context of the present disclosure, the term "immunologically equivalent" is preferably used with respect to the immunological effect or properties of an antigen or antigen variant used for immunization. For example, an amino acid sequence is immunologically equivalent to a reference amino acid sequence if, when exposed to a subject's immune system, such as T cells that bind to the reference amino acid sequence or cells that express the reference amino acid sequence, it induces an immune response with specificity that reacts with the reference amino acid sequence. Thus, a molecule that is immunologically equivalent to an antigen exhibits the same or essentially the same properties and / or exerts the same or essentially the same effect as the antigen targeted by T cells with respect to stimulating, priming, and / or expanding T cells.

[0354] As used herein, "activation" or "stimulation" refers to the state of immune effector cells, such as T cells, that are stimulated sufficiently to induce detectable cell proliferation. Activation can also involve the initiation of signal transduction pathways, the induction of cytokine production, and detectable effector function. The term "activated immune effector cells" refers, among other things, to immune effector cells undergoing cell division.

[0355] The term "priming" refers to the process by which an immune effector cell, such as a T cell, first contacts its specific antigen, leading to differentiation into an effector cell, such as an effector T cell.

[0356] The term "clonal expansion" or "expansion" refers to the process of increasing a specific entity. In the context of the present disclosure, the term is preferably used in the context of an immunological response in which lymphocytes are stimulated by an antigen, proliferate, and the specific lymphocytes that recognize said antigen are amplified. Preferably, clonal expansion results in differentiation of lymphocytes.

[0357] The term "antigen" refers to an agent containing an epitope capable of generating an immune response. The term "antigen" particularly includes proteins and peptides. In one embodiment, an antigen is presented or present on the surface of a cell of the immune system, such as an antigen-presenting cell, such as a dendritic cell or macrophage. In one embodiment, an antigen or its processing product, such as a T cell epitope, is bound by an antigen receptor. Thus, the antigen or its processing product can specifically react with immune effector cells, such as T lymphocytes (T cells). In one embodiment, the antigen is a disease-related antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, and the epitope is derived from such an antigen.

[0358] The term "disease-associated antigen" is used in its broadest sense to refer to any antigen associated with a disease. A disease-associated antigen is a molecule containing an epitope that stimulates the host's immune system to generate a cellular antigen-specific immune response and / or a humoral antibody response against the disease. Thus, a disease-associated antigen or its epitope can be used for therapeutic purposes. A disease-associated antigen can be associated with infection by a microorganism, typically a microbial antigen, or can be associated with cancer, typically a tumor.

[0359] The term "tumor antigen" or "tumor-associated antigen" refers to a component of a cancer cell that can originate from the cytoplasm, cell surface, and cell nucleus. In particular, this term refers to an antigen produced intracellularly or as a surface antigen on a tumor cell. Tumor antigens are typically selectively expressed by cancer cells (e.g., expressed at higher levels in cancer cells than in non-cancerous cells), and in some cases, are expressed only by cancer cells. Examples of tumor antigens include, but are not limited to, p53, ART-4, BAGE, β-catenin / m, Bcr-abL, CAMEL, CAP-1, CASP-8, CDC27 / m, CDK4 / m, CEA, cell surface proteins of the claudin family, such as claudin 6, claudin 18.2, and claudin 12, c-MYC, CT, Cyp-B, DAM, ELF2M, ETV6-AML1, G250, GAGE, GnT-V, Gap 100, HAGE, HER-2 / neu, HPV-E7, HPV-E6, HAST-2, hTERT (or hTRT), LAGE, LDLR / FUT, MAGE-A, preferably MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, or MAGE-A12, MAGE-B, MAGE-C, MART-1 / MelanA, MC1R, myosin / m, MUC 1, MUM-1, MUM-2, MUM-3, NA88-A, NF1, NY-ESO-1, NY-BR-1, p190 minor BCR-abL, Pml / RARa, PRAME, proteinase 3, PSA, PSM, RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, SCGB3A2, SCP1, SCP2, SCP3, SSX, survivin, TEL / AML1, TPI / m, TRP-1, TRP-2, TRP-2 / INT2, TPTE, WT, and WT-1. Particularly preferred tumor antigens are proteins of the claudin family, such as CLAUDIN-6 or CLAUDIN-18.2.

[0360] The term "viral antigen" refers to any viral component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. A viral antigen can be a viral ribonucleoprotein or an envelope protein.

[0361] The term "bacterial antigen" refers to any bacterial component that has antigenic properties, i.e., is capable of eliciting an immune response in an individual. Bacterial antigens can be derived from the bacterial cell wall or cytoplasmic membrane.

[0362] The terms "expressed on the cell surface" or "associated with the cell surface" mean that a molecule, such as a receptor or antigen, is located in association with the plasma membrane of a cell, with at least a portion of the molecule facing the extracellular space of the cell and accessible from the outside of the cell, for example, by an antibody located on the outside of the cell. In this context, a portion is preferably at least 4, preferably at least 8, preferably at least 12, more preferably at least 20 amino acids. The association can be direct or indirect. For example, the association can be through one or more transmembrane domains, one or more lipid anchors, or through interaction with any other protein, lipid, saccharide, or other structure that can be found on the outer leaflet of the plasma membrane of the cell. For example, a molecule associated with the surface of a cell can be a transmembrane protein having an extracellular portion, or a protein that associates with the surface of a cell by interacting with another protein that is a transmembrane protein.

[0363] "Cell surface" or "surface of a cell" is used according to its usual meaning in the art and thus includes the outside of a cell that is accessible 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 is accessible to binding by, for example, an antigen-specific antibody added to the cell. In one embodiment, the antigen expressed on the surface of a cell is an integral membrane protein with an extracellular portion that is recognized by a CAR.

[0364] The term "extracellular portion" or "exodomain" in the context of the present invention refers to a part of a molecule, such as a protein, that faces the extracellular space of a cell and is preferably accessible from the outside of said cell, for example by binding to a molecule, such as an antibody, that is located on the outside of said cell. Preferably, the term refers to one or more extracellular loops or domains or fragments thereof.

[0365] The term "epitope" refers to a portion or fragment of a molecule, such as an antigen, that is recognized by the immune system. For example, an epitope can be recognized by T cells, B cells, or an antibody. An epitope of an antigen can include a continuous or discontinuous portion of the antigen and can be about 5 to about 100, e.g., about 5 to about 50, more preferably about 8 to about 30, and most preferably about 10 to about 25 amino acids in length. For example, an epitope can be preferably 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In one embodiment, an epitope is about 10 to about 25 amino acids in length. The term "epitope" includes T cell epitopes.

[0366] The term "T cell epitope" refers to a portion or fragment of a protein that is recognized by T cells when presented in the context of an MHC molecule. The terms "major histocompatibility complex" and the abbreviation "MHC" refer to a complex of genes present in all vertebrates, including MHC class I and MHC class II molecules. MHC proteins or molecules are important in signaling between lymphocytes and antigen-presenting or diseased cells during the immune response; they bind to peptide epitopes and present them for recognition by T cell receptors on T cells. Proteins encoded by MHC are expressed on the surface of cells and display both self-antigens (peptide fragments from the cell itself) and non-self-antigens (e.g., fragments of invading microorganisms) to T cells. For class I MHC / peptide complexes, the binding peptide is typically about 8 to about 10 amino acids long, although longer or shorter peptides can also be effective. For class II MHC / peptide complexes, the binding peptide is typically about 10 to about 25 amino acids long, particularly about 13 to about 18 amino acids long, although longer and shorter peptides can also be effective.

[0367] In one embodiment, the target antigen is a tumor antigen, and the vaccine antigen or a fragment thereof (e.g., epitope) is derived from the tumor antigen. The tumor antigen may be a "standard" antigen generally known to be expressed in various cancers. The tumor antigen may also be a "neoantigen" that is specific to an individual's tumor and has not previously been recognized by the immune system. The neoantigen or neoepitope may result from one or more cancer-specific mutations in the genome of a cancer cell that result in amino acid changes. When the tumor antigen is a neoantigen, the vaccine antigen preferably comprises an epitope or fragment of the neoantigen containing one or more amino acid changes.

[0368] Cancer mutations vary from individual to individual. Therefore, cancer mutations encoding novel epitopes (neoepitopes) are attractive targets for the development of vaccine compositions and immunotherapies. The effectiveness of tumor immunotherapy depends on the selection of cancer-specific antigens and epitopes that can induce a strong immune response in the host. RNA can be used to deliver patient-specific tumor epitopes to patients. Dendritic cells (DCs) present in the spleen are particularly interesting antigen-presenting cells for RNA expression of immunogenic epitopes or antigens, such as tumor epitopes. The use of multiple epitopes has been shown to enhance therapeutic efficacy in tumor vaccine compositions. Rapid sequencing of tumor mutagenesis can provide multiple epitopes for personalized vaccines that can be encoded by the RNA described herein, for example, as a single polypeptide in which the epitopes are optionally separated by linkers. In certain embodiments of the present disclosure, the RNA encodes at least 1 epitope, at least 2 epitopes, at least 3 epitopes, at least 4 epitopes, at least 5 epitopes, at least 6 epitopes, at least 7 epitopes, at least 8 epitopes, at least 9 epitopes, or at least 10 epitopes. Exemplary embodiments include RNA encoding at least 5 epitopes (termed "pentatopes") and RNA encoding at least 10 epitopes (termed "decatopes").

[0369] According to various aspects of the present invention, the objective is preferably to provide an immune response against cancer cells expressing a tumor antigen such as CLDN6 or CLDN18.2, and to treat cancer diseases involving cells expressing a tumor antigen such as CLDN6 or CLDN18.2. Preferably, the present invention involves the administration of antigen receptor-engineered immune effector cells, such as T cells, targeted against cancer cells expressing a tumor antigen such as CLDN6 or CLDN18.2.

[0370] Peptide and protein antigens can be 2 to 100 amino acids in length, including, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. In some embodiments, peptides can be greater than 50 amino acids. In some embodiments, peptides can be greater than 100 amino acids.

[0371] According to the present invention, vaccine antigens must be recognizable by immune effector cells. Preferably, when recognized by immune effector cells, the antigen can induce stimulation, priming, and / or expansion of immune effector cells bearing antigen receptors that recognize the antigen in the presence of appropriate costimulatory signals. In the context of embodiments of the present invention, the antigen is preferably present on the surface of a cell, preferably on the surface of an antigen-presenting cell. Recognition of the antigen on the surface of diseased cells can result in an immune response against the antigen (or cells expressing the antigen).

[0372] In one embodiment of all aspects of the present invention, the antigen is expressed on a diseased cell, such as a cancer cell. In one embodiment, the antigen is expressed on the surface of a diseased cell, such as a cancer cell. In one embodiment, the antigen receptor is a CAR that binds to the extracellular domain of the antigen or an epitope of the extracellular domain. In one embodiment, the CAR binds to a native epitope of the antigen present on the surface of a living cell. In one embodiment, binding of a CAR, when expressed by and / or present on a T cell, to an antigen present on a cell, such as an antigen-presenting cell, results in stimulation, priming, and / or expansion of the T cell. In one embodiment, binding of a CAR, when expressed by and / or present on a T cell, to an antigen present on a diseased cell, such as a cancer cell, results in cytolysis and / or apoptosis of the diseased cell, and the T cell preferably releases cytotoxic factors, such as perforin and granzymes.

[0373] chemotherapy In certain embodiments, additional treatments may be administered to the patient in combination with the treatments described herein, including classical cancer treatments such as radiation therapy, surgery, hyperthermia, and / or chemotherapy.

[0374] Chemotherapy is a type of cancer treatment that typically involves the use of one or more anticancer drugs (chemotherapeutic agents) as part of a standardized chemotherapy regimen. The term chemotherapy has come to connote the nonspecific use of intracellular toxins to inhibit mitosis. This connotation excludes more selective agents that block extracellular signals (signal transduction). The development of specific molecular or genetic targeted therapies that inhibit growth-promoting signals from classical endocrine hormones (primarily estrogen for breast cancer and androgen for prostate cancer) is now referred to as hormone therapy. In contrast, other inhibition of growth signals, such as those associated with receptor tyrosine kinases, is referred to as targeted therapy.

[0375] Importantly, the use of drugs (whether chemotherapy, hormonal therapy, or targeted therapy) constitutes a systemic cancer therapy in that they are introduced into the bloodstream and can therefore, in principle, address cancer at any anatomical location in the body. Systemic therapy is often used in combination with other modalities that constitute local cancer therapy (i.e., treatments whose effectiveness is limited to the anatomical area where they are applied), such as radiation therapy, surgery, or hyperthermia.

[0376] Traditional chemotherapy agents are cytotoxic by interfering with cell division (mitosis), but cancer cells vary widely in their sensitivity to these agents. To a large extent, chemotherapy can be thought of as a way to damage or stress cells, which, if apoptosis is initiated, can result in cell death.

[0377] Chemotherapeutic agents include alkylating agents, antimetabolites, anti-microtubule agents, topoisomerase inhibitors, and cytotoxic antibiotics.

[0378] Alkylating agents have the ability to alkylate many molecules, including proteins, RNA, and DNA. Subtypes of alkylating agents include nitrogen mustards, nitrosoureas, tetrazines, aziridines, cisplatin and derivatives, and nonclassical alkylating agents. Nitrogen mustards include mechlorethamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide, and busulfan. Nitrosoureas include N-nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, and streptozotocin. Tetrazines include dacarbazine, mitozolomide, and temozolomide. Aziridines include thiotepa, mitomycin, and diaziquone (AZQ). Cisplatin and derivatives include cisplatin, carboplatin, and oxaliplatin. They impair cellular function by forming covalent bonds with amino, carboxyl, sulfhydryl, and phosphate groups in biologically important molecules. Non-classical alkylating agents include procarbazine and hexamethylmelamine. In a particularly preferred embodiment, the alkylating agent is cyclophosphamide.

[0379] Antimetabolites are a group of molecules that interfere with DNA and RNA synthesis. Many of them have structures similar to the building blocks of DNA and RNA. Antimetabolites resemble either nucleobases or nucleosides but have altered chemical groups. These drugs exert their effects by blocking enzymes required for DNA synthesis or by incorporating into DNA or RNA. Subtypes of antimetabolites include antifolates, fluoropyrimidines, deoxynucleoside analogs, and thiopurines. Antifolates include methotrexate and pemetrexed. Fluoropyrimidines include fluorouracil and capecitabine. Deoxynucleoside analogs include cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, and pentostatin. Thiopurines include thioguanine and mercaptopurine.

[0380] Antimicrotubule agents block cell division by interfering with microtubule function. Vinca alkaloids prevent microtubule formation, while taxanes prevent microtubule disassembly. Vinca alkaloids include vinorelbine, vindesine, and vinflunine. Taxanes include docetaxel (Taxotere) and paclitaxel (Taxol).

[0381] Topoisomerase inhibitors are drugs that affect the activity of two enzymes: topoisomerase I and topoisomerase II, and include irinotecan, topotecan, camptothecin, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, mervalone, and aclarubicin.

[0382] Cytotoxic antibiotics are a diverse group of drugs with various mechanisms of action. The common theme they share in their chemotherapeutic indications is the disruption of cell division. The most important subgroups are the anthracyclines (e.g., doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, and aclarubicin) and bleomycin; other notable examples include mitomycin C, mitoxantrone, and actinomycin.

[0383] In one embodiment, lymphodepleting therapy may be applied prior to administration of immune effector cells, for example, by administering cyclophosphamide and fludarabine, which may increase cell persistence and the incidence and duration of clinical responses.

[0384] Immune checkpoint inhibitors In certain embodiments, immune checkpoint inhibitors are used in combination with other therapeutic agents described herein.

[0385] As used herein, "immune checkpoint" refers to costimulatory and inhibitory signals that regulate the magnitude and quality of T cell receptor recognition of antigens. In certain embodiments, the immune checkpoint is an inhibitory signal. In certain embodiments, the inhibitory signal is the interaction between PD-1 and PD-L1. In certain embodiments, the inhibitory signal is the interaction between CTLA-4 and CD80 or CD86, which displaces CD28 binding. In certain embodiments, the inhibitory signal is the interaction between LAG3 and an MHC class II molecule. In certain embodiments, the inhibitory signal is the interaction between TIM3 and galectin-9.

[0386] As used herein, "immune checkpoint inhibitor" refers to a molecule that completely or partially reduces, inhibits, prevents, or modulates one or more checkpoint proteins. In certain embodiments, an immune checkpoint inhibitor prevents inhibitory signals associated with an immune checkpoint. In certain embodiments, an immune checkpoint inhibitor is an antibody or fragment thereof that interferes with inhibitory signaling associated with an immune checkpoint. In certain embodiments, an immune checkpoint inhibitor is a small molecule that interferes with inhibitory signaling. In certain embodiments, an immune checkpoint inhibitor is an antibody, fragment thereof, or antibody mimetic that interferes with the interaction between checkpoint blockade proteins, e.g., an antibody or fragment thereof that interferes with the interaction between PD-1 and PD-L1. In certain embodiments, an immune checkpoint inhibitor is an antibody or fragment thereof that interferes with the interaction between CTLA-4 and CD80 or CD86. In certain embodiments, an immune checkpoint inhibitor is an antibody or fragment thereof that interferes with the interaction between LAG3 and its ligand, or TIM-3 and its ligand. A checkpoint inhibitor can also be in the form of a soluble form of the molecule (or variant thereof) itself, e.g., a soluble PD-L1 or a PD-L1 fusion.

[0387] The "programmed death 1 (PD-1)" receptor refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is primarily expressed on pre-activated T cells in vivo and binds to two ligands, PD-L1 and PD-L2. As used herein, the term "PD-1" includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, as well as analogs that share at least one epitope with hPD-1.

[0388] "Programmed death-ligand 1 (PD-L1)" is one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that downregulates T cell activation and cytokine secretion upon binding to PD-1. As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1, and analogs that share at least one epitope with hPD-L1.

[0389] "Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4)" is a T-cell surface molecule and a member of the immunoglobulin superfamily. This protein downregulates the immune system by binding to CD80 and CD86. As used herein, the term "CTLA-4" includes human CTLA-4 (hCTLA-4), variants, isoforms, and species homologs of hCTLA-4, as well as analogs that share at least one epitope with hCTLA-4.

[0390] Lymphocyte activation gene 3 (LAG3) is an inhibitory receptor involved in the inhibition of lymphocyte activity by binding to MHC class II molecules. This receptor enhances the function of Treg cells and inhibits CD8 + Inhibits effector T cell function. As used herein, the term "LAG3" includes human LAG3 (hLAG3), variants, isoforms, and species homologs of hLAG3, and analogs that share at least one common epitope.

[0391] "T-cell membrane protein 3 (TIM3)" is an inhibitory receptor involved in inhibiting lymphocyte activity by inhibiting TH1 cell responses. Its ligand is galectin 9, which is upregulated in various types of cancer. As used herein, the term "TIM3" includes human TIM3 (hTIM3), variants, isoforms, and species homologs of hTIM3, as well as analogs that share at least one common epitope.

[0392] "B7 family" refers to inhibitory ligands with undefined receptors. The B7 family includes B7-H3 and B7-H4, both of which are upregulated in tumor cells and tumor-infiltrating cells.

[0393] In certain embodiments, immune checkpoint inhibitors suitable for use in the methods disclosed herein are antagonists of inhibitory signals, such as antibodies targeting PD-1, PD-L1, CTLA-4, LAG3, B7-H3, B7-H4, or TIM3. These ligands and receptors are reviewed in Pardoll, D., Nature. 12:252-264, 2012.

[0394] In certain embodiments, the immune checkpoint inhibitor is an antibody or antigen-binding portion thereof that disrupts or inhibits signaling from an inhibitory immunoregulator. In certain embodiments, the immune checkpoint inhibitor is a small molecule that disrupts or inhibits signaling from an inhibitory immunoregulator.

[0395] In certain embodiments, the inhibitory immunomodulator is a component of the PD-1 / PD-L1 signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administering to a subject an antibody, or antigen-binding portion thereof, that interferes with the interaction between the PD-1 receptor and its ligand, PD-L1. Antibodies that bind to PD-1 and interfere with the interaction between PD-1 and its ligand, PD-L1, are known in the art. In certain embodiments, the antibody, or antigen-binding portion thereof, specifically binds to PD-1. In certain embodiments, the antibody, or antigen-binding portion thereof, specifically binds to PD-L1 and inhibits its interaction with PD-1, thereby increasing immune activity.

[0396] In certain embodiments, the suppressive immunomodulator is a component of the CTLA4 signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administering to a subject an antibody or antigen-binding portion thereof that targets CTLA4 and interferes with its interaction with CD80 and CD86.

[0397] In certain embodiments, the suppressive immunomodulator is a component of the LAG3 (lymphocyte activation gene 3) signaling pathway. Accordingly, certain embodiments of the present disclosure provide for administering to a subject an antibody or antigen-binding portion thereof that targets LAG3 and disrupts its interaction with MHC class II molecules.

[0398] In certain embodiments, the inhibitory immunomodulator is a component of the B7 family signaling pathway. In certain embodiments, the B7 family members are B7-H3 and B7-H4. Therefore, certain embodiments of the present disclosure provide for administering to a subject an antibody or antigen-binding portion thereof that targets B7-H3 or B7-H4. Although the B7 family does not have a defined receptor, these ligands are upregulated on tumor cells or tumor-infiltrating cells. Preclinical mouse models have shown that blocking these ligands can enhance anti-tumor immunity.

[0399] In certain embodiments, the suppressive 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.

[0400] It will be understood by those skilled in the art that other immune checkpoint targets can also be targeted by antagonists or antibodies, provided that targeting results in stimulation of an immune response, such as an anti-tumor immune response as reflected, for example, in increased T cell proliferation, enhanced T cell activation, and / or increased cytokine production (e.g., IFN-γ, IL2).

[0401] RNA targeting According to the present invention, it is particularly preferred that the peptides, proteins or polypeptides described herein, in particular vaccine antigens, are administered in the form of RNA encoding the peptides, proteins or polypeptides described herein. In one embodiment, different peptides, proteins or polypeptides described herein are encoded by different RNA molecules.

[0402] In one embodiment, the RNA is formulated in a delivery vehicle. In one embodiment, the delivery vehicle comprises a particle. In one embodiment, the delivery vehicle comprises at least one lipid. In one embodiment, the at least one lipid comprises at least one cationic lipid. In one embodiment, the lipid forms a complex with and / or encapsulates the RNA. In one embodiment, the lipid is included in a vesicle that encapsulates the RNA. In one embodiment, the RNA is formulated in a liposome.

[0403] According to the present disclosure, after administering the RNA described herein, at least a portion of the RNA is delivered to target cells.In one embodiment, at least a portion of the RNA is delivered to the cytosol of target cells.In one embodiment, the RNA is translated by target cells to produce the encoded peptide or protein.

[0404] Some embodiments of the present disclosure include targeted delivery of the RNAs disclosed herein (eg, RNAs encoding vaccine antigens).

[0405] In one embodiment, the present disclosure includes targeting the lymphatic system, particularly secondary lymphoid organs, more particularly the spleen.When the administered RNA is an RNA encoding a vaccine antigen, it is particularly preferred to target the lymphatic system, particularly secondary lymphoid organs, more particularly the spleen.

[0406] 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.

[0407] The "lymphatic system" is part of the circulatory system and an important part of the immune system, including the network of lymphatic vessels that transport lymph. The lymphatic system consists of lymphoid organs, the conducting network of lymphatic vessels, and circulating lymph. Primary or central lymphoid organs generate lymphocytes from immature precursor cells. The thymus and bone marrow constitute the primary lymphoid organs. Secondary or peripheral lymphoid organs, including the lymph nodes and spleen, maintain mature naive lymphocytes and initiate adaptive immune responses.

[0408] RNA can be delivered to the spleen using so-called lipoplex formulations, in which RNA is bound to liposomes containing cationic lipids and, optionally, additional lipids or helper lipids, to form injectable nanoparticle formulations. Liposomes can be obtained by injecting a solution of lipids in ethanol into water or a suitable aqueous phase. RNA lipoplex particles can be prepared by mixing liposomes with RNA. RNA lipoplex particles targeted to the spleen are described in International Publication No. 2013 / 143683, which is incorporated herein by reference. It has been found that RNA lipoplex particles with a net negative charge can be used to selectively target spleen tissue or spleen cells, such as antigen-presenting cells, particularly dendritic cells. Thus, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression occurs in the spleen. Thus, the RNA lipoplex particles of the present disclosure can be used to express RNA in the spleen. In one embodiment, after administration of the RNA lipoplex particles, no or essentially no RNA accumulation and / or RNA expression occurs in the lungs and / or liver. In one embodiment, after administration of the RNA lipoplex particles, RNA accumulation and / or RNA expression occurs in antigen-presenting cells, such as professional antigen-presenting cells in the spleen. Thus, the RNA lipoplex particles of the present disclosure can be used to express RNA in such antigen-presenting cells. In one embodiment, the antigen-presenting cells are dendritic cells and / or macrophages.

[0409] In the context of the present disclosure, the term "RNA lipoplex particle" refers to a particle comprising a lipid, particularly a cationic lipid, and RNA. Such cationic lipids are described above. Electrostatic interactions between positively charged liposomes and negatively charged RNA result in complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes can generally be synthesized using a cationic lipid, such as DOTMA, and an additional lipid, such as DOPE. In one embodiment, the RNA lipoplex particle is a nanoparticle.

[0410] Additional lipids may be incorporated to adjust the overall positive-to-negative charge ratio and physical stability of the RNA lipoplex particles. Such additional lipids are described above. In certain embodiments, the additional lipid is a neutral lipid. As used herein, "neutral lipid" refers to a lipid having a net charge of zero. Examples of neutral lipids include, but are not limited to, 1,2-di-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, and cerebroside. In certain embodiments, the additional lipid is DOPE, cholesterol, and / or DOPC.

[0411] In certain embodiments, the RNA lipoplex particles comprise both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.

[0412] In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 10:0 to about 1:9, about 4:1 to about 1:2, or about 3:1 to about 1:1. In certain embodiments, the molar ratio can be about 3:1, about 2.75:1, about 2.5:1, about 2.25:1, about 2:1, about 1.75:1, about 1.5:1, about 1.25:1, or about 1:1. In an exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2:1.

[0413] In one embodiment, the RNA lipoplex particles described herein have an average diameter in the range of about 200 nm to about 1000 nm, about 200 nm to about 800 nm, about 250 nm to about 700 nm, about 400 nm to about 600 nm, about 300 nm to about 500 nm, or about 350 nm to about 400 nm. In certain embodiments, the RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm. In one embodiment, the RNA lipoplex particles have an average diameter in the range of about 250 nm to about 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter ranging from about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.

[0414] The charge of the RNA lipoplex particles of the present disclosure is the sum of the charge present in at least one cationic lipid and the charge present in RNA.The charge ratio is the ratio of the positive charge present in at least one cationic lipid to the negative charge present in RNA.The charge ratio of the positive charge present in at least one cationic lipid to the negative charge present in RNA is calculated by the following formula: charge ratio = [(cationic lipid concentration (mol)) * (total number of positive charges in cationic lipid)] / [(RNA concentration (mol)) * (total number of negative charges in RNA)].

[0415] The spleen-targeted RNA lipoplex particles described herein at physiological pH preferably have a net negative charge, such as a positive to negative charge ratio of about 1.9:2 to about 1:2. In certain embodiments, the positive to negative charge ratio in the RNA lipoplex particles at physiological pH is about 1.9:2.0, about 1.8:2.0, about 1.7:2.0, about 1.6:2.0, about 1.5:2.0, about 1.4:2.0, about 1.3:2.0, about 1.2:2.0, about 1.1:2.0, or about 1:2.0.

[0416] RNA delivery systems have an inherent liver selectivity. This is related to lipid nanoparticles, including lipid-based particles, cationic and neutral nanoparticles, particularly liposomes, nanomicelles, and lipid nanoparticles containing lipophilic ligands in bioconjugates. Liver accumulation is caused by the discontinuous nature of the hepatic vasculature or lipid metabolism (liposomes and lipid or cholesterol conjugates).

[0417] In one embodiment of targeted delivery of a cytokine such as IL2, the target organ is the liver and the target tissue is hepatic tissue. Delivery to such a target tissue is preferred, particularly when the presence of the cytokine in this organ or tissue is desired, and / or when it is desired to express large amounts of the cytokine, and / or when the systemic presence of the cytokine, particularly in significant amounts, is desired or required.

[0418] In one embodiment, the RNA encoding the cytokine is administered in a formulation for targeting to the liver, such formulations being described herein above.

[0419] For in vivo delivery of RNA to the liver, drug delivery systems can be used to transport RNA to the liver by preventing its degradation. For example, polyplex nanomicelles, consisting of a poly(ethylene glycol) (PEG)-coated surface and an mRNA-containing core, are useful systems because the nanomicelles provide excellent in vivo stability of RNA under physiological conditions. Furthermore, the stealth properties provided by the polyplex nanomicelle surface, composed of a dense PEG pallisade, effectively evade the host immune defenses.

[0420] Pharmaceutical Composition The nucleic acids, nucleic acid particles, peptides, proteins, polypeptides, RNA, RNA particles, immune effector cells and additional agents, e.g., immune checkpoint inhibitors, described herein may be administered in pharmaceutical compositions or medicaments for therapeutic or prophylactic treatment, and may be administered in the form of any suitable pharmaceutical composition, which may include a pharmaceutically acceptable carrier, and may optionally include one or more adjuvants, stabilizers, etc. In one embodiment, the pharmaceutical composition is for use in therapeutic or prophylactic treatment, e.g., the treatment or prevention of a disease in which an antigen is involved, such as a cancer disease as described herein.

[0421] The term "pharmaceutical composition" relates to a formulation comprising a therapeutically effective agent, preferably together with a pharmaceutically acceptable carrier, diluent and / or excipient. The pharmaceutical composition is useful for treating, preventing, or reducing the severity of a disease or disorder by administering the pharmaceutical composition to a subject. A pharmaceutical composition is also known in the art as a pharmaceutical formulation. In the context of the present disclosure, a pharmaceutical composition comprises a nucleic acid, nucleic acid particle, peptide, protein, polypeptide, RNA, RNA particle, immune effector cell, and / or additional agent described herein.

[0422] The pharmaceutical compositions of the present disclosure may contain or be administered with one or more adjuvants. The term "adjuvant" refers to a compound that prolongs, enhances, or accelerates an immune response. Adjuvants include a heterogeneous group of compounds, such as oil emulsions (e.g., Freund's adjuvant), inorganic compounds (e.g., alum), bacterial products (e.g., Bordetella pertussis toxin), or immune-stimulating complexes. Examples of adjuvants include, but are not limited to, LPS, GP96, CpG oligodeoxynucleotides, growth factors, and cytokines, such as monokines, lymphokines, interleukins, and chemokines. Cytokines may be IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL12, IFNα, IFNγ, GM-CSF, or LT-α. Further known adjuvants are aluminum hydroxide, Freund's adjuvant, or oils such as Montanide® ISA 51. Other suitable adjuvants for use in the present disclosure include lipopeptides such as Pam3Cys.

[0423] Pharmaceutical compositions according to the present disclosure are generally applied in a "pharmaceutically effective amount" and a "pharmaceutically acceptable formulation."

[0424] The term "pharmaceutically acceptable" refers to the non-toxicity of a substance that does not interact with the action of the active ingredients of the pharmaceutical composition.

[0425] The term "pharmacologically effective amount" or "therapeutically effective amount" refers to an amount that alone or together with further doses achieves a desired response or a desired effect. In the case of the treatment of a specific disease, the desired response preferably relates to the inhibition of the course of the disease. This includes slowing the progression of the disease, particularly halting or reversing the progression of the disease. The desired response in the treatment of a disease can also be delaying or preventing the onset of the disease or condition. The effective amount of the compositions described herein depends on the condition being treated, the severity of the disease, individual patient parameters including age, physiological condition, size, and weight, the duration of treatment, the type of concomitant treatment (if any), the specific route of administration, and similar factors. Thus, the dosage of the compositions described herein can depend on such various parameters. If the patient's response is inadequate with the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) can be used.

[0426] The pharmaceutical compositions of the present disclosure may include salts, buffering agents, preservatives, and optionally other therapeutic agents. In one embodiment, the pharmaceutical compositions of the present disclosure include one or more pharmaceutically acceptable carriers, diluents, and / or excipients.

[0427] Suitable preservatives for use in the pharmaceutical compositions of the present disclosure include, but are not limited to, benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0428] The term "excipient" as used herein refers to a substance that may be present in the pharmaceutical compositions of the present disclosure but is not an active ingredient. Examples of excipients include, but are not limited to, carriers, binders, diluents, lubricants, thickeners, surfactants, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, or coloring agents.

[0429] The term "diluent" refers to an agent that dilutes and / or thins. Furthermore, the term "diluent" includes any one or more of a fluid, liquid, or solid suspension and / or mixture medium. Examples of suitable diluents include ethanol, glycerol, and water.

[0430] The term "carrier" refers to a component, which may be natural, synthetic, organic, or inorganic, with which an active ingredient is combined to facilitate, enhance, or enable administration of a pharmaceutical composition. As used herein, a carrier may be one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to a subject. Suitable carriers include, but are not limited to, sterile water, Ringer's solution, lactated Ringer's solution, sterile sodium chloride solution, isotonic saline, polyalkylene glycols, hydrogenated naphthalenes, and, in particular, biocompatible lactide polymers, lactide / glycolide copolymers, or polyoxyethylene / polyoxypropylene copolymers. In one embodiment, the pharmaceutical composition of the present disclosure comprises isotonic saline.

[0431] Pharmaceutically acceptable carriers, excipients, or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro edit. 1985).

[0432] Pharmaceutical carriers, excipients or diluents can be selected with regard to the intended route of administration and standard pharmaceutical practice.

[0433] In one embodiment, the pharmaceutical compositions described herein may be administered intravenously, intraarterially, subcutaneously, intradermally, or intramuscularly. In certain embodiments, the pharmaceutical compositions are formulated for local or systemic administration. Systemic administration may include enteral administration, including absorption via the digestive tract, or parenteral administration. As used herein, "parenteral administration" refers to administration by any means other than via the digestive tract, such as by intravenous injection. In a preferred embodiment, the pharmaceutical composition is formulated for systemic administration. In another preferred embodiment, the systemic administration is by intravenous administration. In one embodiment of all aspects of the present invention, RNA encoding the antigen is administered systemically.

[0434] As used herein, the term "co-administration" refers to the process of administering different compounds or compositions (e.g., immune effector cells (which may be "administered" by in vivo generation in a subject) and an antigen, a polynucleotide encoding the antigen, or host cells genetically modified to express the antigen) to the same patient. The different compounds or compositions may be administered simultaneously, essentially simultaneously, or sequentially. In one embodiment, the antigen, a polynucleotide encoding the antigen, or host cells genetically modified to express the antigen is administered after the administration or generation of immune effector cells genetically modified to express an antigen receptor, e.g., at least 1 day, e.g., 1-10 days or 1-5 days, after the administration or generation of immune effector cells genetically modified to express an antigen receptor. The antigen, a polynucleotide encoding the antigen, or host cells genetically modified to express the antigen can be administered several times over time, at regular or varying time intervals, e.g., at time intervals of 10 to 40 days after administration or generation of immune effector cells genetically modified to express an antigen receptor, and the first administration of the antigen, a polynucleotide encoding the antigen, or host cells genetically modified to express the antigen can be at least 1 day, e.g., 1 to 10 days or 1 to 5 days, after administration or generation of immune effector cells genetically modified to express an antigen receptor.

[0435] treatment The agents, compositions and methods described herein can be used to treat subjects with diseases, such as diseases characterized by the presence of diseased cells that express antigens.Particularly preferred diseases are cancer diseases.For example, when the antigen is derived from a virus, the agents, compositions and methods can be useful for treating viral diseases caused by the virus.When the antigen is a tumor antigen, the agents, compositions and methods can be useful for treating cancer diseases in which cancer cells express the tumor antigen.

[0436] The agents, compositions, and methods described herein can be used for the therapeutic or prophylactic treatment of various diseases, and the provision of immune effector cells and / or the activity of immune effector cells described herein are beneficial to patients with cancer, infectious diseases, etc. In one embodiment, the agents, compositions, and methods described herein are useful for the prophylactic and / or therapeutic treatment of antigen-mediated diseases.

[0437] The term "disease" refers to an abnormal condition affecting an individual's body. Disease is often interpreted as a medical condition associated with specific symptoms and signs. Diseases can be caused by factors from external sources, such as infections, or by internal malfunctions, such as autoimmune diseases. In humans, "disease" is often used more broadly to refer to conditions that cause pain, impairment, distress, social problems, or death in the affected individual, or that cause similar problems in those who come into contact with the individual. In this broader sense, disease sometimes includes impairment, incapacity, disability, syndrome, infection, isolated symptoms, deviant behavior, and atypical changes in structure and function, although in other contexts and for other purposes, these may be considered distinct categories. Because suffering from and living with many illnesses can alter one's outlook on life and personality, illnesses typically affect individuals not only physically but also emotionally.

[0438] In the present context, the terms "treatment", "treating" or "therapeutic intervention" relate to the management and care of a subject with the aim of combating a condition, such as a disease or disorder. This term is intended to include the full range of treatments for a given condition from which a subject is suffering, such as the administration of therapeutically effective compounds to alleviate symptoms or complications, slow the progression of a disease, disorder or condition, relieve or reduce symptoms and complications, and / or cure or eliminate a disease, disorder or condition, as well as to prevent a condition, where prevention is to be understood as the management and care of an individual with the aim of combating a disease, condition or disorder and includes the administration of active compounds to prevent the onset of symptoms or complications.

[0439] The term "therapeutic treatment" relates to any treatment that improves the health status and / or prolongs (increases) the lifespan of an individual. Said treatment may eliminate the disease in an individual, halt or delay the onset of the disease in an individual, inhibit or delay the onset of the disease in an individual, reduce the frequency or severity of symptoms in an individual, and / or reduce recurrence in an individual who currently has or has previously had the disease.

[0440] The term "prophylactic treatment" or "preventative treatment" relates to any treatment intended to prevent a disease from occurring in an individual. The terms "prophylactic treatment" or "preventative treatment" are used interchangeably herein.

[0441] The terms "individual" and "subject" are used interchangeably herein. They refer to a human or another mammal (e.g., a mouse, rat, rabbit, dog, cat, cow, pig, sheep, horse, or primate) that may or may not have a disease or disorder (e.g., cancer), but that may or may not have the disease or disorder. In many embodiments, the individual is a human. Unless otherwise specified, the terms "individual" and "subject" do not denote a particular age and thus encompass adults, elderly people, children, and newborns. In embodiments of the present disclosure, an "individual" or "subject" is a "patient."

[0442] The term "patient" refers to an individual or subject for treatment, particularly an afflicted individual or subject.

[0443] In one embodiment of the present disclosure, the objective is to provide an immune response against diseased cells that express an antigen, such as cancer cells that express a tumor antigen, to treat a disease, such as a cancer disease, involving cells that express an antigen, such as a tumor antigen.

[0444] The immune response against the antigen can be induced, which can be therapeutic or partially or completely protective.Therefore, the pharmaceutical compositions described herein can be applied to induce or enhance immune response.Therefore, the pharmaceutical compositions described herein are useful for the preventive and / or therapeutic treatment of diseases associated with antigens.

[0445] As used herein, "immune response" refers to the body's orchestrated response to an antigen or a cell expressing an antigen, and refers to a cellular immune response and / or a humoral immune response.

[0446] "Cell-mediated immunity," "cellular immunity," "cellular immune response," or similar terms are intended to include cellular responses directed against cells characterized by the expression of antigens, particularly those characterized by the presentation of antigens by class I or class II MHC. The cellular response involves cells called T cells or T lymphocytes that act as either "helpers" or "killers." Helper T cells (CD4 + T cells (also called T cells) play a central role by regulating the immune response and are known as killer cells (cytotoxic T cells, cytolytic T cells, CD8 + T cells (also called CTLs) kill diseased cells, such as cancer cells, and prevent the production of further diseased cells.

[0447] The present disclosure contemplates an immune response that can be protective, preventative, prophylactic, and / or therapeutic. As used herein, "inducing an immune response (or inducing)" can indicate that an immune response to a particular antigen was not present before induction, or that there was a basal level of immune response to a particular antigen before induction, which was enhanced after induction. Thus, "inducing an immune response (or inducing)" includes "enhancing an immune response (or enhancing)."

[0448] The term "immunotherapy" relates to the treatment of a disease or condition by inducing or enhancing an immune response. The term "immunotherapy" includes antigen immunization or antigen vaccination.

[0449] The terms "immunization" or "vaccination" refer to the process of administering an antigen to an individual with the intent of inducing an immune response, for example, for therapeutic or prophylactic reasons.

[0450] The term "macrophage" refers to a subgroup of phagocytes produced by differentiation of monocytes. Activated by inflammation, immune cytokines, or microbial products, macrophages nonspecifically engulf foreign pathogens within the macrophage and kill them through hydrolytic and oxidative attack, resulting in their degradation. Peptides from degraded proteins are displayed on the macrophage cell surface, where they can be recognized by T cells and interact directly with antibodies on the surface of B cells, leading to activation of T and B cells and further stimulation of the immune response. Macrophages belong to a class of antigen-presenting cells. In one embodiment, the macrophages are splenic macrophages.

[0451] The term "dendritic cell" (DC) refers to another subtype of phagocyte belonging to the class of antigen-presenting cells. In one embodiment, dendritic cells are derived from hematopoietic bone marrow progenitors. These progenitor cells first transform into immature dendritic cells. These immature cells are characterized by high phagocytic activity and low T cell activation capacity. Immature dendritic cells constantly sample the surrounding environment for pathogens, such as viruses and bacteria. Upon contact with presentable antigens, they are activated to become mature dendritic cells and begin migrating to the spleen or lymph nodes. Immature dendritic cells phagocytose pathogens, degrade their proteins into small fragments, and upon maturation, present these fragments on their cell surface using MHC molecules. At the same time, they upregulate cell surface receptors that act as coreceptors in T cell activation, such as CD80, CD86, and CD40, greatly enhancing their ability to activate T cells. They also upregulate CCR7, a chemotactic receptor that directs dendritic cells to migrate through the bloodstream to the spleen or through the lymphatic system to lymph nodes. Here, they act as antigen-presenting cells, activating helper T cells, killer T cells, and B cells by presenting antigens together with non-antigen-specific costimulatory signals. Thus, dendritic cells can actively induce immune responses associated with T cells or B cells. In one embodiment, the dendritic cells are splenic dendritic cells.

[0452] The term "antigen-presenting cell" (APC) refers to any of a variety of cells that can display, acquire, and / or present at least one antigen or antigen fragment on (or at) their cell surface. Antigen-presenting cells can be distinguished into professional and non-professional antigen-presenting cells.

[0453] The term "professional antigen-presenting cells" refers to antigen-presenting cells that constitutively express major histocompatibility complex class II (MHC class II) molecules, which are necessary for interaction with naive T cells. When T cells interact with the MHC class II molecule complex on the membrane of the antigen-presenting cell, the antigen-presenting cell produces costimulatory molecules that induce T cell activation. Professional antigen-presenting cells include dendritic cells and macrophages.

[0454] The term "non-professional antigen-presenting cells" refers to antigen-presenting cells that do not constitutively express MHC class II molecules but do so upon stimulation with certain cytokines, such as interferon gamma. Exemplary non-professional antigen-presenting cells include fibroblasts, thymic epithelial cells, thyroid epithelial cells, glial cells, pancreatic beta cells, or vascular endothelial cells.

[0455] "Antigen processing" refers to the breakdown of an antigen into processing products that are fragments of the antigen (e.g., breakdown of a protein into peptides), and the association (e.g., by binding) of one or more of these fragments with an MHC molecule for presentation to specific T cells by a cell, such as an antigen-presenting cell.

[0456] The terms "antigen-associated disease," "antigen-expressing cell-associated disease," or similar terms refer to any disease associated with an antigen, e.g., a disease characterized by the presence of an antigen. An antigen-associated disease may be an infectious disease, or a cancer disease or simply cancer. As noted above, the antigen may be a disease-associated antigen, such as a tumor-associated antigen, a viral antigen, or a bacterial antigen. In one embodiment, an antigen-associated disease is a disease involving cells that express the antigen, preferably on their cell surface.

[0457] The term "infectious disease" refers to any disease (e.g., the common cold) that can be transmitted from individual to individual or from organism to organism and is caused by a microbial agent. Infectious diseases are known in the art and include, for example, viral diseases, bacterial diseases, or parasitic diseases, which are caused by viruses, bacteria, and parasites, respectively. In this regard, infectious diseases can be, for example, hepatitis, sexually transmitted diseases (e.g., chlamydia or gonorrhea), tuberculosis, HIV / acquired immunodeficiency syndrome (AIDS), diphtheria, hepatitis B, hepatitis C, cholera, severe acute respiratory syndrome (SARS), avian influenza, and influenza.

[0458] The term "cancer disease" or "cancer" refers to or describes a physiological condition in an individual that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia. More specifically, examples of such cancer include bone cancer, blood cancer, lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, prostate cancer, uterine cancer, cancer of the genital and reproductive organs, Hodgkin's disease, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, bladder cancer, kidney cancer, renal cell carcinoma, renal pelvis cancer, neoplasms of the central nervous system (CNS), neuroectodermal carcinoma, spinal axis tumor, glioma, meningioma, and pituitary adenoma. The term "cancer" according to the present disclosure also includes cancer metastasis.

[0459] The term "solid tumor" or "solid cancer" as used herein refers to the appearance of a cancerous mass, as known in the art, for example, in Harrison's Principles of Internal Medicine, 14th edition. Preferably, this term refers to cancer or carcinoma of body tissues other than blood, preferably other than blood, bone marrow, and lymphatic system. For example, but not limited to, solid tumors include cancer of the prostate, lung, colorectal tissue, bladder, oropharynx / larynx tissue, kidney, breast, endometrium, ovary, cervix, stomach, pancreas, brain, and central nervous system.

[0460] Combination strategies in cancer treatment can be desirable due to the resulting synergistic effects, which can be significantly more potent than the effects of monotherapy approaches. In one embodiment, the pharmaceutical composition is administered together with an immunotherapeutic agent. As used herein, "immunotherapeutic agent" refers to any agent that can be involved in activating a specific immune response and / or immune effector function(s). The present disclosure contemplates the use of antibodies as immunotherapeutic agents. Without wishing to be bound by theory, antibodies can achieve therapeutic effects on cancer cells through various mechanisms, including inducing apoptosis, blocking components of signaling pathways, or inhibiting tumor cell proliferation. In certain embodiments, the antibody is a monoclonal antibody. Monoclonal antibodies can induce cell death via antibody-dependent cell-mediated cytotoxicity (ADCC) or can bind to complement proteins, resulting in direct cytotoxicity, known as complement-dependent cytotoxicity (CDC). Non-limiting examples of anti-cancer antibodies and potential antibody targets (in parentheses) that may be used in combination with the present disclosure include abagovomab (CA-125), abciximab (CD41), adecatumumab (EpCAM), afutuzumab (CD20), alacizumab pegol (VEGFR2), altumomab pentetate (CEA), amatuximab (MORAb-009), anatumomab mafenatox (TAG-72), apolizumab (HLA-DR), arcitumomab (CEA), atezolizumab (PD-L1), bavituximab (phosphatidylserine), bectumomab (CD22), belimumab (BAFF), bevacizumab (VEGF-A), bivacizumab mertansine (CD44 v6), blinatumomab (CD19), brentuximab vedotin (CD30TNFRSF8), cantuzumab mertansine (mucin CanAg), cantuzumab mertansine (MUC1), capromab pendetide (prostate cancer cells), carlumab (CNT0888), catumaxomab (EpCAM, CD3), cetuximab (EGFR), sitatuzumab bogatox (EpCAM), cixutumumab (IGF-1 receptor), claudiximab (claudin), clivatuzumab tetraxetan (MUC1), conatumumab (TRAIL-R2), dacetuzumab (CD40), dalotuzumab (insulin -like growth factor I receptor), denosumab (RANKL), detumomab (B lymphoma cells), drozitumab (DR5), ecloneximab (GD3 ganglioside), edrecolomab (EpCAM), elotuzumab (SLAMF7), enavatuzumab (PDL192), ensituximab (NPC-1C), epratuzumab (CD22), ertumaxomab (HER2 / neu, CD3), etaracizumab (integrin ανβ3), farletuzumab (folate receptor 1), FBTA05 (CD20), ficlatuzumab (SCH900105), figitumumab (IGF-1 receptor), framvotumab (glycoprotein 75), fresolimumab (TGF-β), galiximab (CD80), ganitumab (IGF-I), gemtuzumab ozogamicin (CD33), gevokizumab (ILΙβ), girentuximab (carbonic anhydrase 9 (CA-IX)), glembatumumab vedotin ( GPNMB), ibritumomab tiuxetan (CD20), icrucumab (VEGFR-1), igovoma (CA-125), indatuximab ravtansine (SDC1), intetumumab (CD51), inotuzumab ozogamicin (CD22), ipilimumab (CD152), iratumumab (CD30), labetuzumab (CEA), lexatumumab (TRAIL-R2), ribivirumab (hepatitis B surface antigen), lintuzumab (CD33), lorvotuzumab mertansine (CD56), lucatumumab (CD40), rumiliximab (CD23), mapatumumab (TRAIL-R1), matuzumab (EGFR), mepolizumab (IL5), milatuzumab (CD74), mitsumomab (GD3 ganglionic Osido), mogamulizumab (CCR4), moxetumomab pasudotox (CD22), nacolomab butafenatox (C242 antigen), naptumomab estafenatox (5T4), namatumab (RON), necitumumab (EGFR), nimotuzumab (EGFR), nivolumab (IgG4), ofatumumab (CD20), olaratumab (PDGF-Ra), onartuzumab (human scatter factor receptor kinase), oportuzumab monatox (EpCAM), oregovomab (CA-125), oxelumab (OX-40), panitumumab (EGFR), patritumab (HER3), pemtumomab (MUC1), pertuzumab (HER2 / neu), pintumomab (adenocarcinoma antigen), pritumumab (vimentin), racotumomab (N-glycolylneuraminic acid), radletumab (fibronectin extra domain B), rafivirumab (rabies virus glycoprotein), ramucirumab (VEGFR2), rilotumumab (HGF), rituximab (CD20), lobatumumab (IGF-1 receptor), samalizumab (CD200), sinostat Brotuzumab (FAP), siltuximab (IL6), tabalumab (BAFF), tacatuzumab tetraxetan (α-fetoprotein), taplitumomab paptox (CD19), tenatumomab (tenascin-C), teprotumumab (CD221), ticilimumab (CTLA-4), tigatuzumab (TRAIL-R2), TNX-650 (IL13), tositumomab (CD20), trastuzumab (HER2 / neu), TRBS07 (GD2), tremelimumab (CTLA-4), tucotuzumab celmoleukin (EpCAM), ublituximab (MS4A1), urelumab (4-1BB), boroximab (integrin α5β1), votumumab (tumor antigen CTAA 16.88), zalutumumab (EGFR), and zanolimumab (CD4).

[0461] Citation of documents and tests referenced herein is not intended as an admission that any of the foregoing is pertinent prior art. All statements regarding the contents of these documents are based on information available to applicant and do not constitute any admission as to the accuracy of the contents of these documents.

[0462] The following description is presented to enable those skilled in the art to make and use various embodiments. Descriptions of specific devices, techniques, and applications are provided only as examples. Various modifications to the examples described herein will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other examples and applications without departing from the spirit and scope of the various embodiments. Accordingly, the various embodiments are not intended to be limited to the examples described and shown herein, but are to be accorded the scope consistent with the appended claims. [Example]

[0463] Example 1 Identification of CD8-DARPin Ribosome display Ribosome display selection was performed as described by Hartmann and colleagues (Hartmann et al. 2018). Briefly, for the first three selection rounds, the translated VV-N3C DARPin library was subjected to a prepanning step using immobilized neutravidin or streptavidin (both at 20 μM). For on-target selection, the library was incubated with immobilized hCD8αβ-Fc (50 nM). The resulting DARPin library was amplified and used as a template for the next selection round. After three rounds with immobilized target protein, a fourth selection round was performed using the target in solution. Prior to on-target selection, a preselection step using 0.9 pmol of non-biotinylated hCD8αα-Fc was performed. The library was then exposed to biotinylated hCD8αβFc target protein (0.65 pmol) and a 100-fold molar excess (65 pmol) of non-biotinylated hCD8ααFc. The fifth selection round was again performed using immobilized protein, which included a preselection step with immobilized CD8αα-Fc (20 nM) before incubation with hCD8αβ-Fc. Finally, a sixth selection round was performed in solution as follows: after preincubation with 0.9 pmol of soluble CD8αα-Fc, a combined off-rate and counterselection step was performed, in which the library was co-incubated with biotinylated target protein (0.65 pmol), excess non-biotinylated hCD8αα-Fc, and non-biotinylated hCD8αβ-Fc (both 65 pmol). After the fifth and sixth selection rounds, DNA fragments encoding DARPins were analyzed for CD8 binding by single clone analysis.

[0464] DARPin expression and crude lysate preparation To test the selected DARPins for specific binding to CD8 after the fifth and sixth selection rounds, DNA fragments were cloned into the bacterial expression vector pQE-HisHA and transformed into E. coli XL1-blue as previously described (Hartmann et al. 2018). Single clones were picked and grown overnight at 37 °C in 600 μl of 2YT medium (2YT, 1% glucose, 100 μg / ml ampicillin), after which the cultures reached an OD of 0.1. 600 The cells were diluted to 100 mL and expression of DARPin was induced by adding 100 mL of 5.5 mM isopropyl-bD-thiogalactopyranoside (IPTG) to 2YT medium. After 5 h of incubation at 37 °C, the bacteria were harvested by centrifugation, and the pellet was stored overnight at -80 °C. The next day, the pellet was thawed on ice and lysed by adding B_PER solution followed by 2 h of incubation at room temperature. The lysate was pelleted to remove cellular debris, and the supernatant containing crude DARPin was aliquoted and stored at -80 °C until use in the cell-binding assay. Protein content was determined by Bradford assay. Crude lysate preparations were always handled on ice and subjected to up to three freeze-thaw cycles to avoid loss of protein quality. DARPin clones were sequenced using standard sequencing techniques to obtain DNA and protein sequences.

[0465] Cell binding assay To analyze the specific binding of DARPins to human and NHP CD8, cell binding assays were performed using Molt4.8 and J76S8ab cells and primary human and NHP PBMCs (isolated and activated as described above). 5Cells were washed once with wash buffer (PBS, 2% FCS, 0.1% NaN3) and incubated with 10 μl of crude DARPin extract in a total volume of 200 μl for 60 min at 4 °C. After incubation, cells were washed twice with wash buffer, stained with fluorescently labeled antibodies, and analyzed by flow cytometry as described below. Screening for CD8 binding by cell-binding assay using cell lines and validation by binding to human and NHP PBMCs were performed on an n=1 basis.

[0466] Example 2 CD8-specific binding of DARPin The previously described combinatorial DARPin library VV-N3C (Hartmann et al. 2018) was screened for CD8-specific DARPins by ribosome display using generated recombinant CD8 protein as bait. In total, up to six selection rounds were performed. The first three and five rounds were performed using immobilized CD8αβ-Fc as the bait protein, including preselection steps against neutravidin, streptavidin, and immobilized Fc protein to eliminate the selection of DARPins with undesired specificities. The fourth and sixth rounds were performed in solution and included counterselection with non-biotinylated CD8αβ-Fc and CD8αα-Fc to select binders with high affinity for CD8αβ. To identify the best CD8-specific DARPins for targeted gene transfer, the output repertoire was screened in a two-step procedure. First, CD8 binding was assessed in a cell-based assay. In the second step, the gene transfer activity of 10 clones was evaluated. In total, 94 DARPin clones obtained from the fifth and sixth selection rounds were expressed in E. coli and tested for binding to Molt4.8 cells (expressing CD8αα) and J67S8ab cells (expressing CD8αβ). Of these, 31 individual DARPin clones that bound equally well to both cell lines were selected (Figure 4A) and further analyzed for binding to primary human and NHP T cells. All candidates expressed human CD8αα. +Specific binding to CD8 - Cells were not modified above background (Figure 4B). Notably, variation in cell staining intensity across DARPin candidates was observed (Figure 4B). In the next step, the cross-reactivity of these DARPins with macaque PBMCs was assessed. All identified human CD8-specific DARPins also bound to CD8 on NHP PBMCs (Figure 4C). Of the identified CD8 binders, five DARPins that bound human CD8 with intermediate MFI and five candidates that bound with high MFI were selected for further characterization. 28 of the candidates were successfully characterized, and sequencing revealed that each of these candidates (Table 1) had a unique amino acid sequence.

[0467] [Table 2] TIFF2026004425000010.tif182153TIFF2026004425000011.tif100153

[0468] Example 3 Generation of tagged DARPins for nanoparticle functionalization 63H6 DARPin with N-terminal H6 and HA tags (for purification and detection) and C-terminal cysteine ​​or polyglutamic acid tags (E10, E20) was generated for LNP and polyplex conjugation: >H6-HA-63H6-Cys MRGSHHHHHHGSYPYDVPDYAAAQPADLGKKLLEAARAGQDDEVRILMANGADVNASDSVGNTPLHLAAWHGHLEIVDVLLKYGADVNASDVSGQTPMHLAALQGHLEIVEVLLKYGADVNTHDRWGLTPLHLAAHQGHLEIVEVLLKHGADVNAQDKFGKTPFDLAIDNGNEDIAEVLQKAAGGC >H6-HA-63H6-E10 MRGSHHHHHHGSYPYDVPDYAAAQPADLGKKLLEAARAGQDDEVRILMANGADVNASDSVGNTPLHLAAWHGHLEIVDVLLKYGADVNASDVSGQTPMHLAALQGHLEIVEVLLKYGADVNTHDRWGLTPLHLAAHQGHLEIVEVLLKHGADVNAQDKFGKTPFDLAIDNGNEDIAEVLQKAAGGSEEEEEEEEEE >H6-HA-63H6-E20 MRGSHHHHHHGSYPYDVPDYAAAQPADLGKKLLEAARAGQDDEVRILMANGADVNASDSVGNTPLHLAAWHGHLEIVDVLLKYGADVNASDVSGQTPMHLAALQGHLEIVEVLLKYGADVNTHDRWGLTPLHLAAHQGHLEIVEVLLKHGADVNAQDKFGKTPFDLAIDNGNEDIAEVLQKAAGGSEEEEEEEEEEEEEEEEEEEE

[0469] For this purpose, the corresponding gene fusions were cloned into the pET-21a expression vector. Recombinant protein production was carried out at a 1 L scale using E. coli BL21(DE3) cells carrying the pET-21a vector encoding each tagged DARPin variant at 37 °C and 120 rpm. Cultures reached an OD of approximately 0.5–0.7. 600After reaching a pH of 10, protein expression was induced by adding 1 mL of 1 M IPTG and incubating at 37°C and 120 rpm for an additional 3 h. E. coli cells were then harvested by centrifugation, resuspended in 25 mL of IMAC equilibration buffer, and lysed by five consecutive sonication cycles. After centrifugation of the cell debris (15,000 × g for 30 min at 4°C), the supernatant was purified by IMAC using an AKTAprime™ plus system and a 1 mL HisTrap column with a linear gradient of 10 to 500 mM imidazole in 20 min. DARPin protein-containing fractions were collected and dialyzed against PBS or 25 mM HEPES, pH 7.5, 10% trehalose. The purity of all tagged DARPin proteins was >90%, as judged by SDS-PAGE analysis (Figure 6). An additional band corresponding to the S-linked dimeric species is visible in the non-reduced H6-HA-63H6-Cys sample. In the functionalization step, these dimers are removed by TCEP incubation.

[0470] To assess the retained functional properties of the tagged 63H6 DARPin variants and their binding to CD8, a cell binding assay was performed using human PBMCs. 6 Human PBMCs were washed once with wash buffer (DPBS, 5% FCS, 5 mM EDTA), collected by centrifugation at 300 × g for 5 minutes, and incubated with 2 μM or 1 μM DARPin in 100 μL at 4 °C for 1 hour. Cells were then washed twice with wash buffer, centrifuged (300 × g for 5 minutes), and stained in 100 μL of a fluorescently labeled antibody mixture (anti-CD4-BV421, anti-CD3-FITC, anti-his-APC) for 1 hour at 4 °C. Cells were washed twice with wash buffer and once with DPBS. Dead cell staining was performed in 100 μL of the fixable dye eFluor780™ diluted 1:750 in DPBS for 20 minutes at 4 °C. Cells were then washed twice with wash buffer and resuspended in 100 μL of wash buffer for flow cytometry analysis. Measurements were performed on a BD FACSCanto II and data were analyzed using FlowJo X. Human CD8 +Specific binding of H6-HA-63H6-Cys, H6-HA-63H6-E10, and H6-HA-63H6-E20 to T cell populations was demonstrated in three independent PBMC donors (Figure 7).

[0471] Example 4 CD8-specific transfection with DARPin-functionalized LNPs LNPs consist of various lipids that can self-assemble into NPs with diameters of approximately 50–150 nm. Cargos such as RNA or DNA for gene delivery purposes can be encapsulated in these NPs by mixing them with a lipid mixture during the self-assembly process. So-called PEG-lipids have a stabilizing function and are exposed on the exterior of LNPs. Therefore, they are optimal candidates for the attachment of targeting ligands such as DARPins to achieve functionalization of LNPs. To achieve this attachment, click chemistry represents a promising approach. The cysteine / maleimide reaction is well known from antibody-drug conjugation, which has already been applied clinically. Therefore, a CD8-DARPin construct with a terminal cysteine ​​(CD8-DARPinSH) was produced in E. coli, and a PEG-lipid with a terminal maleimide group was also synthesized. Next, LNPs with exposed maleimides were generated (LNP-Mal) and functionalized in a second step by the addition of CD8-specific DARPinSH (clones 63H6 and 63A4). Gel electrophoresis was performed on free DARPin, LNP-Mal alone, and DARPin plus LNP-Mal or LNPs with exposed azides (LNP-N3) as a control (Figure 8A). Free DARPin could only be detected in the case of the incompatible reactive group (LNP-N3), and it can be assumed that all CD8-DARPin was conjugated to the maleimide group on the LNPs. Functionalization with DARPin slightly increases the diameter of the LNPs without affecting the particle size distribution as measured by the coherence or polydispersity index of the LNPs (Figure 8B). These DARPin-functionalized LNPs were subsequently used to deliver CD8 luciferase-mRNA. + and CD8 - Jurkat cell line (Fig. 8C, D). +Only in Jurkat cells could we detect a 10- to 100-fold higher signal with CD8-DARPin-functionalized LNPs compared to non-functionalized LNPs or LNP-N3 controls. Functionalization of LNPs with CD8-DARPinSH similarly demonstrated improved transfection of primary human T cells (Figure 8E).

[0472] Example 5 CD8-specific transfection with DARPin-functionalized PLX PLX consists of a cationic core polymer that encapsulates nucleic acid cargo. This core complex is stable and has a transfection capacity comparable to that of LNPs. Adding an anionic polymer to the core complex to shield the positive charge can reduce transfection potential. Functionalization of PLX with a targeting ligand can restore transfection potential and improve its specificity. Binding of targeting ligands, such as CD8-DARPin, to PLX can be achieved through electrostatic attraction between the cationic core polymer and an anionic polymer linked to the targeting ligand. Coupling of targeting ligands to polyglutamic acid (PGA) via reactive ester chemistry has previously been described as a viable approach (Smith et al., 2017). To avoid further chemical modification of the ligand, we generated a CD8-specific DARPin (CD8-DARPinE20) (clone 63H6) with a tag consisting of 20 glutamic acid repeats. PLX was incubated with different amounts of CD8-DARPinE20 (shown as w / w ratios) to track changes in physicochemical properties in response to CD8-DARPinE20 modification. Gel electrophoresis revealed that free DARPinE20 was undetectable in the presence of core PLX at any of the w / w ratios tested, demonstrating that all E20-tagged DARPins were bound to the core particles (Figure 9A). DLS measurements revealed that modification of core PLX with CD8-DARPinE20 was accompanied by a significant size increase of the particles, more pronounced at higher w / w ratios (Figure 9B). Furthermore, a concomitant decrease in particle surface charge (expressed as zeta potential) could be observed (Figure 9C). These results are consistent with the expectation that absorption of CD8-DARPinE20 on the particle surface should result in a size increase and screening of the positive charge of the core particles. Subsequently, the DARPin-modified PLX was also tested in human T cells, where the delivery of luciferase-mRNA and Thy1.1-mRNA (mixed in a 50 / 50 ratio) encapsulated in the same PLX was tested.Here, not only the luciferase signal but also the surface expression of the mouse marker Thy1.1, detected by flow cytometry, allowed for the determination of successful T cell transfection. CD8-DARPinE20-modified PLX transfected CD8 cells with both RNA cargoes. + showed enhanced delivery to CD8 - No enhanced delivery to Jurkat cells was observed (Figure 9D, E). Importantly, no increased transfection was observed for all control PLX (non-functionalized, irrelevant DARPin modification, no CD8-DARPin conjugation). Functionalization of PLX with CD8-DARPin E20 similarly improved transfection of primary human T cells without affecting viability (Figure 9F). Flow cytometry analysis of transfection also demonstrated improved transfection of CD4 + T cells and CD8 + This further demonstrated that CD8-DARPinE20-modified PLX specifically transfects only CD8+ T cells, enabling differentiation from T cells (Figure 9G).

[0473] Example 6 CD8-specific transfection of DARPin-modified LNPs in vivo As a next step, we evaluated the potential of LNPs functionalized by cysteine / maleimide reaction to target human T cells in vivo. Therefore, immunodeficient mice were transplanted with human PBMCs and treated 21 days later with 20 μg of mRNA (luciferase and Thy1.1, 50 / 50) encapsulated in either non-functionalized or CD8-DARPin-functionalized LNPs. One day after LNP administration, luciferase signals were detected by in situ bioluminescence imaging, indicating that the LNPs had transfected spleen-resident cells following functionalization-induced hepatocyte targeting (Figure 10A). These data indicate that LNPs can be retargeted to transfect secondary lymphoid tissues where T cells reside. Analysis of Thy1.1 expression by flow cytometry analysis of peripheral blood revealed that human (CD45 + )CD8 +T cells were transfected but CD4 + It was further revealed that T cells were not transfected (Fig. 10B).

[0474] Example 7 CD8-specific transfection of DARPin-modified PLX in vivo Furthermore, we evaluated the potential of CD8-DARPinE20-modified PLX to target human T cells in vivo. Therefore, immunodeficient mice were transplanted with human PBMCs and, 21 days later, treated with 20 μg of mRNA (luciferase and Thy1.1, 50 / 50) encapsulated in either non-functionalized or CD8-DARPinE20-modified PLX. One day after PLX administration, luciferase signals were detected by in situ bioluminescence imaging, indicating that PLX had acquired transfection of spleen-resident cells due to functionalization. This indicates that PLX can also be retargeted to transfect secondary lymphoid tissues where T cells reside. Analysis of Thy1.1 expression by flow cytometry analysis of peripheral blood revealed that human (CD45 + )CD8 + T cells were transfected but CD4 + It was further revealed that T cells were not transfected.

[0475] Example 8 Functionalized nanoparticles as vehicles for delivering mixed RNA / DNA cargoes As mentioned above, in vivo genome manipulation requires the delivery of gene editing tools. However, to date, such tools have relied on DNA templates, and enzymes can be encoded as mRNA. We demonstrate that our NPs, modified with CD8-DARPin via our in-house developed functionalization strategy, can effectively transduce human CD8 in vitro and in vivo. +We were able to clearly demonstrate that T cells can be targeted. To enable genome manipulation, we tested whether a mixed cargo of DNA and RNA could be sufficiently packaged and delivered to T cells. Therefore, we isolated CD8 T cells from peripheral blood of healthy donors as before. + T cells were isolated and cultured at 1 × 10 with 50 ng of mixed cargo (minicircle DNA encoding Venus, a modified form of yellow fluorescent protein from Aequorea Victoria, and Thy1.1-mRNA, 50 / 50) encapsulated in either non-functionalized or CD8-DARPinE20-modified PLX. 6 Target cells were treated. One day after PLX administration, RNA expression was detected by flow cytometry (Figure 11). After CD3 / CD28 bead stimulation, proliferating human T cells showed strong expression of the gene of interest encoded in the minicircle DNA. These findings demonstrate that a single CD8-DARPin-functionalized NP batch can simultaneously deliver DNA and RNA.

Claims

1. 1. A method for preparing immune effector cells genetically modified to express an antigen receptor, the method comprising contacting the immune effector cells with particles comprising a nucleic acid encoding the antigen receptor and a targeting molecule for targeting the immune effector cells, wherein the targeting molecule is an ankyrin repeat protein.

2. 10. The method of claim 1, wherein contacting the immune effector cell with the particle delivers the nucleic acid to the immune effector cell.

3. 3. The method of claim 1 or 2, wherein the immune effector cells to be genetically modified are present in vivo or in vitro.

4. The method of any one of claims 1 to 3, wherein the immune effector cells to be genetically modified are present in vivo.

5. The method of any one of claims 1 to 4, wherein the immune effector cells to be genetically modified are present in vivo in a subject, and the method comprises administering the particles to the subject.

6. 1. A method for treating a subject, comprising: (i) preparing in vitro immune effector cells genetically modified to express an antigen receptor, using a method comprising contacting the immune effector cells with particles comprising a nucleic acid encoding the antigen receptor and a targeting molecule for targeting the immune effector cells, wherein the targeting molecule is an ankyrin repeat protein; and (ii) administering to the subject the immune effector cells genetically modified to express an antigen receptor. A method comprising:

7. 7. The method of claim 6, wherein contacting the immune effector cell with the particle delivers the nucleic acid to the immune effector cell.

8. 1. A method for treating a subject, comprising:

1. A method comprising administering to the subject particles comprising a nucleic acid encoding an antigen receptor and a targeting molecule for targeting immune effector cells, wherein the targeting molecule is an ankyrin repeat protein.

9. 9. The method of claim 8, wherein the particles deliver the nucleic acid to immune effector cells of the subject.

10. 10. The method of claim 8, wherein delivering the nucleic acid to immune effector cells generates immune effector cells in the subject that are genetically modified to express an antigen receptor.

11. The method according to any one of claims 5 to 9, which is a method for inducing an immune response in the subject.

12. 12. The method of claim 11, wherein the immune response is a T cell-mediated immune response.

13. 13. The method of claim 11 or 12, wherein the immune response is an immune response against a target cell population or target tissue that expresses an antigen.

14. 14. The method of claim 13, wherein the target cell population or target tissue is a cancer cell or tissue.

15. The method of claim 14, wherein the cancer cells or cancer tissue is a solid cancer.

16. 1. A method for treating a subject having a disease, disorder, or condition associated with expression or up-regulation of an antigen, comprising: (i) preparing in vitro immune effector cells genetically modified to express an antigen receptor that targets the antigen associated with said disease, disorder or condition, or cells that express said antigen associated with said disease, disorder or condition, using a method comprising contacting said immune effector cells with particles comprising a nucleic acid encoding said antigen receptor and a targeting molecule for targeting said immune effector cells, wherein said targeting molecule is an ankyrin repeat protein; and (ii) administering to the subject the immune effector cells genetically modified to express an antigen receptor. A method comprising:

17. 17. The method of claim 16, wherein contacting the immune effector cell with the particle delivers the nucleic acid to the immune effector cell.

18. 1. A method for treating a subject having a disease, disorder, or condition associated with expression or up-regulation of an antigen, comprising: administering to the subject particles comprising a nucleic acid encoding an antigen receptor that targets the antigen associated with the disease, disorder, or condition or a cell expressing the antigen associated with the disease, disorder, or condition, and a targeting molecule for targeting the immune effector cells, the targeting molecule being an ankyrin repeat protein. A method comprising:

19. 20. The method of claim 18, wherein the particles deliver the nucleic acid to immune effector cells of the subject.

20. 20. The method of claim 19, wherein delivering the nucleic acid to immune effector cells generates immune effector cells in the subject that are genetically modified to express an antigen receptor.

21. 21. The method of any one of claims 16 to 20, wherein the disease, disorder or condition is cancer and the antigen associated with the disease, disorder or condition is a tumor antigen.

22. The method of any one of claims 16 to 21, wherein the disease, disorder or condition is a solid cancer.

23. The method of any one of claims 6 to 22, which is a method for treating or preventing cancer in a subject.

24. 24. The method of claim 23, wherein the cancer is a solid tumor.

25. 25. The method of claim 23 or 24, wherein the cancer is associated with expression or increased expression of a tumor antigen targeted by the antigen receptor.

26. 26. The method of any one of claims 5 to 25, further comprising administering to the subject an antigen targeted by the antigen receptor, a polynucleotide encoding the antigen, or a host cell genetically modified to express the antigen.

27. 27. The method of claim 26, wherein the polynucleotide is RNA.

28. 27. The method of claim 26, wherein the host cell comprises a polynucleotide encoding the antigen.

29. The method of any one of claims 1 to 28, wherein the antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

30. The method of any one of claims 1 to 29, wherein the nucleic acid is RNA.

31. The method of any one of claims 1 to 29, wherein the nucleic acid is DNA.

32. The method of any one of claims 1 to 7, 10 to 17 and 20 to 31, wherein the genetic modification is transient or stable.

33. 33. The method of any one of claims 1 to 7, 10 to 17 and 20 to 32, wherein the genetic modification is carried out by a virus-based method, a transposon-based method, or a gene editing-based method.

34. 34. The method of Claim 33, wherein the gene editing-based method comprises CRISPR-based gene editing.

35. 35. The method of any one of claims 1 to 34, wherein the particle is a non-viral particle.

36. 36. The method of any one of claims 1 to 35, wherein the particles are lipid-based and / or polymer-based particles.

37. The method of any one of claims 1 to 36, wherein the particles are nanoparticles.

38. The method of any one of claims 1 to 37, wherein the particles are functionalized with the targeting molecule on their surface.

39. 39. The method of any one of claims 1 to 38, wherein the particles are functionalized with the targeting molecule by linking the targeting molecule to at least one particle-forming component.

40. 40. The method of any one of claims 1 to 39, wherein the immune effector cells are T cells.

41. The method of any one of claims 1 to 40, wherein the immune effector cells are CD8+ T cells.

42. 42. The method of any one of claims 1 to 41, wherein the targeting molecule targets CD8.

43. The targeting molecule comprises a repeating consensus sequence: NX １ X ２ DX ３ X ４ X ５ X ６ TPX ７ HLX ８ X ９ X １０ X １１ X １２ HX １３ X １４ IVX １５ VLLKX １６ X １７ X １８ DX １９ a repeat module including where: X １ is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, X ２ is any amino acid, X ３ is any amino acid, X ４ is any amino acid, X ５ is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably G; X ６ is any amino acid, X ７ is any amino acid, preferably an amino acid selected from the group consisting of A, C, F, G, H, I, K, L, M, R, T, V, W, and Y, more preferably L; X ８ is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably A or V; X ９ is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably A; X １０ is any amino acid, X １１ is any amino acid, X １２ is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably G; X １３ is any amino acid, preferably L, X １４ is any amino acid, preferably an amino acid selected from the group consisting of D, E, H, K and R, more preferably E; X １５ is any amino acid, preferably D or E, X １６ is any amino acid, X １７ is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably an amino acid selected from the group consisting of A, G and S, more preferably G; X １８ is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably A; X １９ is any amino acid, preferably an amino acid selected from the group consisting of I, L and V, more preferably V; 43. The method of any one of claims 1 to 42.

44. The targeting molecule comprises a repeating consensus sequence: ︚ １ ︸ ２ ︤ ３ ︸ ４ ︹ ６ !! ８ ︸ ９ ︸ １０ ︸ １１ H １３ ︸ １４ ︩︹ １５ ________________ １６ .. a repeat module including where: X １ is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, X ２ is any amino acid, X ３ is any amino acid, X ４ is any amino acid, X ６ is any amino acid, X ８ is A or V, X ９ is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably A; X １０ is any amino acid, X １１ is any amino acid, X １３ is any amino acid, preferably L, X １４ is any amino acid, preferably an amino acid selected from the group consisting of D, E, H, K and R, more preferably E; X １５ is any amino acid, preferably D or E, X １６ is any amino acid, 44. The method according to any one of claims 1 to 43.

45. The targeting molecule comprises a repeating consensus sequence: NX １ X ２ DX ３ X ４ GX ６ TPLHLX ８ AX １０ X １１ GHLEIVX １５ VLLKX １６ GADV a repeat module including where: X １ is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V; X ２ is any amino acid, X ３ is any amino acid, X ４ is any amino acid, X ６ is any amino acid, X ８ is A or V, X １０ is any amino acid, X １１ is any amino acid, X １５ is D or E, X １６ is any amino acid, 44. The method according to any one of claims 1 to 43.

46. 46. ​​The method of any one of claims 43 to 45, wherein said targeting molecule comprises at least two repeat modules, which may be identical or different, each comprising said repeat consensus sequence.

47. 47. The method of any one of claims 43 to 46, wherein the targeting molecule comprises 2 to 20 repeat modules, which may be identical or different, each comprising the repeat consensus sequence.

48. 48. The method of any one of claims 43 to 47, wherein the targeting molecule comprises three repeat modules, which may be identical or different, each comprising the repeat consensus sequence.

49. The targeting molecule comprises three repeat modules, wherein: The first repeat module of the targeting molecule has the consensus sequence: NAX ２ DX ３ X ４ GX ６ TPLHLX ８ AWHGHLEIVX １５ VLLKX １６ GADV、 Including, The second repeat module of the targeting molecule has the consensus sequence: !?? ２ ︤ ３ ︸ ４ ︹ ６ _________________________ １０ ︸ １１ _|||||||||_______ １６ ..|..、 and The third repeat module of the targeting molecule has the consensus sequence: NX １ X ２ DX ３ X ４ GX ６ TPLHLAAX １０ X １１ GHLEIVEVLLKX １６ GADV、 Including, where: X １ is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V; X ２ is any amino acid, X ３ is any amino acid, X ４ is any amino acid, X ６ is any amino acid, X ８ is A or V, X １０ is any amino acid, X １１ is any amino acid, X １５ is D or E, X １６ is any amino acid, preferably an amino acid selected from the group consisting of Y, H and N, 49. The method of any one of claims 1 to 48.

50. 50. The method of any one of claims 1 to 49, wherein said targeting molecule comprises at least one repeat module comprising a sequence selected from the group of repeat modules 1, 2 and 3 of SEQ ID NOs: 1 to 28, respectively, as shown in Figure 5.

51. 51. The method of any one of claims 1 to 50, wherein said targeting molecule comprises three repeat modules, wherein repeat module 1 is selected from the group of repeat module 1 of SEQ ID NOs: 1 to 28 as shown in Figure 5, repeat module 2 is selected from the group of repeat module 2 of SEQ ID NOs: 1 to 28 as shown in Figure 5 and repeat module 3 is selected from the group of repeat module 3 of SEQ ID NOs: 1 to 28 as shown in Figure 5.

52. 52. The method of any one of claims 1 to 51, wherein said targeting molecule comprises three repeat modules, wherein repeat module 1, repeat module 2 and repeat module 3 are repeat module 1, repeat module 2 and repeat module 3 of a sequence selected from the group consisting of SEQ ID NOs: 1 to 28 as shown in Figure 5.

53. The method of any one of claims 43 to 52, wherein said repeat module is present in a repeat domain.

54. 54. The method of claim 53, wherein the repeat domain further comprises an N-terminal and / or C-terminal capping module.

55. 55. The method of any one of claims 1 to 54, wherein the targeting molecule comprises a sequence selected from the group consisting of SEQ ID NOs: 1 to 28.

56. Ankyrin repeat protein-containing molecules that target immune effector cells.

57. 57. The molecule of claim 56, wherein the immune effector cell is a T cell.

58. 58. The molecule of claim 56 or 57, wherein the immune effector cell is a CD8+ T cell.

59. 59. The molecule of any one of claims 56 to 58, which targets CD8.

60. The ankyrin repeat protein comprises a repeating consensus sequence: NX １ X ２ DX ３ X ４ X ５ X ６ TPX ７ HLX ８ X ９ X １０ X １１ X １２ HX １３ X １４ IVX １５ VLLKX １６ X １７ X １８ DX １９ a repeat module including where: X １ is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, X ２ is any amino acid, X ３ is any amino acid, X ４ is any amino acid, X ５ is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably G; X ６ is any amino acid, X ７ is any amino acid, preferably an amino acid selected from the group consisting of A, C, F, G, H, I, K, L, M, R, T, V, W, and Y, more preferably L; X ８ is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably A or V; X ９ is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably A; X １０ is any amino acid, X １１ is any amino acid, X １２ is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably G; X １３ is any amino acid, preferably L, X １４ is any amino acid, preferably an amino acid selected from the group consisting of D, E, H, K and R, more preferably E; X １５ is any amino acid, preferably D or E, X １６ is any amino acid, X １７ is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably an amino acid selected from the group consisting of A, G and S, more preferably G; X １８ is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably A; X １９ is any amino acid, preferably an amino acid selected from the group consisting of I, L and V, more preferably V; 60. A molecule according to any one of claims 56 to 59.

61. The ankyrin repeat protein comprises a repeating consensus sequence: ︚ １ ︸ ２ ︤ ３ ︸ ４ ︹ ６ !! ８ ︸ ９ ︸ １０ ︸ １１ H １３ ︸ １４ ︩︹ １５ ________________ １６ .. a repeat module including where: X １ is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, X ２ is any amino acid, X ３ is any amino acid, X ４ is any amino acid, X ６ is any amino acid, X ８ is A or V, X ９ is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V, more preferably A; X １０ is any amino acid, X １１ is any amino acid, X １３ is any amino acid, preferably L, X １４ is any amino acid, preferably an amino acid selected from the group consisting of D, E, H, K and R, more preferably E; X １５ is any amino acid, preferably D or E, X １６ is any amino acid, A molecule according to any one of claims 56 to 60.

62. The ankyrin repeat protein comprises a repeating consensus sequence: NX １ X ２ DX ３ X ４ GX ６ TPLHLX ８ AX １０ X １１ GHLEIVX １５ VLLKX １６ GADV a repeat module including where: X １ is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V; X ２ is any amino acid, X ３ is any amino acid, X ４ is any amino acid, X ６ is any amino acid, X ８ is A or V, X １０ is any amino acid, X １１ is any amino acid, X １５ is D or E, X １６ is any amino acid, A molecule according to any one of claims 56 to 61.

63. 63. The molecule of any one of claims 60 to 62, wherein the ankyrin repeat protein comprises at least two repeat modules, which may be identical or different, each comprising the repeat consensus sequence.

64. 64. The molecule of any one of claims 60 to 63, wherein the ankyrin repeat protein comprises 2 to 20 repeat modules, which may be identical or different, each comprising the repeat consensus sequence.

65. 65. The molecule of any one of claims 60 to 64, wherein the ankyrin repeat protein comprises three repeat modules, which may be identical or different, each comprising the repeat consensus sequence.

66. The ankyrin repeat protein comprises three repeat modules, wherein: The first repeat module of the targeting molecule has the consensus sequence: NAX ２ DX ３ X ４ GX ６ TPLHLX ８ AWHGHLEIVX １５ VLLKX １６ GADV、 Including, The second repeat module of the targeting molecule has the consensus sequence: !?? ２ ︤ ３ ︸ ４ ︹ ６ _________________________ １０ ︸ １１ _|||||||||_______ １６ ..|..、 and The third repeat module of the targeting molecule has the consensus sequence: NX １ X ２ DX ３ X ４ GX ６ TPLHLAAX １０ X １１ GHLEIVEVLLKX １６ GADV、 Including, where: X １ is any amino acid, preferably an amino acid selected from the group consisting of A, C, D, G, N, P, S, T and V; X ２ is any amino acid, X ３ is any amino acid, X ４ is any amino acid, X ６ is any amino acid, X ８ is A or V, X １０ is any amino acid, X １１ is any amino acid, X １５ is D or E, X １６ is any amino acid, preferably an amino acid selected from the group consisting of Y, H and N, A molecule according to any one of claims 56 to 65.

67. 67. The molecule of any one of claims 56 to 66, wherein said ankyrin repeat protein comprises at least one repeat module comprising a sequence selected from the group of repeat modules 1, 2 and 3 of SEQ ID NOs: 1 to 28, respectively, as shown in Figure 5.

68. 68. The molecule of any one of claims 56 to 67, wherein said ankyrin repeat protein comprises three repeat modules, wherein repeat module 1 is selected from the group of repeat module 1 of SEQ ID NOs: 1 to 28 as shown in Figure 5, repeat module 2 is selected from the group of repeat module 2 of SEQ ID NOs: 1 to 28 as shown in Figure 5 and repeat module 3 is selected from the group of repeat module 3 of SEQ ID NOs: 1 to 28 as shown in Figure 5.

69. 69. The molecule of any one of claims 56 to 68, wherein said ankyrin repeat protein comprises three repeat modules, wherein repeat module 1, repeat module 2 and repeat module 3 are repeat module 1, repeat module 2 and repeat module 3 of a sequence selected from the group consisting of SEQ ID NOs: 1 to 28 as shown in Figure 5.

70. 70. The molecule of any one of claims 60 to 69, wherein said repeat module is present in a repeat domain.

71. 71. The molecule of claim 70, wherein the repeat domain further comprises an N-terminal and / or C-terminal capping module.

72. 72. The molecule of any one of claims 56 to 71, wherein the ankyrin repeat protein comprises a sequence selected from the group consisting of SEQ ID NOs: 1 to 28.

73. 73. The molecule of any one of claims 56 to 72, further comprising another peptide or protein moiety, optionally fused to said ankyrin repeat protein.

74. 74. The molecule of any one of claims 56 to 73, which is a polypeptide compound.

75. 74. The molecule of any one of claims 56 to 73, further comprising a lipid or lipid-like component or another non-peptide component.

76. A nucleic acid encoding the molecule of any one of claims 56 to 74.

77. 77. A host cell comprising the nucleic acid of claim 76, optionally expressing said molecule.

78. A particle comprising a molecule according to any one of claims 56 to 75.

79. 79. The particle of claim 78, further comprising a nucleic acid encoding an antigen receptor.

80. 80. The particle of claim 79, wherein the antigen receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

81. 81. The particle of claim 79 or 80, wherein the antigen is associated with a disease, disorder or condition.

82. The particle of any one of claims 79 to 81, wherein the antigen is a tumor-associated antigen.

83. 83. The particle of any one of claims 79 to 82, wherein the nucleic acid is RNA.

84. 83. The particle of any one of claims 79 to 82, wherein the nucleic acid is DNA.

85. 85. The particle of any one of claims 78 to 84, which is a non-viral particle.

86. 86. The particle of any one of claims 78 to 85, which is a lipid-based and / or polymer-based particle.

87. 87. The particle of any one of claims 78 to 86, which is a nanoparticle.

88. 88. The particle of any one of claims 78 to 87, wherein the particle is functionalized with the ankyrin repeat protein on its surface.

89. 89. The particle according to any one of claims 78 to 88, wherein the particle is functionalized with the ankyrin repeat protein by linking the ankyrin repeat protein to at least one particle-forming component.

90. 90. A composition comprising a molecule according to any one of claims 56 to 75, a particle according to any one of claims 78 to 89, or a plurality thereof.

91. 90. A pharmaceutical composition comprising a molecule according to any one of claims 56 to 75, a particle according to any one of claims 78 to 89, or a plurality thereof.

92. 92. A kit comprising a molecule according to any one of claims 56 to 75, a nucleic acid according to claim 76, a host cell according to claim 77, a particle according to any one of claims 78 to 89, a composition according to claim 90, or a pharmaceutical composition according to claim 91.

93. 93. The kit of claim 92, further comprising instructions for using the kit in the method of any one of claims 1 to 55.

94. 90. A particle or a plurality thereof according to any one of claims 78 to 89 for use in a method according to any one of claims 1 to 55.

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